JP2019108371A - Targeted nanoparticles - Google Patents

Abstract

To provide nanoparticles for effective and specific delivery of drugs.SOLUTION: A carrier nanoparticle comprises a polyol-containing polymer coupled through a reversible covalent linkage to a boronic acid-containing polymer, particularly a polymer containing a nitrophenylboronic acid group. The nanoparticle is configured to present the boronic acid-containing polymer to an external environment. The boronic acid-containing polymer is conjugated to a targeting ligand at its terminal end opposite the nanoparticle.SELECTED DRAWING: Figure 16

Description

Government Rights This invention was made with Government support under Grant CA 119347 awarded by the National Cancer Institute. The government has certain rights in the invention.

  The present disclosure relates to carrier nanoparticles, particularly nanoparticles suitable for delivering the subject compounds, as well as related compositions, methods, and systems.

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

  Several methods are known, whether for pathological examination, for therapeutic treatment, or for basic biological studies, and typically the biological and / or chemical properties of interest. It has been used to deliver various classes of biomaterials and biomolecules related to biological activity.

  As the number of molecules suitable for use as chemical or biological agents (eg, drugs, biologics, therapeutics, or imaging agents) increases, it is suitable for use with compounds of varying complexity, size, and chemistry. Development of such delivery systems has proved particularly difficult.

  Nanoparticles are structures useful as carriers for delivering agents using various delivery methods. Several nanoparticle delivery systems exist, which utilize a variety of different strategies to package, transport, and deliver agents to specific targets.

  In some embodiments, provided herein are nanoparticles that provide multifunctional tools for the effective and specific delivery of a subject compound, as well as related compositions, methods, and systems. Specifically, in some embodiments, the nanoparticles described herein are flexible systems for delivering and delivering a wide range of molecules of various sizes, sizes, and chemistries to a given target. It can be used as

  According to one aspect, nanoparticles comprising a polyol containing polymer and a boronic acid containing polymer are described. In the nanoparticles, the boronic acid containing polymer is coupled to a polyol containing polymer, and the nanoparticles are adapted to present the boronic acid containing polymer in the external environment of the nanoparticles. One or more target compounds may be carried by the nanoparticles as part of or combined with a polyol containing polymer and / or a boronic acid containing polymer.

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

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

  According to another aspect, a system for delivering a compound to a target is described. The system comprises at least a polyol-containing polymer and a boronic acid-containing polymer capable of being linked to one another through a reversible covalent bond to construct the nanoparticles described herein comprising the compound.

  According to another aspect, a method of administering a compound to an individual is described. The method comprises the step of administering to the individual an effective amount of the nanoparticles described herein, the compound being contained 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. The above system comprises a polyol containing polymer capable of being linked together through a reversible covalent bond that constructs the nanoparticles described herein attached to a compound to be administered to an individual according to the methods described herein and It contains at least a boronic acid-containing polymer.

  According to another aspect, a method of preparing nanoparticles comprising a polyol containing polymer and a boronic acid containing polymer is described. The method comprises the steps of contacting a polyol containing polymer with a boronic acid containing polymer under certain conditions for coupling the polyol containing polymer and 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, some polyol-containing polymers described in detail in the following sections of the disclosure are described.

  Also described herein are nanoparticles having a polyol containing polymer conjugated to a polymer having a nitrophenylboronic acid group, which enhances the stability of the nanoparticle by reducing its pKa.

  Another aspect of the present disclosure is that, in some embodiments, the targeting ligand can be one that can facilitate delivery of the nanoparticle to a particular target, such as a cell expressing a binding partner of the target ligand of the particle. Provides a description of targeting nanoparticles that can only have one. Targeted nanoparticles of this kind are superior to, for example, nanoparticles comprising multiple target ligands with smaller overall size by having less surface ligands, and not affinity based interactions There is less ligand to mediate nonspecific binding through binding based interactions. Furthermore, nanoparticles containing or carrying a therapeutic agent can be successfully targeted to the target location (such as cells or tissues) with only a single targeting ligand, thereby providing a very high targeting ligand pair The therapeutic agent is delivered to the target at a therapeutic agent ratio. This aspect of the nanoparticles described can significantly increase the efficiency of producing such therapeutic agents while reducing the need to use multiple expensive antibodies to mediate targeting.

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

  The nanoparticles described herein, as well as related compositions, methods, and systems, protect the transported compounds from degradation, recognition by the immune system, and loss due to combination with serum proteins or blood cells. Can be used in some embodiments as a delivery system capable of

  The nanoparticles described herein, as well as related compositions, methods, and systems, are steric stabilization and / or tissues, particular cell types within tissues, and even particular cells within particular cell types. It can be used in some embodiments as a delivery system characterized by the ability to deliver a compound to a specific target, such as an internal site.

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

  The nanoparticles described herein, as well as related compositions, methods, and systems, have enhanced specificity and / or selectivity during targeting and / or targeting relative to certain systems in the art. Can be used in some embodiments to deliver the compound with enhanced recognition of the compound.

  The nanoparticles described herein, as well as related compositions, methods, and systems, include medical applications such as, but not limited to, therapeutic, diagnostic, and clinical applications, where controlled delivery of a subject compound is desirable Can be used in some embodiments. Further applications include biological analysis, veterinary applications, as well as delivery of the subject compounds in organisms other than animals, in particular plants.

  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, the examples, and the drawings, and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated in and form a part of the specification, illustrate one or more embodiments of the disclosure, together with the detailed description and examples, to explain the principles and practices of the disclosure. To illustrate.
FIG. 1 shows a schematic of nanoparticles and related formation methods in the absence of boronic acid containing compounds. Panel A shows a schematic of a polyol (MAP, 4) containing polymer and a subject compound (nucleic acid) according to one embodiment described herein. Panel B shows nanoparticles formed by constructing the polyol-containing polymer and compound 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 together with a molecule of interest (nucleic acid) according to an embodiment of the present disclosure. Panel B shows BA-pegylated stabilized nanoparticles formed by constructing the polymers and compounds shown in panel A. FIG. 7 shows the formation of a complex comprising a polyol containing polymer according to one embodiment described herein and a subject compound. Specifically, FIG. 3 shows the results of a MAP gel retardation assay using plasmid DNA according to an embodiment of the present disclosure. Load the DNA ladder into lane 1. Lanes 2-8 show plasmid DNA combined with MAP with gradually increasing charge ratio. The charge ratio is defined as the amount of positive charge in the MAP divided by the amount of negative charge in the nucleic acid. FIG. 7 shows the formation of a complex comprising a polyol containing polymer according to one embodiment described herein and a subject compound. Specifically, FIG. 4 shows the results of a MAP gel retardation assay using siRNA according to an embodiment of the present disclosure. Load the DNA ladder into lane 1. Lanes 2-8 show siRNA combined with MAP with gradually increasing charge ratio. 8 illustrates the properties of nanoparticles according to some embodiments described herein. Specifically, FIG. 5 shows the particle size (as determined from dynamic light scattering (DLS) measurements) to the charge ratio, and the zeta potential to the charge ratio of the MAP-plasmid nanoparticles according to one embodiment of the present disclosure ( FIG. 2 shows a plot of the properties related to the surface charge of the nanoparticles). 8 illustrates the properties of nanoparticles according to some embodiments described herein. Specifically, FIG. 6 shows a plot of particle size (DLS) versus charge ratio, and zeta potential versus charge ratio of BA-pegylated MAP-plasmid nanoparticles according to one embodiment of the present disclosure. 16 shows salt stability of BA-pegylated MAP-plasmid nanoparticles according to one embodiment disclosed herein. Plot A: 5: 1 BA-PEG + np + 1 × PBS after 5 minutes; Plot B: 5: 1 BA-PEG + np, dialysis 3 × w / 100 kDa + 1 × PBS after 5 minutes; plot C: 5: 1 pre-pegylated w / BA-PEG + 1 X PBS 5 min; plot D: 5: 1 pre-pegylated w / BA-PEG, dialysis 3 x w / 100 kDa + PBS 5 min. FIG. 16 shows in vitro delivery of agents to human cells using nanoparticles according to one embodiment described herein. Specifically, FIG. 8 is a relative light indicator indicative of the amount of luciferase protein expressed from pGL3 plasmid delivered to cells for transfection of HeLa cells with MAP / pGL3 according to an embodiment of the present disclosure FIG. 6 shows a plot of units (RLU) to charge ratio. FIG. 16 shows delivery of an agent to a target using nanoparticles according to one embodiment described herein. Specifically, FIG. 9 shows a plot of cell viability versus charge ratio after MAP / pGL3 transfection according to one embodiment of the present disclosure. Survival data are for the experiment shown in FIG. FIG. 7 shows delivery of multiple compounds to a target using nanoparticles according to one embodiment described herein. Specifically, FIG. 10 shows relative light units for co-transfection of MAP particles containing HeLa cells with pGL3 and siGL3 with charge ratios +/- 5 according to one embodiment described herein. FIG. 7 shows a plot of (RLU) versus particle type. The expression siCON denotes an siRNA with a control sequence. FIG. 16 shows delivery of a compound to a target using nanoparticles according to one embodiment described herein. Specifically, FIG. 11 shows relative light units (RLU) versus siGL3 for delivery of MAP / siGL3 to HeLa-Luc cells with charge ratio +/− 5 according to one embodiment described herein. FIG. 6 shows a plot of the concentration of FIG. 1 shows a schematic diagram of the synthesis of a boronic acid containing polymer presenting a target ligand according to some embodiments described herein. Specifically, FIG. 12 shows an outline of the synthesis of boronic acid-PEG disulfide-transferrin according to one embodiment of the present disclosure. FIG. 1 shows a schematic of the synthesis of nanoparticles according to some embodiments described herein. Specifically, FIG. 13 shows an outline of the formation of nanoparticles comprising camptothecin munic acid polymer (CPT-munic 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. Specifically, 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 one embodiment of the present disclosure. Figure 20 shows the salt stability of MAP-4 / siRNA stabilized with nitroPEG-PEG and with 0.25 mol% Herceptin-PEG-nitroPBA without PEG. For formulations at a charge ratio of 3 (+/−), 10 × PBS was added in 5 minutes so that the resulting solution is 1 × PBS. (A) Targeted MAP-CPT nanoparticles with increasing ratio of HERCEPTIN®-PEG-nitro PBA to nanoparticles in BT-474, (B) BT-474 (black bar) and MCF- FIG. 7 shows cellular uptake of MAP-CPT nanoparticles or targeted MAP-CPT nanoparticles in or without free Herceptin® (10 mg / mL) in 7 (white bar) cell lines. A and B show plasma pharmacokinetics of short, medium and long MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles in BALB / c mice injected with 10 mg CPT / kg. Free CPT injected at 10 mg / kg into CD2F1 mice is shown as a comparison. (A) Plasma concentration of CPT bound to polymer as a function of time. (B) Plasma concentrations of unconjugated CPT as a function of time. Figure 7 shows the biodistribution of MAP-CPT (5 mg CPT / kg) and targeted MAP-CPT (5 mg CPT / kg, 29 mg HERCEPTIN® / kg) nanoparticles after 4 and 24 hours of treatment. (A) Tumor, heart, liver, spleen, kidney, or total CPT injected rate (%) per gram of lung (B) Conjugated in tumor, heart, liver, spleen, kidney, and lung Percentage of total CPT in each organ not present (C) Total concentration of CPT in plasma (D) Percentage of total unconjugated CPT in plasma. Figure 7 shows the biodistribution of MAP-CPT (5 mg CPT / kg) and targeted MAP-CPT (5 mg CPT / kg, 29 mg HERCEPTIN® / kg) nanoparticles after 4 and 24 hours of treatment. (A) Tumor, heart, liver, spleen, kidney, or total CPT injected rate (%) per gram of lung (B) Conjugated in tumor, heart, liver, spleen, kidney, and lung Percentage of total CPT in each organ not present (C) Total concentration of CPT in plasma (D) Percentage of total unconjugated CPT in plasma. Figure 7 shows the biodistribution of MAP-CPT (5 mg CPT / kg) and targeted MAP-CPT (5 mg CPT / kg, 29 mg HERCEPTIN® / kg) nanoparticles after 4 and 24 hours of treatment. (A) Tumor, heart, liver, spleen, kidney, or total CPT injected rate (%) per gram of lung (B) Conjugated in tumor, heart, liver, spleen, kidney, and lung Percentage of total CPT in each organ not present (C) Total concentration of CPT in plasma (D) Percentage of total unconjugated CPT in plasma. Figure 7 shows the biodistribution of MAP-CPT (5 mg CPT / kg) and targeted MAP-CPT (5 mg CPT / kg, 29 mg HERCEPTIN® / kg) nanoparticles after 4 and 24 hours of treatment. (A) Tumor, heart, liver, spleen, kidney, or total CPT injected rate (%) per gram of lung (B) Conjugated in tumor, heart, liver, spleen, kidney, and lung Percentage of total CPT in each organ not present (C) Total concentration of CPT in plasma (D) Percentage of total unconjugated CPT in plasma. (A) MAP-CPT at 4 hours (5 mg CPT / kg), (B) MAP-CPT at 24 hours (5 mg CPT / kg), (C) Targeted MAP-CPT at 4 hours (5 mg CPT / kg, 29 mg Herceptin (registration (DTM) / (D) Confocal immunofluorescence of BT-474 tumor sections taken from NCr nude mice treated with targeted MAP-CPT (5 mg CPT / kg, 29 mg HERCEPTIN® / kg) at 24 hours The microscope image is shown. Left panel: emission 440 nm (CPT, pink), center left panel: emission 519 nm (MAP, green), center right panel: emission 647 nm (Herceptin®, blue), right panel: overlay of images. 12 shows anti-tumor efficacy tests in NCr nude mice bearing BT-474 xenograft tumors. Mean tumor volume as a function of time, groups receiving Herceptin twice weekly, all other groups receiving three times weekly. Show anti-tumor efficacy test in NCr nude mice treated with (A) 5.9 mg / kg Herceptin, (B) targeted MAP-CPT nanoparticles (5.9 mg Herceptin® / kg and 1 mg CPT / kg) .

  Nanoparticles that can be used in connection with the delivery of a subject compound (also referred to herein as cargo), and related compositions, methods, and systems, methods of making such nanoparticles, methods of treatment using such nanoparticles And kits for constructing the above-described nanoparticles are provided herein.

  The term "nanoparticle" as used herein refers to composite structures of nanoscale dimensions. Specifically, the nanoparticles are typically particles in the size range of about 1 to about 1000 nm and are usually spherical, but different forms are possible depending on the composition of the nanoparticles. The portion of the nanoparticle that contacts the external environment of the nanoparticle is generally identified as the surface of the nanoparticle. In the nanoparticles described herein, size limitations may be limited to two dimensions, such that the nanoparticles described herein comprise composite structures having a diameter of about 1 to about 1000 nm. In the case, the specific diameter depends on the composition of the nanoparticles and the intended use of the nanoparticles according to the experimental design. For example, nanoparticles used in some therapeutic applications typically have a size of about 200 nm or less, and in particular, those used for delivery related to cancer treatment are typically about 1 With a diameter of about 100 nm. The term "targeted nanoparticle" refers to a nanoparticle that is conjugated to a targeting agent or ligand.

Also, further desirable properties of the nanoparticles, such as surface charge and steric stabilization, may vary in view of the particular target application. Some of the exemplary properties that may be desirable in clinical applications, such as cancer treatment, are described in the scientific literature. Additional features can be identified 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 well known in the art. Exemplary techniques for detecting particle dimensions include dynamic light scattering (DLS) and various microscopy methods such as transmission electron microscopy (TEM) and atomic force microscopy (AFM). Not limited to these. Exemplary techniques for detecting particle morphology include, but are not limited to, TEM and AFM. Exemplary techniques for detecting the surface charge of nanoparticles include, but are not limited to, zeta potential methods. Additional techniques suitable for detecting other chemical properties include ¹ H, ¹¹ B, and ¹³ C, and ¹⁹ F NMR, UV / Vis and infrared / Raman spectroscopy, and fluorescence spectroscopy (nanoparticles When used in conjunction with fluorescent labels), as well as additional techniques identifiable by one skilled in the art.

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

  The terms "deliver" and "delivery" as used herein refer to an activity that affects the spatial location of a compound, in particular, an activity that controls said location. Thus, delivery of a compound in the sense of the present disclosure affects the arrangement and movement of the compound for a specific time under a specific set of conditions, so that the arrangement and movement of the compound under these conditions is not In some cases, it refers to the ability of the compound to change with respect to the configuration and movement.

  Specifically, delivery of a compound to a reference endpoint refers to the ability to control the placement and movement of the compound such that the compound is ultimately placed at the selected reference endpoint. In in vitro systems, delivery of a compound is usually associated with the corresponding modification of the chemical and / or biological detectable property and activity of the compound. In in vivo systems, delivery of a compound is typically associated with modification of the pharmacokinetics and optionally the pharmacodynamics of the compound.

  The pharmacokinetics of a compound typically refers to the absorption, distribution, metabolism, and excretion of the compound from the system provided by the individual's body. In particular, the term "absorption" refers to the process by which substances enter the body, the term "distribution" refers to the distribution or interspersion of substances throughout fluids and tissues of the body, and the term "metabolism" It refers to the irreversible conversion of the parent compound to the daughter metabolite, and the term "excretion" refers to the elimination of matter from the body. If the compound is present in a formulation, pharmacokinetics also includes the release of the compound from the formulation, which is typically indicative of the process of release from the formulation of the drug. The term "pharmacodynamics" refers to the physiological effect of the compound on the body, or in a microorganism or parasite on the body, and the mechanism of action of the drug, and the relationship between drug concentration and effect. One of ordinary skill in the art can identify techniques and procedures suitable for detecting the pharmacokinetics and pharmacodynamic characteristics and properties of the subject compounds, specifically, the subject agents such as drugs.

  The term "agent" as used herein refers to a compound capable of exhibiting chemical or biological activity associated with a target. The term "chemical activity" as used herein refers to 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 the organism. Exemplary chemical activities of the agent include the formation of covalent or electrostatic interactions. Exemplary 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, refers to a subject biological system comprising a unicellular or multicellular organism or part thereof, including in vitro or in vivo biological systems or parts thereof.

  The nanoparticles herein are described as being arranged in the nanoparticles such that the boronic acid containing polymer coupled to the polyol containing polymer is presented to the external environment of the nanoparticles.

  The term "polymer" as used herein typically refers to large molecules consisting of repeating structural units connected by covalent chemical bonds. Suitable polymers may be linear and / or branched and may take the form of homopolymers or copolymers. If a copolymer is used, the copolymer may be a random copolymer or a branched copolymer. Exemplary polymers include water dispersible, especially 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 medical applications and applications, the polymer should have a low toxicity profile, in particular neither toxic nor cytotoxic. 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 refer to a polymer presenting a plurality of hydroxyl functional groups. Specifically, a polyol-containing polymer suitable for forming the nanoparticles described herein comprises at least a portion of hydroxyl functional groups for coupling interaction with at least one boronic acid of a boronic acid-containing polymer Containing a polymer that presents

  The term "present", as used herein in connection with a compound or functional group, refers to a bond that is carried out to maintain the chemical reactivity of the compound or functional group when bound. Thus, the functional groups presented on the surface can perform one or more chemical reactions that chemically characterize the functional group under appropriate conditions.

The structural units that form the polyol containing polymer include pentaerythritol, ethylene glycol, and monomeric polyols such as glycerin. Exemplary polyol containing polymers include polyesters, polyethers, and polysaccharides. Exemplary suitable polyethers include diols such as polyethylene glycol, polypropylene glycol, and poly (tetramethylene ether) glycol, and in particular, the general formula HO- (CH ₂ CH ₂ O) _p -H (formula Among them, diols having p of 1 or more) can be mentioned, but not limited thereto. Exemplary suitable polysaccharides include, but are not limited to, cyclodextrin, starch, glycogen, cellulose, chitin, and β-glucan. Exemplary suitable polyesters include, but are not limited to, polycarbonate, polybutyrate, and polyethylene terephthalate (all terminated with hydroxyl end groups). Exemplary polyol-containing polymers include polymers having a molecular weight of about 500,000 or less, and particularly about 300 to about 100,000.

  Polyol containing polymers are commercially available and / or can be produced using techniques and procedures that can be identified by one skilled in the art. Exemplary procedures for synthesizing exemplary polyol polymers have already been described in the scientific literature, and other procedures are illustrated in Examples 1-4. Additional procedures for making 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 refers to a polymer containing at least one boronic acid group present for attachment to a hydroxyl group of a polyol containing polymer. Specifically, the boronic acid-containing polymer of the nanoparticles described herein comprises, in at least one structural unit, a polymer comprising an alkyl or aryl substituted boronic acid containing a carbon-boron chemical bond. Suitable BA polymers include polymers in which the boronic acid is present at the terminal structural unit or any other suitable position in order to render the resulting polymer hydrophilic. Exemplary polyol containing polymers include polymers having a molecular weight of about 40,000 or less, in particular about 20,000 or less or about 10,000 or less.

  Boronic acid 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. Exemplary procedures for synthesizing exemplary polyol polymers are already described in the scientific literature, and other novel procedures are illustrated 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 to the BA polymer. The terms "couple" or "coupling" as used herein in the context of binding between two molecules refer to an interaction that forms a reversible covalent bond. Specifically, in the presence of a suitable medium, the boronic acid presented on the BA polymer interacts with the hydroxyl group of the polyol via rapid and reversible pair-covalent interactions, which is preferred Form boronate esters in various media. Suitable media include water and some aqueous solutions, as well as additional organic media that can be identified by one skilled in the art. Specifically, when contacted in an aqueous medium, the BA polymer and the polyol polymer react to form water as a by-product. It is also known that the boronic acid-polyol interaction generally proceeds in an organic medium, although it is more preferred in aqueous solution. Furthermore, cyclic esters formed with 1,2 and 1,3 diols are generally more stable than their non-cyclic ester counterparts.

Thus, in the nanoparticles described herein, at least one boronic acid of the boronic acid containing polymer is reversibly covalently linked to the hydroxyl group of the polyol containing polymer. Formation of boronic esters between BA polymers and polyol polymers can be done by methods and techniques identifiable by those skilled in the art, such as boron-11 nuclear magnetic resonance ( ¹¹ B NMR), potentiometric titration, UV / Vis and fluorescence detection techniques. It can be detected, whereby the technology of choice depends on the specific chemistry and properties of the nanoparticles, including the boronic acid and the polyol.

  The nanoparticles resulting from the coupling interaction of the BA polymer described herein with the polyol polymer described herein present the BA polymer on the surface of the particle. In some embodiments, the nanoparticles may have a diameter of about 1 to about 1000 nm and a spherical morphology, but the size and morphology of the particles are, for the most part, specific BAs used to form the nanoparticles. It is determined by the polymer and the polyol polymer, as well as by the compounds carried on the nanoparticles according to the present disclosure.

In some embodiments, the target compound carried by the nanoparticles forms part of the BA polymer and / or the polyol polymer. An example of such an embodiment is for imaging a target as provided by nanoparticles in which one or more atoms of the polymer are replaced by particular isotopes (eg ¹⁹ F and ¹⁰ B) and It is suitable as an agent for subjecting the target to radiation treatment.

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

  The terms "couple," "couple," or "couple" as used herein are a joint, connection, force, or linkage connection or integral to hold two or more components together. (For example, the first compound is directly conjugated to the second compound, and one or more intermediate compounds, in particular molecules, are arranged between the first compound and the second compound). Including direct or indirect binding as in the embodiments described above.

Specifically, in some embodiments, the compound can be attached to the polyol polymer or BA polymer via covalent attachment of the compound to a suitable portion of the polymer. An exemplary covalent attachment is illustrated in Example 19 where attachment of the drug camptothecin to the mucic acid polymer is carried out via biodegradable ester attachment, and attachment of transferrin to BA-PEG ₅₀₀₀ via PEGylation of transferrin. It is illustrated in Example 9 to be implemented.

  In some embodiments, the polymer is designed to be capable of binding to a particular target compound, eg, by adding one or more functional groups capable of specifically binding to the corresponding functional group on the target compound. Or can be modified. For example, in some embodiments, BA-PEG-X, wherein X is a maleimide or iodoacetyl group or any leaving group that specifically reacts with thiols or non-specifically with amines The nanoparticles can be pegylated with The compound to be bound can then be reacted to a maleimide or iodoacetyl group after being modified to express a thiol functional group. The compound to be bound may also be modified with an aldehyde or ketone group, which can react with the diol in the polyol via a condensation reaction to give an acetal or ketal.

  In some embodiments, the compound of interest may be 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 the suitable moieties of the polymer. it can. An exemplary noncovalent bond is illustrated in Example 10.

  The compounds of interest can be attached to the nanoparticles before, during or after formation of the nanoparticles, for example, via modification of the polymer and / or any attached compounds in the particulate composite. An exemplary procedure for carrying out the conjugation of compounds in nanoparticles is illustrated in the example section. Additional procedures for attaching compounds to the BA polymers, polyol polymers or other components of the nanoparticles described herein (e.g., previously introduced target compounds) are identified by those skilled in the art when reading this disclosure It is possible.

  In some embodiments, at least one subject compound attached to a BA polymer presented on a nanoparticle described herein is an agent that can be used as a targeting ligand. Specifically, in some embodiments, nanoparticles are delivered to selected targets in one or more agents used as targeting ligands in BA polymers, and in polyol polymers and / or BA polymers 1 Combine with one or more agents.

  The terms "targeting ligand" or "targeting agent" as used in the present disclosure, are specified specifically to allow the nanoparticle to bind to a cell receptor, for example to associate with a specific target. Refers to any molecule that can be presented on the surface of a nanoparticle for the purpose of recognizing cells of Examples of suitable ligands include, but are not limited to, vitamins (eg, folic acid), proteins (eg, transferrin and monoclonal antibodies), monosaccharides (eg, galactose), peptides, and polysaccharides. Specifically, targeting ligands may be antibodies to specific surface cell receptors such as anti-VEGF, small molecules such as folate, and other proteins such as holotransferrin.

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

  In some embodiments, at least one of the compounds contained or bound to the polyol polymer and / or the BA polymer (including the targeting ligand) is a target, eg, chemical or biological activity, For example, it may be an agent, in particular a drug, delivered to the individual in which the therapeutic activity is exerted.

  Selection of polyol polymers and BA polymers suitable for forming the nanoparticles described herein may be performed in view of the subject compounds and targets. Specifically, the selection of suitable polyol containing polymers and suitable BA polymers to form the nanoparticles described herein provides candidate polyol polymers and BA polymers, and in the sense of the present disclosure, coupling The selection can be carried out by selecting a polyol polymer and a BA polymer capable of forming an action, wherein the selected BA polymer and the polyol polymer are contained in or bound to the polyol polymer and / or the BA polymer In view of the target compound and the target ligand, the polyol polymer has a chemical composition lower than that of the BA polymer. The detection of the BA polymer on the surface of the nanoparticles and the relevant presentation in the external environment of the nanoparticles is carried out by detection of the zeta potential which can indicate a modification of the surface of the nanoparticles, as illustrated in example 12. Can. (Specifically, refer to FIG. 6). As a further procedure for detecting the surface charge of the particles and the stability of the particles in the salt solution, detection of the change of the particle size such as that exemplified in Example 12 (specifically, FIG. 7 Reference) and additional procedures which can be identified by the person skilled in the art.

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

[In the formula,
A is the organic moiety of the following formula:

(In the formula,
R ¹ and R ₂ are independently selected from any carbon-based or organic group having a molecular weight of about 10 kDa or less
X is independently selected from aliphatic groups containing one or more of -H, -F, -C, -N, or -O;
Y is independently, -OH, or _{-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 Hydroxyl (-OH) groups including, but not limited to, G ₃ ) OH, wherein R ₁ G ₁ , R ₁ G ₂ and R ₁ G ₃ are independently organic based functional groups Is selected from organic moieties having
B is one of ^{R 1} and _{R 2} of the first part A, which is one organic moiety linking of ^{R 1} and _{R 2} of the second A portion.

  The term "moiety" as used herein refers to a group of atoms that make up a larger molecule or part of a molecular species. Specifically, a moiety refers to a component of a repeating polymer structural unit. Exemplary moieties include acid or base species, sugars, carbohydrates, alkyl groups, aryl groups, and constituents of any other molecule useful in forming a polymer structural unit.

  The term "organic moiety" as used herein refers to a moiety containing a carbon atom. Specifically, organic groups 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 target compounds may be linked to (A), (B), or (A) and (B).

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

(In the formula,
d is 0 to 100,
e is 0 to 100,
f is 0 to 100,
Z is a covalent bond linking one organic moiety to another, in particular another A or B as defined herein,
Z ₁ is independently selected from -NH ₂ , -OH, -SH, and -COOH).

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

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

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 .

  In some embodiments, for the polyol-containing polymer of the particles described herein, (A) may be independently selected from the following formula:

(In the formula,
The spacer may independently be selected from any organic moiety, and in particular may comprise an alkyl, phenyl or alkoxy group optionally containing a heteroatom such as sulfur, nitrogen, oxygen or fluorine.
The amino acids are selected from free amines and any organic groups having free carboxylic acid groups,
n is 1 to 20,
Z ₁ is independently selected from -NH ₂ , -OH, -SH, and -COOH).

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

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

  In some embodiments, (B) may 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) is a neutral, cationic or anionic organic group whose nature and composition depend on the chemical nature of the compound to be covalently or noncovalently tethered. Contains

  Exemplary cationic moieties of (B) for use with anionic cargoes include amidine groups, quaternary ammonium, primary amine groups, secondary amine groups, tertiary amine groups (protonated below its pKa), and Although the organic group which has imidazolium is mentioned, it is not limited to these.

  Exemplary anionic moieties contained in (B) for use with cationic cargos include, but are not limited to, sulfonates of the formula, nitrates of the formula, carboxylates of the formula, and organic groups having a phosphonate. I will not.

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

(Wherein R ₅ is an electrophilic group capable of covalently bonding to A when A contains a nucleophilic group). Examples of R ₅ in this case include, but are not limited to:
In particular, -C (= O) OH, -C (= O) Cl, -C (= O) NHS, -C (= NH ₂ ⁺ ) OMe, -S (= O) OCl-, -CH ₂ Br, Alkyl and aromatic esters, terminal alkynes, tosylates, and mesylates. When moiety A contains an electrophilic end group, R ₅ has a nucleophilic group such as -NH ₂ (primary amine), -OH, -SH, N ₃ , and a secondary amine.

Specifically, when the moiety (B) is a cationic moiety (B) for use with an anionic cargo, the "organic group" consists of C _m H _{2 m} (m is 1 or more) in general An amidine of formula (XIII), a quaternary ammonium of formula (XIV), a primary amine group of formula (XV), a dibasic amine of formula (XVI) It must contain at least one of a functional group including a secondary amine group, a tertiary amine group of formula (XVII) (protonated below its pKa), and an imidazolium of formula (XVIII):

In an embodiment, where the moiety (B) is an anionic moiety (B) for use with a cationic cargo, the “organic group” comprises a general formula consisting of C _m H _{2 m} (m is 1 or more) Of a sulfonate of formula (XIX), a nitrate of formula (XX), a carboxylate of formula (XXI), and a phosphonate of formula (XXII). Must contain at least one:

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

  In embodiments where (B) consists of a primary amine group of formula (XV) and / or a secondary amine group of formula (XVI), compounds containing a carboxylic acid group may also be linked via the formation of a peptide bond. it can.

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

(In the formula,
q may be 1 to 20, specifically, 5;
p is 20 to 200,
L is a leaving group).

The term "leaving group" as used herein refers to molecular fragments that leave with an electron pair in anisotropic bond cleavage. Specifically, the leaving group may be anionic or neutral molecules, ability leaving group leaves correlates with pK _a of the conjugate acid, pK _a is that lower is better desorption It is related to the basic ability. Exemplary anionic leaving group, Cl ^-, Br ^-, and I ^- halides such, and para - toluenesulfonate or "tosylate" (TsO ^-) include sulfonate esters such. Exemplary neutral molecular leaving groups are water (H ₂ O), ammonia (NH ₃ ), and alcohols (ROH).

  Specifically, in some embodiments, L may be chloride (Cl), methoxy (OMe), t-butoxy (OtBU), or N hydroxysuccinimide (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:

(In the formula,
n is 1 to 20, and specifically, may be 1 to 4).

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

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

(In the formula,
R ₃ and R ₄ may be independently selected from any hydrophilic organic polymer, specifically, independently, any poly (ethylene oxide), and a zwitterionic polymer,
X ₁ may be an organic moiety containing one or more of -CH, -N, or -B,
Y ¹ is an alkyl group having —C _m H _{2 m} — (m is 1 or more) optionally containing an olefin or alkynyl group, or an alkynyl group, or an aromatic such as phenyl, biphenyl, naphthyl or anthracenyl May be
r is 1 to 1000,
a is 0 to 3,
b is 0 to 3,
Functional group 1 and functional group 2 can be the same or different and can be attached to a target ligand, specifically 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 , wherein t is 2-2000, especially 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 may be a phenyl group.

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

In some embodiments, the functional group 1 and functional group 2 are the same or different, and _{independently, -B (OH) 2, -OCH} 3, is selected from -OH.

Specifically, the functional groups 1 and / or 2 of the formula (XXXI) may be cargo, in particular a functional group capable of binding to a target ligand such as a protein, an antibody or a peptide, or -OH, -OCH _3, or - (X) - (Y) -B (OH) may be a terminal group of _2, and the like.

  The term "functional group" as used herein refers to a particular group of atoms within its molecular structure or moiety that participates in the characteristic chemical reaction of that structure or moiety. Exemplary 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. is there. Specifically, functional groups in the sense of the present disclosure include carboxylic acids, amines, triarylphosphines, azides, acetylenes, sulfonyl azides, thio acids and aldehydes. Specifically, for example, the functional groups capable of binding to the corresponding functional groups in the target ligand may be selected to include the following binding partners: carboxylic acid group and amine group, azide and acetylene group, Azide and triaryl phosphine groups, sulfonyl azide and thio acids, and aldehydes and primary amines. Additional functional groups can be identified by one of ordinary skill in the art upon 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 may be referred to as corresponding functional group pairs.

The end group refers to a constituent unit present at the end of a macromolecule or oligomer molecule. For example, the terminal group of the PET polyester may be an alcohol group or a carboxylic acid group. End groups can be used to determine molecular weight. Exemplary end groups include -OH, -COOH, a NH _2, and OCH _3.

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

(In the formula, s is 20 to 300).

  Exemplary agents and target ligands capable of binding to the nanoparticles of the present disclosure include polynucleotides, nucleotides, aptamer polypeptides, proteins, polysaccharides, macromolecular complexes (protein and polynucleotides, saccharides and / or polysaccharides And mixtures thereof, including, but not limited to, organic or inorganic molecules including viruses, molecules including radioactive isotopes, antibodies, or antibody fragments.

  The term "polynucleotide" as used herein refers to 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 or pyrimidine base and a ribose or deoxyribose sugar conjugated to a phosphate group, and which is the basic structural unit of a nucleic acid. The term "nucleoside" refers to a compound consisting of a purine or pyrimidine base combined with deoxyribose or ribose, and in particular found in nucleic acids, such as guanosine or adenosine. The terms "nucleotide analogue" or "nucleoside analogue" refer to a nucleotide or nucleoside, respectively, in which one or more individual atoms are replaced by 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, nucleic acid aptamers are iterative rounds of in vitro selection to bind to various molecular targets such as, for example, small molecules, proteins, nucleic acids and even cells, tissues and organisms, or equivalently SELEX (test May contain nucleic acid species that have been modified through in vitro evolution). Aptamers are useful in biotechnological and therapeutic applications because they confer molecular recognition properties that compete with antibodies. Peptide aptamers are peptides that are designed to specifically bind to and interfere with protein-protein interactions inside cells. Specifically, peptide aptamers can be obtained, for example, according to a selection strategy derived from the yeast two hybrid (Y2H) system. Specifically, according to this strategy, variable peptide aptamer loops bound to the transcription factor binding domain are screened against target proteins bound 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 the downstream yeast marker gene.

  The term "polypeptide" as used herein refers to an organic linear, cyclic or branched polymer consisting of two or more amino acid monomers and / or their analogues. The term "polypeptide" includes analogs and fragments of these in addition to amino acid polymers of any length, including full length proteins and peptides. Polypeptides of three or more amino acids are also referred to as protein oligomers, peptides or oligopeptides. Specifically, the terms "peptide" and "oligopeptide" usually refer to a polypeptide comprising 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; D and L Includes both optical isomers. Specifically, non-naturally occurring amino acids include the D stereoisomers of naturally occurring amino acids (which are less susceptible to enzymatic degradation and, therefore, contain useful ligand building blocks). The term "artificial amino acids" refers to molecules having a molecular structure that can be easily coupled to one another using standard amino acid coupling chemistry, but that are not similar to natural amino acids. The term "amino acid analogue" refers to one or more of the individual atoms being substituted by different atoms, isotopes or different functional groups, but otherwise being identical to the original amino acid from which the analogue is derived Refers to an amino acid. All these amino acids can be synthetically incorporated into a peptide or polypeptide using standard amino acid coupling chemistry. The term "polypeptide" as used herein includes polymers comprising one or more monomers or basic units other than amino acid monomers. The terms monomer, subunit or basic unit refers to a chemical compound capable of chemically bonding with another monomer of the same or different chemical nature to form a polymer under appropriate conditions. The term "polypeptide" is further intended to include polymers in which one or more of the basic units are covalently linked to another basic unit by a chemical bond other than an amide or peptide bond.

  The term "protein" as used herein is not limited to other proteins, DNA, RNA, lipids, metabolites, hormones, chemokines, and other biomolecules including small molecules. Refers to a polypeptide with specific secondary and tertiary structure that may be involved in the interaction. An exemplary protein described herein is an antibody.

  The term "antibody" as used herein is a type of protein capable of specifically binding to an antigen generated by activated B cells and stimulating an immune response in a biological system after stimulation with the antigen. Point to A complete antibody typically consists of four subunits, including two heavy chains and two light chains. The term "antibody" includes natural and synthetic antibodies, including, but not limited to, monoclonal antibodies, polyclonal antibodies, or fragments thereof. Exemplary antibodies include IgA, IgD, IgG1, IgG2, IgG3, IgM and the like. Exemplary fragments include Fab Fv, Fab'F (ab ') 2 and the like. A monoclonal antibody is an antibody that specifically binds to, and is thereby complementary to, a single specific spatial and polar arrangement of another biomolecule called an "epitope." In some forms, the monoclonal antibodies may have the same structure. Polyclonal antibody refers to a mixture of different monoclonal antibodies. In some forms, the polyclonal antibody may be a mixture of monoclonal antibodies in which at least two of the monoclonal antibodies bind to different antigenic epitopes. Different antigenic epitopes may be present on the same target, on different targets, or a combination thereof. Antibodies can be prepared by techniques well known in the art such as host immunity and serum recovery (polyclonal) or preparing continuous hybridoma cell lines and recovering secreted proteins (monoclonal).

  In some embodiments, the polyol polymer forms a non-covalent complex or linkage with one or more target compounds to be delivered according to the schematics of FIGS.

  In some embodiments, the nanoparticle structure comprises an agent and a polyol-containing polymer, wherein the agent is covalently linked to the polyol polymer. Examples of polyol polymers conjugated to agents are detailed in Examples 16-21. In these embodiments, the polyol polymer conjugated to the agent (referred to herein as a "polyol polymer-agent conjugate") has a structure whose site on the surface is for interaction with the BA molecule. Form the nanoparticles presented.

  In some of these embodiments, the nanoparticles further comprise a BA polymer adapted to impart a steric stabilization and / or targeting function to the nanoparticles. Specifically, in these embodiments, the addition of the BA polymer can minimize self-aggregation and unwanted interactions with other nanoparticles to enhance salt and serum stability. For example, steric stabilization is provided by a BA-polymer with PEG, as exemplified herein by the exemplary nanoparticles described in Example 12.

  In such embodiments, the nanoparticle structure offers several advantages over prior art agent delivery methods, such as the ability to provide controlled release of one or more agents. This feature can be provided, for example, by the use of biodegradable ester linkages between the agent and the polyol polymer. One skilled in the art will recognize other possible linkages suitable for this purpose. In these embodiments, another advantage is the ability to provide specific targeting of agents through portions of the BA polymer.

  In some embodiments, BA polymers include fluorinated boronic acids (Example 7) or fluorinated cleavable boronic acids (Example 8) that can be used as contrast agents in MRI or other similar techniques. Good. Such contrast agents may be useful to track the pharmacokinetics or pharmacodynamics of the agent delivered by the nanoparticles.

  In some embodiments, the nanoparticle structure comprises an agent and a polyol polymer, wherein the nanoparticles are modified liposomes. In these embodiments, the modified liposome comprises a lipid that is conjugated to the polyol polymer via a covalent bond such that the surface of the liposome presents the polyol polymer. In these embodiments, the modified liposome is formed such that the agent to be delivered is contained within the liposomal nanoparticles.

  The term "liposome" as used herein refers to a porous structure consisting of lipids. Lipids typically have tail groups that include long hydrocarbon chains and hydrophilic head groups. The lipids are arranged to form a lipid bilayer with an internal aqueous environment suitable for containing the agent to be delivered. Such liposomes present an outer surface which may contain suitable targeting ligands or molecules for specific recognition by cell surface receptors or other targets of interest.

  The term “conjugate” as used herein refers to one molecule forming a covalent bond with a second molecule, wherein the atoms are another single and multiple (eg, double) Included are bonds that bind covalently (eg, C = C-C = C-C) and affect one another to delocalize the electrons.

  In yet another embodiment of the present disclosure, the nanoparticle structure comprises an agent and a polyol, wherein the nanoparticles are modified micelles. In these embodiments, the modified micelle comprises a polyol polymer that has been modified to contain a hydrophobic polymer block.

  The term "hydrophobic polymer block" as used in the present disclosure refers to a segment of a polymer that becomes naturally hydrophobic.

  The term "micelle" as used herein refers to an aggregate of molecules dispersed in a liquid. Typical micelles form aggregates with a hydrophilic "head" region in contact with the surrounding solvent in an aqueous solution, isolating a 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, a polyol polymer having a hydrophobic polymer block, when mixed with the delivered agent, forms a nanoparticle that is a modified micelle comprising the delivered agent contained within the nanoparticle. Be placed. Such nanoparticle embodiments present a polyol polymer on the surface suitable to interact with the BA polymer with or without the targeting function according to the previous embodiments. In these embodiments, BA polymers that can be used for this purpose include those having hydrophilic A and hydrophobic B in formula (I) or (II). This interaction provides the same or similar advantages as the other nanoparticle embodiments described above.

  In some embodiments, the nanoparticles or related components can be included in the composition with an acceptable vehicle. The term "vehicle" as used herein, for the nanoparticles contained in the composition as the active ingredient, usually acts as a solvent, carrier, binder, excipient or diluent Point to any of the following media.

  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, the nanoparticles can be included in the pharmaceutical composition with an excipient or diluent. Specifically, in some embodiments, the nanoparticles are combined with one or more compatible and pharmaceutically acceptable vehicles, specifically, a pharmaceutically acceptable diluent or excipient. Disclosed is a pharmaceutical composition containing the same.

  The term "excipient" as used herein refers to an inert substance used as a carrier for the active ingredient of a medicament. Suitable excipients for the pharmaceutical compositions disclosed herein include any substance that enhances the individual's ability to absorb nanoparticles. Also, suitable excipients include any material that can be used to bulk the formulation comprising the nanoparticles to allow for convenient and accurate administration. In addition to single dose use, excipients can be used in the manufacturing process to aid in the handling of the nanoparticles. Various excipients may be used depending on the route of administration and the form of medication. Exemplary excipients include anti-adherents, binders, coatings, disintegrants, fillers, flavors (eg, sweeteners), and colorants, glidants, lubricants, preservatives, adsorbents. Examples include, but are not limited to.

  The term "diluent" as used herein refers to a diluent for diluting or carrying the active ingredient of the composition. Suitable diluents include any substance that can reduce the viscosity of the pharmaceutical formulation.

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

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

  The term "lyophilization" (also known as freeze-drying) refers to a dehydration process commonly used to preserve perishable material or to make it more convenient to transport the material. Freeze drying is performed by freezing the material, then reducing the ambient pressure and applying sufficient heat to sublime the frozen water in the material directly from the solid phase to a gas.

  In pharmaceutical applications, freeze-drying is often used to extend the shelf life of products such as vaccines and other injectables. By removing the water from the material and sealing the material in a vial, the material can be easily stored, transported, and later reconstituted into the original injectable form.

  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 the step of contacting the target with the nanoparticles described herein.

  In some embodiments, provided is a method of delivering an agent to an individual comprising the steps of formulating suitable nanoparticles according to the various disclosed embodiments. Nanoparticles may also be formulated into pharmaceutically acceptable compositions according to some disclosed embodiments. The method further comprises the step of delivering the nanoparticles to the subject. The nanoparticles or nanoparticle formulations may be administered 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 can be administered in tablets or capsules, by injection, inhalation, eye drops, ointments, suppositories, infusions; topically by lotions or ointments; and rectally by suppositories.

  The term "individual" as used herein includes plants or animals, in particular higher animals, in particular vertebrates such as mammals, in particular humans, including Including single organisms without limitation.

  The terms "parenteral administration" and "administered parenterally" as used herein mean, but not limited to, methods of administration other than enteral and topical administration, usually by injection. Intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal space Internal and intrastemal, injection, and infusion.

  The phrases "systemic administration", "systemically administered", "peripheral administration" and "peripherally administered" as used herein refer to the system of an individual to enter into metabolism and other similar As used in the process, it means administering the nanoparticles or compositions thereof to the central nervous system in a manner other than direct, eg subcutaneous administration.

  The actual dosage level of the active ingredient or agent in the pharmaceutical composition described herein is not toxic to the individual to obtain the desired therapeutic response for the particular individual, composition, and method of administration. May be varied to obtain an effective amount of active ingredient.

  These therapeutic polymer conjugates can be used orally, nasally, eg, as a spray, rectally, vaginally, parenterally, in a large bath, and topically (including buccal and sublingual) in powders, ointments, or drops. It can be administered to humans and other animals for treatment by any suitable route of administration, including.

  Regardless of the route of administration chosen, the therapeutic polymer conjugate which may be used in a suitable hydrated form and / or the pharmaceutical composition of the present invention may be pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. Formulated into

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

  The terms "drug" or "therapeutic agent" can be used in the treatment, prevention or diagnosis of a condition in an individual, or otherwise used to enhance the individual's physical or mental health. Refers to an active agent that can

  The term "state" as used herein generally relates to a complete physical, mental, and sometimes social health condition, in whole or in part. Refers to the physical condition of the body of one or more of the individual as a whole or portions thereof that are not consistent with the individual's physical condition. The conditions described herein include, but are not limited to diseases and disorders, where the term "disease" refers to the condition of the body associated with any functional abnormality of the body or parts thereof. The term "disease" refers to the condition of the body that impairs the normal function of the body or any of its parts, and is typically manifested by identifying signs and symptoms. Exemplary conditions include injuries, disorders, diseases (including mental and physical illness), syndromes, infections, deviant behavior of an individual, and atypical variations in the structure and function of an individual's body or parts thereof. But not limited thereto.

  The term "treatment" as used herein refers to any activity that is part of medical care or treatment of a medical or surgical condition.

  The term "prevention" as used herein refers to any activity that reduces the burden of death or morbidity from a condition in an individual. This is done at the primary, secondary and tertiary prevention levels, where: a) primary prevention avoids the onset of the disease b) secondary prevention activity treats the disease early, thereby causing the disease C) Tertiary prevention aims to increase the chances of interventions to prevent the progression of the disease and the appearance of symptoms, c) tertiary prevention restores function and reduces the complications associated with the disease Reduce the negative impact.

Exemplary compounds that can be delivered by the nanoparticles described herein and suitable as a drug can emit electromagnetic radiation (eg, ¹⁰ B isotopes) used in radiation therapy (eg, boron neutron capture) Containing compounds. Additional therapeutic agents include any lipophilic or hydrophilic, synthetic or natural bioactive therapeutic agent, including those known in the art. The Merck Index, An Encyclopedia of Chemicals, Drugs, and Biologicals, 13th Edition, 2001, Merck and Co. , Inc. , Whitehouse Station, N.S. J. Examples of such therapeutic agents include small molecule drugs, antibiotics, steroids, polynucleotides (eg, genomic DNA, cDNA, mRNA, siRNA, shRNA, miRNA, antisense oligonucleotides, viruses, and chimeric polynucleotides), Plasmids, peptides, peptide fragments, small molecules (eg, doxorubicin), chelating agents (eg, deferoxamine (DESFERAL), ethylenediaminetetraacetic acid (EDTA)), natural products (eg, taxol, amphotericin), and other biologically active compounds Molecules include, but are 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 therapeutics with the nanoparticles described herein. Not only may low molecular weight therapeutic agents be therapeutic agents within composite particles, but in further embodiments may be covalently attached to polymers in composites. In some embodiments, the covalent bond is reversible (eg, through a prodrug form or a biodegradable bond such as a disulfide) to provide another method of delivering a therapeutic agent. In some embodiments, therapeutic agents that can be delivered with the nanoparticles described herein include epothilones, camptothecins, chemotherapeutic agents such as taxol, or plasmids, siRNAs, shRNAs, miRNAs, antisense oligonucleotides Included are nucleic acids such as aptamers, or combinations thereof, and additional drugs that can be identified by one of ordinary skill in the art upon reading the present disclosure.

In some embodiments, the compound to be delivered is a compound suitable for imaging cells or tissues of an individual. Exemplary compounds that can be delivered by the nanoparticles described herein and suitable for imaging include isotopes such as ¹⁹ F isotopes for MR imaging, ¹⁸ F or ⁶⁴ Cu isotopes for PET imaging, etc. Containing compounds.

Specifically, the nanoparticles described herein may be adapted to contain a ¹⁹ F-containing BA polymer. For example, ¹⁹ F atoms can be incorporated into non-cleavable or cleavable BA polymer compounds. Other positions of the ¹⁹ F atom can be on the BA polymer component, the polyol polymer component, or the agent to be delivered. These and other variations will be apparent to those skilled in the art.

  In some embodiments, the nanoparticles described herein can be used to deliver chemicals used in agriculture. In another embodiment of the present invention, the agent delivered by the nanoparticles described herein is a biologically active compound having biocidal and agricultural utility. These biologically active compounds include those known in the art. For example, suitable agriculturally biologically active compounds include, but are not limited to, fertilizers, fungicides, herbicides, insecticides, and fungicides. Microbicides are also used in water treatment to treat municipal water supply and in industrial water systems such as cold water in papermaking, white water systems and the like. Water systems susceptible to microbial attack or degradation are also found in the leather industry, textile industry, and the paint or paint industry. Examples of such microbicides and their use are described in US Pat. Nos. 5,693,631, 6,034,081, and 6,060,466, which are incorporated herein by reference. Are listed individually and in combination. Compositions containing active agents such as those discussed above may be used as known for the active ingredient itself. Furthermore, since such use is not a pharmacological use, the polymer of the composite does not necessarily have to meet the required toxicity profile in pharmaceutical use.

  In certain embodiments, nanoparticles comprising a polyol polymer and a BA polymer can be included in any system suitable for delivering any of the compounds presented herein, particularly agents using the nanoparticles. In some embodiments of the system, nanoparticles are provided that include components suitable 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 included in the molecule alone or in the presence of a suitable excipient, vehicle or diluent.

  In the kit of parts, the polyol polymer, the BA polymer, and / or the agent to be delivered are independently included in a kit that can be included in the composition along with a suitable vehicle carrier or adjuvant. For example, a polyol polymer and / or a BA polymer may be included alone in one or more compositions and / or in a suitable vector. Also, the agent to be delivered may be included in the composition with a suitable vehicle carrier or adjuvant. Alternatively, the agent may be supplied by the end user and may not be present 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.

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

  In the kit of parts disclosed herein, kit components can be provided along with suitable instructions and other necessary reagents to practice the methods disclosed herein. In some embodiments, the kit can contain the composition in a separate container. Instructions for performing the assay may also be included in the kit, for example, written or audio instructions in electronic assistance, such as a document or tape or CD-ROM. Also, the kit can include other packaged reagents and materials (eg, wash buffer, etc.), depending on the particular method used.

  The identification of suitable carrier agents or adjuvants of the composition, as well as generally further details regarding the manufacture and packaging of the kit, may be identifiable by one of ordinary skill in the art upon reading the present disclosure.

  In some embodiments, nanoparticles can be prepared by preparing the individual components of the nanoparticles and then mixing the components in various orders to achieve the desired composite nanoparticle structure. 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 another solution containing another molecule of interest to one solution containing the molecule of interest. For example, an aqueous solution of polyol polymer may be mixed with an aqueous solution of BA polymer in the context of the present disclosure.

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

  In some embodiments, nanoparticles can be prepared by mixing a polyol polymer and an agent to be delivered (Figures 1 and 2) to form a polyol polymer-agent nanoparticle. In another embodiment, the nanoparticles can be prepared by further mixing the BA polymer and the polyol polymer-agent nanoparticles. In another embodiment, the nanoparticles are prepared by mixing the polyol polymer and the BA polymer and then mixing the agent to be delivered. In yet another embodiment, the nanoparticles are prepared by simultaneously mixing the polyol polymer, the BA polymer, and the agent to be delivered.

  In some embodiments, the nanoparticles are prepared by forming polyol polymer-agent conjugates in accordance with various embodiments of the present disclosure to prepare nanoparticles of polyol polymer-agent conjugates. In other embodiments, nanoparticles consisting of polyol polymer-agent conjugates can be prepared by dissolving the nanoparticles in a suitable aqueous solution. In a further embodiment, nanoparticles consisting of polyol polymer-agent conjugates can be prepared by mixing a polyol polymer-agent conjugate with a BA polymer that provides or does not provide a targeting ligand.

  In some embodiments, nanoparticles can be prepared by mixing a polyol polymer having a hydrophobic polymer block and an agent to be delivered to prepare a modified micelle according to an embodiment of the present disclosure. In another embodiment, the nanoparticles can be prepared by further mixing the modified micelles and the BA polymer. In yet another embodiment, the nanoparticles can be prepared by mixing a polyol polymer and a BA polymer and then mixing the delivered agent to prepare nanoparticles that are modified micelles.

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

  The formation of nanoparticles according to some embodiments of the present disclosure can be analyzed using techniques and procedures known to those skilled in the art. For example, in some embodiments, gel retardation assay is used to monitor and measure incorporation of nucleic acid agents within nanoparticles (Example 10). In some embodiments, suitable nanoparticle size and / or zeta potential can be selected using known methods (Example 11).

  The identification of suitable carrier agents or adjuvants of the composition, as well as generally further details regarding the manufacture and packaging of the kit, may be identifiable by one of ordinary skill in the art upon reading the present disclosure.

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

[Wherein, A is an organic moiety of the following formula:

Wherein R ₁ and R ₂ are independently selected from any carbon-based or organic group having a molecular weight of about 10 kDa or less; X is independently -H, -F, -C,- N, or selected from aliphatic groups containing one or more of -O; Y is independently selected from organic moieties presenting -OH or -OH), B is in the polymer it is an organic moiety linking the one of R ₁ and R ₂ of R ₁ and one second part a of R ₂ of the first portion a]. In some embodiments, X can be C _n H _{2n + 1} , where n is any single number from 0 to 5 and Y is -OH. In some embodiments, A can be any one of the following:

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

Where d is any single number from 0 to 100, e is any single number from 0 to 100, and 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); May be any one of the following:

(Wherein q is any single number from 1 to 20; p is any single number from 20 to 200; L is a leaving group), these B subunits Is paired with any one of the above-mentioned A subunits]. In a more specific embodiment, the polyol-containing polymeric nanoparticle segment of the targeting nanoparticle shown in the structural unit of formula (I) may be:

The polyol-containing polymeric nanoparticle segment of the targeted nanoparticle shown in the structural unit of formula (II) may be:

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

(Wherein n is any single number from 1 to 20). In some embodiments of the targeted nanoparticles described, the polyol containing polymer is:

  In summary, any one of the formulas for subpart A (formula VI, VII or VIII) is described in combination with any one of the formulas for subpart B (formula XXIII or XIV) Nanoparticulate polyol-containing polymers can be formed. In certain embodiments described herein, the nanoparticle is any one of the formulas for subpart A (IX, X, or XI) and any one of the formulas for subpart B (formula It may have a polyol containing polymer formed from a combination with XXIII or XIV).

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

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

(Wherein s is any single number from 20 to 300). The polymers of Formulas XXXIII, XXXIV, and XXXV can be further modified to change the position of PEG on the phenyl ring such that it is in the ortho, meta or para position to the boronic acid group.

  In summary, any one of the formulas for subpart A (formula VI, VII or VIII) is described in combination with any one of the formulas for subpart B (formula XXIII or XIV) The polyol-containing polymer nanoparticle segments of the nanoparticles can be formed, and the resulting polyol-containing polymer may then be coupled to a nitrophenylboronic acid-containing polymer. In some embodiments, the conjugate of 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 embodiments described herein, the nanoparticle is any one of the formulas for subpart A (IX, X, or XI) and any one of the formulas for subpart B (formula It may have a polyol containing polymer formed from a combination with XXIII or XIV), which may then be coupled to a boronic acid containing polymer having the formula XXX. In some embodiments described herein, the nanoparticle is any one of the formulas for subpart A (IX, X, or XI) and any one of the formulas for subpart B It may have a polyol containing polymer formed from a combination with (Formula XXIII or XIV), which is then coupled to a boronic acid containing polymer corresponding to any one of Formulas XXXIII, XXXIV, or XXXV You may

  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 (such as shRNA, siRNA, or miRNA). In some embodiments, the small molecule chemotherapeutic agent can be one or more of camptothecin, epothilone, or taxane. Also, the nanoparticles described herein can comprise a combination of one or more polynucleotides and one or more small molecule chemotherapeutic agents. In this regard, the nanoparticles described in combination with any one of the polymers of subpart A (formula VI, VII, or VIII) in combination with any one of the polymers of subpart B (formula XXIII or XIV) The resulting polyol-containing polymer can then be coupled to a nitrophenylboronic acid-containing polymer, which can form a polyol-containing polymer nanoparticle segment of the polymer, subpart A, subpart B, or nitrophenyl boron of said polymer Polymers with acids may be formed with one or more therapeutic agents (eg, 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 subpart B polymers (Formula XXIII or XIV) to contain the described polyol of the nanoparticles A polymer nanoparticle segment can be formed, and the resulting polyol-containing polymer may then be coupled to a nitrophenylboronic acid-containing polymer, having subpart A, subpart B or nitrophenylboronic acid of said polymer The polymer may be formed with one or more of DNA, RNA, or interfering RNA (such as shRNA, siRNA, or miRNA). In some embodiments, the polymer of Subpart A (Formula VI, VII, or VIII) is combined with any one of the subpart B polymers (Formula XXIII or XIV) to contain the described polyol of the nanoparticles A polymer nanoparticle segment can be formed, and the resulting polyol-containing polymer may then be coupled to a nitrophenylboronic acid-containing polymer, having subpart A, subpart B or nitrophenylboronic acid of said polymer The polymer 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 subpart B polymers (Formula XXIII or XIV) to contain the described polyol of the nanoparticles A polymer nanoparticle segment can be formed, and the resulting polyol-containing polymer may then be coupled to a nitrophenylboronic acid-containing polymer, having subpart A, subpart B or nitrophenylboronic acid of said polymer The polymer may be formed with one or more of DNA, RNA, or interfering RNA (such as shRNA, siRNA, or miRNA). In some embodiments, conjugation of the described polyol containing polymer to 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 incorporating a therapeutic agent or polynucleotide is any one of the formulas for subpart A (IX, X, or XI) and for subpart B And a polyol-containing polymer formed from a combination of any one of the formulas (Formula X XIII or XIV) with one of the formulas XXXIII, XXXIV, or XXXV. It may be coupled to the corresponding nitrophenylboronic acid containing polymer.

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

[Wherein, A is an organic moiety of the following formula:

Wherein R ₁ and R ₂ are independently selected from any carbon-based or organic group having a molecular weight of about 10 kDa or less; X is independently -H, -F, -C,- N, or selected from aliphatic groups containing one or more of -O; Y is independently selected from organic moieties presenting -OH or -OH), B is in the polymer it is an organic moiety linking the one of R ₁ and R ₂ of R ₁ and one second part a of R ₂ of the first portion a]. In some embodiments, X can be C _n H _{2n + 1} , where n is any single number from 0 to 5 and Y is -OH. In some embodiments, A can be any one of the following:

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

Where d is any single number from 0 to 100, e is any single number from 0 to 100, and 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); May be any one of the following:

(Wherein q is any single number from 1 to 20; p is any single number from 20 to 200; L is a leaving group), these B subunits Is paired with any one of the above-mentioned A subunits]. In a more specific embodiment, the polyol-containing polymeric nanoparticle segment of the targeting nanoparticle shown in the structural unit of formula (I) may be:

  The polyol-containing polymer nanoparticle segment of the targeted nanoparticle shown in the structural unit of formula (II) may be:

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

(Wherein n is any single number from 1 to 20). In some embodiments of the targeted nanoparticles described, the polyol containing polymer is:

In summary, any one of the formulas for subpart A (formula VI, VII or VIII) is described in combination with any one of the formulas for subpart B (formula XXIII or XIV) Polyol-containing polymers of targeted nanoparticles can be formed. In certain embodiments described herein, the targeting nanoparticle is any one of the formulas for subpart A (IX, X, or XI) and any one of formulas for subpart B It may have a polyol containing polymer formed from a combination with (Formula XXIII or XIV).

  Also, the targeted nanoparticles described can have a boronic acid containing polymer coupled to the polyol containing polymer through a reversible covalent bond. In some embodiments, the nanoparticles will present the boronic acid containing polymer to the external environment of the nanoparticles. In a further embodiment, the boronic acid containing polymer is conjugated to the target ligand at the 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:

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

(Wherein s is any single number from 20 to 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:

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

(Wherein s is any single number from 20 to 300). The polymers of Formulas XXXIII, XXXIV, and XXXV can be further modified to change the position of PEG on the phenyl ring such that it is in the ortho, meta or para position to the boronic acid group.

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

  In some aspects described herein, nanoparticles formed from the polyol-containing polymeric nanoparticle segments described herein or from the combination of the polyol-containing polymeric nanoparticle segments and the boronic acid-containing polymer are targeted ligands To form a 3: 1 ratio of targeted nanoparticles to nanoparticles with the target ligand. In some aspects described herein, nanoparticles formed from the polyol-containing polymeric nanoparticle segments described herein or from the combination of the polyol-containing polymeric nanoparticle segments and the boronic acid-containing polymer are targeted ligands To form a 1: 1 ratio of targeted nanoparticles to nanoparticles with the target ligand. In some aspects described herein, the nanoparticles formed from the polyol containing polymer nanoparticle segments described herein or from the combination of the polyol containing polymer nanoparticle segments and the boronic acid containing polymer are single Conjugated to a target ligand of to form a targeting nanoparticle. In some embodiments, the described target ligands are conjugated to the boronic acid containing polymer at the end opposite to the boronic acid. The targeting ligands conjugated to the targeting nanoparticles described are proteins, protein fragments, amino acid peptides or aptamers from amino acids or polynucleotides or other high affinity known to bind to the target of interest It may be any one of the molecules. In some embodiments, the targeting ligand which is a protein or protein fragment is any one of: an antibody, a cell receptor, a ligand of a cell receptor, eg, transferrin, or a protein or chimeric protein having a portion thereof It may be one. Where the targeting ligand is an antibody, the antibody may be a human, mouse, rabbit, non-human primate, dog or rodent antibody, or a chimera consisting of any two such antibodies. May be In addition, antibodies may be humanized such that only a small portion of the variable regions comprising CDR segments or CDR segments are non-human and the remainder of the antibody is human. The antibodies described herein may be any isotype, such as IgG, IgM, IgA, IgD, IgE, IgY, or another type of isotype that is understood to be produced by a mammal. In some embodiments, the targeting ligand may comprise only an antibody involved in binding to its target, a cellular receptor, an amino acid peptide derived from a ligand of the cellular receptor.

  In some embodiments, the nanoparticles are combined with any one of the formulas for subpart B (Formula XXIII or XIV) to form the polyol-containing polymer nanoparticle segment of the targeted nanoparticle described Formed from any one of the formulas for subpart A (formula VI, VII or XIV) and further containing a boronic acid such as phenylboronic acid or nitrophenylboronic acid conjugated to a target ligand Coupling to any of the polymers 5, 4, 3, 2 or 1.

  In some embodiments, the nanoparticles are combined with any one of the formulas for subpart B (Formula XXIII or XIV) to form the polyol-containing polymer nanoparticle segment of the targeted nanoparticle described Formed from any one of the formulas for subpart A (formula VI, VII or XIV) and further containing a boronic acid such as phenylboronic acid or nitrophenylboronic acid conjugated to a target ligand Couple to one polymer.

  In certain embodiments described herein, the targeting nanoparticle is any one of the formulas for subpart A (IX, X, or XI) and any one of formulas for subpart B It may have a polyol containing polymer formed from a combination with (Formula XXIII or XIV), which comprises a boronic acid containing polymer 5, 4 having Formula XXX coupled to the target ligand at the end opposite to the boronic acid It may be coupled to any of three, two or one.

  In certain embodiments described herein, the targeting nanoparticle is any one of the formulas for subpart A (IX, X, or XI) and any one of formulas for subpart B It may have a polyol containing polymer formed from a combination with (Formula XXIII or XIV), which is coupled to one boronic acid containing polymer having Formula XXX coupled to the target ligand at the end opposite to the boronic acid It can ring.

  In some embodiments, the nanoparticles are combined with any one of the formulas for subpart B (Formula XXIII or XIV) to form the polyol-containing polymer nanoparticle segment of the targeted nanoparticle described A boronic acid containing polymer 5 such as phenylboronic acid or nitrophenylboronic acid formed from any one of the formulas for subpart A (formula VI, VII or XIV) and further conjugated to a target ligand , 4, 3, 2 or any one of which one, wherein the resulting targeting nanoparticles are conjugated to only a single targeting ligand.

  In some embodiments, the nanoparticles are combined with any one of the formulas for subpart B (Formula XXIII or XIV) to form the polyol-containing polymer nanoparticle segment of the targeted nanoparticle described Boronic acid-containing polymers such as phenylboronic acid or nitrophenylboronic acid formed from any one of the formulas for subpart A (formula VI, VII or XIV) and further conjugated to a target ligand 1 Coupled to one another, where the resulting targeting nanoparticles are conjugated to only a single target ligand.

  In certain embodiments described herein, the targeting nanoparticle is any one of the formulas for subpart A (IX, X, or XI) and any one of formulas for subpart B It may have a polyol containing polymer formed from a combination with (Formula XXIII or XIV), which comprises a boronic acid containing polymer 5, 4 having Formula XXX coupled to the target ligand at the end opposite to the boronic acid It can be coupled to any of three, two, or one, where the resulting targeted nanoparticles are conjugated to only a single target ligand.

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

  In some embodiments, the targeting nanoparticles described herein are conjugated to any target ligand. In some embodiments, the targeting nanoparticles described herein are conjugated to a single targeting ligand. In some embodiments described herein, the targeting nanoparticle is any one of the formulas for subpart A (IX, X, or XI) and any of the formulas for subpart B It may have a polyol containing polymer formed from a combination with one (formula XXIII or XIV), which is then selected as a target ligand selected from one or more of a protein, a protein fragment, an amino acid peptide, or an aptamer To a boronic acid containing polymer corresponding to any one of formulas XXXI, XXXIII, XXXIV, or XXXV, further conjugated to You may

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

  In some embodiments, the targeting nanoparticles described herein are conjugated to a single targeting ligand. In some embodiments described herein, the targeting nanoparticle is any one of the formulas for subpart A (IX, X, or XI) and any of the formulas for subpart B It may have a polyol containing polymer formed from a combination with one (formula XXIII or XIV), which in turn is a protein or a chimera with an antibody, a cell receptor, a ligand of a cell receptor, or a portion of these A boronic acid containing polymer corresponding to any one of Formulas XXXI, XXXIII, XXXIV, or XXXV, further conjugated to a protein, coupled to any of 5, 4, 3, 2, or 1 You may

  In some embodiments described herein, the targeting nanoparticle is any one of the formulas for subpart A (IX, X, or XI) and any of the formulas for subpart B It may have a polyol containing polymer formed from a combination with one (formula XXIII or XIV), which in turn has a single antibody, a cell receptor, a ligand for a cell receptor, or a portion of these It may be coupled to one boronic acid containing polymer corresponding to any one of Formulas XXXI, XXXIII, XXXIV, or XXXV, further conjugated to the protein or chimeric protein.

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

  In some embodiments, the targeting nanoparticles described herein are conjugated to a single targeting ligand. In some embodiments described herein, the targeting nanoparticle is any one of the formulas for subpart A (IX, X, or XI) and any of the formulas for subpart B It may have a polyol containing polymer formed from a combination with one (formula XXIII or XIV), which in turn is a protein or a chimera with an antibody, a cell receptor, a ligand of a cell receptor, or a portion of these A boronic acid containing polymer corresponding to any one of Formulas XXXI, XXXIII, XXXIV, or XXXV, further conjugated to a protein, coupled to any of 5, 4, 3, 2, or 1 Here, the obtained targeting nanoparticles are conjugated to only a single target ligand.

  In some embodiments described herein, the targeting nanoparticle is any one of the formulas for subpart A (IX, X, or XI) and any of the formulas for subpart B It may have a polyol containing polymer formed from a combination with one (formula XXIII or XIV), which in turn has a single antibody, a cell receptor, a ligand for a cell receptor, or a portion of these It may be coupled to a boronic acid containing polymer corresponding to any one of Formulas XXXI, XXXIII, XXXIV, or XXXV, further conjugated to the protein or chimeric protein, wherein the resulting targeting The nanoparticles are conjugated to only a single 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 (such as shRNA, siRNA, or miRNA). In some embodiments, the small molecule chemotherapeutic agent can be one or more of camptothecin, epothilone, or taxane. Also, the targeted nanoparticles described herein may comprise a combination of one or more polynucleotides and one or more small molecule chemotherapeutic agents.

  Having discussed the various types of nanoparticles and targeting nanoparticles that can be produced using the components described herein, the following specific embodiments can be made. In one embodiment, the targeted nanoparticles described are coupled to the mucic acid polymer in a reversible covalent linkage with a mucic acid containing polymer and a therapeutic agent selected from camptothecin, epothilone, taxanes, or interfering RNA sequences And said targeted nanoparticle is adapted to present said phenylboronic acid containing polymer in the external environment of the nanoparticle, and said phenylboronic acid containing polymer having formula XXXI, XXXIII, XXXIV, or XXXV. Here, the phenylboronic acid containing polymer is conjugated to the target ligand at the end opposite to the nanoparticle, and the targeting nanoparticle comprises a single target ligand.

  In one embodiment, the targeted nanoparticles described are coupled to the mucic acid polymer in a reversible covalent linkage with a mucic acid-containing polymer and a therapeutic agent selected from camptothecin, epothilone, taxanes, or interfering RNA sequences. And said targeted nanoparticle is adapted to present said phenylboronic acid containing polymer in the external environment of the nanoparticle, and said phenylboronic acid containing polymer having formula XXXI, XXXIII, XXXIV, or XXXV. Here, the phenylboronic acid containing polymer is conjugated to the antibody at the end opposite to the nanoparticle, and the targeting nanoparticle comprises a single antibody.

  In one embodiment, the targeted nanoparticles described are coupled to the mucic acid polymer in a reversible covalent linkage with a mucic acid-containing polymer and a therapeutic agent selected from camptothecin, epothilone, taxanes, or interfering RNA sequences. And said targeted nanoparticle is adapted to present said phenylboronic acid containing polymer in the external environment of the nanoparticle, and said phenylboronic acid containing polymer having formula XXXI, XXXIII, XXXIV, or XXXV. Wherein the phenylboronic acid containing polymer is a human, mouse, rabbit, non-human primate, dog, or rodent antibody, or a chimeric antibody consisting of any two such antibodies (wherein The antibody is any one of the IgG, IgD, IgM, IgE, IgA, or IgY isotype. To any one of) the nanoparticles are conjugated at the opposite end, the targeted nanoparticles comprise a single antibody.

  In one embodiment, the targeted nanoparticles described are coupled to the mucic acid polymer in a reversible covalent linkage with a mucic acid-containing polymer and a therapeutic agent selected from camptothecin, epothilone, taxanes, or interfering RNA sequences. And said targeted nanoparticle is adapted to present said phenylboronic acid containing polymer in the external environment of the nanoparticle, and said phenylboronic acid containing polymer having formula XXXI, XXXIII, XXXIV, or XXXV. Here, the phenylboronic acid containing polymer is conjugated to a cell receptor at the end opposite to the nanoparticle, and the targeting nanoparticle comprises a single cell receptor.

  In one embodiment, the targeted nanoparticles described are coupled to the mucic acid polymer in a reversible covalent linkage with a mucic acid-containing polymer and a therapeutic agent selected from camptothecin, epothilone, taxanes, or interfering RNA sequences. And said targeted nanoparticle is adapted to present said phenylboronic acid containing polymer in the external environment of the nanoparticle, and said phenylboronic acid containing polymer having formula XXXI, XXXIII, XXXIV, or XXXV. Here, the phenylboronic acid containing polymer is conjugated to the receptor ligand at the end opposite to the nanoparticle, and the targeting nanoparticle comprises a single receptor ligand.

  The method systems described herein are further described in the following examples, which are provided for the purpose of illustration and are not intended to be limiting. One skilled in the art will understand the applicability of the features described in detail with respect to methods of detecting nucleic acids and methods of detecting other targets (such as proteins, antigens, eukaryotic or prokaryotic cells).

  All chemical reagents were obtained from commercial sources 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 small angle light scattering detector. A 7.5% acetic acid solution was used as the eluent at a flow rate of 1 mL / min.

  The plasmid containing the firefly luciferase gene was extracted and purified from bacteria expressing pGL3 and pGL3. siGL3 was purchased from Integrated DNA Technologies (sequences shown below). siCON1 (sequence shown below) was purchased from Dharmacon. HeLa cells were used to determine the effect of pDNA or siRNA delivery by cationic mumate diamine-DMS polymer.

Example 1: Synthesis of Mucic Acid Dimethyl Ester (1)
5 g (22.8 moles) of mucinic 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 under vacuum overnight, 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: Synthesis of 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 constant stirring for 500 hours with 500 mL of round bottom After heating to reflux at 85 ° C. in a flask, 14.2 g (88.6 mmol) of N-BOC diamine (Fluka) dissolved in methanol (32 mL) was added. The reaction suspension was then refluxed back. After refluxing overnight, the mixture is filtered, washed with methanol, recrystallized from methanol and then 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), 85 under constant stirring. Reflux at <0> C overnight. The precipitate was then filtered, washed with methanol and vacuum dried overnight to give 5.7 g (15.6 mmol, 96%) of a mucic acid diamine. 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)
In 1.5mL Eppendorf tubes were filled with a solution of bis Example 3 in 0.1 M NaHCO ₃ in 0.8 mL 85.5 mg (0.233 mmol) (hydrochloride) salt (3). Sverimino acid dimethyl 2 HCl (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 to 8 mL with water and brought to pH 4 by the addition of 1 N HCl. The solution was then dialyzed for 24 hours in ddH ₂ O using a 3500 MWCO dialysis membrane (Pierce pleated dialysis tubing). The dialyzed solution was lyophilized to dryness to give 49 mg of white fluffy powder. ¹ H NMR (500 MHz, dDMSO) δ 9.15 (bs), 7.92 (bs), 5.43 (bs), 4.58 (bs), 4.17 (bs), 3.82 (bs) , 3.37 (bs), 3.28 (bs), 2.82 (bs), 2.41 (bs), 1.61 (bs), 1.28 (bs). ¹³ C NMR (126 MHz, dDMSO) δ 174.88 (s, 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.

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

Example 5: Boronic acid-amide-PEG ₅₀₀₀ (5)
When using the nucleic acid, eg, siRNA, to construct the polymer of Example 4, they form nanoparticles. These nanoparticles need to have steric stabilization in order to be used in mammals, and may optionally include targeting agents. To perform these two functions, the nanoparticles may be modified with PEG for steric stabilization and a PEG targeting ligand. To do this, a boronic acid containing PEG compound is prepared. For example, PEG containing boronic acid can be synthesized according to the following example.

332mg of 4-carboxyphenyl boronic acid (2 mmol) was dissolved in SOCl ₂ of 8 mL. To this, a few drops of DMF were added and the mixture was allowed to reflux for 2 hours under argon. The excess SOCl ₂ was removed under reduced pressure and the resulting solid was dissolved in 10 mL of anhydrous dichloromethane. To this solution was added 500 mg of PEG ₅₀₀₀ -NH ₂ (2 mmol) and 418 μL of triethylamine (60 mmol) dissolved in 5 mL of 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 lipid was precipitated with 20 mL of diethyl ether. The precipitate was filtered off, dried and redissolved in ddH ₂ O. The aqueous solution was then filtered through a 0.45 μm filter and dialyzed for 24 h with dd3500 MWCO dialysis membrane in ddH ₂ O. 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 of the PEG compound of Example 5 (under reducing conditions) 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 stir bar. To this was added 110 mg of aldolithiol-2 (0.5 mmol, Aldrich) dissolved in 4 mL of methanol. The solution was stirred at room temperature for 2 hours, after which 77 mg of mercaptophenylboronic acid (0.5 mmol, Aldrich) in 1 mL of methanol was added. The resulting solution was stirred at room temperature for a further 2 hours. The methanol was removed under vacuum and the residue was redissolved 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 for 15 hours with a 3500 MWCO dialysis membrane in dd H ₂ O. The dialyzed solution was lyophilized to dryness. ¹ H NMR (300 MHz, dDMSO) δ 8.12-8.00 (m), 7.83-7.72 (m), 7.72-7.61 (m), 7.61-7.43 (M), 3.72 (d, J = 5.4), 3.68-3.15 (m), 3.01-2.83 (m).

Example 7: Synthesis of (2,3,5,6) -tetrafluorophenylboronic acid-PEG ₅₀₀₀ (7)
The fluorinated version of the boronic acid containing PEG compound of Example 5 can be synthesized and used as a contrast agent with therapeutic nanoparticles. Fluorine atoms for imaging can be incorporated as described and as exemplified below.

Dissolve (2,3,5,6) -fluorocarboxyphenylboronic acid in excess SOCl ₂ (̃100 equivalents), to which a few drops of DMF are added. The mixture was refluxed for 2 hours under argon. The excess SOCl ₂ is removed under reduced pressure and the resulting residue is dissolved in anhydrous dichloromethane. To this solution is added PEG ₅₀₀₀ -NH ₂ (1 equivalent) and triethylamine (30 equivalents) dissolved in dichloromethane at 0 ° C. under argon. The resulting mixture is allowed to warm to room temperature and stirring is continued overnight. The dichloromethane solvent is removed under reduced pressure and the lipid obtained is precipitated with diethyl ether. The precipitate is filtered off, dried and redissolved in ddH ₂ O. The aqueous solution is then filtered through a 0.45 μm filter and dialyzed for 24 hours with 3500 MWCO dialysis membranes in dd H ₂ O. The dialyzed solution is lyophilized to dryness.

Fluorine-containing compounds are useful to provide ¹⁹ F in nanoparticles. ¹⁹ F can be detected by magnetic resonance spectroscopy using standard patient MRI. Nanoparticles can be imaged by the addition of ¹⁹ F (which may be done for imaging only, and therapeutic agents may be added to enable imaging and treatment).

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 a contrast agent with therapeutic nanoparticles. Fluorine atoms for imaging can be incorporated as described and as exemplified below.

Add 250 mg of PEG ₅₀₀₀ -SH (0.05 mmol, Lay San Bio Inc.) to a glass vial equipped with a stir bar. To this is added 110 mg of aldolithiol-2 (0.5 mmol, Aldrich) dissolved in 4 mL of methanol. The solution is stirred for 2 hours at room temperature and then 77 mg of (2,3,5,6) -fluoro-4-mercaptophenylboronic acid (0.5 mmol) in 1 mL of methanol are added. The resulting solution is stirred at room temperature for a further 2 hours. The methanol is removed under vacuum and the residue is redissolved 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 for 15 hours with a 3500 MWCO dialysis membrane in dd H ₂ O. The dialyzed solution is lyophilized to dryness.

Example 9 Synthesis of Boronic Acid-PEG ₅₀₀₀ -Transferrin (9)
For example, a targeting agent could be placed at the other end of the boronic acid in the compounds of Examples 5-8 according to the approach schematically illustrated in FIG. 12 in connection with transferrin binding.

  Thus, the components of the system containing the nucleic acid as a therapeutic agent may be (a targeting ligand is a protein such as transferrin (FIG. 12), an antibody or antibody fragment, a peptide such as RGD or LHRH, a small molecule such as folic acid or galactose, etc.) May be there. 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 schematically illustrated in FIG. A solution of 10 mg (0.13 μmol) of human holo-transferrin (iron rich) (Sigma Aldrich) in 1 mL of 0.1 M PBS buffer (pH 7.2), 3.2 mg OPSS-PEG ₅₀₀₀ -SVA (5 equivalents), 0.64 μmol, added to Laysan Bio Inc.). The resulting solution was stirred at room temperature for 2 hours. Pegylated transferrin was purified from unreacted OPSS-PEG ₅₀₀₀ -SVA using Ultracel 50,000 MWCO (Amicon Ultra-4, Millipore) and from unreacted transferrin using gel filtration column G3000SWxl (Tosoh Biosep) (HPLC) And confirmed by MALDI-TOF analysis). Then, 100 μg of OPSS-PEG ₅₀₀₀ pegylated transferrin in 100 μL was incubated with 20 μL of 4-mercaptophenylboronic acid (1 μg / μL, 20 μg, 100 equivalents) at room temperature for 1 hour. After incubation, the solution was dialyzed twice using a YM-30,000 NMWI apparatus (Millipore) to remove excess 4-mercaptophenylboronic acid and pyridyl-2-thione side products.

Example 10: Construction of MAP-nucleic acid particles-gel retardation assay As illustrated in Figure 1, 1 μg of plasmid DNA or siRNA (0.1 μg / μL, 10 μL) in water without DNAse and RNASe, DNAse and RNASe Charge ratios of 0.5, 1, 1.5, 2, 2.5, and 5 mixed with 10 μL of different concentrations of MAP in water ("+" charge on polymer vs. "-" charge on nucleic acid) I got The resulting mixture was incubated for 30 minutes at room temperature. 10 μL of the 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 as shown in FIGS. The nucleic acids not contained in the nanoparticles move on the gel. These results provide a guide to the charge ratio needed to entrap the nucleic acid 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 without DNAse and RNASe is mixed with 10 μL of various concentrations of MAP in water without DNAse and RNASe Then, charge ratios of 0.5, 1, 1.5, 2, 2.5 and 5 were obtained. The resulting mixture was incubated for 30 minutes at room temperature. Then, for particle size determination, 20 μL of the mixture was diluted to 70 μL with DNAse and RNASe free water. The 70 μL solution was then diluted to 1400 μL with 1 mM KCl for zeta potential measurements. Particle size and zeta potential measurements were made on a ZetaPals dynamic light scattering (DLS) instrument (Brookhaven Instruments). The results are shown in FIG.

Example 12 Stabilization of Particle Size by PEGylation with Boronic Acid PEG _{5k As} illustrated in FIG. 2, 2 μg of plasmid DNA (0.45 μg / μL, 4.4 μL) in water without DNAse and RNASe It was diluted to 80 μL with water without DNAse and RNASe. This plasmid solution is mixed with 4.89 μg of MAP (0.5 μg / μL, 9.8 μL) and diluted to 80 μL with DNAse- and RNASe-free water as well, to a charge ratio of 3 +/− and 0. A final plasmid concentration of 0125 μg / μL was obtained. The resulting mixture was incubated for 30 minutes at room temperature. 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 a further 30 minutes, dialyzed twice with 0.5 ml of a 100,000 MWCO membrane (BIOMAX, Millipore Corporation) in water without DNAse and RNASe and free of 160 μl DNAse and RNASe Reconstituted with water. Half of this solution was diluted with 1.4 mL of 1 mM KCl for zeta potential measurement (FIG. 6). In addition, the zeta potential of the BA-containing nanoparticles exhibits a lower zeta potential than the non-containing nanoparticles. These results support the conclusion that BA-containing nanoparticles have BA localized to the exterior of the nanoparticles. The other half was used to measure particle size. Particle size was measured every minute for 5 minutes, after which 10.2 μL of 10 × PBS was added to give a final solution of 90.2 μL in 1 × PBS. The particle size was then measured again every minute for an additional 10 minutes, as shown in FIG. BA-containing nanoparticles separated from non-particulate components (by filtration) are stable in PBS, but particles that do not contain BA are not. These data support the conclusion that BA-containing nanoparticles have a BA localized outside the nanoparticles as they are stabilized against aggregation in PBS.

Example 13 Transfection of MAP / p DNA Particles into HeLa Cells 48 hours prior to transfection, HeLa cells were seeded at 20,000 cells / well in a 24-well plate and grown in medium supplemented with 10% FBS The MAP particles were formulated to contain 1 μg of pGL3 in 200 μL of Opti-MEM I at various charge ratios of polymer to pDNA (see Example 9). The growth media was removed, cells were washed with PBS and particle formulation added. Cells were then incubated for 5 hours at 37 ° C. and 5% CO ₂ before 800 μL of growth medium supplemented with 10% FBS was added. After incubation for 48 hours, one fraction of cells was analyzed for cell viability using the MTS assay. The remaining cells were lysed in 100 μl of 1 × luciferase cell culture lysis reagent. Luciferase activity was determined by adding 100 μL of luciferase assay reagent to 10 μL of cell lysate, and bioluminescence 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: Co-transfection of MAP / pDNA and / or siRNA particles into HeLa cells 48 hours before transfection HeLa cells are seeded at 20,000 cells / well in a 24-well plate, and 10% FBS is added It was grown in the culture medium. 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. The growth media was removed, cells were washed with PBS and particle formulation added. Cells were then incubated for 5 hours at 37 ° C. and 5% CO ₂ before 800 μL of growth medium supplemented with 10% FBS was added. After incubation for 48 hours, cells were assayed for luciferase activity and cell viability as described in Example 12. The results are shown in FIG. Because transfection is with siGL3 (the correct sequence) and RLU is lower, siGL3 and pGL3 have to be co-delivered.

Example 15: Transfection of MAP / siGL3 Particles into HeLa-LUC Cells 48 hours before transfection, 20,000 HeLa-LUC cells (containing the gene encoding firefly luciferase protein) in a 24-well plate Cells / well were seeded and grown in medium supplemented with 10% FBS. MAP particles were formulated to contain 50 and 100 nM siGL3 in 200 μL Opti-MEM I at a charge ratio of 5 +/−. The growth media was removed, cells were washed with PBS and particle formulation added. Cells were then incubated for 5 hours at 37 ° C. and 5% CO ₂ before 800 μL of growth medium supplemented with 10% FBS was added. After incubation for 48 hours, cells were assayed for luciferase activity and cell viability as described in Example 12. The results are shown in FIG. These data suggest that inhibition of endogenous genes can occur as RLU decreases with increasing concentrations of siGL3.

Example 16 Synthesis of Mucic Acid Diiodide (10)
In a 250 mL round bottom flask, 1 g (2.7 mmol) of mucoic 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 were added dropwise. The mixture was allowed to react overnight under constant stirring at room temperature. The solvent was then removed by vacuum pump, the product was filtered, washed with methanol and dried under vacuum to give 0.8 g (1.3 mmol, 46%) of mutic acid diiodide. 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)
To 7 mL of 0.1 M degassed sodium carbonate, 17 mg of L-cysteine and 0.4 g of mutic acid diiodide were added. 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 via 1N HCl. Then acetone was slowly added to form a precipitate. After filtration, washing with acetone and vacuum drying, 60 mg of crude product was obtained.

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

Example 18: Synthesis of polymer (poly (muc acid-dicysteine-PEG)) (12)
After 12 mg (21.7 μmol) of dicysteine mucinate and 74 mg (21.7 μmol) of PEG-DiSPA 3400 are dried under vacuum, 0.6 mL of argon in a 10 mL two-necked round bottom flask is used. Anhydrous DMSO was added. 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 solution containing the polymer was then dialyzed using a 10 kDa membrane centrifuge and lyophilized to give 47 mg (58%) of poly (mucic acid-dicysteine-PEG).

The polyol containing polymer is an anionic AB polymer.

Example 19: covalent attachment of drug (camptothecin, CPT) to mucic acid polymer (13)
10 mg (2.7 μmol of repeating unit) of poly (mucic acid-dicysteine-PEG) was dissolved in 1.5 mL of anhydrous DMSO in a glass jar. 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 insoluble material was removed by filtration through a 0.2 μm filter. The polymer solution was then dialyzed against water through a 10 kDa membrane and then lyophilized to obtain a poly (mucic acid-dicysteine-PEG) -CPT conjugate.

Example 20: Formation of nanoparticles comprising CPT-munic acid polymer (13) in water (20)
By blending the effective diameter of poly (muc acid-dicysteine-PEG) and poly (muc acid-dicysteine-PEG) -CPT conjugates into redistilled water (0.1-10 mg / mL) It measured and evaluated via dynamic light scattering (DLS) using ZetaPALS (Brookhaven Instrument Co) apparatus. Then, 3 consecutive runs of 1 minute each were recorded and averaged. The zeta potentials of both components were measured in 1.1 mM KCl solution using a ZetaPALS (Brookhaven Instrument Co) instrument. Then, 10 consecutive automation runs at a target residue of 0.012 were performed and the results were averaged (Figure 14). Specifically, these two distributions were measured for the poly (mucic acid-dicysteine-PEG) -CPT conjugate, the main distribution being 57 nm (60% of the total particle population). Also, the second smallest distribution was measured at 233 nm.

Example 21: Formulation of boronic acid-pegylated nanoparticles comprising CPT-muchic acid polymer (13) and boronic acid-disulfide-PEG ₅₀₀₀ (6) in water Polymer in redistilled water at a concentration of 0.1 mg / mL Polymer 6 (BA-) in water, so that it is dissolved and then the ratio of PBA-PEG to diol in the mumate sugar in the poly (mutuate-dicysteine-PEG) -CPT conjugate is 1: 1. Boronic acid PEGylated poly (mucic acid-dicysteine-PEG) -CPT nanoparticles are formulated by adding PEG). The mixture is incubated for 30 minutes, after which the effective diameter and zeta potential are measured using a ZetaPALS (Brookhaven Instrument Co) apparatus.

Example 22 Targeting Nanoparticles for Delivering pDNA in Mice The plasmid pApoE-HCRLuc contains a gene that expresses luciferase and is under the control of a liver specific promoter. Polymer (MAP) 4 (0.73 mg), Polymer 6 (73 mg), and Polymer 9 (0.073 mg) were combined in 5 mL water, then 1.2 mL water containing the pApoE-HCRLuc plasmid was added (polymer The charge ratio for the 4 plasmid will be +3). The particles were placed in D5W (5% glucose in water) by continuous spin filtration, then D5W was added (starting from the initial formulation that was in water). Nude mice were implanted with Hepa-1 to 6 hepatoma cells and tumors were allowed to grow to approximately 200 mm ³ in size. An amount of targeting nanoparticles equivalent to 5 mg plasmid / kg mouse was injected i. v. Was injected. 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 but not in the area of the liver.

Example 23 Synthesis of PBA-PEG and Nitro-PBA-PEG, and Determination of pKa A covalent, reversible bond between boronic acid and MAP (containing diol) to prepare stabilized targeted nanoparticles. The characteristics were used. The pKa of phenylboronic acid (PBA) is as high as 8.8. In order to lower the pKa, PBA having an electron withdrawing group on the phenyl ring was used to raise 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. The addition of the bases DMF and DIPEA was necessary to allow the synthesis reaction to proceed. However, the presence of these bases results in the formation of tetrahedral adducts with acidic PBA. Worked up with 0.5 N HCl to remove these adducts, then equilibrated to neutral pH by dialysis against water.

  To synthesize PBA-PEG, add 200 mg (1.21 mM) of 3-carboxyphenylboronic acid dissolved in 5 mL of anhydrous tetrahydrofuran to 18.7 μL (0.24 mM) of anhydrous dimethylformamide under argon did. The reaction vessel was transferred to an ice bath and 195 μL (2.89 mM) of oxalyl chloride was added slowly under argon. The reaction was allowed to proceed for 2 hours at room temperature under aeration and constant agitation. The solvent and excess reagents were removed under vacuum. 37 mg (0.2 mM) of the obtained dried acyl chloride compound were dissolved in 15 mL of anhydrous dichloromethane under argon. Then 500 mg (0.1 mM) of NH2-PEG5kDa-CO2H and 52 μL (0.3 mM) of dry DIPEA were added under argon. After reacting overnight under constant stirring, the solvent was removed under vacuum and the dry product was reconstituted with 0.5 N HCl. This solution was passed through a 0.2 μm filter (Acrodisc®) and dialyzed against water using a 3 kDa MWCO membrane filter device (Amicon®) until a constant pH is obtained. The supernatant was then filtered through 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 to 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 in the same manner as PBA-PEG-CO2H except that the starting material was 3-carboxy 5-nitrophenylboronic acid. The 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 to 3.62 (PEG peak). MALDI-TOF (m / z): 5600 g / mol (NH2-PEG5 kDa-CO2H, 5400 g / mol).

  The pKa of the synthesized PBA compounds was found by measuring the change in absorbance of PBA when converting from rhombohedra (at low pH) to tetrahedral structure (at high pH). 1 N NaOH was titrated to a solution of 10-3 M PBA in 0.1 M PBS (pH 7.4). The pH was recorded and the corresponding sample was removed from the 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) nitro PBA-PEG-CO 2 H, 12.3 mg (64 μM) 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), and 11.1 mg ( 96 μM) NHS was dissolved in 2.4 mL of 0.1 M MES buffer (pH 6.0). The mixture was allowed to react for 15 minutes on a rotary shaker at room temperature. Excess reactant was removed by dialysis using a 3 kDa molecular weight cut-off membrane filter device (AmiconTM). The activated carboxylic acid PEG compound was added to 20 mg (0.14 mM) of a 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 (pH 7.4) using a 50 kDa molecular weight cut-off membrane filter device (AmiconTM). Analysis of the conjugate by mass spectrometry (MALDI-TOF) indicated that an average of one or two nitro PBA-PEG compounds were conjugated per antibody.

Example 26 Formation and Characterization of Targeted MAP Nanoparticles The effect of PBA-PEG and nitro PBA-PEG on nanoparticle formation and stability was investigated. A solution of PBA-PEG was added to the MAP solution 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 complexation of PBA-PEG with diol-containing MAP. It was then added to an equal volume of siRNA in water and mixed by pipetting. After 10 minutes, tests and characterizations were performed. The formulation of nitro-PBA-PEG stabilized particles of various charge ratios was carried out as well. Conjugation 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 with MAP-2. However, when the salt is added, all particles aggregate, which indicates that PBA-PEG can not provide sufficient stability to the nanoparticles. When the particles at all charge ratios were tested with nitro PBA-PEG, 1 and 3 (+/−) indicated stabilized nanoparticles. The difference in particle stability conferred by PBA-PEG and nitro PBA-PEG demonstrates the importance of modifying the PEGylated boronic acid compound to be more strongly bound by the polyol.

  For siRNA condensed with MAP-4 in the absence of the stabilizing agent PEG, a particle size of 640 nm was observed with a high zeta potential of +32 mV. When nitro PBA-PEG was added to stabilize the particles, the diameter decreased to 130 nm with a negative surface charge of -4 mV. This negative charge is the result of the bond between the boronic acid and the diol. Interestingly, when introducing nitro PBA-PEG conjugated to 0.25 mol% IgG (Herceptin®), the particle size is further reduced to 82 nm, but the zeta potential remains the same Met.

  The salt stability of MAP-4 / siRNA nanoparticles with different PEG groups attached was evaluated. Particle sizes were recorded for 5 runs of 1 minute using a zeta potential analyzer (DLS ZetaPALS instrument, Brookhaven Instruments (Holtsville, NY)). The measurement was stopped to allow a 10 × PBS solution to be added to the particle formulation so that the resulting particle formulation contained 1 × PBS. The measurement was then started again and 10 consecutive runs of 1 minute each were recorded to investigate 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 in salt conditions.

Example 27 Formation and Characterization of Targeted MAP-CPT Nanoparticles To prepare targeted MAP-CPT nanoparticles, using the covalent, reversible binding property between boronic acid and MAP (containing diol) . The association constant between PBA and MAP is as low as about 20 M ⁻¹ . In order to raise the coupling constant, the acidity of the boron atom was raised to raise the coupling constant with MAP, using PBA having an electron withdrawing group on the phenyl ring. 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. The addition of the bases DMF and DIPEA was necessary to allow the synthesis reaction to proceed. However, the presence of these bases results in the formation of tetrahedral adducts with acidic PBA. Worked up with 0.5 N HCl to remove these adducts, then equilibrated to neutral pH by dialysis against water.

The pKa value of modified PBA was determined by the change in absorbance due to the conformational change of PBA from rhombohedral to tetrahedral form as the pH was raised. Lower pKa is obtained from the PBA having an electron withdrawing group, nitro PBA-PEG-CO ₂ H had the lowest pKa of 6.8. Thus, at physiological pH, most of PBA was 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). The low pKa value observed for nitro PBA-PEG-CO ₂ H and the high binding constant with MAP make it selected as a linker between IgG (Herceptin®) and MAP-CPT nanoparticles .

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

^a Equal amount of N, N-diisopropylethylamine (DIPEA) was added per amine group in mucinic acid di (aspartic acid-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 particle size of about 40 nm was observed for targeted MAP-CPT nanoparticles by DLS and cryo-EM (Table 3). An increase of about 10 nm from this non-targeted nanoparticle indicates binding of IgG (Herceptin®) to the nanoparticle (the amount is about 1 HERCEPTIN® antibody per nanoparticle) Met). In fact, from the cryo-EM image, the targeted nanoparticles appeared to be non-spherical and to have protrusions indicating antibody binding. The surface charge of the targeted MAP-CPT nanoparticles is higher than that of MAP-CPT nanoparticles due to the presence of positively charged Herceptin (pH of Herceptin® 9.2, 9.2) Also slightly higher.

Example 28: Cellular Uptake Test HER2 overexpressing human breast cancer cell line (BT-474) was used to investigate the cellular uptake of HERCEPTIN <(R)> targeted MAP-CPT nanoparticles relative to non-targeted nanoparticles. The HER2-negative breast cancer cell line (MCF-7) was used as a negative control. For these tests, 20,000 cells of BT-474 (in RPMI-1640 medium supplemented with 10% fetal bovine serum) or MCF-7 (Dulbecco supplemented with 10% fetal calf serum) per well in a 24-well plate Seed in modified Eagle's medium (Cellgro (Manassas, Va.)) And maintained at 37 ° C. in a humidified oven containing 5% CO ₂ . After 40 hours, medium MAP-CPT (40 μg CPT / mL), medium MAP-CPT containing 10 mg / mL free HERCEPTIN®, targeted MAP-CPT with various target densities (40 μg CPT / mL), or 10 mg The medium was changed to 0.3 mL of fresh media containing any of the targeted MAP-CPTs containing / mL of free Herceptin®. Transfection was performed for 30 minutes at 37 ° C. The formulation was then removed and cells were washed twice with cold PBS. In order to detach and lyse the cells, 200 μL of 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 via BCA assay. To another portion, an equal volume of 0.1 N NaOH was added, which was incubated overnight at room temperature and fluorescence was measured at 370/440 nm using a known concentration of MAP-CPT as a standard.

  One HERCEPTIN®-PEG-nitro PBA per nanoparticle is sufficient to achieve approximately 70% more uptake into the BT-474 cell line as compared to non-targeted nanoparticles Was observed (FIG. 17A). Uptake of targeted MAP-CPT into BT-474 cells showed inhibition in the presence of free Herceptin® (FIG. 17B). In contrast, uptake of MAP-CPT nanoparticles into MCF-7 showed no dependence either on targeting or on the presence of free HERCEPTIN® (FIG. 2B). These data indicate that there is receptor mediated uptake into BT-474 cells by targeted MAP-CPT nanoparticles via association of HER2 receptors. Cellular uptake of targeted and non-targeted MAP-CPT nanoparticles also occurred via non-specific fluid phase endocytosis.

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

The medium, pipetted into 96 well plates, was incubated for 2 hours at 37 ° C. in a humidified oven to equilibrate. The formulation was mixed into the relevant medium and returned to the oven. Samples were taken at predetermined times and immediately frozen at -80 ° C until time of analysis. For release in BALB / c mouse plasma and human plasma, carboxylic acid / bicarbonate buffer system in plasma at physiological pH levels, main pH, incubated in a 37 ° C. humidified oven containing 5% CO ₂ The buffer system was maintained.

  The amount of unconjugated CPT was determined by first mixing 10 μL of the sample with 10 μL of 0.1 N HCl and incubating for 30 minutes at room temperature. Then, 80 μL of methanol was added and the mixture was incubated for 3 hours at room temperature to precipitate proteins. 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 10 μL of the obtained solution are subjected to HPLC. Injected. The peak area of eluted CPT (at 7.8 minutes) was compared to the control. To measure the total amount of CPT, 10 μL of sample was mixed with 6.5 μL of 0.1 N NaOH. This solution was incubated for 1 hour at room temperature to release CPT from the parent polymer. The carboxylate CPT form was then converted to the lactone form by the addition of 10 μL of 0.1 N HCl. Then 73.5 μL of methanol was added and the mixture was incubated for 3 hours at room temperature. The samples were then centrifuged and processed as described above. From the difference between the total CPT concentration and the concentration of unconjugated CPT, the concentration of CPT bound to the polymer was determined.

  The release of CPT from short, medium and long MAP-CPT nanoparticles and from targeted MAP-CPT nanoparticles all showed first order kinetics. The release half-lives of CPT from short, medium and long MAP-CPT nanoparticles were very similar in all conditions tested, but longer half-lives were observed for targeted MAP-CPT ( Table 4). A strong dependence of release rate on pH was observed. When the pH was raised from 6.5 to 7.4, the emission half life was reduced from 338 hours to 58 hours for short, medium and long MAP-CPT nanoparticles. These data indicate that hydrolysis plays an important role in the release of CPT. A release half-life of 59 hours was observed in BALB / c mouse plasma, but for short, medium and long MAP-CPT nanoparticles, a significantly shorter half-life of 38 hours was obtained in human plasma. Mouse plasma has more esterase activity than human plasma, but human plasma is known to contain more "fat" than mouse plasma. Thus, to understand the difference in release rates between human and mouse plasma, the contribution of esterase and fat to the release rate of CPT was examined individually. Butyrylcholinesterase (BCHE) is present in mouse and human plasma and was used to test the contribution of esterase to the release rate. Low density lipoprotein (LDL) was chosen to test the contribution of "fat" to the release rate. These components were composed in PBS (pH 7.4). The presence of BCHE was found to have no effect on the release rate (61 h halving compared to 58 h in PBS (pH 7.4) for short, medium and long MAP-CPT nanoparticles Period). However, the addition of LDL dramatically increased the release rate. This effect is likely due to the destruction of the nanoparticles by competing hydrophobic interactions. Thus, destruction of the nanoparticles due to the presence of "fat" and subsequent hydrolysis CPT cleavage appears to be another major mechanism of CPT release from MAP-CPT nanoparticles. In vitro release from HERCEPTIN® targeted MAP-CPT nanoparticles exhibits a longer half life than the non-targeted version. The presence of HERCEPTIN® with pI 9.2 increases the stability of negatively charged nanoparticles by electrostatic interaction, thereby causing the nanoparticles to be from some of the competing hydrophobic interactions It is possible to protect.

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

Example 30: Cytotoxicity assay Medium MAP, nitro PBA-PEG, CPT, medium MAP-CPT nanoparticles, targeted MAP-CPT nanoparticles, and in vitro cytotoxicity of HERCEPTIN®, two HER2 + breast cancer cell lines (BT-474, SKBR-3) and two HER2-breast cancer cell lines (MCF-7, MDA-MB-231) were evaluated (Table 5). In these tests, cells were maintained at 37 ° C. in a humidified oven containing 5% CO ₂ . BT-474, MCF-7, SKBR-3, and MDA-MB-231 cell lines, respectively, RPMI-1640 medium, Dulbecco's modified Eagle's medium, McCoy's 5A modified medium, and Dulbecco's modified Eagle's medium (all 10) % Fetal bovine serum was added). 3,000 cells per well were plated in 96 well plates. After 24 hours, the medium is removed and fresh medium containing various concentrations of MAP, nitro PBA-PEG, CPT, medium MAP-CPT nanoparticles, targeted MAP-CPT nanoparticles, or HERCEPTIN® I replaced it. 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. After incubating this for 2 hours in a 37 ° C. humidified oven containing 5% CO ₂ , the absorbance was measured at 490 nm. Wells containing untreated cells were used as a control.

Medium MAP and nitro PBA-PEG give IC ₅₀ values greater than 500 μM and 1000 μM (highest concentration tested), respectively, which show minimal toxicity. The IC ₅₀ concentrations of CPT, medium MAP-CPT nanoparticles, and targeted MAP-CPT nanoparticles were based on the content of CPT. It should be noted that CPT is gradually released from the medium MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles (see in vitro release test). Furthermore, due to the acidic nature of endosomes, the uptake of nanoparticles by cells prolongs the release time. These factors contribute to the higher IC ₅₀ values observed for medium MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles compared to CPT (Table 5). In HER2 + cell lines, targeted MAP-CPT nanoparticles gave lower IC ₅₀ values compared to MAP-CPT alone, but in HER2- cell lines, targeting does not affect IC ₅₀ The BT-474 was the cell line most resistant to CPT and thus to medium MAP-CPT nanoparticles. When HERCEPTIN at a concentration of 0.001 to 0.5 μM was added to BT-474 or SKBR-3 cells, a constant cell viability of about 60% was obtained compared to untreated controls. It has already been observed that there is no true IC ₅₀ value over this concentration range for these cell lines (Phillips, et al., Cancer Res. 68, 9280-9290 (2008)). No effect was observed in HER2- cell lines at concentrations of Herceptin less than 0.5 μM.

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

Example 31: Pharmacokinetic studies Plasma pharmacokinetic studies of short, medium and long MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles at 10 mg / kg (CPT reference) injection are conducted in female BALB / c mice (Figure 18). In these tests, nanoparticles formulated in 0.9% by weight NaCl (non-targeted nanoparticles) or PBS (targeted MAP-CPT nanoparticles) were used as tails of 12-16 week old female BALB / c mice. Administration was via intravenous bolus administration. At predetermined time points, blood was collected into blood collection tubes (Microvette CB 300 EDTA, Sarstedt) via bleeding in the saphenous vein. The samples were immediately centrifuged at 10,000 g for 15 minutes at 4 ° C., the supernatant removed and stored at -80 ° C. until time of analysis. Analysis of CPT unconjugated and conjugated to polymer was as follows. The amount of unconjugated CPT was determined by first mixing 10 μL of the sample with 10 μL of 0.1 N HCl and incubating for 30 minutes at room temperature. Then, 80 μL of methanol was added and the mixture was incubated for 3 hours at room temperature to precipitate proteins. 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 10 μL of the obtained solution are subjected to HPLC. Injected. The peak area of eluted CPT (at 7.8 minutes) was compared to the control. To measure the total amount of CPT, 10 μL of sample was mixed with 6.5 μL of 0.1 N NaOH. This solution was incubated for 1 hour at room temperature to release CPT from the parent polymer. The carboxylate CPT form was then converted to the lactone form by the addition of 10 μL of 0.1 N HCl. Then 73.5 μL of methanol was added and the mixture was incubated for 3 hours at room temperature. The samples were then centrifuged and processed as described above. From the difference between the total CPT concentration and the concentration of unconjugated CPT, the concentration of CPT bound to the polymer was determined. Non-compartmental modeling software PK Solutions 2.0 (Summit Research Services (Montrose, CO)) was used for pharmacokinetic data analysis. All animals were processed according to the National Institute of Health Guidelines for Animal Care and approved by the California Institute of Technology Institutional Animal Care and Use Committee.

For all nanoparticles, CPT attached to the polymer showed a biphasic profile with fast redistribution phase (α) and long ejection phase (β) (FIG. 18A). The efflux phases for medium and long MAP-CPT nanoparticles are particularly long with half-lives of 16.6 and 17.6 hours, respectively, with high area under the curve of 2298 and 3636 μg ^* h / mL (AUC ) Value was obtained. Furthermore, they exhibited low volume distribution and clearance rates. Twenty-four hours after injection, 11.3% of the injected volume of medium MAP-CPT nanoparticles and 20.5% of the injected volume of long MAP-CPT nanoparticles still in plasma as CPT bound to polymer It was circulating. In contrast, mice injected with 10 mg / kg CPT alone show fast clearance, AUC is only 1.6 μg ^* h / mL and 0.01% of the injected volume is in circulation after 8 hours It was left. Targeting of medium MAP-CPT nanoparticles affected the pharmacokinetic profile by increasing the redistribution phase and prolonging the excretory phase to 21.2 hours, and the AUC value was as high as 2766 μg ^* h / 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 The MTD value was determined in female NCr nude mice and defined as the highest dose that reduced body weight by less than 15% and did not die from treatment. . In these experiments, 12-week-old female NCr nude mice were randomly divided into 13 groups, each containing 5 mice. 200 mg / kg medium MAP or nitro PBA-PEG, 10, 15, or 20 mg / kg (based on CPT) short MAP-CPT nanoparticles, 8, 10 or 15 mg / kg (based on CPT) medium MAP-CPT nano Formulation of targeted MAP-CPT nanoparticles of particles, 5, 8 or 10 mg / kg (CPT basis) and 8 or 10 mg / kg (CPT basis) targeted MAP-CPT nanoparticles via tail vein injection It was administered on day 0 and day 7. All injections were formulated in 0.9 w / v% saline, except for targeted MAP-CPT formulated in PBS (pH 7.4). The weight and health of the mice were recorded and monitored daily for two weeks after the start of treatment. MTD was defined as the highest dose that resulted in less than 15% body weight loss and was not associated with treatment. Animals were euthanized by CO ₂ asphyxiation when the criteria for MTD were exceeded or at the end of the study.

  About the weight of mice treated with medium MAP, nitro PBA-PEG, short, medium, and long MAP-CPT, and targeted MAP-CPT after weighing and twice weekly on days 0 and 7 It was monitored (Table 6). Mouse weight and health are not affected by treatment with high doses of medium MAP or nitro PBA-PEG, which indicates minimal toxicity of these polymer components. In the group containing CPT, maximal weight loss was seen 3-5 days after each treatment. Most of the groups returned to their reduced weight. The study was terminated if weight loss continued and exceeded an average of 15%. Several mice in the group treated with 15 mg / kg of medium MAP-CPT and 10 mg / kg of long MAP-CPT showed diarrhea and appeared to be weakened. All other mice looked healthy. MTD values were found to be 20, 10, 8 and 8 mg / kg (CPT reference) for short, medium and long MAP-CPT, and targeted MAP-CPT, respectively.

^a All groups containing MAP-CPT are based on mg CPT / kg. ^b Maximum weight loss rate (%) ^{c The} animal was discarded because weight loss exceeded 15%.

Example 33: Biodistribution in Nude Mice To assess biodistribution of targeted nanoparticles in mice, 7 week old NCr nude mice were subcutaneously implanted with 17β-estradiol pellets. Two days later, BT-474 cancer cells suspended in RPMI-1640 medium were injected subcutaneously into the right front abdomen at 10 million cells / animal. Treatment started one day after the average size of the tumors reached 260 mm ³ . Animals are randomized into two groups of 6 mice per group, 5 mg CPT / kg MAP-CPT nanoparticles (PBS, in pH 7.4), or 5 mg CPT / kg and 29 mg HERCEPTIN® / Treated with kg of targeted MAP-CPT nanoparticles (PBS, in pH 7.4) via intravenous injection into the tail vein. After 4 and 24 hours, blood was collected from 3 animals in each group via bleeding in the saphenous vein. The animals were then euthanized by CO ₂ asphyxiation and perfused with PBS. Tumor, lung, heart, spleen, kidney and liver were removed and two equal sized sections were made. One section was embedded in Tissue-Tek® OCT (Sakura), another was collected in an eppendorf tube, and both were immediately frozen at -80 ° C until processing time.

  Organs were weighed and 100 mg of each were placed in Lysing Matrix A homogenizer tubes supplemented with 0.64 cm (1/4 inch) ceramic spheres (MP Biomedicals, Solon, Ohio). One mL of RIPA lysis buffer (Thermo Scientific) was added and tissues were homogenized at 6 m / s for 30 seconds using a FastPrep®-24 homogenizer (MP Biomedicals, Solon, Ohio). This was repeated 3 times, cooling on ice for 1 minute between each round. The samples were then centrifuged at 14,000 g for 15 minutes at 4 ° C. The amount of unconjugated CPT was determined by first mixing 10 μL of the supernatant with 10 μL of 0.1 N HCl and incubating for 30 minutes at room temperature. Then, 80 μL of methanol was added and the mixture was incubated for 3 hours at room temperature to precipitate proteins. 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 minutes) was compared to the control. To measure the total amount of CPT, 10 μL of sample was mixed with 6.5 μL of 0.1 N NaOH. This solution was incubated for 1 hour at room temperature to release CPT from the parent polymer. The carboxylate CPT form was then converted to the lactone form by the addition of 10 μL of 0.1 N HCl. Then 73.5 μL of methanol was added and the mixture was incubated for 3 hours at room temperature. The samples were then centrifuged and processed as described above.

  The mean percent injected (ID) percentage of total CPT per gram of tumor, heart, liver, spleen, kidney, and lung is shown in FIG. 19A. Four hours after dosing, there were 5.3 and 5.2% ID total CPT per gram of tumor in mice treated with MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles, respectively. Twenty-four hours after treatment, 2.6% ID of total CPT remained per gram of tumor in mice treated with MAP-CPT nanoparticles, but per gram of tumor in mice that received targeted MAP-CPT nanoparticles All CPT with 3.2% ID was found. These data show that targeted nanoparticles of essentially the same size and surface charge as the non-targeted version have been reported by others without increasing tumor localization over the non-targeted version. Indicates that it matches. Similar CPT distributions were obtained in the heart, liver, kidney and lung in animals treated with MAP-CPT nanoparticles and targeted MAP-CPT nanoparticles. However, at 24 hours targeting MAP-CPT accumulated relatively large amounts of total CPT in the spleen relative to the non-targeted version. This effect has already been observed for humanized antibodies in mice.

  FIG. 19B shows the mean percentage of unconjugated CPT in the organs of the tumor, heart, liver, spleen, kidney and lung. The proportion of unconjugated CPT in heart, liver, spleen, kidney and lung is low at both 4 and 24 hours, indicating that free CPT is rapidly cleared from these organs. The percentage of unconjugated CPT for both MAP-CPT and targeted MAP-CPT nanoparticles was significantly higher in the 24-hour tumor compared to 4 hours. Retention of free CPT in tumors suggests intracellular accumulation of CPT. Targeted nanoparticles show a slightly higher percentage of unconjugated CPT than non-targeted nanoparticles in mouse tumors at both 4 hours and 24 hours.

  The total concentration of CPT in plasma and the percentage of total unconjugated CPT in plasma were similar for both MAP-CPT and targeted MAP-CPT nanoparticles (FIG. 19C). After 4 hours, 17 and 20 μg / mL total CPT remained in plasma for MAP-CPT and targeted MAP-CPT nanoparticles, respectively. After 24 hours, the amount of total CPT remaining in plasma was the same at 12 μg / mL for both MAP-CPT and targeted MAP-CPT nanoparticles. The percentage of total unconjugated CPT in plasma is less than 3% for all conditions, indicating that the non-conjugated CPT is slightly released from the plasma and cleared rapidly ( Figure 19D).

Example 33 Treatment of Tumors with Targeted Nanoparticles NCr nude mice bearing BT-474 tumors with MAP-CPT (5 mg CPT / kg) or targeted MAP-CPT (5 mg CPT / kg, 29 mg Herceptin / kg) It was processed. After 4 and 24 hours, mice were euthanized, tumors were removed, and sections of 20-30 μm thickness were made using a clearstat. Tumor sections were placed on Superfrost Plus slides (Fisher Scientific, Hampton, NH) and stored at -80 ° C until processing time. The slides were thawed and tissue sections were directly fixed on the slides with 10% formalin solution for 15 minutes. The slides were then washed 3 times with PBS for 5 minutes each and blocked for 1 hour in 5% goat serum blocking buffer. CPT is naturally fluorescent that emits at 440 nm. To identify the location of the MAP, PEG within the MAP was stained with a rat anti-PEG primary antibody that recognizes 14 μg / mL of internal PEG units and incubated overnight at 4 ° C. This was followed by three washes with PBS and incubation for 1 hour with 2 μg / mL Alexa Fluor® 488 goat anti-rat IgM secondary antibody. The slides were then washed three times with PBS and incubated with 2 μg / mL Alexa Fluor® 633 goat anti-human IgG secondary antibody for 1 hour to visualize Herceptin®. Slides were washed three more times with PBS, loaded with Prolong Gold Antifade reagent and stored at 4 ° C. until imaging time. Images were acquired with a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss, Germany) using a 63 × Plan-Neofluar oil objective. CPT was detected using two photon excitation at 720 nm (emission filter BP 390-465 nm). PEG and Herceptin were detected using excitation at 488 nm (emission filter LP 530) and 633 nm (emission filter BP 645-700 nm), respectively. All laser and gain settings were set at the beginning of imaging and did not change. Image analysis was performed with the Zeiss lsm image browser.

  FIG. 20A shows tumor sections of mice treated with MAP-CPT nanoparticles after 4 hours. CPT signal was dispersed. CPT and MAP signals appeared to be co-localized in the merged image. Twenty-four hours after injection, CPT signal had accumulated in a punctate form (FIG. 20B). Merged images suggest co-localization of CPT and MAP. In mice treated with targeted MAP-CPT nanoparticles, spots showing accumulation of CPT were observed after 4 and 24 hours of treatment (FIGS. 20C and 20D). In the merged image, CPT and MAP signals were colocalized. 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: Anti-tumor efficacy test in nude mice BT-474 is one of the most resistant breast cancer to anticancer agents including camptothecin. To promote tumor growth in nude mice, 7 week old NCr nude mice were implanted with 17β-estradiol pellets two days prior to implantation of BT-474 tumor cells. At day 0 (5 days after transplantation), the tumor size for each group averaged 250 nm. The treatment started on the first day. Animals are randomly divided into 9 groups, each containing 6-8 mice, 1 mg or 8 mg / kg MAP-CPT nanoparticles (CPT standard, in PBS (pH 7.4)), 8 mg / kg CPT (dissolved in 20% DMSO, 20% PEG 400, 30% ethanol, and 30% 10 mM pH 3.5 phosphoric acid), 80 mg / kg irinotecan (5 w / v% dextrose solution in D5 W), 2.9 or 5.9 mg / kg HERCEPTIN® (in PBS (pH 7.4), 0.5 mg CPT / kg and 2.9 mg HERCEPTIN® / kg or 1 mg CPT / kg and 5.9 mg Herceptin ( (Trademark) / kg of targeted MAP-CPT nanoparticles (in PBS (pH 7.4)) or saline was treated. All treatments were freshly prepared and administered via tail vein intravenous injection. Injections were standardized at 150 μL per 20 g of mouse weight. Treatment containing HERCEPTIN® was performed once a week for 2 weeks, and for all other groups once a week for 3 weeks. It recorded three times a week the tumor size using caliper measurements (length × width ^2/2), and continuously monitor the health of the animals. Animals were euthanized when tumor volume exceeded 1000 mm ³ . Six weeks after treatment initiation, animals were euthanized by CO ₂ asphyxiation. The results of this test are presented in FIG. 21 and Table 7.

Tumors in the control group that received saline grew rapidly (FIG. 21). After 28 days, five of eight mice had tumors that exceeds the size limit of 1000 mm ^3. All animals in this group were euthanized at this time.

  In the group treated with CPT (8 mg / kg), the tumor was not inhibited compared to the control group (P> 0.05). In addition to 1 and 3 treatment-related deaths being recorded on day 9 and day 16 respectively, 4 were euthanized on day 28 because the size limit of the tumor was exceeded. None of the animals were alive until the end of the study.

Mice treated with 80 mg / kg irinotecan showed no significant tumor inhibition as compared to the control group (P> 0.05). One treatment-related death occurred on day 11. Tumor size reached an average of 575 mm ³ by the end of the study.

Animals receiving 8 mg CPT / kg of MAP-CPT nanoparticles showed highly significant tumor inhibition compared to the control group (P <0.01). By the end of the study, the average tumor size had decreased to 63 mm ³ and 3 out of 8 treated mice had a tumor size that regressed to zero. No deaths occurred in the MTD test using NCr nude mice without tumors treated with 8 mg CPT / kg MAP-CPT, but one died on day 21 due to weight loss in this test. Because this may be due to increased tumor burden in this test and / or that the mice used in this test were 7 weeks old, 12 week old mice were used in the MTD test.

Animals treated with 5.9 mg / kg HERCEPTIN® showed highly significant tumor inhibition as compared to the control group (P <0.01). On day 11 of treatment, seven of the eight animals treated had tumors that had regressed to zero. However, by the end of the study, two of the regressed tumors had relapsed, giving a total of 5 animals with zero tumor volume, and the group had an average tumor volume of 60 mm ³ (FIG. 22). Mice treated with 1 mg CPT / kg MAP-CPT nanoparticles terminated early as no anti-tumor effect was observed. Nevertheless, 5.9 mg / kg HERCEPTIN® is targeted at a low CPT dose of 1 mg / kg MAP-CPT nanoparticles (5.9 mg HERCEPTIN® / kg and 1 mg CPT / kg When added to MAP-CPT nanoparticles) via nitro PBA-BA linker as a standardizing agent, all tumors regressed to zero on day 9 of treatment and remained zero until the end of the study (FIG. 22) ). One non-treatment-related death occurred at day 37. This result is highly significant (P <0.01) compared to the control group. The combination of the results observed in these three groups shows that the addition of HERCEPTIN® standard agent in addition to the HERCEPTIN® intrinsic activity improves the effect of MAP-CPT nanoparticles .

In animals treated with 2.9 mg / kg HERCEPTIN®, two animals had tumors that had regressed to zero by the end of the study. However, the mean tumor size increased to 278 mm ³ from the start of the study. This result is significant as compared to the control group (0.01 ≦ P ≦ 0.05). When the animals were treated with targeted MAP-CPT nanoparticles containing 0.5 mg CPT / kg and 2.9 mg HERCEPTIN® / kg, two tumors regressed to zero. Average tumor size decreased to 141 mm ³ by the end of the study. This result is highly significant (P <0.01) compared to the control group. These results further suggest the benefit of normalization in tumor inhibition.

  Five weeks after transplantation of the 17β-estradiol pellet, it was observed that several mice, regardless of treatment group, had bladder bloating and abdominal bloating. This could be due to the use of 17β-estradiol pellets causing hydronephrosis and urine retention in athymic nude mice. After 6 weeks of treatment, the condition worsened and more mice were observed to develop bladder and abdominal distension, so animals were euthanized and the experiment ended.

^a N _begin is the number of animals at the beginning of _the test, and N _end is the number of animals surviving to the _end of the test. ^b N _TRD is the number of deaths associated with the treatment, N _NTRD is the number of deaths not associated with the treatment, _Neuthan is the animal that was euthanized because it exceeded the 1000 mm ³ tumor size limit It is a number. ^c N _{reg to zero} is the number of animals with tumors that have regressed to zero at the end of the study.

  The above examples are provided to give those skilled in the art a complete disclosure and description of methods of making and using embodiments of the particles, compositions, systems, and methods of the present disclosure, which the inventor is a disclosure of. It is not intended to limit the range considered as Variations of the above-described method for practicing the present disclosure which are obvious to one skilled in the art 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 skill of those skilled in the art to which the present disclosure pertains. All references cited in this disclosure are incorporated by reference to the same extent as if the full text of each reference was individually incorporated by reference.

  Reference is made to the background art, the summary of the invention, the detailed description of the invention and the complete disclosure of each document cited in the examples, including patents, patent applications, articles, abstracts, laboratory manuals, books or other disclosures. Is hereby incorporated by reference.

  It is to be understood that this 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 context clearly dictates otherwise. Include. The term "plural" includes two or more referents 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 invention belongs.

  Any methods and materials similar or equivalent to those described herein may be used in the practice for testing the appropriate materials and method embodiments described herein.

  A number of embodiments of the present disclosure have been described. Nevertheless, it will be 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 (87)

  Targeted nanoparticles comprising a polyol containing polymer, wherein the targeted nanoparticles are conjugated to a single target ligand.   The targeted nanoparticle of claim 1, wherein the target ligand comprises a protein, a protein fragment, or an amino acid peptide.   The protein comprises an antibody, transferrin, a ligand for a cell receptor, or a cell receptor protein, and the targeting ligand alternatively comprises a fragment of an antibody, transferrin, a ligand for a cell receptor, an aptamer, or a cell receptor protein 3. The targeted nanoparticle of claim 2, which may be included.   4. The targeted nanoparticle of claim 2 or 3, wherein the protein fragment comprises an antibody fragment.   The targeting nanoparticle according to claim 2 or 3, wherein the amino acid peptide comprises an amino acid sequence derived from an antibody.   6. The targeted nanoparticle of any one of claims 1 to 5, further comprising a therapeutic agent.   7. The targeted nanoparticle of claim 6, wherein the therapeutic agent is a small molecule chemotherapeutic agent or polynucleotide.   8. The targeted nanoparticle of claim 7, wherein the polynucleotide is an interfering RNA.   9. The any one of claims 6-8, wherein the targeting ligand is transferrin or an antibody, and the nanoparticles further comprise a therapeutic agent selected from camptothecin, epothilone, taxanes, and polynucleotides, or any combination thereof. Targeted nanoparticles according to one of the claims.   A method of delivering nanoparticles to a target in a subject, comprising the steps of obtaining a nanoparticle conjugated to a single target ligand that specifically binds to said target, and conjugating to said single target ligand Administering the nanoparticles to the subject.   11. The method of claim 10, wherein the target ligand comprises a protein, a protein fragment, or an amino acid peptide.   The protein comprises an antibody, transferrin, a ligand for a cell receptor, an aptamer, or a cell receptor protein, and the targeting ligand alternatively comprises a fragment of an antibody, a transferrin, a ligand for a cell receptor, or a cell receptor protein The method of claim 11, which may include.   The method according to claim 11 or 12, wherein the protein fragment comprises an antibody fragment.   The method according to claim 11 or 12, wherein the amino acid peptide comprises an amino acid sequence derived from an antibody.   15. The method of any one of claims 10-14, wherein the nanoparticle conjugated to the single targeting ligand further comprises a therapeutic agent.   16. The method of claim 15, wherein the therapeutic agent is a small molecule chemotherapeutic agent or polynucleotide.   17. The method of claim 16, wherein the polynucleotide is an interfering RNA.   18. The targeting ligand is transferrin or an antibody, and the nanoparticle further comprises a therapeutic agent selected from camptothecin, epothilone, taxanes, and polynucleotides, or any combination thereof. The method described in.   Targeted nanoparticles comprising a polyol containing polymer and a boronic acid containing polymer, wherein the boronic acid is reversibly covalently coupled to the polyol containing polymer, the nanoparticles being the external environment of the nanoparticles A targeted nanoparticle, adapted to present the boronic acid containing polymer, wherein the boronic acid containing polymer is conjugated to the target ligand at the end opposite to the nanoparticle. 20. The targeted nanoparticle of claim 19, wherein the polyol containing polymer comprises one or more of at least one of the following structural units:
[In the formula,
A is the organic moiety of the following formula:
(In the formula,
R ₁ and R ₂ are independently selected from any carbon-based or organic group having a molecular weight of about 10 kDa or less
X is independently selected from aliphatic groups containing one or more of -H, -F, -C, -N, or -O;
Y is independently selected from organic moieties presenting -OH or -OH)
B, in the polymer, one of R ₁ and R ₂ of the first of said portions A, a one organic moiety linking of R ₁ and R ₂ of the second of said portions A] . 21. The targeted nanoparticle of claim 20, wherein R ₁ and R ₂ independently have the following formula:
(In the formula,
d is 0 to 100,
e is 0 to 100,
f is 0 to 100,
Z is a covalent bond linking one organic moiety to another,
Z ₁ is independently selected from -NH ₂ , -OH, -SH, and -COOH). X is C _n H _{2n + 1} (wherein n is 0 to 5), and
22. The targeted nanoparticle of claim 20 or 21, wherein Y is -OH. 21. The targeted nanoparticle of claim 20, wherein A is independently selected from the group consisting of:
(In the formula,
The spacer is independently selected from any organic group
The amino acids are selected from free amines and any organic groups having free carboxylic acid groups,
n is 1 to 20,
Z ₁ is independently selected from -NH ₂ , -OH, -SH, and -COOH). 24. Targeted nanoparticle according to claim 23, wherein A is independently selected from the group consisting of:
The targeted nanoparticle according to any one of claims 20 to 24, wherein B is
(In the formula,
q is 1 to 20,
p is 20 to 200,
L is a leaving group). 21. Targeted nanoparticle according to claim 20, wherein the structural unit of formula (I) is as follows:
21. Targeted nanoparticle according to claim 20, wherein the structural unit of formula (II) is as follows:
21. Targeted nanoparticle according to claim 20, wherein the structural unit of formula (III) is as follows:
(In the formula,
n is 1 to 20). 20. The targeted nanoparticle of claim 19, wherein the polyol containing polymer is as follows:
30. The targeted nanoparticle of any one of claims 19-29, wherein the boronic acid containing polymer comprises at least one terminal boronic acid group and has the following general formula:
(In the formula,
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 an alkyl group or an aromatic group of the formula —C _m H _{2 m} — (wherein m is 1 or more),
r is 1 to 1000,
a is 0 to 3,
b is 0-3). R _{3 and R 4} (wherein, t is a is _{2~2000) (CH 2 CH 2 O} ) t is, targeted nanoparticles of claim 30. X ₁ is —NH—C (= O) —, —S—S—, —C (= O) —NH—, —O—C (= O) —, or —C (= O) —O— 32. The targeted nanoparticle of claim 30 or 31, wherein Y ₁ is a phenyl group.   33. The targeted nanoparticle of any one of claims 30-32, wherein r = 1, a = 0 and b = 1. Functional group 1 and functional group 2 are the same or different, and _{independently, -B (OH) 2, -OCH} 3, is selected from -OH, in any one of claims 30 to 33 Targeted nanoparticles as described. 35. The targeted nanoparticle of any one of claims 19-34, wherein the boronic acid containing polymer is as follows:
(In the formula, s = 20 to 300).   36. The targeted nanoparticle of any one of claims 19-35, wherein the boronic acid containing polymer is conjugated to the target ligand at the end opposite to the boronic acid.   The target ligand is any one of an antibody, transferrin, a ligand for a cell receptor, an aptamer, or a cell receptor protein, and the target ligand is alternatively a fragment of an antibody, transferrin, a cell receptor 37. The targeted nanoparticle according to any one of claims 19 to 36, which may comprise a ligand or a cell receptor protein.   38. The targeted nanoparticle of any one of claims 19-37, further comprising a therapeutic agent.   39. The targeted nanoparticle of claim 38, wherein the therapeutic agent is a small molecule chemotherapeutic agent or polynucleotide.   40. The targeted nanoparticle of claim 39, wherein the polynucleotide is an interfering RNA.   40. The targeted nanoparticle of claim 39, wherein the small molecule chemotherapeutic agent is selected from camptothecin, epothilone, taxanes, and polynucleotides, or any combination thereof. 30. The targeted nanoparticle of any one of claims 19-29, wherein the boronic acid containing polymer comprises a nitrophenylboronic acid group and has the following general formula:
(In the formula,
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 a nitro group,
r is 1 to 1000,
a is 0 to 3,
b is 0-3). R _{3 and R 4} (wherein, t is a is _{2~2000) (CH 2 CH 2 O} ) t is, targeted nanoparticles of claim 42. X ₁ is —NH—C (= O) —, —S—S—, —C (= O) —NH—, —O—C (= O) —, or —C (= O) —O— 44. The targeted nanoparticle of claim 42 or 43, wherein Y ₁ is a phenyl group having a nitro group.   45. The targeted nanoparticle of any one of claims 42-44, wherein r = 1, a = 0 and b = 1. Functional group 1 and functional group 2 are the same or different, and _{independently, -B (OH) 2, -OCH} 3, is selected from -OH, in any one of claims 42-45, Targeted nanoparticles as described. 47. The targeted nanoparticle of any one of claims 19-29 or 42-46, wherein the nitrophenylboronic acid containing polymer is any one of the following:
(In the formula, s = 20 to 300).   48. The targeted nanoparticle of any one of claims 42-47, wherein the nitrophenylboronic acid containing polymer is conjugated to the target ligand at the opposite end as the nitrophenylboronic acid.   The target ligand is any one of an antibody, transferrin, a ligand for a cell receptor, an aptamer, or a cell receptor protein, and the target ligand is alternatively a fragment of an antibody, transferrin, a cell receptor 49. The targeted nanoparticle of any one of claims 42 to 48, which may comprise a ligand or a cell receptor protein.   50. The targeted nanoparticle of any one of claims 42-49, further comprising a therapeutic agent.   51. The nanoparticle of claim 50, wherein the therapeutic agent is a small molecule chemotherapeutic agent or polynucleotide.   52. The targeted nanoparticle of claim 51, wherein the polynucleotide is an interfering RNA.   52. The targeted nanoparticle of claim 51, wherein the small molecule chemotherapeutic agent is selected from camptothecin, epothilone, taxanes, and polynucleotides, or any combination thereof.   56. A method of delivering nanoparticles to a target in a subject, comprising the steps of obtaining a targeted nanoparticle according to any one of claims 19 to 53, wherein said nanoparticle is specifically directed to said target. A method comprising the steps of: conjugating to a single target ligand that binds; and administering to the subject nanoparticles that are conjugated to the single target ligand.   The target ligand is any one of an antibody, transferrin, a ligand for a cell receptor, an aptamer, or a cell receptor protein, and the target ligand is alternatively a fragment of an antibody, transferrin, a cell receptor 55. The method of claim 54, which may comprise a ligand or a cell receptor protein.   56. The method of claim 55, wherein the targeting nanoparticle further comprises a therapeutic agent.   57. The method of claim 56, wherein the therapeutic agent is a small molecule chemotherapeutic agent or polynucleotide.   58. The method of claim 57, wherein the polynucleotide is an interfering RNA.   58. The method of claim 57, wherein the small molecule chemotherapeutic agent is selected from camptothecin, epothilone, taxanes, and polynucleotides, or any combination thereof.   60. A method of delivering nanoparticles to a target in a subject comprising obtaining a nanoparticle according to any one of claims 19-59, wherein said nanoparticle specifically binds to said target And D. administering a single targeting ligand, and administering to the subject a nanoparticle conjugated to the single targeting ligand.   The target ligand is any one of an antibody, transferrin, a ligand for a cell receptor, an aptamer, or a cell receptor protein, and the target ligand is alternatively a fragment of an antibody, transferrin, a cell receptor 61. The method of claim 60, which may comprise a ligand or a cell receptor protein.   62. The method of claim 61, wherein the targeting nanoparticle further comprises a therapeutic agent.   63. The method of claim 62, wherein the therapeutic agent is a small molecule chemotherapeutic agent or polynucleotide.   64. The method of claim 63, wherein the polynucleotide is an interfering RNA.   64. The method of claim 63, wherein the small molecule chemotherapeutic agent is selected from camptothecin, epothilone, taxanes, and polynucleotides, or any combination thereof.   A targeted nanoparticle comprising a munic acid containing polymer, a therapeutic agent and a phenylboronic acid containing polymer, wherein the phenylboronic acid is reversibly covalently coupled to the munic acid polymer, said targeted nano A particle is adapted to present the phenylboronic acid containing polymer in the external environment of the nanoparticle, wherein the phenylboronic acid containing polymer is conjugated to the target ligand at the end opposite to the nanoparticle Targeted nanoparticles, wherein said targeted nanoparticles comprise a single target ligand. The therapeutic agent is any one of camptothecin, epothilone, taxanes, polynucleotides, or any combination thereof; and the phenylboronic acid containing polymer is any one of the following:
Wherein the targeting ligand comprises any one of an antibody, transferrin, a ligand for a cell receptor, or a cell receptor protein, and the targeting ligand is alternatively 67. The targeted nanoparticle of claim 66, which may comprise a fragment of an antibody, transferrin, a ligand for a cell receptor, or a cell receptor protein. The therapeutic agent is camptothecin and the phenylboronic acid containing polymer is any one of the following:
67. The targeted nanoparticle of claim 66, wherein: s = 20-300, the target ligand comprises an antibody.   Conjugating a nanoparticle containing a polyol containing polymer with a boronic acid containing polymer, and conjugating the boronic acid containing polymer with a target ligand, wherein the targeting nanoparticle of the targeting nanoparticle 70. The targeted nanomer of any one of claims 1-9, 19-53, or 66-68, wherein the ratio to the target ligand is 3: 1 or less, or 2: 1 or less, or 1: 1 or less. How to make particles.   70. The method of claim 69, wherein the ratio of the targeting nanoparticles to the targeting ligands of the nanoparticles is 1: 1.   The polyol-containing polymer is a mutic acid polymer, and the boronic acid-containing polymer comprises phenylboronic acid or nitrophenylboronic acid, and the target ligand is an antibody, transferrin, a ligand of a cell receptor, an aptamer, or a cell receptor 71. The method of claim 69 or 70, comprising a body protein. 72. The method according to any one of claims 69 to 71, wherein said phenylboronic acid containing polymer or said nitrophenylboronic acid containing polymer is one of the following compounds:
(In the formula, s = 20 to 300).   73. The method of any one of claims 69-72, wherein the boronic acid containing polymer is conjugated to the target ligand prior to conjugation to the nanoparticle comprising a polyol containing polymer.   Targeting wherein the ratio of nanoparticles comprising a polyol containing polymer, a boronic acid containing polymer, a target ligand and said nanoparticles to said target ligand is less than 3: 1, or less than 2: 1, or less than 1: 1 69. A kit for producing the targeted nanoparticle according to any one of claims 1-9, 19-53, or 66-68, comprising instructions for producing the nanoparticles.   75. The kit of claim 74, wherein the instructions describe a method of producing targeted nanoparticles wherein the ratio of the nanoparticles to the target ligand is 1: 1.   The polyol-containing polymer is a mutic acid polymer, and the boronic acid-containing polymer contains phenylboronic acid or nitrophenylboronic acid, and the targeting ligand is an antibody, transferrin, a ligand of a cell receptor, or a cell receptor protein 76. The kit of claim 74 or 75, comprising The kit according to any one of claims 74 to 76, wherein said phenylboronic acid containing polymer or said nitrophenylboronic acid containing polymer is one of the following compounds:
(In the formula, s = 20 to 300).   78. The kit of any one of claims 74-77, wherein the boronic acid containing polymer and the target ligand are provided as a single conjugated unit.   79. The kit of any one of claims 74-78, further comprising one or more reagents for conjugating the targeting ligand and the boronic acid containing polymer.   70. A method of treating a subject having a disease state, said method further comprising a therapeutic agent for treating said disease state of any of claims 1-9, 19-37, 40, 42-49, 52 or 66-67 Obtaining the nanoparticles according to any one of the preceding claims, wherein the boronic acid-containing polymer is conjugated to a single targeting ligand which specifically binds to the cells responsible for the disease state, Administering the nanoparticles to the subject.   81. The method of claim 80, wherein the disease state is cancer and the therapeutic agent is a chemotherapeutic compound.   82. The method of claim 81, wherein the chemotherapeutic compound is any one of camptothecin, epothilone, or taxane.   70. A method of treating a subject having a disease state, said method further comprising a therapeutic agent for treating said disease state of any of claims 1-9, 19-37, 40, 42-49, 52 or 66-67 Obtaining the nanoparticles according to any one of the preceding claims, wherein the nitrophenylboronic acid containing polymer is conjugated to a single targeting ligand which specifically binds to the cells responsible for the disease state. And administering the nanoparticles to the subject.   84. The method of claim 83, wherein the disease state is cancer and the therapeutic agent is a chemotherapeutic compound.   85. The method of claim 84, wherein the chemotherapeutic compound is any one of camptothecin, epothilone, or taxane.   73. The method of any one of claims 69-72, wherein the boronic acid containing polymer is conjugated to the target ligand after being conjugated to the nanoparticle comprising a polyol containing polymer.   70. A composition comprising the nanoparticles according to any one of claims 1 to 9, 19 to 53, or 66 to 68, and a suitable vehicle and / or excipient.