Cationic mucic acid polymer-based delivery system

Abstract

Polymer and polymer conjugate-based nanoparticle delivery systems for delivering biological materials, and methods of making and using these compositions, are provided. Certain embodiments of the present disclosure provide a compound comprising the following structural unit of formula (I) or formula (II) or formula (III): TIFF2022191285000090.tif45166 wherein B is an uncharged segment comprising a polyalkylene glycol; and B is a cationic charged segment comprising at least one polyhydroxy linkage, while providing a polymer comprising alternating charged and uncharged segments comprising one or more of the formula:

Description

Cross - Reference to Related Applications

[0001] This application claims the benefit of priority to U.S. Patent Application No. 62 / 187,366, filed on July 1, 2015, the content of which is incorporated herein by reference for all purposes. and is hereby incorporated by reference for all purposes. Government Rights

[0002] This invention was made with government support under Grant No. CA151819 awarded by the National Institutes of Health. The government has certain rights in the invention. Technical Field

[0003] The present disclosure relates to nanoparticle delivery systems based on polymers and polymer conjugates for delivering biological materials, and methods of making and using these compositions. BACKGROUND OF THE INVENTION

[0004] Background Therapies that use RNA interference (RNAi) as their mechanism of action have great promise for the treatment of human diseases. For example, siRNAs have attractive features as therapeutic agents, including: (i) the ability to target essentially any gene (thus, in principle, all targets are druggable for the development of new drugs), (ii) potent single - digit picomolar IC (concentration required for 50% inhibition) for mRNA suppression in well - designed siRNAs, (iii) chemical modifications and sequence designs that can minimize off - target effects and immune stimulation without sacrificing efficacy and target 50 specificity, and (iv) the catalytic RNAi mechanism of action, resulting in extended siRNA suppression of mRNA target expression. Effective and efficient therapies can be achieved. ​​A major obstacle to the translation (also referred to as translocation) of siRNA to a substance is the delivery of the nucleic acid to the target. However, siRNA-based experimental therapeutic substances have reached clinical practice.

Summary of the Invention

Problems to be Solved by the Invention

[0005] Therapeutic substances studied for cancer treatment are mainly administered systemically, and some types of synthetic compounds (positively charged lipids or polymers) are used in these formulations to deliver siRNA. Many of these formulations are currently called nanoparticles (NPs). CALAA-01 was the first siRNA-based therapeutic substance to reach the clinic for the treatment of cancer. This targeted nanoparticle contains a cyclodextrin-based polycation (CDP) assembled by siRNA via electrostatic interactions between the positive charge on the polymer and the negative charge on the siRNA backbone. CALAA-01 was able to deliver siRNA to solid tumors in patients and release functional siRNA that suppresses the target using the RNAi mechanism (the first example in humans). Although CALAA-01 reveals several positive characteristics, one of its drawbacks is that it has an unusually limited circulation time. The rapid clearance of CALAA-01 observed in animals (mice, rats, dogs, and non-human primates) is also observed in humans.

Means for Solving the Problems

[0006] Overview Increase the circulation time of siRNA-containing nanoparticles and the non-siRNA components within the formulation. ​​​​​​The development of a polymeric system for siRNA delivery that reduces the amount would be advantageous. This disclosure is directed to delivery systems that overcome some of the drawbacks of the prior art. This disclosure Among the aspects of, diblock and triblock Copolymers, including cationic mucic acid-based polymers (cMAP), and nanoparticles derived therefrom. These compounds and structures and methods of making and using them will be described in more detail within this specification.

[0007] Certain embodiments of the present disclosure include the following structural units of formula (I) or formula (II) or formula (III) :

Chemical formula

[0008] In certain embodiments, A is polyethylene glycol and a suitable linking group, or Or they are included. In other embodiments, the polyalkylene glycol moiety has a nominal number average molecular weight in the range of about 500 daltons to about 50,000 daltons within these polymers Have.

[0009] In overlapping embodiments, B is a cationic charged segment containing at least one pair of adjacent diols containing a sugar linkage and at least One polyhydroxy is included. In some embodiments ​​​In these polyhydroxy linkages, mucic acid is included. B may further include at least one repeating subunit containing the structure of formula (V) :

Chemical formula

[0010] In other embodiments, in these polymers, B may include at least one repeating subunit containing cMAP, and the subunit structure is formula (VI):

Chemical formula

[0011] In some specific embodiments, the polymer may be described by the structure of formula (VII):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0012] Other structures contemplated within the scope of the present disclosure include structures of formula (VIII):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0013] Still other polymers can be described by the structure of formula (IX):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0014] Also contemplated within the scope of the present disclosure are polymers of any one of the preceding structures, and the structure of formula (X)

Chemical formula

[0015] The present disclosure also provides nanoparticles comprising any of the polymers or polymer composites described herein. The nanoparticles may include a single nanoparticle or nanoparticles. Preferably, the nanoparticles are monodisperse. The nanoparticles may include: It may further comprise an encapsulated biological material, e.g., siRNA, and / or one or It may be further conjugated to more targeting ligands. When administered by itself, the bioavailability of a biological substance is It has better bioavailability than the same biological substance.

[0016] In some additional embodiments, the polymers, polymer composites, and nanoparticles are Optionally, the biological material and / or the targeting ligand are included and formulated in a pharmaceutical composition. Other embodiments may incorporate these formulated compositions into the respective biological material required. The patient treatment is provided by administering the same to the patient.

[0017] Yet a further embodiment provides a method of making the inventive polymer. [Brief description of the drawings]

[0018] The present application will be better understood when read in conjunction with the accompanying drawings, in which: Although exemplary embodiments of the subject matter are shown in these figures; The problems are not limited to the disclosed specific methods, devices, and systems. In addition, the drawings are not necessarily drawn to scale. The drawings are as follows:

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Mode for Carrying Out the Invention

[0019] Detailed Description of Exemplary Embodiments The present disclosure is directed to delivery systems that overcome some of the drawbacks of the prior art.

[0020] The inventors have investigated the cause of the short circulation time mentioned elsewhere herein and have shown that CALA A - 01 is degraded by the glomerular basement membrane (GBM) in the kidney. The inventors have speculated that this clearance mechanism may affect any NP formulation primarily assembled by electrostatic interaction between the cationic delivery component and the anionic nucleic acid. Other siRNA delivery systems using either a cationic polymer or lipid have shown similar short circulation times and renal clearance. polymer or lipid circulation time and renal clearance.

[0021] Numerous current polymers and liposome systems used to deliver siRNA in vivo tem contains, in addition to large amounts of substances such as poly(ethylene glycol) (PEG), their formu lation contains an excess of cationic components in the agent (the positive-to-negative charge ratio is usually greater than 1) and is used to sterically stabilize the formed NPs. The excess cationic components may have undesirable side effects in vivo and can cause adverse reactions such as platelet aggregation, complement activation, and inflammatory reactions.

[0022] It would be advantageous to develop a polymer system for siRNA delivery that both increases the circulation time of siRNA-containing nanoparticles and reduces the amount of non-siRNA components in the formulation. A family of cationic mucic acid-based polymers (cMAP) for siRNA delivery in vivo is described herein. This polymer delivery system has several features similar to the CDP system, because the latter system has functioned in humans. The cationic polymers developed here use simpler sugars and are embodied as mucic acid (also called mucin acid or mucic acid) rather than cyclodextrin, and enable alternative strategies for surface functionalization. Instead of nanoparticle surface functionalization via inclusion complex (also called inclusion complex) formation with adamantane (AD) (CDP), cMAP contains adjacent diols that are binding sites for boronic acids that can be used to PEGylate and target cMAP-based nanoparticles. Nanoparticles formed by mucic acid-containing polymers for the delivery of small molecule drugs incorporated targeting agents via this assembly method. Also, the basic cMAP is functionalized It was further reacted to PEG and linear block copolymers. The carboxylic acid-PEG, or activated carboxylic acid-PEG-methoxy (PEGm), which was activated at the end group of cMAP, reacted with either of them to form two possible copolymers (also referred to as copolymers): cMAP-PEG copolymer or mPEG-cMAP-PEGm triblock polymer. The cMAP-PEG copolymer can be combined with siRNA to form a PEG loop on the surface and stabilize the nanoparticles. On the other hand, the mPEG-cMAP-PEGm triblock can form a PEG brush structure on the nanoparticle surface. The latter triblock approach has been previously investigated with CDP and plasmid DNA (pDNA), and the triblock polymer did not have the ability to encapsulate pDNA. It has been shown that polymers that encapsulate pDNA may not be good in concentrated siRNA, and vice versa. Here, the inventors show that the mPEG-cMAP-PEGm triblock polymer can form siRNA-containing nanoparticles (which can have ca. (about) 30 wt% of the formulation which is siRNA) with an increase in circulation time in mice. Furthermore, the nanoparticles can be readily assembled directly in phosphate-buffered saline (PBS) without the use of any additional 5-nPBA-PEGm to stabilize the NPs. The reaction with either of the carboxylic acid-PEG, which was activated at the end group of cMAP, or activated carboxylic acid-PEG-methoxy (PEGm) leads to two possible copolymers (also referred to as copolymers): cMAP-PEG copolymer or mPEG-cMAP-PEGm triblock polymer. The cMAP-PEG copolymer can be combined with siRNA to form a PEG loop on the surface and stabilize the nanoparticles. On the other hand, the mPEG-cMAP-PEGm triblock can form a PEG brush structure on the nanoparticle surface. The latter triblock approach has been previously investigated with CDP and plasmid DNA (pDNA), and the triblock polymer did not have the ability to encapsulate pDNA. It has been shown that polymers that encapsulate pDNA may not be good in concentrated siRNA, and vice versa. Here, the inventors show that the mPEG-cMAP-PEGm triblock polymer can form siRNA-containing nanoparticles (which can have ca. (about) 30 wt% of the formulation which is siRNA) with an increase in circulation time in mice. Furthermore, the nanoparticles can be readily assembled directly in phosphate-buffered saline (PBS) without the use of any additional 5-nPBA-PEGm to stabilize the NPs. This disclosure can be more readily understood by referring to the following description in connection with the accompanying figures and examples, all of which form a part of this disclosure. This disclosure is not limited to the specific products, methods, conditions or parameters described or shown herein, and

[0023] ​ The terms used herein are for the purpose of describing particular embodiments only as examples and are not intended to limit any claimed invention. Similarly, unless otherwise specified, any description of possible mechanisms or modes of action or reasons for improvements are meant to be exemplary only and the disclosure herein is not restricted by the accuracy or inaccuracy of any such proposed mechanisms or modes of action or reasons for improvements. Throughout this specification it will be recognized that the description relates to compositions and methods of making and using said compositions. That is, where the disclosure describes or claims features or embodiments related to a composition or a method of making or using a composition such description or claims are intended to extend these features or embodiments to embodiments (i.e., compositions methods of making, and methods of using) in each of these contexts as is recognized. In this disclosure, the singular forms "a", "an", and "the" include plural references and, unless the context clearly dictates otherwise references to a particular numerical value include at least that particular value. Thus, for example a reference to "a substance" is a reference to at least one of such a substance and its equivalents known to those skilled in the art (also referred to as those of ordinary skill in the art) in that technology .

[0024] When a value is expressed as an approximation by use of the descriptor "about" it will be understood that that particular value forms another embodiment. In general, the use of the term "about" is inclusive and the disclosure herein is not restricted by the accuracy or inaccuracy of any such proposed mechanisms or modes of action or reasons for improvements. Throughout this specification

[0025] it will be recognized that the description relates to compositions and methods of making and using said compositions. That is, where the disclosure describes or claims features or embodiments related to a composition or a method of making or using a composition such description or claims are intended to extend these features or embodiments to embodiments (i.e., compositions An approximation that may vary depending on the desired characteristics sought to be obtained by the disclosed subject matter is indicated and, based on its functionality, is interpreted in the particular context in which it is used. One of ordinary skill in the art will be able to interpret this as an everyday matter. In some cases , the number of significant digits used for a particular value may be one of the non-limiting ways of determining the degree of the word "about". In other cases, the gradual change used in a series of values can be used to define the intended range within which the term "about" can be used for each value. Where ranges exist, all ranges are inclusive and combinable. That is, a reference to a value within a range includes all values within that range.

[0026] For clarity, certain features of the present disclosure described herein in the context of separate embodiments should also be recognized as being capable of being provided in combination in a single embodiment. That is, each individual embodiment, unless clearly incompatible or clearly excluded, is capable of being combined with any other embodiment(s) (the term "embodiment(s)" indicating that a plurality may also be included) and such combinations are considered to be another embodiment. Conversely, for the sake of brevity, the various features of the present disclosure described in the context of a single embodiment may be provided separately or in any sub-combination. Finally, an embodiment may be described as part of a series of steps or a more general structure, while each of the steps may be considered an independent embodiment capable of being combined with others.

[0027] The transitional terms "comprising", "consisting essentially of", and "consisting of" are intended to embrace their generally accepted meanings in patent parlance, i.e., (i) "comprising" is synonymous with "including", "containing", or "characterized by", is inclusive or open-ended, and does not exclude additional, recited elements or method steps; (ii) "consisting of" excludes any element, step, or ingredient not specified in the claim; and (iii) "consisting essentially of" limits the scope of the claim to those that do not "materially affect" the "basic and novel characteristics (s)" of the claimed invention. Embodiments described by the phrase "comprising" (or its equivalents) also provide, as embodiments, those described indifferently with respect to "consisting of" and "consisting essentially of". For those embodiments provided by "consisting essentially of", the basic and novel characteristics (s) are the inventive substances, and the ease of operation of the methods (and the systems used in such methods and the compositions derived therefrom) for preparing and using the substances themselves, where the methods and substances are capable of providing the highlighted properties using only the elements provided in the claims. That is, other materials are also inventive combinations are intended, and, in particular, (i) "comprising" is synonymous with "including", "containing", or "characterized by", is inclusive or open-ended, and does not exclude additional, recited elements or method steps; (ii) "consisting of" excludes any element, step, or ingredient not specified in the claim; and (iii) "consisting essentially of" limits the scope of the claim to those that do not "materially affect" the "basic and novel characteristics (s)" of the claimed invention. Embodiments described by the phrase "comprising" (or its equivalents) also provide, as embodiments, those described indifferently with respect to "consisting of" and "consisting essentially of". For those embodiments provided by "consisting essentially of", the basic and novel characteristics (s) are the inventive substances, and the ease of operation of the methods (and the systems used in such methods and the compositions derived therefrom) for preparing and using the substances themselves, where the methods and substances are capable of providing the highlighted properties using only the elements provided in the claims. That is, other materials are also inventive combinations are intended, and, in particular, (i) "comprising" is synonymous with "including", "containing", or "characterized by", is inclusive or open-ended, and does not exclude additional, recited elements or method steps; (ii) "consisting of" excludes any element, step, or ingredient not specified in the claim; and (iii) "consisting essentially of" limits the scope of the claim to those that do not "materially affect" the "basic and novel characteristics (s)" of the claimed invention. Embodiments described by the phrase "comprising" (or its equivalents) also provide, as embodiments, those described indifferently with respect to "consisting of" and "consisting essentially of". For those embodiments provided by "consisting essentially of", the basic and novel characteristics (s) are the inventive substances, and the ease of operation of the methods (and the systems used in such methods and the compositions derived therefrom) for preparing and using the substances themselves, where the methods and substances are capable of providing the highlighted properties using only the elements provided in the claims. That is, other materials are also inventive combinations (including), "containing" (containing), or "characterized by" (characterized by are intended, and, in particular, (i) "comprising" is synonymous with "including", "containing", or "characterized by", is inclusive or open-ended, and does not exclude additional, recited elements or method steps; (ii) "consisting of" excludes any element, step, or ingredient not specified in the claim; and (iii) "consisting essentially of" limits the scope of the claim to those that do not "materially affect" the "basic and novel characteristics (s)" of the claimed invention. Embodiments described by the phrase "comprising" (or its equivalents) also provide, as embodiments, those described indifferently with respect to "consisting of" and "consisting essentially of". For those embodiments provided by "consisting essentially of", the basic and novel characteristics (s) are the inventive substances, and the ease of operation of the methods (and the systems used in such methods and the compositions derived therefrom) for preparing and using the substances themselves, where the methods and substances are capable of providing the highlighted properties using only the elements provided in the claims. That is, other materials are also inventive combinations (consisting of), and (ii) "consisting of" (consisting of are intended, and, in particular, (i) "comprising" is synonymous with "including", "containing", or "characterized by", is inclusive or open-ended, and does not exclude additional, recited elements or method steps; (ii) "consisting of" excludes any element, step, or ingredient not specified in the claim; and (iii) "consisting essentially of" limits the scope of the claim to those that do not "materially affect" the "basic and novel characteristics (s)" of the claimed invention. Embodiments described by the phrase "comprising" (or its equivalents) also provide, as embodiments, those described indifferently with respect to "consisting of" and "consisting essentially of". For those embodiments provided by "consisting essentially of", the basic and novel characteristics (s) are the inventive substances, and the ease of operation of the methods (and the systems used in such methods and the compositions derived therefrom) for preparing and using the substances themselves, where the methods and substances are capable of providing the highlighted properties using only the elements provided in the claims. That is, other materials are also inventive combinations are intended, and, in particular, (i) "comprising" is synonymous with "including", "containing", or "characterized by", is inclusive or open-ended, and does not exclude additional, recited elements or method steps; (ii) "consisting of" excludes any element, step, or ingredient not specified in the claim; and (iii) "consisting essentially of" limits the scope of the claim to those that do not "materially affect" the "basic and novel characteristics (s)" of the claimed invention. Embodiments described by the phrase "comprising" (or its equivalents) also provide, as embodiments, those described indifferently with respect to "consisting of" and "consisting essentially of". For those embodiments provided by "consisting essentially of", the basic and novel characteristics (s) are the inventive substances, and the ease of operation of the methods (and the systems used in such methods and the compositions derived therefrom) for preparing and using the substances themselves, where the methods and substances are capable of providing the highlighted properties using only the elements provided in the claims. That is, other materials are also inventive combinations ", and the "basic and novel characteristics (group)" of the claimed invention are not materially affected. Embodiments described by the phrase "comprising" (or its equivalents) also provide, as embodiments, those described indifferently with respect to "consisting of" and "consisting essentially of". For those embodiments provided by "consisting essentially of", the basic and novel characteristics (s) are the inventive substances, and the ease of operation of the methods (and the systems used in such methods and the compositions derived therefrom) for preparing and using the substances themselves, where the methods and substances are capable of providing the highlighted properties using only the elements provided in the claims. That is, other materials are also inventive combinations ", and the "basic and novel characteristics (group)" of the claimed invention are not materially affected. Embodiments described by the phrase "comprising" (or its equivalents) also provide, as embodiments, those described indifferently with respect to "consisting of" and "consisting essentially of". For those embodiments provided by "consisting essentially of", the basic and novel characteristics (s) are the inventive substances, and the ease of operation of the methods (and the systems used in such methods and the compositions derived therefrom) for preparing and using the substances themselves, where the methods and substances are capable of providing the highlighted properties using only the elements provided in the claims. That is, other materials are also inventive combinations are intended, and, in particular, (i) "comprising" is synonymous with "including", "containing", or "characterized by", is inclusive or open-ended, and does not exclude additional, recited elements or method steps; (ii) "consisting of" excludes any element, step, or ingredient not specified in the claim; and (iii) "consisting essentially of" limits the scope of the claim to those that do not "materially affect" the "basic and novel characteristics (s)" of the claimed invention. Embodiments described by the phrase "comprising" (or its equivalents) also provide, as embodiments, those described indifferently with respect to "consisting of" and "consisting essentially of". For those embodiments provided by "consisting essentially of", the basic and novel characteristics (s) are the inventive substances, and the ease of operation of the methods (and the systems used in such methods and the compositions derived therefrom) for preparing and using the substances themselves, where the methods and substances are capable of providing the highlighted properties using only the elements provided in the claims. That is, other materials are also inventive combinations ing essentially of" are also provided as embodiments. For those embodiments provided by "consisting essentially of", the basic and novel characteristics (s) are the inventive substances, and the ease of operation of the methods (and the systems used in such methods and the compositions derived therefrom) for preparing and using the substances themselves, where the methods and substances are capable of providing the highlighted properties using only the elements provided in the claims. That is, other materials are also inventive combinations ing essentially of", the basic and novel characteristics (s) are the inventive substances, and the ease of operation of the methods (and the systems used in such methods and the compositions derived therefrom) for preparing and using the substances themselves, where the methods and substances are capable of providing the highlighted properties using only the elements provided in the claims. That is, other materials are also inventive combinations (group) are the inventive substances, and the ease of operation of the methods (and the systems used in such methods and the compositions derived therefrom) for preparing and using the substances themselves, where the methods and substances are capable of providing the highlighted properties using only the elements provided in the claims. That is, other materials are also inventive combinations and the ease of operation of the methods (and the systems used in such methods and the compositions derived therefrom) for preparing and using the substances themselves, where the methods and substances are capable of providing the highlighted properties using only the elements provided in the claims. That is, other materials are also inventive combinations and the substances are capable of providing the highlighted properties using only the elements provided in the claims. That is, other materials are also inventive combinations and the substances are capable of providing the highlighted properties using only the elements provided in the claims. That is, other materials are also inventive combinations may be present in the product, but the presence of these additional materials is not necessary to provide the stated advantages of their compositions (i.e., the effects may be additive) and / or these additional substances do not weaken the performance of the product composition. Similarly, if additional steps can also be used in the present method, their presence is not necessary to achieve the stated effects or advantages and / or does not impair the stated effects or benefits. When a list is presented, unless otherwise stated, each individual element of that list and any combination of that list should be understood to be a separate embodiment. For example, a list of embodiments presented as "A, B, or C" includes the embodiments, "A", "B"

[0028] ", "C", "A or B", "A or C", "B or C", or "A, B, or C" and should be interpreted as such. Similarly, terms such as C alkyl also include, as separate embodiments, C1 alkyl, C2 alkyl, C3 alkyl, C alkyl, and C2 alkyl. 1-3 Throughout this specification, as would be understood by one of ordinary skill in the relevant art, words should be given their ordinary meaning. However, to avoid misunderstanding, the meaning of certain terms is defined or clarified. 1-2 references to alcohols, aldehydes, amines, carboxylic acids, ketones, or other similarly reactive functional -3 groups also include their protected analogs (also referred to as analogs). For example,

[0029] Throughout this specification, as would be understood by one of ordinary skill in the relevant art, words should be given their ordinary meaning. However, to avoid misunderstanding, the meaning of certain terms is defined or clarified. To avoid misunderstanding, the meaning of certain terms is defined or clarified.

[0030] references to alcohols, aldehydes, amines, carboxylic acids, ketones, or other similarly reactive functional groups also include their protected analogs (also referred to as analogs). For example, For example, references to hydroxy or alcohol also include their substitutions, where hydroxy is acetyl (Ac), benzoyl (Bz), benzyl (Bn, Bnl), β-methoxy ethoxymethyl ether (MEM), dimethoxytrityl, [bis-(4-methoxyphenyl) phenylmethyl](DMT), methoxymethyl ether (MOM), methoxytrityl [(4-meth oxyphenyl)diphenylmethyl, MMT), p-methoxybenzyl ether (PMB), methyl thiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydro furan (THF), trityl (triphenylmethyl, Tr), silyl ether (the most prevalent ones include trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri- iso-propylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS) eth er), ethoxyethyl ether (EE). References to amines also include their substitutions, where amines are BOC glycine, carbobenzyloxy (Cbz ), p-methoxybenzylcarbonyl (Moz or MeOZ), tert-butyloxycarbonyl ( BOC), 9-fluorenylmethyloxycarbonyl (FMOC), acetyl (Ac), benzoyl (Bz), benzyl (Bn), carbamate, p-methoxybenzyl (PMB), 3,4-dimethoxy benzyl (DMPM), p-methoxyphenyl (PMP), tosyl (Ts) group, or sulfonam ide (Nosyl&Nps) group. References to substitutions containing a carbonyl group also include their substitutions, where carbonyl is acetal or ketal, acylal, or or is protected by a diatan group. Substitution to include a carboxylic acid or carboxylate group in the reference to, the carboxylic acid or carboxylate group, its methyl ester, benzyl ester, tert-butyl ester, ester of 2,6-disubstituted phenol (e.g., 2,6-dimethyl phenol, 2,6-diisopropylphenol, 2,6-di-tert-butylphenol), silyl esters, orthoesters, or substituents protected by oxazoline are included.

[0031] Embodiments of the present disclosure include the following structural units of formula (I) or formula (II) or formula (III): [Chemical formula] polymers containing alternating charged and uncharged segments, including one or more of wherein, A is an uncharged segment containing a polyalkylene glycol; B is a cationic charged segment containing at least one polyhydroxy linkage including at least a pair of adjacent diols is included.

[0032] In the polymers of the present disclosure, the term polyalkylene glycol refers to a functional linkage: [Chemical formula] refers to any polymer including, wherein x ranges from 1 to about 6, still, in practice, and if possible, either 2 or 3, preferably 2 if possible (the higher the value of x, the lower the hydrophilicity of the polyalkylene glycol is shown). Thus, in a preferred embodiment, A is polyethylene glycol and an optional (but preferably) suitable linking group or includes them, and the linking group is necessary to bind to other components of the overall polymer . Suitably, in each instance, polyalkylene glycol, generally, and polyethylene glycol, specifically, has a nominal (also referred to as nominal) number-averaged molecular weight (MW ) in the range of from about 500 Daltons to about 50,000 Daltons. In a more specific embodiment, a portion of the polyalkylene / polyethylene glycol n can have an MW value of from about 500 Da to about 1 kDa, from about 1 kDa to about 5 kDa, from about 5 kDa to about 10 kDa, from about 10 kDa to about 15 kDa, from about 15 kDa to about 20 kDa, from about 20 kDa to about 30 kDa, from about 30 kDa to about 40 kDa, from about 40 kDa to about 50 kDa, or any combination of two or more of these ranges of MW n .

[0033] In some of these embodiments, B is a cationic charged segment containing at least one polyhydroxy linkage containing at least a pair of adjacent diols. Polyhydroxy sucrose or carbohydrate (also referred to as carbohydrate) linkages are preferred for their biocompatibility, but chiral and achiral synthetic polyhydroxy linkages can also be employed (e.g., polyhydroxy (meth)acrylic acid). In certain preferred embodiments, the polyhydroxy linkage contains mucin acid, where B contains at least one repeating subunit having a structure represented by formula (IV), formula (IV) or (IVA): [Chemical formula] These structures are rotational iosomers of each other and, for the purposes of this, are functionally are energetically equivalent. As used herein, one representative of these structures encompasses any one or both of these structures in connection with their use.

[0034] In other embodiments, B further comprises at least one repeating subunit having the structure of formula (V): [Chemical formula] wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 4 - 6 or 5 if possible. Such linkages are useful for both their cationic properties and the convenience of their use in linking a portion of a polyhydroxy (e.g., mucic acid) to a polyalkylene glycol. When combined with the aforementioned mucic acid linkage, the combination of linkages (IV) and (V) presents a substructure (also referred to as a basic structure) containing at least one repeating subunit comprising cMAP, and their subunit structures are represented by formula (VI): wherein m is, independently of each occurrence, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 4 - 6 or 5; and n is, independently of each occurrence, 1, 2, 3, 4, or 5, preferably 1 if possible. It should be noted that m and n are not necessarily limited to these values, and larger values for these variables are also contemplated within the scope of the present disclosure. [Chemical formula] as represented by; wherein m is, independently of each occurrence, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 4 - 6 or 5; and n is, independently of each occurrence, 1, 2, 3, 4, or 5, preferably 1 if possible. It should be noted that m and n are not necessarily limited to these values, and larger values for these variables are also contemplated within the scope of the present disclosure. are not necessarily limited to these values, and larger values for these variables are also contemplated within the scope of the present disclosure. With these building blocks (also referred to as basic units), even more specific tri

[0035] Along with these building blocks (also referred to as basic units), even more specific tri It is possible to describe the scope of block and diblock polymers. Again, the structures described below can be prepared using the methods described in the examples and using homologs (also referred to as congeners) of the reactants described therein. For example, in certain embodiments, the polymers of the present disclosure have the structure of formula (VII): The structures described below can be prepared using the methods described in the examples and using homologs (also referred to as congeners) of the reactants described therein. For example, in certain embodiments, the polymers of the present disclosure have the structure of formula (VII): The structures described below can be prepared using the methods described in the examples and using homologs (also referred to as congeners) of the reactants described therein. For example, in certain embodiments, the polymers of the present disclosure have the structure of formula (VII): The polymers of the present disclosure have the structure of formula (VII):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0036] Here again, the values of p and q may be the same or different for each occurrence and may be the same or different from each other and may be the same or different from each other. The same applies to n and r, i.e., the values for n and r may be the same or different for each occurrence and may be the same or different from each other. In certain embodiments, when m is 5 and n is 1, the numerical values for p correspond to a range from about 1 to about 100, and preferably from about 10 to about 100 if possible. In certain embodiments, p is about 1 to about 10, about 10 to about 25, about 25 to about 50, about 50 to about 75, about 75 to about 100, or any combination of two or more of these ranges. The MW values for q corresponding to the cited range include those in the range from about 12 to about 1200 . In certain embodiments, q is about 12 to about 100, about 100 to about 400 n , about 400 to about 800, about 800 to about 1200, or any combination of two or more of these ranges. In certain embodiments, q can also be within the range of about 100 to about 500. In other subsets of this embodiment, X1 and X2 are each independently of their respective occurrences, -(CH2) -COOH and / or -(CH2) -NH2. Considering the nature of chains A and B, such structures can also be represented as PEG-cMAP-PEG triblock polymers 1-4 -COOH and / or -(CH2) 1-4 -NH2.

[0037] Considering the nature of chains A and B, such structures can also be represented as PEG-cMAP-PEG triblock polymers -COOH and / or -(CH2)

[0038] In other embodiments, the polymers of the present disclosure have the structure of formula (VIII):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0039] Again, the values of p and q may be the same or different for each occurrence and may be the same or different from each other. The same is true for n and r; that is, the values of n and r may be the same or different for each occurrence and may be the same or different from each other. In certain embodiments, when m is 5 and n is 1, the numerical values for p correspond to a range of from about 1 to about 100, and preferably from about 10 to about 100 if possible. In certain embodiments, when m is 5 and n is 1, the numerical values for p correspond to a range of from about 1 to about 100, and preferably from about 10 to about 100 if possible. In certain embodiments, when m is 5 and n is 1, the numerical values for p correspond to a range of from about 1 to about 100, and preferably from about 10 to about 100 if possible. In certain embodiments, p is about 1 to about 10, about 10 to about 25, about 25 to about 50, about 50 to about 75, about 75 to about 100, or any combination of two or more of these ranges. In certain embodiments, p is about 1 to about 10, about 10 to about 25, about 25 to about 50, about 50 to about 75, about 75 to about 100, or any combination of two or more of these ranges. n The numerical values for q corresponding to the cited MW range include those ranging from about 12 to about 1200. In certain embodiments, q is about 12 to about 100, about 100 to about 400, about 400 to about 800, about 800 to about 1200, or any combination of two or more of these ranges. In certain embodiments, q is about 12 to about 100, about 100 to about 400, about 400 to about 800, about 800 to about 1200, or any combination of two or more of these ranges. In certain embodiments, q can also be in the range of about 100 to about 500. In other subsets of this embodiment, X1 and X2 are, independently of each other, -(CH2)4-COOH and / or -(CH2)-NH2. 1- In other subsets of this embodiment, X1 and X2 are, independently of each other, -(CH2)4-COOH and / or -(CH2)-NH2. 1-4 In other subsets of this embodiment, X1 and X2 are, independently of each other, -(CH2)4-COOH and / or -(CH2)-NH2.

[0040] Considering the nature of the various chains and end group elements, such structures are cMAP-PEG diblocks or It can also be referred to as a PEG-cMAP diblock polymer.

[0041] In yet other embodiments, the polymers of the present disclosure include the structure of formula (IX):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0042] Again, the values of p and q may be the same or different for each occurrence and may be the same or different from each other. The same is true for n and r; that is, the values of n and r may be the same or different for each occurrence and may be the same or different from each other. In certain embodiments, when m is 5 and n is 1, the numerical value for p corresponds to a range of from about 1 to about 100, and preferably from about 10 to about 100 if possible. In certain embodiments, p is from about 1 to about 10, from about 10 to about 25, from about 25 to about 50, from about 50 to about 75, from about 75 to about 100, or any combination of two or more of these ranges. The numerical values for q corresponding to the cited MW range include those ranging from about 12 to about 1200. In certain embodiments, q is from about 12 to about 100, from about 100 to about 400, from about 400 to about 800, from about 800 to about 1200, or any combination of two or more of these ranges. In certain embodiments, q can also be in the range of from about 100 to about 500. In other subsets of such embodiments, X1 and X2, independently of their respective presence, are -(CH2)4-COOH and / or -(CH2)-NH2. Considering the nature of the various chains and end group elements, such structures are cMAP-PEG-cMAP triblock polymers. n polymers. In certain embodiments, q is from about 12 to about 100, from about 100 to about 400, from about 400 to about 800, from about 800 to about 1200, or any combination of two or more of these ranges. In certain embodiments, q can also be in the range of from about 100 to about 500. In other subsets of such embodiments, X1 and X2, independently of their respective presence, are -(CH2)4-COOH and / or -(CH2)-NH2. Considering the nature of the various chains and end group elements, such structures are cMAP-PEG-cMAP triblock polymers. 1- 4-COOH and / or -(CH2)-NH2. 1-4 Considering the nature of the various chains and end group elements, such structures are cMAP-PEG-cMAP triblock

[0043] polymers. It can also be referred to as a block polymer.

[0044] In each of the structures presented, in certain non - relevant embodiments, m is 4, 5 or 6 without relevance. In certain embodiments, m is 5 in each occurrence.

[0045] In each of the structures presented, certain non - relevant embodiments include those where n is 1 .

[0046] In each of the structures presented, in certain non - relevant embodiments, r is 2, 3 or 4 without relevance. In some of these embodiments, r is 3 in each occurrence .

[0047] In each of the structures presented, in certain non - relevant embodiments, p provides an average molecular weight sufficient for a subunit containing cMAP in the range of about 5 kDa to about 15 kDa, about 6 kDa to about 14 kDa, 7 kDa to about 13 kDa, about 8 kDa to about 12 kDa, 9 kDa to about 11 kDa, or about 10 kDa. (When the cMAP fragments (also called fragments) each have an MW of about 420 Da), this corresponds to a value of p in the range of about 12 to about 36, about 14 to about 33, about 17 to about 31, about 19 to about 29, about 22 to about 26, or about 24. In each of the structures presented, in certain non - relevant embodiments, q is in the range of about 500 Da to about 50 kDa, about 1 kDa to about 40 kDa, 5 kDa to about 30 kDa, or about 5 kDa to about 20 kDa .

[0048] In each of the structures presented, in certain non - relevant embodiments, q is in the range of about 500 Da to about 50 kDa, about 1 kDa to about 40 kDa, 5 kDa to about 30 kDa, or about 5 kDa to about 20 kDa when it is sufficient to provide the number average molecular weight of the sub-units containing PEG in the range of are included. When the polyalkylene glycol moiety is polyethylene glycol , and assuming that the ethylene glycol fragment has an MW of about 44 Da, this corresponds to about 11 to about 12 00, in the range of about 23 to about 910, about 110 to about 680, or about 110 to about 450 corresponding to the value of q.

[0049] Each combination of values for m, n, p, q , r, or z according to any suitable definition of X1, X2, X3, and / or X4 described herein represents a separate embodiment, and any combination of these embodiments provides the definition of another embodiment.

[0050] Exemplary and non-limiting schemes for preparing the various polymers described herein are provided in the Examples . Each of these synthetic routes, as well as those using homologs of the specifically described reagents , is considered to be within the scope of the present disclosure. As used herein, the term homolog refers to a compound that differs from the model by one or more methylene groups. In one method , the polymer can be prepared by connecting at least one non-charged segment containing a polyalkylene glycol , through the use of at least one linking group, to at least one cationic charged segment containing at least one polyhydroxy linkage . In the case where the cationic charged segment is one of the above-mentioned mucic acid derivatives, this method involves chemically reacting two PEG polymers with a mucic acid polymer, two mucic acid polymers with one PEG polymer, or one mucic acid polymer with one PEG polymer, stoichiometrically, and this includes Each having an appropriate linking group to form the desired diblock or triblock polymer. Such coupling reactions can be effected using carboxylic acid / amino condensation reactions and can form amide bonds as described herein.

[0051] The formation of nanoparticles according to some embodiments of the present disclosure can be analyzed by techniques and procedures known to those skilled in the art in order.

[0052] Still other embodiments of the present disclosure include polymer conjugates (also referred to as polymer complexes) of the cMAP / PEG-containing polymers described herein. Such polymer conjugates include any cMAP / PEG-containing polymer and a structure of formula (X):

Chemical formula

[0053] In certain of these embodiments, n is 1. Among these embodiments, exemplary structures include: :

Chemical formula

[0054] In some embodiments of the polymer conjugate, s is from 20 to about 120, about 12 0 to about 240, about 240 to about 480, about 480 to about 720, about 720 to about 960, about 96 0 to about 1200, or in the range of any combination of two or more of these ranges.

[0055] In other embodiments of the polymer conjugate, L is -(C 0-2 alkylene-)NH-C(=O)-(C 0- 2alkylene)-, -(C 0-2 alkylene)-C(=O)-NH-(C 0-2 alkylene)-, -(C 0-2 alkylene)-O -C(=O)-(C 0-2 alkylene)- or -(C 0-2 alkylene)-C(=O)-O-(C 0-2 alkylene)-. . In these subsets, L is -NH-C(=O)-, -C(=O)-NH-, -OC(=O)-, or -C(=O)-O-. The single or multiple linking groups of L (also referred to as multi-linking groups) can be employed by any polymer. .

[0056] So far, the present disclosure has been described with respect to polymers or polymer conjugates However, important elements of the present disclosure include nanoparticles derived from these polymers or polymer conjugates, and the provisions provided for these polymers and polymer conjugates are equally useful in the description of the relevant nanoparticles. These nanoparticles tend to be substantially similar and depend on the size of various cMAP or PEG fragments and / or the length of the chains associated with the boronic acid-containing polymers, having cross-sectional dimensions (i.e., diameters) in the range of about 20 nm to about 300 nm. Certain embodiments also describe that these nanoparticles have diameters in the range of about 20 nm to about 40 nm (hereinafter referred to as about 20 nm to about 40 nm), about 40 nm to about 80 nm, about 80 nm to about 120 nm, about 120 nm to about 180 nm, about 180 nm to about 240 nm, about 240 nm to about 300 nm, or combinations of two or more of these ranges and can be said. These nanoparticles tend to be substantially similar and, depending on the size of various cMAP or PEG fragments and / or the length of the chains associated with the boronic acid-containing polymers, have cross-sectional dimensions (i.e., diameters) in the range of about 20 nm to about 300 nm. Certain embodiments also describe that these nanoparticles have diameters in the range of about 20 nm to about 40 nm (hereinafter referred to as about 20 nm to about 40 nm), about 40 nm to about 80 nm, about 80 nm to about 120 nm, about 120 nm to about 180 nm, about 180 nm to about 240 nm, about 240 nm to about 300 nm, or combinations of two or more of these ranges and can be said. These nanoparticles tend to be substantially similar and, depending on the size of various cMAP or PEG fragments and / or the length of the chains associated with the boronic acid-containing polymers, have cross-sectional dimensions (i.e., diameters) in the range of about 20 nm to about 300 nm. Certain embodiments also describe that these nanoparticles have diameters in the range of about 20 nm to about 40 nm (hereinafter referred to as about 20 nm to about 40 nm), about 40 nm to about 80 nm, about 80 nm to about 120 nm, about 120 nm to about 180 nm, about 180 nm to about 240 nm, about 240 nm to about 300 nm, or combinations of two or more of these ranges and can be said. These nanoparticles tend to be substantially similar and, depending on the size of various cMAP or PEG fragments and / or the length of the chains associated with the boronic acid-containing polymers, have cross-sectional dimensions (i.e., diameters) in the range of about 20 nm to about 300 nm. Certain embodiments also describe that these nanoparticles have diameters in the range of about 20 nm to about 40 nm (hereinafter referred to as about 20 nm to about 40 nm), about 40 nm to about 80 nm, about 80 nm to about 120 nm, about 120 nm to about 180 nm, about 180 nm to about 240 nm, about 240 nm to about 300 nm, or combinations of two or more of these ranges and can be said. Similarly, reference to a single nanoparticle should be considered to include alternative embodiments encompassing a population or plurality of nanoparticles. In certain embodiments, the plurality of nanoparticles are substantially monodisperse, and in unrelated embodiments, a standard deviation in cross-sectional dimensions among the nanoparticles of less than 20%, 30%, 40%, 50%, or 60% is provided, on average, as measured by cryo-transmission electron microscopy (cryo-TEM). Particle size (also referred to as particle diameter, particle size) and distribution can be defined by various methods including cryo-TEM micrograph analysis. In this method, representative cryo-transmission electron micrographs are

[0057] Similarly, reference to a single nanoparticle should be considered to include alternative embodiments encompassing a population or plurality of nanoparticles. In certain embodiments, the plurality of nanoparticles are substantially monodisperse, and in unrelated embodiments, a standard deviation in cross-sectional dimensions among the nanoparticles of less than 20%, 30%, 40%, 50%, or 60% is provided, on average, as measured by cryo-transmission electron microscopy (cryo-TEM). Particle size (also referred to as particle diameter, particle size) and distribution can be defined by various methods including cryo-TEM micrograph analysis. In this method, representative cryo-transmission electron micrographs are Similarly, reference to a single nanoparticle should be considered to include alternative embodiments encompassing a population or plurality of nanoparticles. In certain embodiments, the plurality of nanoparticles are substantially monodisperse, and in unrelated embodiments, a standard deviation in cross-sectional dimensions among the nanoparticles of less than 20%, 30%, 40%, 50%, or 60% is provided, on average, as measured by cryo-transmission electron microscopy (cryo-TEM). Particle size (also referred to as particle diameter, particle size) and distribution can be defined by various methods including cryo-TEM micrograph analysis. In this method, representative cryo-transmission electron micrographs are Similarly, reference to a single nanoparticle should be considered to include alternative embodiments encompassing a population or plurality of nanoparticles. In certain embodiments, the plurality of nanoparticles are substantially monodisperse, and in unrelated embodiments, a standard deviation in cross-sectional dimensions among the nanoparticles of less than 20%, 30%, 40%, 50%, or 60% is provided, on average, as measured by cryo-transmission electron microscopy (cryo-TEM). Particle size (also referred to as particle diameter, particle size) and distribution can be defined by various methods including cryo-TEM micrograph analysis. In this method, representative cryo-transmission electron micrographs are are used True (typically, in more than 3 randomly selected liquid samples frozen in liquid ethane from which), the average diameter of the particles is measured, the particles within a predetermined size fraction gradient ( also referred to as a particle size gradient) are counted, and by statistically correlating those numbers, a predetermined number of particles (more than 100) are analyzed. For additional information, see also the Examples and Appendix (also referred to as the appendix).

[0058] These nanoparticles (including any one or more inventive polymers or polymer conjugates are particularly attractive for their ability to carry biological "cargo", and in certain embodiments, these nanoparticles further comprise an encapsulated biological material (also referred to as an encapsulated biologically derived material). These biological materials may be covalently bound within or by the nanoparticles, or otherwise contained. In certain embodiments, the biological materials are polynucleotides or small molecule therapeutic agents. Examples of such therapeutic agents include, but are not limited to, small molecule formulations, antibiotics, steroids, polynucleotides (e.g., genomic DNA, cDNA, mRNA, siRNA, shRNA, miRNA, antisense oligonucleotides, viruses and chimeric polynucleotides), plasmids, peptides, peptide fragments, small molecules (e.g., doxorubicin), chelating agents (e.g., deferoxamine (DESFERAL) ethylene diamine tetraacetic acid (EDTA)), natural products (e.g., taxol, amphotericin B), and other biologically active macromolecules such as proteins and enzymes. See also U.S. Patent No. 6,048,736, which is incorporated herein by reference. Active agents (therapeutic agents) that can be used as therapeutic agents together with the nanoparticles are listed Small molecule therapeutic agents are not only therapeutic agents within the composite particles, but in further embodiments may also be covalently bound to the polymer in the complex. In some embodiments, the covalent bond is reversible (e.g., through a prodrug form or biodegradable linkage, e.g., a disulfide or any such), and provides another way to deliver the therapeutic agent. In some embodiments the therapeutic agents that can be delivered with the nanoparticles described herein include chemotherapeutic agents such as epothilone, camptothecin-based drugs, taxol, etc., or nucleic acids such as plasmids, siRNA, shRNA, miRNA, antisense oligonucleotide aptamers or combinations thereof such as those, and additional drugs that can be identified by those skilled in the art upon reading this disclosure.

[0059] In certain preferred embodiments, the biological substance is a polynucleotide which is an RNA molecule. In some of these embodiments, the RNA molecule is a siRNA molecule.

[0060] While not intending to be bound by the accuracy of any particular theory, when dispersed in an aqueous medium the nanoparticles are thought to organize themselves by presenting hydrophilic linkages to their aqueous environment and maintaining cationic species in the internal cavity (see, e.g., FIGS. 16A and 16B). Negatively charged cargo includes nucleic acids and associates with the positive charges in the cMAP structure, and in some cases is aided by self-assembly of the nanoparticles.

[0061] When the nanoparticles include a functionalized (nitro)boronic acid-containing polymer linkage, the nanoparticles 、may also be further conjugated to one or more targeting (also referred to as target-directed) ligands In such cases, the conjugation occurs through a condensation linkage between the distal end of the boronic acid-containing polymer and the target-directed ligand. In some embodiments, this target-directed ligand includes an antibody, transferrin, a ligand for a cell receptor, or a cell receptor protein, an aptamer, or a fragment of an antibody, transferrin, a ligand for a cell receptor, or a cell receptor protein. In certain embodiments, a single type of target-directed ligand is conjugated to each polymer or nanoparticle, or a population of nanoparticles. In other embodiments, multiple types of target-directed ligands are conjugated to each polymer or nanoparticle, or within a population of nanoparticles. In yet other embodiments, a single molecular entity of the target-directed ligand is conjugated to each individual nanoparticle. The ability to conjugate a single molecular entity to individual nanoparticles is described in U.S. Patent Application No. 13 / 782,458, filed March 1, 2013, which is incorporated herein by reference at least for this purpose. In other embodiments, multiple molecules of the target-directed ligand are conjugated to each individual nanoparticle. As suggested above, in certain embodiments, the polymer, polymer conjugate, and / or nanoparticle may be present as a dispersion in an aqueous medium, which aqueous medium may also optionally include a buffer, surfactant, or other modifier. The present disclosure also includes one or more biologically active agents and those described herein

[0062] As indicated above, in certain embodiments, the polymer, polymer conjugate, and / or nanoparticle may be present as a dispersion in an aqueous medium, and the aqueous medium may also optionally include a buffer, surfactant, or other modifier. The present disclosure also includes one or more biologically active agents and those described herein (also referred to as modifiers). The present disclosure also encompasses one or more biologically active agents and those described herein either the polymer or polymer conjugate or nanoparticle or plurality of nanoparticles and contemplate a pharmaceutical composition comprising a pharmaceutically acceptable vehicle, carrier or excipient .

[0063] As used herein, the term "vehicle" typically acts as a solvent, carrier, binder, excipient or diluent for the nanoparticles included in the composition as an active ingredient for any of a variety of media and indicates any of them.

[0064] As used herein, the term "excipient" indicates an inert substance used as a carrier for the active ingredient of a drug treatment For the pharmaceutical compositions disclosed herein, suitable excipients include any substance that enhances the ability of the body of an individual to absorb the nanoparticles. Suitable excipients also include any substance that can be used to bulk up the formulation with the nanoparticles and enable a convenient and accurate dosage In addition to their use in single doses, excipients can be used in the manufacturing process to assist in the handling of the nanoparticles Depending on the route of administration and the form of the drug treatment, different excipients can be used. Exemplary excipients include, but are not limited to, antiadherents (also called antiadhesives), binders , coatings disintegrants, fillers, flavoring agents (such as any sweetener whatever), and coloring agents, glidants (also called lubricants), lubricants, preservatives (also called preservatives), adsorbents are included. .

[0065] As used herein, the term "diluent" dilutes the active ingredient of the composition or The diluting agent (also referred to as excipient) administered to be effective is shown. Suitable diluents include any substance capable of reducing the viscosity of a pharmaceutical preparation .

[0066] Further details regarding the identification of suitable carrier agents or adjuvants for the composition, and the comprehensive manufacture and packaging aging of the kit can be ascertained by those skilled in the art by reading this disclosure.

[0067] The compositions of the present disclosure containing biologically active agents and polymers, polymer conjugates, and / or nanoparticles are useful for treating patients (often referred to as patients) in need of treatment, particularly due to the enhanced bioavailability (also referred to as biological availability) generated by the inventive polymers, polymer conjugates, and / or nanoparticles, including such pharmaceutical compositions. The degree of improvement in the bioavailability of such compositions, whether by themselves or together with cMAP, is surprisingly high compared to the delivery of the same biologically active agent or group of agents. See the examples. Accordingly, important embodiments include compositions containing the biologically active agents and polymers, polymer conjugates, and / or nanoparticles of the present disclosure, including such pharmaceutical compositions, that are administered to patients in need of administration of a biologically active agent.

[0068] The following list of embodiments is intended to supplement rather than replace or supersede the above description.

[0069] Embodiment 1. The following structural units of formula (I), formula (II), or formula (III): [Chemical formula] including one or more of: wherein: A is an uncharged segment containing a polyalkylene glycol; B is a cationic charged segment containing at least one polyhydroxy linkage including at least a pair of adjacent diols and is a polymer containing alternating charged and uncharged segments. In certain subsets of embodiments, A and B independently have a number average molecular weight in the range of 500 Da to about 5000 Da, greater than 5 000 Da to about 10 kDa, greater than 10 kDa to about 20 kDa, greater than 20 kDa to about 30 kDa, greater than 30 kDa to about 40 kDa, greater than 40 kDa to about 50 kDa, or greater than that or any combination thereof. In other subsets, either A or B or both A and B have a number average molecular weight in the range of greater than 5000 Da to about 50,000 Da.

[0070] Embodiment 2. The polymer of Embodiment 1, wherein A is polyethylene glycol and a suitable linking group or contains them.

[0071] Embodiment 3. The polymer of Embodiment 1 or 2, wherein the polyalkylene glycol has a nominal number average molecular weight in the range of about 500 daltons to about 50,000 daltons. In certain subsets of this embodiment, the polyalkylene glycol is from about 500 Da to about 1 kDa, greater than 1 kDa to about 5 kDa, greater than 5 kDa to about 10 kDa, greater than 10 kDa to about 15 kDa, greater than 15 kDa to about... and so on. ​​​from about 20 kDa to about 20 kDa, greater than 20 kDa to about 30 kDa, greater than 30 kDa to about 40 kDa, greater than 40 kDa to about 50 kDa, or having a number average molecular weight in any combination of two or more of these ranges is included.

[0072] Embodiment 4. B is a cationic charged segment containing at least one polyhydroxy sugar linkage containing at least one pair of adjacent diols, a polymer of any one of Embodiments 1 to 3. is included.

[0073] Embodiment 5. B includes at least one repeating subunit having the structure of formula (IV): [Chemical formula] is included, a polymer of any one of Embodiments 1 to 4. The following [Chemical formula] [Chemical formula] is functionally equivalent to that shown in formula (IV), and it is noted that this representative is intended to refer to both. is included.

[0074] Embodiment 6. B further includes at least one repeating subunit having the structure of formula (V): [Chemical formula] is included, where m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 4 - 6 or 5 if possible, a polymer of any one of Embodiments 1 to 5. is included. is included.

[0075] Embodiment 7. B includes at least one repeating subunit containing cMAP, and the subunit structure is formula (VI): [Chemical formula] [Chemical formula]​ represented by, wherein, m is, regardless of each presence, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 4 - 6 or 5; and n is, regardless of each presence, 1, 2, 3, 4, or 5, any one of Embodiments 1 to 6 of the polymer. In other related embodiments, m and n are even larger, for example it can be up to about 10.

[0076] Embodiment 8. The structure of formula (VII):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0077] Embodiment 9. The structure of formula (VIII):

Chemical formula

Chemical formula

[0078] Again, as in embodiment 9, to meet the molecular weight limit, the value for p is about​​​ corresponds to a range from 1 to about 100, and preferably from about 10 to about 100 if possible, and the numerical value for q is corresponds to a range from about 12 to about 1200. In a subset of these embodiments, q can also be in the range from about 100 to about 500. In a certain subset of this embodiment, p and q are, independently of each other, about 500 Da to about 1000 Da, greater than 1000 Da and up to about 5000 Da, greater than about 5000 Da and up to about 10,000 Da, greater than 10,000 and up to about 25,000 Da, greater than 25,000 Da and up to about 50,000 Da for the number average molecular weight of the subunits containing cMAP and PEG, respectively, or any combination of two or more of these ranges, and are sufficient to provide in the corresponding numerical range of these MW n ranges. In other subsets of this embodiment, X1 and X2 are, independently of each other, -(CH2) -COOH and -(CH2) 1-4 -COOH and -(CH2) 1-4 -NH2.

[0079] Embodiment 10. Structure of formula (IX):

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0080] Again, as in embodiments 9 and 10, to meet the molecular weight limitations, the value for p corresponds to the range of from about 1 to about 100, preferably from about 10 to about 100, and the value for q corresponds to the range of from about 12 to about 1200. In a subset of these embodiments, q can also be in the range of from about 100 to about 500. In a certain subset of this embodiment, p and q are, independent of each other, for subunits containing cMAP and PEG, the number average molecular weights are in the range of from about 500 Da to about 1000 Da, greater than 1000 Da to about 5000 Da, greater than about 5000 Da to about 10,000 Da, greater than 10,000 to about 25,000 Da, greater than 25,000 Da to about 50 ,000 Da, or any combination of 2 or more of these ranges, and of these MW ranges In a subset of these embodiments, q can also be in the range of from about 100 to about 500. In a certain subset of this embodiment, p and q are, independent of each other, for subunits containing cMAP and PEG, the number average molecular weights are in the range of from about 500 Da to about 1000 Da, greater than 1000 Da to about 5000 Da, greater than about 5000 Da to about 10,000 Da, greater than 10,000 to about 25,000 Da, greater than 25,000 Da to about 50 In a subset of these embodiments, q can also be in the range of from about 100 to about 500. In a certain subset of this embodiment, p and q are, independent of each other, for subunits containing cMAP and PEG, the number average molecular weights are in the range of from about 500 Da to about 1000 Da, greater than 1000 Da to about 5000 Da, greater than about 5000 Da to about 10,000 Da, greater than 10,000 to about 25,000 Da, greater than 25,000 Da to about 50 ,000 Da, or any combination of 2 or more of these ranges, and of these MW ranges ,000 Da, or any combination of two or more of these ranges, and of these MW n ranges of is sufficient to provide in the corresponding numerical range. In other subsets of this embodiment wherein X1 and X2 are, independently, -(CH2) 1-4 -COOH and -(CH2) 1-4 -NH2.

[0081] Embodiment 11. m is 4, 5, or 6, preferably 5, of any one of Embodiments 6 to 10 polymer.

[0082] Embodiment 12. n is 1, of any one of Embodiments 7 to 11 polymer.

[0083] Embodiment 13. r is 2, 3, or 4, preferably 3, of any one of Embodiments 7 to 12 polymer.

[0084] Embodiment 14. p is such that the number average molecular weight for the subunit containing cMAP is from about or above 5 kDa to about 15 kDa, from about or above 6 kDa to about 14 kDa, from about or above 7 kDa to 13 kDa, from about or above 8 kDa to about 12 kDa, from about 9 kDa to about 11 kDa or is sufficient to provide in the range of about 10 kDa, of any one of Embodiments 8 to 13 according polymer. In some subsets of embodiments, for example, the cMAP fragment has an MW of about 420 Da n in which case this corresponds to a p having a numerical value in the range of from about 12 to about 36, from about 14 to about 33, from about 17 to about 31 to about 29, from about 19 to about 29, from about 22 to about 26, or about 24. corresponds to.

[0085] Embodiment 15. q is such that the number average molecular weight for the subunit containing PEG is from about or above 500 Da from above about 50 kDa, about or from above about 1 kDa to about 40 kDa, about or from above about 5 kDa to about 30 kDa, or from above about 5 kDa to about 20 kDa in the range provided for by any one of embodiments 8 to 13. A polymer according to any one of these embodiments is sufficient. Some of these embodiments are such that, for example, assuming that the ethylene glycol fragment has an MWn of about 44 Da this corresponds to a q having a numerical value in the range from about 11 to about 1200, from about 23 to about 910, from about 110 to about 680, or from about 110 to about 450.

[0086] Embodiment 16. A polymer according to any one of embodiments 1 to 15 and a structure of formula (X)

Chemical Formula

Chemical formula

[0087] Embodiment 17. L is -(C 0-2 alkylene-)NH-C(=O)-(C 0-2 alkylene)-, -(C 0-2 alkylene n)-C(=O)-NH-(C 0-2 alkylene)-, -(C 0-2 alkylene)-O-C(=O)-(C 0-2 alkylene)- or also -(C 0-2 alkylene)-C(=O)-O-(C 0-2 alkylene)-, the polymer complex of Embodiment 16 .

[0088] Embodiment 18. L is -NH-C(=O)-, -C(=O)-NH-, -OC(=O)-, or -C(=O)-O-, the polymer complex of Embodiment 17.

[0089] Embodiment 19. Nanoparticles comprising the polymer of any one of Embodiments 1 to 15.

[0090] Embodiment 20. Nanoparticles comprising the polymer complex of any one of Embodiments 16 to 18.

[0091] Embodiment 21. The nanoparticles are substantially spherical and have a cross-sectional dimension in the range of about 20 nm to about 300 nm, the nanoparticles of any one of Claims 10 to 16 at the time of filing.

[0092] Embodiment 22. A plurality of nanoparticles, each nanoparticle being described by the composition of any one of Embodiments 19 to 21.

[0093] Embodiment 23. Each nanoparticle is described by the composition of any one of Embodiments 19 to 22, and the plurality of nanoparticles are substantially monodisperse, showing a standard deviation in cross-sectional dimensions (i.e., diameter) between nanoparticles of less than 20%, 30%, 40%, 50%, or 60% as measured by cryo-transmission electron microscopy (cryo-TEM).

[0094] Embodiment 24. Nanoparticles further comprising an encapsulated biological agent and comprising a polymer of any one of Embodiments 1 to 15 or a polymer complex of claims 16 to 18 at the time of filing.

[0095] Embodiment 25. The nanoparticles of Embodiment 24, wherein the biological agent is covalently bound to the polymer or polymer complex.

[0096] Embodiment 26. The nanoparticles of Embodiment 24 or 25, wherein the biological agent is a polynucleotide or a small molecule therapeutic agent.

[0097] Embodiment 27. The nanoparticles of Embodiment 24 or 25, wherein the biological agent is a polynucleotide that is an RNA molecule.

[0098] Embodiment 28. The nanoparticles of Embodiment 27, wherein the RNA molecule is a siRNA molecule.

[0099] Embodiment 29. Further conjugated to a targeting ligand (also referred to as a conjugate), Conjugation occurs through a condensation linkage between the distal end of the boronic acid-containing polymer and the targeting ligand, a nanoparticle of any one of embodiments 20 to 28.

[0100] Embodiment 30. A nanoparticle of embodiment 29, wherein a single targeting ligand is conjugated to each polymer.

[0101] Embodiment 31. A nanoparticle of embodiment 29, wherein a plurality of targeting ligands are conjugated to each polymer.

[0102] Embodiment 32. A pharmaceutical composition comprising a biologically active agent and a polymer or polymer conjugate of any one of claims 1 to 18 at the time of filing, and a pharmaceutically acceptable carrier or excipient.

[0103] Embodiment 33. A pharmaceutical composition comprising a biologically active agent and any one nanoparticle of embodiments 19 to 31 or a plurality of nanoparticles and a pharmaceutically acceptable carrier or excipient.

[0104] Embodiment 34. A method comprising administering to a patient any one nanoparticle of embodiments 24 to 28, wherein the bioavailability of the biological substance is improved as compared to administration by the biological substance itself.

[0105] Embodiment 35. A method for preparing a polymer of any one of embodiments 1 to 15, comprising covalently connecting at least one uncharged segment comprising a polyalkylene glycol to at least one cationic charged segment comprising at least one polyhydroxy linkage by use of at least one linking group. Exemplary methods are provided in the examples and the appendix. These methods involve reacting homologs of the specific reactants cited and are also considered within the scope of the present disclosure and are considered to be within the scope of the present disclosure.

[0106] Embodiment 36. The method of embodiment 35, wherein at least one polyhydroxy linkage comprises mucic acid and at least one linking group is an amide.

Examples

[0107] Example

[0108] The following examples are provided to illustrate some of the concepts described within this disclosure. Each example is considered to provide specific individual embodiments of compositions, methods of preparation, and uses, while none of the examples should be considered to limit the more comprehensive embodiments described herein .

[0109] In the following examples, efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be taken into account. Unless otherwise indicated , temperatures are in °C and pressures are at or near atmospheric pressure. Unless otherwise noted, references to molecular weight are intended to refer to the number average molecular weight.

[0110] Summary of Experimental Results

[0111] A new cationic polymer comprising repeating units based on mucic acid and dimethyl suberimidate was synthesized and designated as cMAP. Further modification of cMAP to a triblock polymer having cMAP adjacent to mPEG, mPEG-cMAP-PEGm resulted in a well-defined polymer with a molecular weight of ca. (approximately) 20 kDa . This triblock polymer has a charge of 2+ / - or more The triblock complex was able to fully encapsulate siRNA at a loading ratio of 1:1. Stable NPs composed of polymer and siRNA were obtained with a diameter of approximately 30 nm (by both DLS and CryoTEM). ) and approximately 0.4 mV in both 10 mM phosphate buffer pH 7.4 and 1 mM KCl pH 5.5. Upon injection into mice, mPE These NPs formed with G-cMAP-PEG triblock polymers were The NPs formulated with riboflavin showed prolonged circulation compared to those formulated with riboflavin, with 5-fold more of the formulation in circulation after 1 hour. When a portion of the excess triblock polymer was removed from the formulation, 10% remained. The absence of any excess cationic polymer resulted in these This is advantageous in order to minimize any adverse effects that the entity may cause in vivo.

[0112] Example 1. Materials and Methods.

[0113] Mucic acid and oxalyl chloride were from Sigma-Aldrich. Boc-ethylenediamine was purchased from AK Scientific and dimethylsulfoxide was purchased from Verimidart is a product of Thermo Fisher Scientific. or Sigma-Aldrich, and 3-carboxyl-5-nitrophenylboronic acid was from Alfa Polyethylene glycol reagent was purchased from Jenkem. Jenkem Technology USA or Laysan Bio, Inc. Dimethyl suberimidate was purchased from either ethylenediaminetetraacetate or ethylenediaminetetraacetate. It is a polymerized amine-based charged monomer and can be purchased from Thermo Scientific or Sigma-Aldrich. To assign peaks to the proton and carbon spectra of cMAP, The NMR spectrum of dimethyl berimidate was obtained. Both the proton and carbon NMR of DMS were The R spectrum was more complex than expected and some hydrolysis was present in the newly opened bottle. This suggests that: see Table 2.

[0114] Nuclear magnetic resonance (NMR) spectra were obtained without spinning at 500 MHz or 600 MHz using Varian (Varian) At 25 degrees Celsius (also called 25°C) for 300MHz, 500MHz, or 600MHz equipment Most of the 1 For H proton spectra, a delay time of 1-1.5 seconds was used; For quantitative accumulation of the polymer, a delay of 25 seconds was used. 13 The carbon spectrum of C is the default The default setting was 500 MHz. 1 H- 13 C heteronuclear single quantum coherence (HSQC), 1 H- 1 H Correlation spectroscopy (COSY), and 1 H- 13 C heteronuclear multiple bond correlation spectroscopy (HMBC) spectra were The data were acquired using the default VNMRJ3.0 HSQCAD, COSY, and HMBC settings. In addition, the diffusion gradient length was set to 4 In VNMRJ3.0 with 100.0 ms and 100.0 ms spread delay, bipolar pulse pair stimulated echo (bip Diffusion order using convection compensation (Dbppste_cc) method with olar pulse pair stimulated echo The diffusion-ordered spectroscopy (DOSY) spectrum was obtained for the synthesized polymer.

[0115] The electrospray ionization mass of small molecules was obtained using a Finnigan LCQ ion trap mass spectrometer. The matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) mass spectrum for the polymer was obtained using a 10 mg / mL α-cyano-4-hydroxycinnamic acid matrix with an Applied Biosystems Voyager DE-PRO. (Matrix-assisted laser desorption / ionization-time of flight) (MALDI-TOF) mass spectrum was obtained using a 10 mg / mL α-cyano-4-hydroxycinnamic acid matrix with an Applied Biosystems Voyager DE-PRO. using an Applied Biosystems Voyager DE-PRO. -PRO.

[0116] Example 2: Synthesis of mucic acid-containing polymers.

[0117] Example 2.1. Synthesis of cationic mucic acid polymer (cMAP) (Figure 1). Methanol (360 mL) was added to mucic acid (15 g, 71 mmol, 1 equivalent) in a 500 mL round-bottom flask containing a stir bar. Concentrated sulfuric acid (1.2 mL, 22.5 mmol, 0.3 equivalent) was added to this suspension, stirred overnight, and refluxed at 85 °C. The mixture was cooled to room temperature and filtered through a Buchner funnel using Whatman #5 filter paper. The solid was washed with 600 mL of methanol and then returned to a 500 mL round-bottom flask. 240 mL of methanol and 1.5 mL of triethylamine were added, and the solid was recrystallized by refluxing at 85 °C for 1 hour. The mixture was cooled to room temperature, filtered through a Buchner funnel, and washed with 600 mL of methanol. The solid was dried under reduced pressure at 75 °C overnight to give dimethyl mucate (13.72 g, 80% yield), a white solid. filtered through a Buchner funnel and washed with 600 mL of methanol. The solid was dried under reduced pressure at 75 °C overnight, giving dimethyl mucate (13.72 g, 80% yield), a white solid.1 1H NMR (300M Hz, DMSO-d6): 4.91 (d, 2H), 4.80 (q, 2H), 4.29 (d, 2H), 3.76 (q, 2H), 3.62 (s, 6H).

[0118] In a 500 mL round-bottom flask containing a stir bar, methanol (220 mL) was added to dimethyl mucate ester (13.72 g, 57.6 mmol, 1 equivalent). Triethylamine (20.9 mL, 150 mmol , 2.6 equivalents) was added, and the mixture was stirred and refluxed at 85 °C for 30 minutes, during which time a yellow suspension formed. N-boc-ethylenediamine (23 .7 mL, 150 mmol, 2.6 equivalents) in methanol (55 mL) was added to the suspension, and the mixture was stirred and reflux was resumed at 85 °C overnight . The mixture was cooled to room temperature and filtered through a Buchner funnel using Whatman #5 filter paper . The solid was washed with methanol (750 mL) and recrystallized from methanol (350 mL) at 85 °C for 1. 5 hours. The mixture was cooled to room temperature again, filtered through a Buchner funnel, and washed with methanol (750 mL). The solid was dried under reduced pressure at 75 °C overnight to give N-boc-protected ethylenediamine mucate (19.27 g, 68% yield), a white solid. 1 1H NMR (300 MHz, DMSO-d6): 7.71 (t, 2H), 6.81 (t, 2H), 5.13 (d, 2H), 4.35 (q, 2H), 4.10 (d, 2H), 3.77 (q, 2H), 3.13 (m, 4H), 2.97 (m, 4H), 1.36 (s, 18H). ESI 495.1 [M+H + , 517.4 [M+Na] + .

[0119] ​N-boc protected ethylenediamine mucate (19 .2 g) in a 500 mL round bottom flask containing a stir bar was placed in a water bath. Methanol (260 mL) was then added to the flask , followed by concentrated 12 N hydrochloric acid (65 mL) to produce 3 N HCl in methanol. The reaction flask was sealed with a septum (also called a partition) and vented with a needle. The water bath was set to 25 °C and the suspension was stirred for 6 - 8 hours . The reaction was monitored by thin layer chromatography (TLC) using a mobile phase of 1% methanol in CH2Cl2 and the spots were visualized in an iodine tank. Completion of the reaction was also confirmed by ESI. The slurry was filtered through a glass frit into fine grains and washed with methanol (750 mL) until the filtrate approached neutral pH. The solid was dried overnight at 80 °C under reduced pressure to give ethylenediamine mucate (12.96 g, 91% yield) as a white solid . 1 1H NMR (500 MHz, DMSO-d6): 7.97 - 7.83 (m, 8H), 5.30 (d, 2H), 4.55 (d, 2H ), 4.16 (d, 2H), 3.82 (m, 2H), 2.85 (m, 4H). 13 13C NMR (500 MHz, DMSO-d ￢ 6): 1 74.79, 71.39, 70.98, 39.25, 36.76. ESI 295.1 [M + H] + , 588.93 [2M + H] + .

[0120] Ethylenediamine mucate (100 mg, 0.3 mmol, 1 equiv) was added to a 4 mL glass vial with a stir bar. A 0.5 M sodium carbonate solution in nanopure water (1 mL) was added to the vial and the solution was stirred for 5 minutes. Then, dimethyl suberimidate (DMS) (74.4 mg, 0.3 mmol ) was added to the vial , 1 equivalent) was added to the mixture, and the reactants were stirred at 25 °C overnight for 16 hours. The reactants were diluted with nanopure water (10 mL), and 1 N HCl was added dropwise to adjust the pH to 4. The resulting solution was dialyzed against 15 mL of Amicon Ultra (Amicon Ultra) 3 kD spin filter for nanopure water until the pH of the filtrate became neutral. The polymer solution was concentrated to 3 - 4 mL and filtered into a pre-weighed 20 mL glass vial through a 0.2 um PVDF syringe filter, and freeze-dried to give the cationic mucic acid polymer as a white solid (29.2 mg, yield 16%), which was stored under argon at -20 °C. The filtrate was diluted with nanopure water (10 mL), and 1 N HCl was added dropwise to adjust the pH to 4. The resulting solution was dialyzed against 15 mL of Amicon Ultra (Amicon Ultra) 3 kD spin filter for nanopure water until the pH of the filtrate became neutral. The polymer solution was concentrated to 3 - 4 mL and filtered into a pre-weighed 20 mL glass vial through a 0.2 um PVDF syringe filter, and freeze-dried to give the cationic mucic acid polymer as a white solid (29.2 mg, yield 16%), which was stored under argon at -20 °C. The polymer solution was concentrated to 3 - 4 mL and filtered into a pre-weighed 20 mL glass vial through a 0.2 um PVDF syringe filter, and freeze-dried to give the cationic mucic acid polymer as a white solid (29.2 mg, yield 16%), which was stored under argon at -20 °C. to give the cationic mucic acid polymer as a white solid (29.2 mg, yield 16%), which was stored under argon at -20 °C. and stored under argon at -20 °C. 1 1H NMR (600 MHz, DMSO-d6): 9.59 - 8.74, 7.92, 5.40 , 4.53, 4.16, 3.82, 3.55, 3.26, 2.86 - 2.00, 1.60, 1.28. 13 13C NMR (125 MHz, DMSO-d ￢ 6): 174.61, 168.12, 71.19, 70.96, 51.67, 42.09, 36.71, 32.48, 27.84, 26.65.

[0121] Example 2.2. Synthesis of cMAP - PEG copolymer (Figure 2). After taking out the starting materials from a -20 °C freezer, they were equilibrated to room temperature for 1 hour. cMAP (50 mg, 0.009 mmol, 2 equivalents) and di-SPA-PEG-3.5 kD (succinimidyl propionate, 15.7 mg, 0.0046 mmol, 1 equivalent) were weighed into an oven-dried 10 mL flask equipped with a stir bar. The flask was capped with a septum, the two solids were dried under vacuum for 1 hour, and then the flask was filled with argon. Anhydrous DMSO (2 mL) was added using a needle and syringe to dissolve the two white solids, and the solution was stirred for 24 hours. After taking out the starting materials from a -20 °C freezer, they were equilibrated to room temperature for 1 hour. An oven-dried 10 mL flask equipped with a stir bar was charged with cMAP (50 mg, 0.009 mmol, 2 equivalents) and di-SPA-PEG-3.5 kD (succinimidyl propionate, 15.7 mg, 0.0046 mmol, 1 equivalent). g, 0.009 mmol, 2 equivalents) and di-SPA-PEG-3.5 kD (succinimidyl propionate, 15.7 mg, 0.0046 mmol, 1 equivalent). The flask was capped with a septum, the two solids were dried under vacuum for 1 hour, and then the flask was filled with argon. The two solids were dried under vacuum for 1 hour, and then the flask was filled with argon. Anhydrous DMSO (2 mL) was added using a needle and syringe to dissolve the two white solids, and the solution was stirred for 24 hours. It was stirred. Nanopure water (20 mL) was added to dilute DMSO, and the solution was dialyzed against nanopure water using a 10 kD MWCO Amicon Ultra filter more than 8 times. The residue, cMAP- PEG3.4k copolymer was filtered through a 0.2 um PVDF membrane and lyophilized to a white powder (29.6 mg , yield 45%). 1 H NMR (600 MHz, DMSO-d6): 9.84 - 8.48, 7.90, 5.41, 4.53, 4.15 , 3.82, 3.55, 3.49 (PEG), 3.26, 2.86 - 2.00, 1.59, 1.27. 13 C NMR (125 MHz, DMSO-d6 ): 174.66, 168.17, 71.24, 71.00, 70.24, 67.22, 51.69, 42.11, 36.75, 32.58, 27.8 9, 26.66. The same procedure was used to synthesize the cMAP-PEG5k copolymer using a 15 kD SpectraPor 7 MWCO membrane (Spectrum Labs (S pectrum Labs)) for dialysis and 5kD di-SVA- PEG (succinimidyl valerate).

[0122] The cMAP-PEG-cMAP triblock polymer was separated from the cMAP-PEG copolymer by fractionation with centrifugal spin filters of various MWCOs. The cMAP-PEG3.4k copolymer (also referred to as the copolymer) was dialyzed using a 20 kD MWCO centrifugal spin filter, and then the filtrate was dialyzed through a 10 kD MWCO s pin filter to separate cMAP-PEG3.4K-cMAP, which was filtered through a 0.2 um PVDF membrane and lyophilized to a white powder (10.6 mg, yield 16%). cMAP-PEG5k-cMA P was separated in the same manner.

[0123] ​​​ Example 2.3. Synthesis of mPEG-cMAP-PEGm triblock polymer (Figure 3). After taking out the starting materials from a freezer at -20 °C (also referred to as a refrigerator), they were equilibrated to room temperature for 1 hour. Into an oven-dried 10 mL flask equipped with a stir bar, cMAP (40 mg, 0.006 mmol, 2 equivalents) and mPEG 5k-SVA (85.7 mg, 0.017 mmol, 3 equivalents) were weighed. The flask was capped with a septum, the two solids were dried under vacuum for 1 hour, and then the flask was filled with argon. Using a needle and syringe, anhydrous DMSO (4 mL) was added to dissolve the two white solids, and the solution was stirred for 48 hours. Nanopure water (40 mL) was added to dilute the DMSO, and the solution was dialyzed >8 times using a 20 kD MWCO centrifugal spin filter. The residue, mPEG5k-cMAP-PEG5km, was filtered through a 0.2 um PVDF membrane and lyophilized to a white powder (11.3 mg, yield 9%). After taking out the starting materials from a freezer at -20 °C (also referred to as a refrigerator), they were equilibrated to room temperature for 1 hour. Into an oven-dried 10 mL flask equipped with a stir bar, cMAP (40 mg, 0.006 mmol, 2 equivalents) and mPEG 5k-SVA (85.7 mg, 0.017 mmol, 3 equivalents) were weighed. The flask was capped with a septum, the two solids were dried under vacuum for 1 hour, and then the flask was filled with argon. Using a needle and syringe, anhydrous DMSO (4 mL) was added to dissolve the two white solids, and the solution was stirred for 48 hours. Nanopure water (40 mL) was added to dilute the DMSO, and the solution was dialyzed >8 times using a 20 kD MWCO centrifugal spin filter. The residue, mPEG5k-cMAP-PEG5km, was filtered through a 0.2 um PVDF membrane and lyophilized to a white powder (11.3 mg, yield 9%). After taking out the starting materials from a freezer at -20 °C (also referred to as a refrigerator), they were equilibrated to room temperature for 1 hour. Into an oven-dried 10 mL flask equipped with a stir bar, cMAP (40 mg, 0.006 mmol, 2 equivalents) and mPEG 5k-SVA (85.7 mg, 0.017 mmol, 3 equivalents) were weighed. The flask was capped with a septum, the two solids were dried under vacuum for 1 hour, and then the flask was filled with argon. Using a needle and syringe, anhydrous DMSO (4 mL) was added to dissolve the two white solids, and the solution was stirred for 48 hours. Nanopure water (40 mL) was added to dilute the DMSO, and the solution was dialyzed >8 times using a 20 kD MWCO centrifugal spin filter. The residue, mPEG5k-cMAP-PEG5km, was filtered through a 0.2 um PVDF membrane and lyophilized to a white powder (11.3 mg, yield 9%). After taking out the starting materials from a freezer at -20 °C (also referred to as a refrigerator), they were equilibrated to room temperature for 1 hour. Into an oven-dried 10 mL flask equipped with a stir bar, cMAP (40 mg, 0.006 mmol, 2 equivalents) and mPEG 5k-SVA (85.7 mg, 0.017 mmol, 3 equivalents) were weighed. The flask was capped with a septum, the two solids were dried under vacuum for 1 hour, and then the flask was filled with argon. Using a needle and syringe, anhydrous DMSO (4 mL) was added to dissolve the two white solids, and the solution was stirred for 48 hours. Nanopure water (40 mL) was added to dilute the DMSO, and the solution was dialyzed >8 times using a 20 kD MWCO centrifugal spin filter. The residue, mPEG5k-cMAP-PEG5km, was filtered through a 0.2 um PVDF membrane and lyophilized to a white powder (11.3 mg, yield 9%). After taking out the starting materials from a freezer at -20 °C (also referred to as a refrigerator), they were equilibrated to room temperature for 1 hour. Into an oven-dried 10 mL flask equipped with a stir bar, cMAP (40 mg, 0.006 mmol, 2 equivalents) and mPEG 5k-SVA (85.7 mg, 0.017 mmol, 3 equivalents) were weighed. The flask was capped with a septum, the two solids were dried under vacuum for 1 hour, and then the flask was filled with argon. Using a needle and syringe, anhydrous DMSO (4 mL) was added to dissolve the two white solids, and the solution was stirred for 48 hours. Nanopure water (40 mL) was added to dilute the DMSO, and the solution was dialyzed >8 times using a 20 kD MWCO centrifugal spin filter. The residue, mPEG5k-cMAP-PEG5km, was filtered through a 0.2 um PVDF membrane and lyophilized to a white powder (11.3 mg, yield 9%). After taking out the starting materials from a freezer at -20 °C (also referred to as a refrigerator), they were equilibrated to room temperature for 1 hour. Into an oven-dried 10 mL flask equipped with a stir bar, cMAP (40 mg, 0.006 mmol, 2 equivalents) and mPEG 5k-SVA (85.7 mg, 0.017 mmol, 3 equivalents) were weighed. The flask was capped with a septum, the two solids were dried under vacuum for 1 hour, and then the flask was filled with argon. Using a needle and syringe, anhydrous DMSO (4 mL) was added to dissolve the two white solids, and the solution was stirred for 48 hours. Nanopure water (40 mL) was added to dilute the DMSO, and the solution was dialyzed >8 times using a 20 kD MWCO centrifugal spin filter. The residue, mPEG5k-cMAP-PEG5km, was filtered through a 0.2 um PVDF membrane and lyophilized to a white powder (11.3 mg, yield 9%). After taking out the starting materials from a freezer at -20 °C (also referred to as a refrigerator), they were equilibrated to room temperature for 1 hour. Into an oven-dried 10 mL flask equipped with a stir bar, cMAP (40 mg, 0.006 mmol, 2 equivalents) and mPEG 5k-SVA (85.7 mg, 0.017 mmol, 3 equivalents) were weighed. The flask was capped with a septum, the two solids were dried under vacuum for 1 hour, and then the flask was filled with argon. Using a needle and syringe, anhydrous DMSO (4 mL) was added to dissolve the two white solids, and the solution was stirred for 48 hours. Nanopure water (40 mL) was added to dilute the DMSO, and the solution was dialyzed >8 times using a 20 kD MWCO centrifugal spin filter. The residue, mPEG5k-cMAP-PEG5km, was filtered through a 0.2 um PVDF membrane and lyophilized to a white powder (11.3 mg, yield 9%). After taking out the starting materials from a freezer at -20 °C (also referred to as a refrigerator), they were equilibrated to room temperature for 1 hour. Into an oven-dried 10 mL flask equipped with a stir bar, cMAP (40 mg, 0.006 mmol, 2 equivalents) and mPEG 5k-SVA (85.7 mg, 0.017 mmol, 3 equivalents) were weighed. The flask was capped with a septum, the two solids were dried under vacuum for 1 hour, and then the flask was filled with argon. Using a needle and syringe, anhydrous DMSO (4 mL) was added to dissolve the two white solids, and the solution was stirred for 48 hours. Nanopure water (40 mL) was added to dilute the DMSO, and the solution was dialyzed >8 times using a 20 kD MWCO centrifugal spin filter. The residue, mPEG5k-cMAP-PEG5km, was filtered through a 0.2 um PVDF membrane and lyophilized to a white powder (11.3 mg, yield 9%). After taking out the starting materials from a freezer at -20 °C (also referred to as a refrigerator), they were equilibrated to room temperature for 1 hour. Into an oven-dried 10 mL flask equipped with a stir bar, cMAP (40 mg, 0.006 mmol, 2 equivalents) and mPEG 5k-SVA (85.7 mg, 0.017 mmol, 3 equivalents) were weighed. The flask was capped with a septum, the two solids were dried under vacuum for 1 hour, and then the flask was filled with argon. Using a needle and syringe, anhydrous DMSO (4 mL) was added to dissolve the two white solids, and the solution was stirred for 48 hours. Nanopure water (40 mL) was added to dilute the DMSO, and the solution was dialyzed >8 times using a 20 kD MWCO centrifugal spin filter. The residue, mPEG5k-cMAP-PEG5km, was filtered through a 0.2 um PVDF membrane and lyophilized to a white powder (11.3 mg, yield 9%). 1 H NMR (600 MHz, D MSO-d6): 9.84 - 8.48, 7.90, 5.41, 4.53, 4.15, 3.82, 3.55, 3.49 (PEG), 3.26, 3.20, 2.86 - 2.00, 1.59, 1.27. MSO-d6): 9.84 - 8.48, 7.90, 5.41, 4.53, 4.15, 3.82, 3.55, 3.49 (PEG), 3.26, 3.20, 2.86 - 2.00, 1.59, 1.27.

[0124] The same procedure was continued using 2kD mPEG-SVA to synthesize mPEG-cMAP-PEGm with a 2kD block. For the case of 2kD PEG, the triblock polymer was separated using a 10kD MWCO centrifugal spin filter. The same procedure was continued using 2kD mPEG-SVA to synthesize mPEG-cMAP-PEGm with a 2kD block. For the case of 2kD PEG, the triblock polymer was separated using a 10kD MWCO centrifugal spin filter. The same procedure was continued using 2kD mPEG-SVA to synthesize mPEG-cMAP-PEGm with a 2kD block. For the case of 2kD PEG, the triblock polymer was separated using a 10kD MWCO centrifugal spin filter.

[0125] Example 2.4. Synthesis of 5-nitrophenylboronic acid-PEGm (5-nPBA-PEGm) (Figure 4).

[0126] An oven-dried two-necked 10 mL round-bottom flask containing a dry stirrer bar (also referred to as a drying stir bar) was charged with 3-carboxyl-5-nitrophenylboronic acid (200 mg, 0.95 mmol, 1 equiv). The flask was evacuated with argon and sealed with a rubber septum. BHT inhibitor (5 mL) was added followed by anhydrous tetrahydrofuran and then anhydrous DMF (14.7 uL, 0.19 mmol , 0.2 equiv) to dissolve the boronic acid. The flask was cooled to 0 °C in an ice-water bath. Then , oxalyl chloride (195.4 uL, 2.28 mmol, 2.4 equiv) was added dropwise to the reaction mixture. After the addition of oxalyl chloride was complete, the ice-water bath was removed and the reaction was stirred at room temperature for 2 h while allowing volatile materials to escape through an argon vent. The solvent and DMF were removed via a rotary evaporator and then removed under vacuum for 2 days under dark conditions to afford 3-acyl chloride-5-nitrophenylboronic acid (217.5 mg, 100% yield) as a yellow solid . To an oven-dried 25 mL round-bottom flask containing a dry stirrer bar was added 3-acyl chloride-5 -nitrophenylboronic acid (27.5 mg, 0.12 mmol, 2 equiv). The flask was sealed with a rubber septum, given an argon purge, and cooled to 0 °C in an ice-water bath. Anhydrous dichloromethane (4 mL) was added to dissolve the boronic acid. 5kD mPEG-amine (300 mg, 0.06 mmol, 1 equiv) in an oven-dried 10 mL round-bottom flask purged with argon was dissolved in anhydrous dichloro methane (5 mL) and diisopropylethylamine (DIPEA, 20.9 uL, 0.12 mmol, 2 equiv) and slowly added to the boronic acid solution. The reaction flask was left in the ice-water bath and It was placed and slowly warmed to room temperature, and the reactants were stirred overnight under dark conditions. The solvent and DIPE A was removed via a rotary evaporator and then removed under vacuum for 2 days in the dark done. The solid residue was reconstituted in 0.5N HCl (5 mL) and stirred for 15 minutes. The resulting suspension was filtered through a 0.2 μm Supor syringe filter, and the resulting clear solution was dialyzed against nanopure water using a 15 mL Amicon Ultra 3kD spin fil ter until the pH became constant. The polymer solution was concentrated to 3 - 4 mL and filtered through a 0.2 μm PVDF sy ringe filter into a pre-weighed 20 mL glass vial and lyophilized to dryness to give a fluffy white solid of 5-nitrophenylboronic acid-PEGm (219.2 mg, 70% yield). 1 H NMR (600 MHz, DMSO-d6): 8.89 (t, 1H), 8.72 (m, 1H), 8.68 (m, 1H), 8.64 (m, 1H), 8.60 (s, 2H), 3.5 (s-PEG, 510H), 3.22 (s, 3H). 11 B NMR (160 MHz, 10 mM phosphate buffer, pH 7.4 in D2O): 11.26 (broad s). MALDI: 5825.5 .

[0127] Example 3. Polymer properties

[0128] Example 3.1. Gel permeation chromatography. An Agilent(アジレント) 1100 HPLC equipped with a binary pump and an injector was used, with Wyatt DAWN HELEOS(ワイアット·ドーン·ヘレオス) light scattering and Wyatt Optilab Rex(ワイアット·オプティラボ·レックス) refractive index detection ​​Connected to a Tosoh TSKgel G3000PWXL-CP size exclusion column. The freeze-dried polymer was dissolved in 0.1 M NaNO3 at six different concentrations and directly injected into a refractive index detector via a syringe pump for dn / dc measurement. For absolute molecular weight measurement by light scattering, 100 μL of the polymer solution was injected onto the column and the detected polymer peaks were

[0129] Example 3.2. TNBSA assay of cMAP for primary amines. 5% w / v 2,4,6-trinitrobenzene sulfonic acid in a methanol stock solution was used following the instructions of Thermo Scientific. Briefly, cMAP and glycine were each dissolved in a reaction buffer and serially diluted for concentration ranges of 2 to 0.0039 mg / mL and 20 to 0.00195 mg / mL, respectively. 100 μL of each sample concentration and 50 μL of the TNBSA working solution (also called the dilution standard solution) were added in triplicate to a 96-well plate and shaken briefly. Absorbance was read at 335 nm with a Tecan infinite M200 plate reader, incubated at 37 degrees Celsius for 2 hours and read again. Glycine was used as a positive control.

[0130] Example 3.3. Polymer siRNA encapsulation assay. The ability of cMAP polymers to encapsulate siRNA was determined by two methods: a gel retardation For the gel retardation assay, 0.5 mg / mL polyclonal antibody was used. Increasing volumes of the polymer were measured at 0, 0.5, 1, 1.5, 2, 2.5 for a total volume of 15 μL in water. The mixtures were mixed with 1 μL of 1 mg / mL siRNA at + / - charge ratios of 1, 3, and 5. The mixtures were briefly vol. Mix, centrifuge, and then drop. The mixture was then incubated at room temperature for 15 minutes. 3 μL of 6× DNA loading dye was added to each mixture, and then This was loaded onto a 1 wt% agarose gel and run at 95 V for 1.5 h in 0.5× TBE buffer. The gel was then imaged using a UVP BioDoc-It Imaging System. The images were captured using a 3D microscope (Stem).

[0131] The RiboGreen assay was performed in a similar manner to the gel retardation assay, but in a 96-well plate. In the 100 μL total volume, 0.1 mg / mL polymer and 1 μL of 0.1 mg / mL s The use of increasing amounts of iRNA was excluded. For each of these mixtures, 100 Add 1 μL of Quant-iT RiboGreen RNA Reagent working solution according to the kit protocol. The plates were briefly shaken and incubated in the dark at room temperature for 5 minutes. The plate was incubated with 100 µL of ... and emission wavelength of 520 nm. Measurements were performed in triplicate.

[0132] Example 4. Nanoparticle formulation and characterization.

[0133] Example 4.1. Nanoparticle formulations. cMAP NPs were first mixed in 10 mM phosphate buffer pH 7.4 at a 1:1 molar ratio of cMAP adjacent diol pair to 5-nPBA-PEGm (1 mg of cMAP to 22 mg of 5-nPBA-mPEG), vortexed briefly, spun down by centrifugation, and the mixture was left at room temperature for 15 minutes. Next, siRNA in an equal volume of RNAse-free water was added at a charge ratio of cMAP to siRNA of 3:1 and at a concentration of siRNA up to 0.8 mg / mL. cMAP-PEG copolymers, cMAP-PEG-cMAP triblocks, and mPEG-cMAP-PEGm triblock formulations were also made in a similar manner, but the charge ratio was decreased from 3:1 to 1:1 of polymer to siRNA charge ratio and the concentration of siRNA was varied up to 1 mg / mL. For any formulation without 5-nPBA-PEGm, equal volumes of polymer and siRNA were simply mixed at the appropriate charge ratio. For injection into mice, 0.1 volume of 10× phosphate buffered saline (PBS) solution was added to achieve a 1× PBS solution with siRNA at a final concentration of 0.73 mg / mL. For cMAP-PEG copolymers and mPEG-cMAP-PEGm NPs formulated in PBS, both the polymer and siRNA solutions were in PBS and then mixed together; this could be injected directly into mice. For removal of excess components (i.e., polymer, PEG), the NP formulations were placed in a 0.5 mL 30 kD MWCO Amicon Ultra spin filter and dialyzed 5 - 10 times with PBS at 2000 rpm for 10 minutes. and mixed at a 1:1 molar ratio of cMAP adjacent diol pair to 5-nPBA-PEGm (1 mg of cMAP to 22 mg of 5-nPBA-mPEG) in 10 mM phosphate buffer pH 7.4, vortexed briefly, centrifuged down, and the mixture was left at room temperature for 15 minutes. Next, siRNA in an equal volume of RNAse-free water was added at a charge ratio of cMAP to siRNA of 3:1 and at a concentration of siRNA up to 0.8 mg / mL. cMAP-PEG copolymers, cMAP-PEG-cMAP triblocks, and mPEG-cMAP-PEGm triblock formulations were also made in a similar manner, but the charge ratio was decreased from 3:1 to 1:1 of polymer to siRNA charge ratio and the concentration of siRNA was varied up to 1 mg / mL. For any formulation without 5-nPBA-PEGm, equal volumes of polymer and siRNA were simply mixed at the appropriate charge ratio. For injection into mice, 0.1 volume of 10× phosphate buffered saline (PBS) solution was added to achieve a 1× PBS solution with siRNA at a final concentration of 0.73 mg / mL. For cMAP-PEG copolymers and mPEG-cMAP-PEGm NPs formulated in PBS, both the polymer and siRNA solutions were in PBS and then mixed together; this could be injected directly into mice. For removal of excess components (i.e., polymer, PEG), the NP formulations were placed in a 0.5 mL 30 kD MWCO Amicon Ultra spin filter and dialyzed 5 - 10 times with PBS at 2000 rpm for 10 minutes. Example 4.2. Nanoparticle size and zeta potential. NP size was determined by two different methods: dynamic light scattering and nanoparticle formulations were placed in a 0.5 mL 30 kD MWCO Amicon Ultra spin filter and dialyzed 5 - 10 times with PBS at 2000 rpm for 10 minutes.

[0134] Example 4.2. Nanoparticle size and zeta potential. NP size was determined by two different methods: dynamic light scattering Determined using dynamic light scattering (DLS) and cryogenic transmission electron microscopy (cryoTEM). DLS was performed with Zeta-PALS from Brookhaven Instruments Corporation together with BIC Particle Sizing Software (BIC). Particles were diluted to a concentration of 0.2 mg / mL siRNA depending on the formulation until a stable size was recorded for 10 measurements of 1 minute each . The results of at least 10 measurements were averaged .

[0135] Blotted on filter paper using an FEI Mark IV Vitrobot with a blot time of 2 s (blot force 6) and a drain time of 1 s, then imaged for particles in the frozen solution on a Quantifoil R2 / 2 grid in liquid ethane . CryoTEM imaging was performed. Images were collected on a Tecnai 120-keV transmission electron microscope equipped with a Gatan 2k x 2k UltraScan CCD camera and Serial EM automation software . The acquired images were analyzed to measure the NP diameter using ImageJ software .

[0136] The surface charge, or zeta potential, of the NPs was measured using the same Zeta-PALS used for DLS with the addition of a Brookhaven aqueous electrode assembly. 10 μL of the particle formulation was placed in a cuvette containing 10 mM Li ​​​​​​It was mixed with 1.5 mL of either phosphate buffer (pH 7.4) or 1 mM potassium chloride (pH 5.5). The electrodes were inserted into the cuvette, and the zeta potential was measured with 0.012 of the target residue using BIC PALS Zeta Potential Analyzer (BIC PALS zeta ·potential·analyzer) software. The results of at least 10 measurements were averaged.

[0137] Example 4.3. Nanoparticle Stoichiometry.

[0138] Example 4.3.1. Quantification of 5-nPBA-PEGm Bound to NPs. NPs were formulated with 5-nPBA-PEGm, and the excess components were removed as described above. 50 μL of the filtrate (containing the excess components) from a 30 kD MWCO spin filter was injected into an Agilent 1200 HPLC equipped with a Phenomenex Gemini C18 (Phenomenex Gemini C18) reverse-phase column, a quaternary pump, and an autosampler connected to a multi-wavelength detector. The absorbance at 254 nm was recorded and compared to the calibration curve of 5-nPBA-PEGm.

[0139] Example 4.3.2. Quantification of Cationic Polymer Bound to NPs. NPs were formulated in the absence of 5-nPBA-PEGm. For cMAP, the excess cationic polymer was removed from the aggregated NPs as described above. The cationic polymer bound to the NPs was taken out as the residue (also referred to as the retention solution) from a 30 kD MWCO spin filter containing the NPs, and BcMag TM (BcMag 商品名 ) SAX (Strong Anion Exchange (strong anion exchange)) magnetic beads (Bioclone Inc​​​​​​​ (BioClone) to directly quantify by degrading and blocking siRNA 50 μL of the liquid containing cMAP was injected into the above GPC setup, and the amount of polymer bound to the NPs was directly determined using the refractive index signal by comparison with the cMAP calibration curve. For the cMAP-PEG copolymer and mPEG-cMAP-PEGm, 50 μL of the formulation was injected into the above GPC setup. The refractive index signal corresponding to the polymer not bound to the NPs was recorded and compared with the calibration curve of the same cationic polymer This amount was subtracted from the total amount of polymer used for the formulation to determine the percentage of polymer bound to the NPs

[0140] Example 5. In Vivo Mouse Pharmacokinetics (PK) Study

[0141] All animal studies were approved by the Institutional Animal Care and Use Committee (also referred to as the Animal Experiment Committee) at Caltech (California Institute of Technology). NPs were formulated as described above, except that 20% of the siRNA was replaced with Cy3-fluorophore-labeled siRNA. The NP formulation was intravenously injected via the mouse tail vein at a dose of 5 mg of siRNA per kg of mouse The hindlimbs of Balst / c mice (Taconic and Jackson Labs) were collected using a Sarstedt Microvette CB300 capillary tube containing a red top clot activator ​​​​​​​​(also referred to as the agent) was shaved to draw blood from the underlying vein. Blood was collected at various time points starting 2 minutes after NP injection, up to a maximum of 6 points per mouse. The tubes were centrifuged at 14,000 x g for 15 minutes at 4 °C, and the serum at the top of the tubes was used for the analysis of Cy3 fluorescence by comparing the standard curve of the NP preparation in mouse serum at an excitation wavelength of 530 nm and an emission wavelength of 570 nm The fraction of Cy3-siRNA remaining in the serum was calculated using the serum volume based on the mouse weight and the amount of the preparation injected. The data points are from 3 mice per preparation .

[0142] Example 6. Results and Discussion

[0143] Example 6.1. cMAP Synthesis, NMR Characterization, and Determination of the Terminal Groups. Cationic mucic acid polymer ( cMAP) was synthesized by using a series of reactions schematically illustrated in Figure 1. The intermediate reaction products leading to the preparation of mucic acid and ethylenediamine mucate were fully characterized (Table 1) .

[0144] [Table 1] The condensation reaction of ethylenediamine mucate and dimethyl suberimidate (DMS) produced the cMA P substance. Since DMS can be hydrolyzed under conditions similar to those used for polymerization, the inventors studied the reaction pathway for this reaction and the product formed (Table 2) .

[0145] [Table 2] This information was supported in the characterization of the cMAP product. ​​​​

[0146] NMR analysis of cMAP( 1 H- 13 C HSQC NMR, 1 H- 1 H COSY NMR, and 1 H- 13 C HMBC NMR data( contained)(Tables 3-4) enabled the assignment of all the resonances to various carbon and hydrogen environments in the polymer. These functionalities were utilized in subsequent reactions involving functionalized PEG to form the cMAP-PEG copolymer or the mPEG-cMAP-PEGm triblock polymer and thus, the identification of the terminal group composition of cMAP was important.

[0147]

Table 3

[0148]

Table 4

[0149] The cMAP terminal groups contain the methoxy of the methoxy ester, amine, and a small amount of carboxylic acid (Figure 10). The 1 H NMR analysis of cMAP shows the presence of a characteristically sharp methoxy peak at 3.55 ppm (Figure 11), and this assignment is 1 H- 13 supported by C HSQC NMR measurements (not shown). The methoxy group results from the loss of ammonia through hydrolysis of the imidate group of DMS (Table 2, Figures 6-8), and has been reported previously. The methylene group adjacent to the methoxy is observed as a triplet (also called a triplet of triplets) at 2.25 ppm in the 1 H NMR spectrum ​​It can be done (Figure 11). The amine terminal groups derived from ethylene diamine mucate are 1 H N not directly observable by MR. However, from the analysis of the NMR spectrum of the monomer and the HMBC NMR spectrum of cMAP an assignment was made possible from the methylene group adjacent to the triplet amine functional group at 2.85 ppm. Furthermore, the TNBSA assay for primary amines was positive, and it was thus confirmed that cMAP has a terminal primary amine as a terminal group . Finally, there was sufficient hydrolysis of the methyl ester or a small amount of carboxylic acid as a terminal group resulting from impurities in the starting DMS. The methylene group adjacent to the carboxylic acid is observed as a small triplet at 2.00 ppm in the 1H NMR spectrum ( Figure 11). The ratio of these terminal groups in a batch of cMAP can be determined by comparing the integrals of the triplets at 2.85 (amine), 2.25 (methoxy 1 ), and 2.00 (carboxylate) ppm, and is shown for 8 batches in Table 5. The average values for % amine , % methoxy, and % carboxylate are 49%, 42%, and 9%, respectively.

Table 5

[0150] Example 6.1. cMAP-PEG copolymers and mPEG-cMAP-PEGm triblocks. cMAP was reacted with activated carboxylic acid terminal groups such as PEG, for example, succinimidyl propionate (SPA) or succinimidyl valerate ester (SVA). cMAP has a PEG length ​​​​Di-SPA-PEG or mPEG-SVA generating copolymers having sagas of 2, 3.4, or 5 kD respectively, or reacted with triblock polymers.

[0151] Since a significant amount of diamine-terminated polymer chains are present in the cMAP mixture, reaction with Di-SPA-PEG ( Figure 2) resulted in cMAP-PEG copolymers having a large size distribution (from diblock cMAP-PEG copolymers just slightly larger than 1 0 kD to cMAP-PEG-cMAP triblock polymers terminated with methyl esters or carboxylic acids in cMAPs, to polymers over 100 kD in length; various copolymers from the polymer yields obtained by fractionating the crude polymer through a continuously smaller molecular weight cut-off centrifugal spin filter; reported in Table 6-7). The size distribution is from the polymer yields obtained by fractionating the crude polymer through a continuously smaller molecular weight cut-off centrifugal spin filter). ).

[0152]

Table 6

[0153]

Table 7

[0154] Polymers having such large molecular weights may impose substantial toxicity in vivo Therefore, to synthesize well-defined polymers of moderate length, cMAP-PEG-cMAP triblock polymer species were separated from the copolymers using this fractionation method. Other triblock polymers with this repeating structure of cationic polymers adjacent to PEG or PLA polymers have been previously investigated for gene and iron oxide-carbon nanotube delivery. ).

[0155] When cMAP is reacted with mPEG-SVA, the structure of the resulting product is limited to the mPEG-cMAP-PEGm triblock polymer (Figure 3). Some cMAP-PEGm diblock polymers are also present and separated from the desired triblock by fractionation.

[0156] Example 6.2. Molecular weight of the polymer by GPC. The molecular weight of cMAP was characterized using gel permeation chromatography. The elution time of the polymer can be correlated with its size, but for the new cationic polymer, there is no ideal size standard for calibration. Therefore, the inventors determined the absolute molecular weight of the polymer using a multi-angle light scattering detector. The advantage of this method is that it depends only on the scattering ability of the polymer and its concentration; it does not require a standard for comparison. The differential refractive index was determined for the concentration of cMAP, dn / dc (Table 8), and used to measure the molecular weight. The average molecular weight of 9 batches of cMAP was around 6 kD, with a polydispersity index (PDI) of less than 1.1 (Table 8). The results for individual batches can be found in Table 9. and it was measured. The average molecular weight of 9 batches of cMAP was around 6 kD, with a polydispersity index (PDI) of less than 1.1 (Table 8). The results for individual batches can be found in Table 9. it depends only on the scattering ability of the polymer and its concentration; it does not require a standard for comparison. The differential refractive index was determined for the concentration of cMAP, dn / dc (Table 8), and used to measure the molecular weight. The average molecular weight of 9 batches of cMAP was around 6 kD, with a polydispersity index (PDI) of less than 1.1 (Table 8). The results for individual batches can be found in Table 9. and it was measured. The average molecular weight of 9 batches of cMAP was around 6 kD, with a polydispersity index (PDI) of less than 1.1 (Table 8). The results for individual batches can be found in Table 9. was around 6 kD, with a polydispersity index (PDI) of less than 1.1 (Table 8). The results for individual batches can be found in Table 9. from individual batches can be found in Table 9.

[0157] [Table 8]

[0158] [Table 9]

[0159] Using a similar method, the 5k cMAP-PEG copolymer had a PDI of 1.4, and an Mw of 42 kD and 29 k ​It had an even larger size distribution with Mn of D (Table 8). 5k mPEG-cMAP-PEGm tri The triblock was about 21kD and the PDI was less than 1.1 (Table 8). Furthermore, 3.4kD PEG cMAP-PEG copolymer and 2kD PEG mPEG-cMAP-PEGm triblock, as well as cMAP-PEG co All the results of the cMAP-PEG-cMAP triblock derived from the fraction of the polymer are reported in Table 10 .

[0160]

Table 10

[0161] Example 6.3. siRNA encapsulation by cMAP-based polymers. The ability of cMAP, c MAP-PEG copolymer, and mPEG-cMAP-PEGm triblock polymer to encapsulate siRNA was confirmed using both the RiboGreen a ssay and the gel retardation assay. cMAP can encapsulate siRNA at a charge ratio of 1+ / - (+ / -) , and both the cMAP-PEG5k copolymer and the mPEG5k-cMAP -PEG5km triblock can each encapsulate siRNA sufficiently as determined by the fluorescent RiboGreen assay at charge ratios of 3 or 2, respectively (Figure 12). Similar siRNA encapsulation data are reported for the copolymers and triblock polymers of other PEG lengths in Figures 13-14 . The results of the RiboGreen assay are probably more sensitive but are equivalent to those of the gel retardation assay.

[0162] Example 7. Nanoparticle formulations and properties.

[0163] Example 7.1. Formulation. 5-Nitrophenylboronic acid-PEGm (5-nPBA-PEGm) has a boronic acid group that enables the steric stabilization of siRNA containing NPs such that one end of this 5 kD PEG binds to adjacent diol groups on the mucic acid of cMAP at a pH above 6.8 as shown in Figure 15. Various NP formulations using cMAP, cMAP-PEG copolymers, and mPEG-cMAP-PEGm triblock polymers with or without 5-nPBA-PEGm are shown in Figure 16 (A - B). NPs prepared by mixing cMAP and siRNA at a 3+ / - charge ratio without the addition of 5-nPBA-PEGm are stable in water but unstable in PBS (one 5-nPBA-PEGm per diol added to the formulation, Figure 17). In contrast to cMAP alone, cMAP-PEG copolymers and mPEG-cMAP-PEGm triblock polymers were able to form stable particles without additional 5-nPBA-PEGm. However, pure cMAP-PEG-cMAP triblock polymers isolated from the cMAP-PEG copolymer probably did not contain enough PEG for sufficient shielding (also called masking) and steric stabilization of the NPs and thus were unable to form stable siRNA-containing NPs without the addition of 5-nPBA-PEGm (Table 11 and Figures 18 - 20). and thus were unable to form stable siRNA-containing NPs without the addition of 5-nPBA-PEGm (Table 11 and Figures 18 - 20).

Table 11

[0164]

[0165]

[0166] ​​​​​​​​​​​ Ps were formed, but additional PEG provides even greater steric stability to NPs when tested in vivo. To test whether the formulation with additional 5-nPBA-PEGm also provides greater stability, it was also prepared. The amount of PEG bound to the NPs was approximately 20% (Table 13). The polymer components of the NPs were mixed with an equal amount of siRNA to form NPs at a concentration of 0.8 - 1 mg of siRNA / mL. Furthermore, the cMAP-PEG copolymer and the mPEG-cMAP-PEGm triblock polymer can directly formulate stable NPs in PBS, eliminating the need to first formulate stable particles in a low-salt buffer and then continue with the addition of PBS (required by cMAP).

[0167] Example 7.2. Nanoparticle size. The size of the formulated NPs was characterized by dynamic light scattering (DLS) and cryogenic transmission electron microscopy (CryoTEM). The diameters of these NPs are ca. 30 - 40 nm as determined by both DLS and CryoTEM (Table 12). The NPs had a spherical morphology (CryoTEM imaging, shown in Figure 21). Additional images and size distributions by both DLS and CryoTEM are reported in Figures 22 and 23.

[0168]

Table 12

[0169] Example 7.3. Nanoparticle zeta potential. The zeta potential of the NPs (a measure of the NP surface charge) was measured in two solutions of different pH: at pH 7.4 when 5-nPBA-PEGm binds to adjacent diols on cMAP Buffered 10 mM phosphate; and when 5-nPBA-PEGm dissociates from the diol of mucic acid, p is 1 mM KCl at H5.5. cMAP-siRNA NPs with 5-nPBA-mPEG have a slightly negative zeta potential of -3 mV in phosphate buffer at pH 7.4 when 5-nPBA-mPEG is present. However, when these NPs were placed in 1 mM KCl at pH 5.5, the zeta potential was approximately +1 mV . These results are consistent with the boronic acid binding to the diol in mucic acid to shield the positive charge on cMAP and form the tetrahedral boronate complex at pH 7.4, and the boronic acid dissociating from the NPs at acidic pH 5.5. Similar effects were observed with cMA P-PEG copolymers and mPEG-cMAP-PEGm triblock polymers with and without 5-nPBA-PEGm (Table 12). 16).

[0170] Example 7.4. Nanoparticle Stoichiometry. The amounts of cMAP and copolymer bound to the NPs are shown in Table 13 . For all three polymers (cMAP, cMAP-PEG copolymer, and mPEG-cMAP-PEGm triblock po lymer), approximately 33% of the total polymer used for the formulation was bound for an effective NP charge ratio of 1+ / -. The amount of 5-nPBA-PEGm present in NP formulations containing excess PEG for stabilization is also shown in Table 13. The amount of 5-nPBA -PEGm bound to cMAP + 5-nPBA-PEGm NPs was approximately 34%, or one PEG per diol (Table 13). Approximately 20% of the PEG was for the cMAP-PEG copolymer and mPEG-cMAP-PEGm triblock polymer NP formulations . was found to bind to NP. When the particles were formulated with a 3+ / - charge ratio, the excess cationic polymer was considered, and since the effective NP charge ratio was 1+ / - this meant that there was less than one PEG per diol in the NP. As shown above in the data regarding siRNA encapsulation, substantially all siRNA was encapsulated in NPs (Figure 12).

[0171]

Table 13

[0172] Example 8. In vivo pharmacokinetic study in mice.

[0173] A stable formulation of NPs was tested in vivo by tail vein injection into Balb / c mice. At the injected dose, no toxicity was observed from any of the formulations. The PKs of various NPs were measured and the results are illustrated in Figure 24(A-C).

[0174] An NP composed of cMAP polymer and siRNA mixed at a 3+ / - charge ratio and stabilized with 5-nPBA-PEGm was tested, this NP formulation being similar to the CDP formulation used in clinical studies (CALAA-01). The cMAP-based NP has a slightly longer circulation time than CALAA-01 (Figure 5A). CALAA-01 used an inclusion complex for the interaction of CDP and adamantane-PEG (AD-PEG), so there was a possibility that AD-PEG could dissociate from the NP during circulation and the NP would lose its stability. Others synthesized AD2-PEG and this compound had a C ​It was shown that the DP-based NPs have a high ability to be stabilized. This is due to the enhanced binding of two adamantanes per one PEG (to two CDs), resulting in more steric stabilization in circulation (Figure 24A). Along with this cMAP boronic acid system, the interaction between the PEG compound and the polymer is successfully terminated through the boronic acid ester formed from the boronic acid and diol in the polymer, with ca. 30% of the PEG bound to the NPs. Since only 1 / 3 of the cMAP used to formulate the NPs was bound to the particles (Table 3), this was roughly equivalent to one PEG present per diol. The boronic acid-diol interaction is stronger than expected compared to the inclusion complex between adamantine and cyclodextrin, and as a result, 5-nPBA-PEGm remained attached to cMAP longer than AD-PEG to CDP and was able to bring about an improvement in higher steric stability and circulation time. This is due to the enhanced binding of two adamantanes per one PEG (to two CDs), resulting in more steric stabilization in circulation. (Figure 24A). Along with this cMAP boronic acid system, the interaction between the PEG compound and the polymer is successfully terminated through the boronic acid ester formed from the boronic acid and diol in the polymer, with ca. 30% of the PEG bound to the NPs. Since only 1 / 3 of the cMAP used to formulate the NPs was bound to the particles (Table 3), this was roughly equivalent to one PEG present per diol. The boronic acid-diol interaction is stronger than expected compared to the inclusion complex between adamantine and cyclodextrin, and as a result, 5-nPBA-PEGm remained attached to cMAP longer than AD-PEG to CDP and was able to bring about an improvement in higher steric stability and circulation time. Since only 1 / 3 of the cMAP used to formulate the NPs was bound to the particles (Table 3), this was roughly equivalent to one PEG present per diol. The boronic acid-diol interaction is stronger than expected compared to the inclusion complex between adamantine and cyclodextrin, and as a result, 5-nPBA-PEGm remained attached to cMAP longer than AD-PEG to CDP and was able to bring about an improvement in higher steric stability and circulation time. The boronic acid-diol interaction is stronger than expected compared to the inclusion complex between adamantine and cyclodextrin, and as a result, 5-nPBA-PEGm remained attached to cMAP longer than AD-PEG to CDP and was able to bring about an improvement in higher steric stability and circulation time. The boronic acid-diol interaction is stronger than expected compared to the inclusion complex between adamantine and cyclodextrin, and as a result, 5-nPBA-PEGm remained attached to cMAP longer than AD-PEG to CDP and was able to bring about an improvement in higher steric stability and circulation time. The boronic acid-diol interaction is stronger than expected compared to the inclusion complex between adamantine and cyclodextrin, and as a result, 5-nPBA-PEGm remained attached to cMAP longer than AD-PEG to CDP and was able to bring about an improvement in higher steric stability and circulation time. The boronic acid-diol interaction is stronger than expected compared to the inclusion complex between adamantine and cyclodextrin, and as a result, 5-nPBA-PEGm remained attached to cMAP longer than AD-PEG to CDP and was able to bring about an improvement in higher steric stability and circulation time.

[0175] The NPs formed using the cMAP-PEG copolymer can be stably formulated with siRNA in PBS at a 3+ / - charge ratio into the NPs without using 5-nPBA-mPEG (see above). In the cMAP-PEG copolymer, PEG is thought to form a PEG loop that shields the NP core. Additional 5-nPBA-mPEG can be used for further stabilization of the NPs. The zeta potential that switches from negative at pH 7.4 to positive at pH 5.5 measures the filtered amount of excess PEG in addition to 20% PEG bound to the particles, by which 5-nPBA-PEGm is in the NP formulation with cMAP-PEG. In the cMAP-PEG copolymer, PEG is thought to form a PEG loop that shields the NP core. Additional 5-nPBA-mPEG can be used for further stabilization of the NPs. It was shown that it was able to interact with the copolymer. The NPs formulated with the cMAP-PEG copolymer did not provide a significantly longer circulation time than the cMAP:5-nPBA-PEGm-based NPs, regardless of whether 5-nPBA-PEGm was added (Figure 24B).

[0176] The NPs formed using the mPEG-cMAP-PEGm triblock formed stable NPs in PBS (see above) and are thought to have a brush-like arrangement of PEG at the surface of the NPs. As shown by the data provided in Figure 24B, injection of these NPs into mice resulted in an improved PK profile compared to all other cMAP-based NPs, leaving approximately 5 - 10% of the NPs in the mouse circulation after 60 minutes (all other formulations were below the limit of detection by 60 minutes). Similar results were observed in nude mice with this formulation (Figure 25). These significantly longer circulation times are consistent with NPs having a greater degree of steric stability, presumably due to the brush arrangement of the PEG polymer at the surface of the NPs. The addition of 5-nPBA-PEGm to the triblock polymer-siRNA NPs did not result in an improvement in circulation time (Figure 24B). Furthermore, a siRNA-containing NP formulation with a charge ratio of 2+ / - obtained by removing some of the 66% excess triblock polymer from the 3+ / - NP formulation did not result in a decrease in circulation time when the formulation was spin-filtered through a 30 kD MWCO membrane (Figure 24C). Using this method of purification, it was difficult to remove all of the excess polymer. These results suggested that polymer not incorporated within the NPs did not alter PK.

[0177] ​​​​​​​​​​​​​​Among the polymer modifications considered here, mPEG-cMAP-PEGm NPs provided the longest circulation time and the circulation time did not increase with the additional conjugate 5-nPBA-PEGm. The interaction of 5-nPBA-PEGm with the diol in cMAP is likely to be stronger than the interaction between adamantane and CDP, but there was still a possibility that some amount of PEG was shed from the NPs. On the other hand, the triblock polymer has one or two PEGs per cMAP unit and may be able to achieve the PEG density on the NP surface required for a good brush layer. However, the amount of covalently bound PEG in this triblock polymer was less than that in cMAP+5-nPBA-PEGm NPs. PK data from these systems suggested that PEG shedding during circulation still occurred, but less than that which occurs in the CDP-adamantane system. Some of the 5-nPBA-PEGm must remain on the NPs during circulation.

[0178] To provide further evidence that the NPs remain intact during circulation, serum collected from mice 20 minutes after dosing was electrophoresed on a gel and the siRNA was visualized either with ethidium bromide or a fluorophore-labeled siRNA using a Typhoon imager. The results from these experiments showed that the siRNA and the fluorescently labeled siRNA remain intact in the NPs while circulating in vivo.

[0179] The following references may be useful in understanding certain aspects of the present disclosure: ​​​​1. Wu, S.Y., Lopez-Berestein, G., Calin, G.A. & Sood, A.K. (2014) RNAi Therapies: Drugging the Undruggable( ). Science Transl. Med. RNAi Therapeutics: Drugging the Undruggable). Science Translational Medicine 6, 240ps7. 2. Kanasty, R., Dorkin, J.R., Vegas, A. & Anderson, D. (2013) Delivery materials for siRNA therapeutics (Delivery materials for siRNA therapeutics) Nat. Mater. 12, 967 - 977. 3. Davis, M.E. (2009) The First Targeted Delivery of siRNA in Humans via a Self-Assembling Cyclodextrin Polymer-Based nanoparticle: From Concept to Clinic. (The First Targeted Delivery of siRNA in Humans via a Self-Assembling Cyclodextrin Polymer-Based nanoparticle: From Concept to Clinic) Mol. Pharm. 6, 659 - 668. 4. Davis, M.E. et al. (2010) Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles. (Evidence of RNAi in humans from systemically Evidence of RNAi in humans from 5. Zuckerman, J.E. et al. (2014) Correlating animal and human phase Ia / Ib clinical data with CALAA-01, a targeted, polymer-based nanoparticle contai ning siRNA. (Correlation of animal and human Ia / Ib clinical data with CALAA-01, a targeted, polymer-based nanoparticle containing siRNA.) Proc. Natl. Acad. Sci. USA · of the National Academy of Sciences of the United · States of America) 111, 11449-11454. 6. Zuckerman, J.E., Choi, C.H.J., Han, H., and Davis, M. E. (2012) Polycation-siRNA nanoparticles can disassemble at the kidney glomerular basement membrane. (Polymer-cation-siRNA nanoparticles can disassemble at the kidney glomerular basement membrane.) Proc. Natl. Acad. Sci. 109, 3137-3142. 7. Naeye, B., Deschout, H., Caveliers, V ., Descamps, B., Braeckmans, K., Vanhove, C ., Demeester, J., Lahoutte, T., De Smedt (De Smedt), S.C., Raemdonck, K. 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[0180] As will be appreciated by those skilled in the art, numerous modifications and variations of the present disclosure are within these teachings. Illumination is possible, and all of it is considered here. For example, as described here In addition to the embodiments described herein, the present disclosure contemplates and claims inventions arising from combinations of the features of the disclosures cited herein that complement the features of the present disclosure and the prior art documents cited. Similarly any described substance, feature, or article may be used in combination with any other substance, feature, or article, and such combinations are considered to be within the scope of the present disclosure.

[0181] The disclosures of each patent, patent application, and publication cited or described in this document are hereby incorporated by reference in their entirety for all purposes. In addition to the references already mentioned, the present disclosure includes subject matter related to U.S. Patent Application No. 12 / 540,319, filed on August 12, 2009, now U.S. Patent No. 8,557,292; Application No. 13 / 782,458, filed on March 1, 2013; Application No. 13 / 782,486, filed on March 1, 2013; Application No. 13 / 852,303, filed on March 28, 2013; and International Application No. PCT / US2009 / 053620, filed on August 12, 2009, the contents of which are hereby incorporated by reference for all purposes, including their teachings regarding chemical substances, applications, and methods of making and using the block copolymers described therein. ​

Claims

1. A method for modifying a biological substance and improving its bioavailability and / or its pharmacokinetics, comprising: a step of incorporating the substance into nanoparticles, wherein the nanoparticles comprise a polymer containing alternating charged segments and uncharged segments, the polymer has a structure of formula (VII): 【Chemical 1】 as described by wherein chain A is [Chemical Formula 2] ; cMAP is 【Chemical Formula 3】 chain B is 【Chemical Formula 4】 ; p and q are each independently sufficient to provide a number average molecular weight in the range of 500 Da to 50,000 Da for the subunits containing cMAP and PEG; m is, independently of each occurrence, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; n and r are, independently of each occurrence, 0, 1, 2, 3, 4, or 5; and X1 and X2 are, independently of each occurrence, C1-6 alkyl optionally substituted by -OH, -COOH, -C(=O)O(alkyl), -C(=O)O(aryl), -NH2, -NH(alkyl), -N(alkyl)2, or their salts or protected analogs, the method comprising the step.

2. The method according to claim 1, wherein the nanoparticles comprise 5-nPBA-PEGm.

3. The method according to claim 1, wherein the biological substance is selected from the group consisting of polynucleotides and chemotherapeutic agents.

4. The method according to claim 3, wherein the polynucleotide is an RNA molecule.

5. The method according to claim 4, wherein the RNA molecule is a messenger RNA (mRNA) molecule or a small interfering RNA (siRNA) molecule.

6. The method according to claim 1, wherein the uncharged segment comprises polyethylene glycol and a suitable linking group.

7. The method according to claim 1, wherein p is sufficient to provide a number average molecular weight in the range of 5 kDa to 15 kDa for the subunit containing cMAP.

8. The method according to claim 1, wherein q is sufficient to provide a number average molecular weight in the range of 500 Da to 50 kDa for the subunit containing PEG.

9. The nanoparticles further comprise a second polymer having a structure of formula (X): 【Chemical Formula 5】 ; The polymer and the second polymer are reversibly connected to each other by a borate condensation linkage between the -B(OH) 2 moiety of formula (X) and at least one pair of adjacent diols of the polyhydroxy linkage of segment B; R A is nitro; n is 0 or 1; s is 20 - 1200; L is a linking group between the phenyl ring and the polyethylene oxide linkage; and X 5 is C 1-6 alkyl, optionally substituted by -OH, -COOH, -C(=O)O(alkyl), -C(=O)O(aryl), -NH 2 , -NH(alkyl), -N(alkyl) 2 substituted or a salt or protected analog thereof The method according to any one of claims 1 to 8.

10. L is -(C 0-2 alkylene)-NH-C(=O)-(C 0-2 alkylene)-, -(C 0-2 alkylene)-C(=O)-NH-(C 0-2 alkylene)-, -(C 0-2 alkylene)-O-C(=O)-(C 0-2 alkylene)- or -(C 0-2 alkylene)-C(=O)-O-(C 0-2 alkylene)-; -NH-C(=O)-, -C(=O)-NH-, -O-C(=O)-, or -C(=O)-O-, the method of claim 9. Claim 11 X 5 The method of claim 9, further comprising the step of conjugating a targeting ligand to the nanoparticles in Claim 12 The method of claim 11, wherein the target-directed ligand is an antibody or a fragment thereof, transferrin or a fragment thereof, a ligand for a cell receptor or a fragment thereof, a cell receptor protein or a fragment thereof, or an aptamer or a fragment thereof. Claim 13 The method according to any one of claims 1 to 8, further comprising the step of combining the nanoparticles with a pharmaceutically acceptable carrier or excipient. Claim 14 The method of claim 9, further comprising the step of combining the nanoparticles with a pharmaceutically acceptable carrier or excipient. Claim 15 The method of claim 11, further comprising the step of combining the nanoparticles with a pharmaceutically acceptable carrier or excipient. Claim 16 The method according to any one of claims 1 to 8, wherein the improvement in pharmacokinetics is an increase in circulation time. Claim 17 The method of claim 9, wherein the improvement in pharmacokinetics is an increase in circulation time. Claim 18 The method of claim 11, wherein the improvement in pharmacokinetics is an increase in circulation time.

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