Cationic mucic acid polymer-based delivery systems

Abstract

To provide polymer and polymer conjugate-based nanoparticle delivery systems for delivering biological agents, and methods of making and using these compositions.SOLUTION: The present disclosure provides polymers comprising alternating charged and uncharged segments comprising one or more of the following structural units of Formula (I) or Formula (II) or Formula (III), where A is an uncharged segment comprising polyalkylene glycol; and B is a cationically charged segment comprising one polyhydroxy linkage including at least a pair of adjacent diols.SELECTED DRAWING: Figure 10

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. Government Rights

[0002] This invention was made with government support under grant number CA151819 awarded by the National Institutes of Health. The government has certain rights in this invention.

Technical Field

[0003] The present disclosure relates to nanoparticle delivery systems based on polymers and polymer - complexes for delivering biological substances, and methods of making and using these compositions.

Background Art

[0004] 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) a strong single - digit picomolar IC 50 (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 compromising efficacy and target specificity, and (iv) the catalytic RNAi mechanism of action, resulting in extended siRNA - mediated suppression of mRNA target expression. The major obstacle to the translation (also referred to as translocation) of siRNAs into effective and efficient therapeutic agents is the delivery of nucleic acids to the target, although siRNA - based experimental therapeutic agents have reached clinical practice.

[0005] Therapeutic substances studied for cancer treatment are mainly administered systemically and use some type of synthetic compound (positively charged lipid or polymer) in their 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 with siRNA via electrostatic interaction 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). CALAA-01 reveals several positive traits, but one of its drawbacks is that it has a very limited circulation time. The rapid clearance of CALAA-01 observed in animals (mice, rats, dogs, and non-human primates) is also observed in humans.

[0006] Schematic It would be advantageous to develop a polymeric system for siRNA delivery that increases the circulation time of siRNA-containing nanoparticles and decreases the amount of non-siRNA components within the formulation. The present disclosure is directed to delivery systems that overcome some of the drawbacks of the prior art. Among the aspects of the present disclosure are families of cationic mucic acid-based polymers (cMAP), including diblock and triblock copolymers for in vivo siRNA delivery, and nanoparticles derived therefrom. These compounds and structures, as well as methods of making and using them, are described in more detail within this specification.

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

Chemical formula

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

[0009] In overlapping embodiments, B is a cationic charged segment comprising at least one polyhydroxy comprising at least one pair of adjacent diols in which a sugar linkage is included. In some embodiments, these polyhydroxy linkages include mucic acid. B has the structure of formula (V):

Chemical formula

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

Chemical formula

[0011] In some specific embodiments, the polymer has the structure of formula (VII):

Chemical formula

Chem.

Chem.

Chem.

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

Chem.

Chem.

Chem.

[0013] Still other polymers have the structure of formula (IX): [Chemical formula] which can be described by wherein the terminal group D is: [Chemical formula] and; cMAP is [Chemical formula] and; chain C is

Chem.

[0014] Also contemplated within the scope of the present disclosure are polymers of any one of the preceding structures, and a polymer complex comprising each of the second boronic acid-containing polymers having the structure of formula (X)

Chem.

[0015] The present disclosure also includes nanoparticles or a plurality of nanoparticles comprising any polymer or polymer complex described herein. If possible, the nanoparticles are monodisperse. The nanoparticles may further comprise an encapsulated biological substance, such as siRNA, and / or may be further conjugated to one or more targeting ligands. When administered to a patient, the bioavailability of the biological substance is better than the bioavailability of the same biological substance when administered by itself.

[0016] In some additional embodiments, the polymer, polymer complex, and nanoparticles optionally contain a biological substance and / or a targeting ligand and are formulated in a pharmaceutical composition. Other embodiments provide treatment of a patient by administering these formulated compositions to a patient in need of each biological substance.

[0017] Yet further embodiments provide a method of creating inventive polymers. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] This application is further understood when read in conjunction with the accompanying drawings. For purposes of illustration of the subject matter, exemplary embodiments of the subject matter are shown in these figures; however, the subject matter disclosed herein is not limited to the specific methods, devices, and systems disclosed. Additionally, the drawings are not necessarily drawn to scale. The drawings are as follows:

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[0019] DETAILED DESCRIPTION OF EXAMPLE 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 at the glomerular basement membrane (GBM) in the kidney. The inventors have speculated that this clearance mechanism may affect any NP formulation that is mainly assembled by electrostatic interactions between cationic delivery components and anionic nucleic acids. Other siRNA delivery systems using either cationic polymers or lipids have shown similar short circulation times and renal clearance.

[0021] Many current polymer and liposome systems used to deliver siRNA in vivo contain an excess of cationic components in their formulations (the positive-to-negative charge ratio is usually greater than 1) in addition to large amounts of substances such as poly(ethylene glycol) (PEG), and are 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 responses.

[0022] The development of polymeric systems for siRNA delivery that both increase the circulation time of siRNA-containing nanoparticles and decrease the amount of non-siRNA components within the formulation would be advantageous. A family of cationic mucic acid-based polymers (cMAP) for in vivo siRNA delivery is described herein. This polymeric delivery system has several features similar to the CDP system, since the latter system has functioned in humans. The cationic polymers developed here use simpler sugars, are embodied as mucic acid (also called mucin acid or mucinic acid) rather than cyclodextrin, and enable alternative strategies for surface functionalization. Instead of nanoparticle surface functionalization via inclusion complex (also called inclusion complex) formation (CDP) with adamantane (AD), 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 was further reacted to functionalized PEG and linear block copolymers. Reaction of either di-activated, carboxylic acid-PEG, or activated, carboxylic acid-PEG-methoxy (PEGm) at the end groups of cMAP leads to two possible copolymers (also called copolymers), namely: cMAP-PEG copolymer or mPEG-cMAP-PEGm triblock polymer. The cMAP-PEG copolymer can be combined with siRNA to form PEG loops on the surface and stabilize the nanoparticles, while the mPEG-cMAP-PEGm triblock can form a PEG brush configuration on the nanoparticle surface. The latter triblock approach has been previously investigated with CDP and plasmid DNA (pDNA), and its triblock polymers did not have the ability to encapsulate pDNA. Polymers that encapsulate pDNA may not be good at condensing siRNA, and vice versa has been shown.Here, the inventors show that the mPEG-cMAP-PEGm triblock polymer can form siRNA-containing nanoparticles with increased circulation time in mice, which can have ca. (about) 30 wt% of the formulation that is siRNA. Further, the nanoparticles can be readily assembled directly in phosphate buffered saline (PBS) without using any additional 5-nPBA-PEGm to stabilize the NPs.

[0023] This disclosure can be more readily understood by reference to the following description in connection with the accompanying figures and examples, all of which form a part of this disclosure. It is understood that this disclosure is not limited to the specific products, methods, conditions or parameters described or shown herein, and that the terms used herein are for the purpose of describing particular embodiments by way of example only and are not intended to limit any claimed invention. Similarly, it should be understood that any explanation regarding possible mechanisms or modes of action or improvements, unless otherwise specified, is illustrative only and that the disclosure herein is not restricted by the accuracy or inaccuracy of any such proposed mechanism or mode of action or improvement. Throughout this text, it is recognized that the description refers to compositions and methods of making and using the said compositions. That is, when this disclosure describes or claims a feature or an embodiment related to a composition or a method of making or using a composition, it is intended that such description or claims extend the feature or embodiment to embodiments (i.e., compositions, methods of making, and methods of using) in each of these contexts.

[0024] 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.

[0025] When values are expressed as approximations using the descriptor "about", it will be understood that the particular value forms another embodiment. In general, the use of the term "about" indicates an approximation that can vary depending on the desired characteristics required by the disclosed subject matter and is to be interpreted in the particular context in which it is used based on its functionality. Those of ordinary skill in the art will be able to interpret this as a matter of routine. In some cases, the number of significant figures used for a particular value may be one non-limiting way of determining the degree of the word "about". In other cases, the gradual changes 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. All ranges, if present, are inclusive and combinable. That is, a reference to a value within a range includes all values within that range.

[0026] For clarity, it should be recognized that certain features of the present disclosure described in the context of separate embodiments may also be provided in combination in a single embodiment. That is, each individual embodiment is considered to be combinable with any other embodiment(s) (the term "s" indicating that a plurality may also be included) unless clearly incompatible or explicitly excluded, 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, while an embodiment may be described as part of a series of steps or a more general structure, each of the steps may be considered as an independent embodiment that can be combined with others.

[0027] The transitional terms "comprising", "consisting essentially of", and "consisting of" are intended to embrace meanings commonly accepted 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, unrecited 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 the specified materials or steps "and those that do not materially affect the basic and novel characteristics" of the claimed invention. Embodiments described by the phrase "comprising" (or its equivalent) also provide, as embodiments, those described with respect to "consisting of" and "consisting essentially of". For those embodiments provided by "consisting essentially of", the basic and novel characteristics are the inventive materials, and the ease of operability of the methods (and the systems used in such methods and the compositions derived therefrom) for preparing and using the materials themselves, where the methods and materials are capable of providing the highlighted properties using only the elements provided in the claims. That is, other materials may be present in the inventive compositions, but the presence of these additional materials is not necessary to provide the described advantages of those compositions (i.e., the effects may be additive) and / or these additional substances do not detract from the performance of the composition of the product. Similarly, if additional steps may also be used in the method, their presence is not necessary to achieve the described effects or advantages and / or does not impair the described effects or benefits.

[0028] When a list is presented, unless otherwise stated, each individual element of that list and any combination of that list should be understood as separate embodiments. For example, a list of embodiments presented as "A, B, or C" should be interpreted as including the embodiments, "A", "B", "C", "A or B", "A or C", "B or C", or "A, B, or C". Similarly, C 1-3 Terms such as alkyl also include, as separate embodiments, C1 alkyl, C2 alkyl, C3 alkyl, C 1-2 alkyl, and C2 -3 alkyl.

[0029] Throughout this specification, words should be given their ordinary meanings as understood by those skilled in the relevant art. However, to avoid misunderstanding, the meanings of certain terms are 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, references to hydroxy or alcohols also include their substitutions, where hydroxy is acetyl (Ac), benzoyl (Bz), benzyl (Bn, Bnl), β - methoxyethoxymethyl ether (MEM), dimethoxytrityl, [bis-(4 - methoxyphenyl)phenylmethyl] (DMT), methoxymethyl ether (MOM), methoxytrityl [(4 - methoxyphenyl)diphenylmethyl, MMT], p - methoxybenzyl ether (PMB), methylthiomethyl ether, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (triphenylmethyl, Tr), silyl ethers (among the most common are trimethylsilyl (TMS), tert - butyldimethylsilyl (TBDMS), tri - iso - propylsilyloxymethyl (TOM), and triisopropylsilyl (TIPS) ethers), ethoxyethyl ether (EE). References to amines also include their substitutions, where amines are protected by 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 - dimethoxybenzyl (DMPM), p - methoxyphenyl (PMP), tosyl (Ts) group, or sulfonamide (Nosyl&Nps) group. References to substitutions containing a carbonyl group also include their substitutions, where the carbonyl is protected by an acetal or ketal, acylal, or dithiane group.References to substitutions containing carboxylic acid or carboxylate groups include substitutions in which the carboxylic acid or carboxylate group is protected by its methyl ester, benzyl ester, tert-butyl ester, ester of 2,6-disubstituted phenol (e.g., 2,6-dimethylphenol, 2,6-diisopropylphenol, 2,6-di-tert-butylphenol), silyl ester, orthoester, or oxazoline.

[0031] Embodiments of the present disclosure include the following structural units of formula (I) or formula (II) or formula (III):

Chemical formula

[0032] In the polymers of the present disclosure, the term polyalkylene glycol refers to a functional linkage:

Chemical formula

[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 sugar or carbohydrate (also referred to as saccharide) 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 mucic acid, where B is of formula (IV), formula (IV) or (IVA):

Chemical formula

[0034] In other embodiments, B has a structure of formula (V):

Chemical formula

[0035] Together with these building blocks (also referred to as basic units), it is possible to describe a more specific range of triblock 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 include a structure of formula (VII): [Chemical formula] as described by wherein chain A is [Chemical formula] and chain B is [Chemical formula] and; cMAP is [Chemical formula] and; p and q are each independently sufficient to provide a number average molecular weight in the range of about 500 Da to about 50,000 Da for the sub-units containing cMAP and PEG; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or even higher), possibly 4 - 6 or 5, regardless of its respective presence; n and r are each independently 0, 1, 2, 3, 4, or 5 (or even higher); and X1 and X2 are each independently C 1-6 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 (also referred to as protected analogues).

[0036] Here too, 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 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 value for p corresponds to the range of about 1 to about 100, possibly about 10 to about 100. Specific embodiments include those where 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 cited MW nFor the numerical values of q corresponding to the range, those in the range from about 12 to about 1200 are included. 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 from about 100 to about 500. In other subsets of this embodiment, X1 and X2 are, regardless of their respective presence, -(CH2) 1-4 -COOH and / or -(CH2) 1-4 -NH2.

[0037] Considering the properties of chain A and chain B, such a structure can also be represented as a PEG-cMAP-PEG triblock polymer.

[0038] In other embodiments, the polymers of the present disclosure include 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 presence and may be the same or different from each other. The same applies to 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 the range of from about 1 to about 100, preferably from about 10 to about 100. Specific embodiments include those where 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 cited MW nThe numerical values for q corresponding to the ranges 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 from about 100 to about 500. In other subsets of this embodiment, X1 and X2 are, independently of their respective presence, -(CH2) 1-4 -COOH and / or -(CH2) 1-4 -NH2.

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

[0041] In still 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 the range of from about 1 to about 100, and if possible from about 10 to about 100. Specific embodiments include those where 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 MW cited nThe numerical values for q corresponding to the range include those ranging from about 12 to about 1200. In certain embodiments, those where q ranges 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 are included. In certain embodiments, q can also be in the range from about 100 to about 500. In other subsets of this embodiment, X1 and X2, regardless of their respective presence, are -(CH2) 1-4 -COOH and / or -(CH2) 1-4 -NH2.

[0043] Considering the nature of the various chains and end group elements, such a structure can also be referred to as a cMAP-PEG-cMAP triblock polymer.

[0044] In each of the presented structures, certain non-relevant embodiments include those where m is 4, 5, or 6 without relation. In certain embodiments, m is 5 for each presence.

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

[0046] In each of the presented structures, certain non-relevant embodiments include those where r is 2, 3, or 4 without relation. In some of these embodiments, r is 3 for each presence.

[0047] In each of the structures presented, certain non-relevant embodiments include those in which p is sufficient to provide a number average molecular weight for a subunit containing cMAP in the range of from about 5 kDa to about 15 kDa, from about 6 kDa to about 14 kDa, from 7 kDa to about 13 kDa, from about 8 kDa to about 12 kDa, from 9 kDa to about 11 kDa, or about 10 kDa. If the cMAP fragment (also referred to as a fragment) has an MW of about 420 Da each, this corresponds to a value of p in the range of from about 12 to about 36, from about 14 to about 33, from about 17 to about 31, from about 19 to about 29, from about 22 to about 26, or about 24.

[0048] In each of the structures presented, certain non-relevant embodiments include those in which q is sufficient to provide a number average molecular weight for a subunit containing PEG in the range of from about 500 Da to about 50 kDa, from about 1 kDa to about 40 kDa, from 5 kDa to about 30 kDa, or from about 5 kDa to about 20 kDa. If the polyalkylene glycol moiety is polyethylene glycol and the ethylene glycol fragment has an MW of about 44 Da, this corresponds to a value of q in the range of 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.

[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 distinct embodiment, and any combination of these embodiments provides a 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 homologues of the specifically described reagents, are considered to be within the scope of the present disclosure. As used herein, the term homologue 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 uncharged 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 mucic acid derivatives, this method involves 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, each having an appropriate linking group to form the desired diblock or triblock polymer. Such coupling reactions are effected using a carboxylic acid / amino condensation reaction 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.

[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 the 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 in the range from 20 to about 120, about 120 to about 240, about 240 to about 480, about 480 to about 720, about 720 to about 960, about 960 to about 1200, or 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 0-2 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)-. In these subsets, L is -NH-C(=O)-, -C(=O)-NH-, -OC(=O)-, or -C(=O)-O-. A single or multiple linking group of L (also referred to as a multiple linking group) can be adopted 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 have cross-sectional dimensions (i.e., diameters) in the range of about 20 nm to about 300 nm, depending on the sizes of the various cMAP or PEG fragments and / or the chain lengths associated with the boronic acid-containing polymers. Certain embodiments can also be described as having 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.

[0057] Similarly, reference to a single nanoparticle should be considered to include discrete embodiments encompassing a population or plurality of nanoparticles. In certain embodiments, the plurality of nanoparticles are substantially monodisperse, such that, in independent embodiments, a standard deviation in cross-sectional dimensions between nanoparticles of less than 20%, 30%, 40%, 50%, or 60% is provided, on average, as measured by cryo-transmission electron microscopy (cryo-TEM). The 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, in a representative cryo-transmission electron micrograph (typically derived from more than 3 randomly selected liquid samples frozen in liquid ethane), the average diameter of the particles is measured, the particles within a predetermined size fraction gradient (also referred to as particle size gradient) are counted, and a predetermined number of particles (more than 100) are analyzed by statistically correlating their numbers. For additional information, see also the Examples and Appendix (also referred to as 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 bio-derived material). These biological materials may be covalently bound within or by the nanoparticles or otherwise contained. In certain embodiments, the biological material is a polynucleotide or a small molecule therapeutic agent. 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), ethylenediaminetetraacetic acid (EDTA)), natural products (e.g., taxol, amphotericin), and other biologically active macromolecules such as proteins and enzymes. See also U.S. Patent No. 6,048,736, which lists active agents (therapeutic agents) that can be used as therapeutic agents with the nanoparticles described herein. Small molecule therapeutic agents are not only therapeutic agents within the composite particles but, in further embodiments, may 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 such as a disulfide) and provides another way to deliver the therapeutic agent. In some embodiments, therapeutic agents that can be delivered with the nanoparticles described herein include chemotherapeutic agents such as, for example, epothilones, camptothecin-based drugs, taxol, etc., or nucleic acids such as, for example, plasmids, siRNA, shRNA, miRNA, antisense oligonucleotide aptamers, or combinations thereof, and additional drugs that can be identified by one of ordinary skill in the art upon reading this disclosure.

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

[0060] Without 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 within internal cavities (see, for example, FIGS. 16A and 16B). Negatively charged cargo contains nucleic acids, 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 contain functionalized (nitro)boronic acid-containing polymer linkages, the nanoparticles may be further conjugated to one or more targeting (also referred to as target-directed) ligands. In such cases, conjugation occurs through a condensation linkage between the distal end of the boronic acid-containing polymer and the target-directed ligand. In some embodiments, the 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 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 。

[0062] As suggested above, in certain embodiments, the polymer, polymer conjugate, and / or nanoparticles may be present as a dispersion in an aqueous medium, which may also optionally contain a buffer, surfactant, or other modifier (also referred to as a modifying factor or modifier). The present disclosure also contemplates a pharmaceutical composition comprising one or more biologically active agents and any of the polymers or polymer conjugates or nanoparticles or plural nanoparticles described herein, and a pharmaceutically acceptable vehicle, carrier, or excipient.

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

[0064] As used herein, the term "excipient" refers to an inert substance used as a carrier for the active ingredient of a drug treatment. Suitable excipients for the pharmaceutical compositions disclosed herein 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 dosages, excipients can be used in the manufacturing process to aid in the handling of the nanoparticles. Different excipients can be used depending on the route of administration and the form of the drug treatment. Exemplary excipients include, but are not limited to, antiadherents (also referred to as antiadhesives), binders, coatings disintegrants, fillers, flavoring agents (such as those like sweeteners), and coloring agents, glidants (also referred to as lubricants), lubricants, preservatives (also referred to as antiseptics), adsorbents.

[0065] As used herein, the term "diluent" refers to a diluting agent (also referred to as an excipient) that is administered to dilute or render active the active ingredient of a composition. Suitable diluents include any substance that can reduce the viscosity of a pharmaceutical preparation.

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

[0067] Compositions comprising the biologically active agents and polymers, polymer conjugates, and / or nanoparticles of the present disclosure, including the pharmaceutical compositions thereof, are useful for treating patients in need of treatment, particularly due to the enhanced bioavailability (also referred to as biological availability) conferred by the inventive polymers, polymer conjugates, and / or nanoparticles. The degree of improvement in the bioavailability of such compositions, whether by themselves or in combination with cMAP, was surprisingly high compared to the delivery of the same biologically active agent or group of agents. See the examples. Accordingly, important embodiments include compositions comprising the biologically active agents and polymers, polymer conjugates, and / or nanoparticles of the present disclosure, including the pharmaceutical compositions thereof, which 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) or formula (II) or formula (III):

Chemical formula

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

[0071] Embodiment 3. The polyalkylene glycol has a nominal number average molecular weight in the range of about 500 daltons to about 50,000 daltons, for the polymer of Embodiment 1 or 2. In certain subsets of this embodiment, the polyalkylene glycol has a nominal number average molecular weight in the range of 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 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 any combination of two or more of these ranges.

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

[0073] Embodiment 5. B includes at least one repeating sub-unit having the structure of formula (IV):

Chemical formula

Chemical formula

[0074] Embodiment 6. B further includes at least one repeating sub-unit having the structure of formula (V):

Chemical formula

[0075] Embodiment 7. B includes at least one repeating sub-unit containing cMAP, and the sub-unit structure is represented by formula (VI):

Chemical formula

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

Chem.

Chem.

Chem.

Chem.

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

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0078] Again, as in embodiment 9, to meet the molecular weight limitations, the value for p corresponds to a range from about 1 to about 100, preferably from about 10 to about 100, and the value for q corresponds to a range 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 certain subsets of this embodiment, p and q are each independently such that the number average molecular weight for the sub-units containing cMAP and PEG is 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 two or more of these ranges, and these MW nis sufficient to provide in the corresponding numerical range of the range. In other subsets of this embodiment, X1 and X2 are, independently, -(CH2) 1-4 -COOH and -(CH2) 1-4 -NH2.

[0079] Embodiment 10. The 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 a range of from about 1 to about 100, preferably from about 10 to about 100 if possible, and the value for q corresponds to a 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 certain subsets of this embodiment, p and q independently provide a number average molecular weight for the subunits containing cMAP and PEG, respectively, in the range of from 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, or any combination of two or more of these ranges, and the corresponding numerical ranges of these MW n are sufficient to provide in the corresponding numerical ranges of. In other subsets of this embodiment, X1 and X2 independently are -(CH2) 1-4 -COOH and -(CH2) 1-4 -NH2.

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

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

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

[0084] Embodiment 14. p is a polymer according to any one of Embodiments 8 to 13 that is sufficient to provide a number average molecular weight for the subunit containing cMAP in the range of about or greater than 5 kDa to about 15 kDa, about or greater than 6 kDa to about 14 kDa, about or greater than 7 kDa to 13 kDa, about or greater than 8 kDa to about 12 kDa, about or from 9 kDa to about 11 kDa, or about 10 kDa. In some subsets of embodiments, for example, when the cMAP fragment has an MW of about 420 Da, this corresponds to a p having a numerical value 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. n When it has, this corresponds to a p having a numerical value 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.

[0085] Embodiment 15. q is a polymer according to any one of Embodiments 8 to 13 that is sufficient to provide a number average molecular weight for the subunit containing PEG in the range of about or greater than 500 Da to about 50 kDa, about or greater than 1 kDa to about 40 kDa, about or greater than 5 kDa to about 30 kDa, or about or greater than 5 kDa to about 20 kDa. In some of these embodiments, for example, assuming the ethylene glycol fragment has an MWn of about 44 Da, this corresponds to a q having a numerical value in the range of about 11 to about 1200, about 23 to about 910, about 110 to about 680, or 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] comprising a second boronic acid-containing polymer containing, wherein, The polymer and the second boronic acid-containing polymer are reversibly connected to each other by a borate condensation linkage between the boronic acid moiety of formula (IX) and at least one pair of adjacent diols of polyhydroxy linkage of formula (I), (II), (III), (IV), (VI), (VII), (VIII), or (IX), and X5 is at the distal end of this connection; R A is nitro (or other electron-withdrawing group); n is 0, 1, 2, 3, or 4, preferably 1 if possible; s is from 20 to 1200; L is a linking group between the phenyl ring and the polyethylene oxide linkage; and X5 is a C 1-6 alkyl, optionally substituted by -OH, -COOH, -C(=O)O(alkyl), -C(=O)O(aryl), -NH2, -NH(alkyl), -N(alkyl)2, or a salt or protected analog thereof, a polymer complex (also referred to as a polymer conjugate). Exemplary structures are as follows:

Chemical formula

[0087] Embodiment 17. 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)-, 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 any one of the polymers of Embodiments 1 to 15.

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

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

[0092] Embodiment 22. A plurality of nanoparticles, wherein each individual nanoparticle is described by any one of the compositions of Embodiments 19 to 21.

[0093] Embodiment 23. Each individual nanoparticle is described by any one of the compositions of Embodiments 19 to 22, and the plurality of nanoparticles are substantially monodisperse and exhibit a standard deviation in the cross-sectional dimension (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 comprising any one of the polymers of Embodiments 1 to 15 or the polymer composites of claims 16 to 18 at the time of filing, further comprising an encapsulated biological agent.

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

[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 nanoparticle of Embodiment 24 or 25, wherein the biological substance is a polynucleotide which is an RNA molecule.

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

[0099] Embodiment 29. The nanoparticle of any one of Embodiments 20 to 28, further conjugated (also referred to as conjugate) to a target-directed ligand, wherein the conjugation occurs through a condensation linkage between the distal end of the boronic acid-containing polymer and the target-directed ligand.

[0100] Embodiment 30. The nanoparticle of Embodiment 29, wherein a single target-directed ligand is conjugated to each polymer.

[0101] Embodiment 31. The nanoparticle of Embodiment 29, wherein a plurality of target-directed 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 one or more nanoparticles of any one of Embodiments 19 to 31 and a pharmaceutically acceptable carrier or excipient.

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

[0105] Embodiment 35. A method for preparing a polymer according to 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 include reacting homologs of the specific reactants cited 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, preparation methods, 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 mentioned, references to molecular weight are intended to refer to the number average molecular weight.

[0110] Summary of Experimental Results

[0111] A new cationic polymer with 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 was able to efficiently encapsulate (also referred to as enclose) siRNA with a charge ratio of 2+ / - or more. Stable NPs composed of this triblock polymer and siRNA had a diameter of approximately 30 nm (by both DLS and CryoTEM) and a slightly positive surface charge of approximately 0.4 mV in both 10 mM phosphate buffer pH 7.4 and 1 mM KCl pH 5.5 and could be formulated directly in PBS. Upon injection into mice, these NPs formed with the mPEG-cMAP-PEGm triblock polymer showed extended circulation compared to NPs formulated with cMAP and cMAP-PEG copolymers, with 5 - 10% of the formulation remaining in circulation after 1 hour. When a portion of the excess triblock polymer was removed from the formulation, the circulation time remained the same. The absence of any excess cationic polymer is advantageous to minimize any adverse effects these entities may cause in vivo.

[0112] Example 1. Materials and methods.

[0113] Mucic acid and oxalyl chloride were purchased from Sigma-Aldrich, N-boc-ethylenediamine was purchased from AK Scientific, dimethyl suberimidate was purchased from Thermo Fisher Scientific or Sigma-Aldrich, and 3-carboxyl-5-nitrophenylboronic acid was purchased from Alfa-Aesar. The polyethylene glycol reagent was purchased from either Jenkem Technology USA or Laysan Bio, Inc. Dimethyl suberimidate is a charged monomer that polymerizes mucic acid ethylenediamine and was purchased and used from Thermo Scientific or Sigma-Aldrich. The NMR spectrum of suberimidate dimethyl was obtained to assign peaks to the proton and carbon spectra of cMAP. The proton and carbon NMR spectra of DMS were more complex than expected, suggesting that some hydrolysis was present in newly opened bottles. See Table 2.

[0114] Nuclear magnetic resonance (NMR) spectra were acquired at 25 degrees Celsius (also referred to as 25 °C) on a Varian 300 MHz, 500 MHz, or 600 MHz instrument without spinning at 500 MHz or 600 MHz. Most 1 For the 1H proton spectrum, a delay time of 1 - 1.5 seconds was used; for quantitative integration of the polymer, a 25-second delay was used. 13 The 13C carbon spectrum was obtained at 500 MHz by default settings. 1 1H- 13 13C heteronuclear single quantum coherence (HSQC), 1 1H- 1 1H correlation spectroscopy (COSY), and 1 1H- 13Heteronuclear multiple bond correlation spectroscopy (HMBC) spectra were acquired using default VNMRJ 3.0 HSQCAD, COSY, and HMBC settings. Additionally, diffusion-ordered spectroscopy (DOSY) spectra using the bipolar pulse pair stimulated echo (Dbppste_cc) method with a diffusion gradient length of 4.0 ms and a diffusion delay of 100.0 ms in VNMRJ 3.0 were acquired for the synthesized polymers.

[0115] Electrospray ionization masses of small molecules were obtained using a Finnigan LCQ ion trap mass spectrometer. Matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) mass spectra for the polymers were obtained using a 10 mg / mL α-cyano-4-hydroxycinnamic acid matrix on an Applied Biosystems Voyager DE-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. 1 1H NMR (300 MHz, 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 (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 stirred and reflux 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 in 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. 11H NMR (300 MHz, DMSO-d6): δ 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 12N hydrochloric acid (65 mL) to produce 3N HCl in methanol. The reaction flask was sealed with a septum (also called a septum wall) 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 to fine grains and washed with methanol (750 mL) until the filtrate approached neutral pH. The solid was dried at 80 °C overnight under reduced pressure to give ethylenediamine mucate (12.96 g, 91% yield) as a white solid. 1 1H NMR (500 MHz, DMSO-d6): δ 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): δ 174.79, 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 equivalent) 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, dimethylsuberimidate (DMS) (74.4 mg, 0.3 mmol, 1 equivalent) was added to the mixture, and the reaction was stirred at 25 °C for 16 hours overnight. The reaction 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 nanopure water using a 15 mL Amicon Ultra 3kD spin filter until the pH of the filtrate was neutral. The polymer solution was concentrated to 3 - 4 mL, filtered through a pre-weighed 20 mL glass vial through a 0.2 um PVDF syringe filter, and lyophilized to give the cationic mucic acid polymer as a white solid (29.2 mg, 16% yield), which was stored under argon at -20 °C. 1 H 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 C 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 freezer at -20 °C, they were equilibrated at room temperature for 1 hour. cMAP (50 mg, 0.009 mmol, 2 equivalents) and di-SPA-PEG-3.5kD (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, and 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. Nanopure water (20 mL) was added to dilute the 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, 45% yield). 1 1H 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 13C 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, 27.89, 26.66. The same procedure was used to synthesize the cMAP-PEG5k copolymer using 5kD di-SVA-PEG (succinimidyl valerate) for dialysis using a 15 kD SpectraPor 7 MWCO membrane (Spectrum Labs).

[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 spin filter to separate cMAP-PEG3.4K-cMAP, which was filtered through a 0.2 um PVDF membrane and freeze-dried to a white powder (10.6 mg, yield 16%). cMAP-PEG5k-cMAP was separated in the same manner.

[0123] Example 2.3. Synthesis of mPEG-cMAP-PEGm triblock polymer (Figure 3). After the starting materials were taken out from a -20 °C freezer (also referred to as a refrigerator), they were equilibrated to room temperature for 1 hour. cMAP (40 mg, 0.006 mmol, 2 equivalents) and mPEG 5k-SVA (85.7 mg, 0.017 mmol, 3 equivalents) 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 (4 mL) was added using a needle and syringe 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 freeze-dried to a white powder (11.3 mg, yield 9%). 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, 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. In the case of 2kD PEG, the triblock polymer was separated using a 10 kD MWCO centrifugal spin filter.

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

[0126] To an oven-dried 2-necked 10 mL round-bottom flask containing a dry stirrer bar (also called a drying stir bar), 3-carboxyl-5-nitrophenylboronic acid (200 mg, 0.95 mmol, 1 equiv) was added. The flask was evacuated with argon and sealed with a rubber septum. Anhydrous tetrahydrofuran was added along with a BHT inhibitor (5 mL), followed by anhydrous DMF (14.7 μL, 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 μL, 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 hours, allowing volatile substances to escape through the argon vent. The solvent and DMF were removed via a rotary evaporator and then removed under vacuum for 2 days under dark conditions, giving 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 drying stir bar, 3-acylchloride-5-nitrophenylboronic acid (27.5 mg, 0.12 mmol, 2 eq) was added. The flask was sealed with a rubber septum, purged with argon, 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 eq) in an oven-dried 10 mL round-bottom flask purged with argon was dissolved in anhydrous dichloromethane (5 mL) and diisopropylethylamine (DIPEA, 20.9 uL, 0.12 mmol, 2 eq), and slowly added to the boronic acid solution. The reaction flask was left in the ice-water bath and slowly warmed to room temperature, and the reactants were stirred overnight in the dark. The solvent and DIPEA were removed via a rotary evaporator and then removed under vacuum for 2 days in the dark. The solid residue was reconstituted in 0.5 N 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 filter until the pH became constant. The polymer solution was concentrated to 3 - 4 mL, filtered through a pre-weighed 20 mL glass vial through a 0.2 μm PVDF syringe filter, and lyophilized to dryness to give 5-nitrophenylboronic acid-PEGm (219.2 mg, 70% yield) as a fluffy white solid. 1 1H 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 11B 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 1100 HPLC equipped with a binary pump and an injector was connected to a Tosoh TSKgel G3000PWXL-CP size exclusion column equipped with Wyatt DAWN HELEOS light scattering and Wyatt Optilab Rex refractive index detection. The lyophilized polymer was dissolved in 0.1 M NaNO3 at six different concentrations and directly injected into the 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 analyzed using ASTRA V software.

[0129] Example 3.2. TNBSA assay of cMAP for primary amines. The instructions of Thermo Scientific for 5% w / v 2,4,6-trinitrobenzenesulfonic acid in a methanol stock solution were followed by the modifications as described below. Briefly, cMAP and glycine were each dissolved in the 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 were added in triplicate to a 96-well plate and shaken briefly. Absorbance was read at a wavelength of 335 nm using 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 the cMAP polymer to encapsulate (also referred to as enclose) siRNA was analyzed using two methods: the gel retardation assay and the RiboGreen assay. For the gel retardation assay, increasing volumes of 0.5 mg / mL polymer were mixed with 1 μL of 1 mg / mL siRNA at (+ / -) charge ratios of 0, 0.5, 1, 1.5, 2, 2.5, 3, and 5 for a total volume of 15 μL in water. The mixtures were vortexed (also referred to as stirred) briefly, centrifuged to bring down (also referred to as precipitate), and left at room temperature for 15 minutes. 3 μL of 6× DNA loading dye was added to each mixture, which was then loaded onto a 1% agarose gel and run at 95 V for 1.5 hours in 0.5× TBE buffer. The gel was imaged using a UVP BioDoc-It Imaging System.

[0131] The RiboGreen assay was performed in a similar manner to the gel retardation assay, except for the use of increasing volumes of 0.1 mg / mL polymer and 1 μL of 0.1 mg / mL siRNA in water for a total volume of 100 μL in a 96-well plate. For each of these mixtures, a working solution of 100 μL of Quant-iT RiboGreen RNA reagent was prepared and added according to the kit protocol. The plates were shaken briefly, incubated in the dark at room temperature for 5 minutes, and the fluorescence intensity was read using a Tecan infinite M200 plate reader at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. The measurements were performed in triplicate.

[0132] Example 4. Nanoparticle Formulations and Characterization.

[0133] Example 4.1. Nanoparticle formulations. cMAP NPs were first formulated by mixing cMAP vicinal diol pairs 5-nPBA-PEGm in a 1:1 molar ratio (1 mg of cMAP to 22 mg of 5-nPBA-mPEG) in 10 mM phosphate buffer pH 7.4, vortexing briefly, centrifuging down, and leaving the mixture at room temperature for 15 minutes. Then, 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 varied from 3:1 to 1:1 of the polymer to siRNA charge ratio and the concentration of siRNA was varied to a maximum of 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 solution (PBS) was added to achieve a 1× PBS solution with a final concentration of siRNA 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.

[0134] Example 4.2. Size and zeta potential of nanoparticles. NP size was determined using two different methods: dynamic light scattering (DLS) and cryogenic transmission electron microscopy (cryoTEM). DLS was performed with the Zeta-PALS from Brookhaven Instruments Corporation (BIC) and BIC Particle Sizing Software. The particles were diluted in siRNA at a concentration of 0.2 mg / mL according to 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] After blotting 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, cryoTEM imaging was performed on the particles in the frozen solution on an R2 / 2 Quantifoil grid in liquid ethane. Images were collected with 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 using ImageJ software to measure the NP diameter.

[0136] The surface charge, or zeta potential, of the NPs was measured using the same Zeta-PALS as that used for DLS with the Brookhaven aqueous electrode assembly. 10 μL of the particle preparation was mixed with 1.5 mL of either 10 mM phosphate buffer (pH 7.4) or 1 mM potassium chloride (pH 5.5) in a cuvette. The electrode was inserted into the cuvette, and the zeta potential was measured with the target residue of 0.012 using the 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 component was removed as described above. 50 μL of the filtrate (containing the excess component) from a 30 kD MWCO spin filter was injected into an Agilent 1200 HPLC equipped with a Phenomenex Gemini C18 reverse-phase column and a quaternary pump and 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 obtained by taking the retentate (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) magnetic beads (Bioclone Inc) were used to degrade and sequester siRNA and directly quantitate it. 50 μL of liquid containing cMAP was injected into the GPC set-up described above and the amount of polymer bound to NPs was directly determined using the refractive index signal compared to a cMAP standard curve. For cMAP-PEG copolymers and mPEG-cMAP-PEGm, 50 μL of the formulation was injected into the GPC set-up described above. The refractive index signal corresponding to polymer not bound to NPs was recorded and compared to a standard 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 NPs.

[0140] Example 5. In vivo mouse pharmacokinetic (PK) study.

[0141] All animal studies were conducted at the Institutional Animal Research Center at Caltech. Approved by the Institutional Animal Care and Use Committee (also referred to as the Animal Experiment Committee). 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 hind limbs of Balst / c mice (Taconic and Jackson Labs) were shaved in a red top clot activator (also referred to as a red top clot activator) containing a Sarstedt Microvette CB300 capillary tube for blood collection from the prone vein. Blood was collected at up to 6 points per mouse at various time points starting 2 minutes after NP injection. The tubes were centrifuged at 14,000 xg for 15 minutes at 4 °C, and the serum in the upper part of the tube was used for the analysis of Cy3 fluorescence by comparing it with the standard curve of the NP formulation 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 injected formulation. The data points are from 3 mice per formulation.

[0142] Example 6. Results and Discussion

[0143] Example 6.1. cMAP Synthesis, NMR Characterization, and Determination of 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 cMAP substance was generated by the condensation reaction of ethylene diamine mucate and dimethyl suberimidate (DMS). 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 included) (Tables 3-4) enabled the assignment of all resonances to various carbon and hydrogen environments in the polymer. Since these functionalities were utilized in subsequent reactions with functionalized PEG to form cMAP-PEG copolymers or mPEG-cMAP-PEGm triblock polymers, it was important to identify the terminal group composition of cMAP.

[0147] [Table 3]

[0148] [Table 4]

[0149] The cMAP terminal groups include 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- 13Supported by (not shown) CHSQC NMR measurements. 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 1 observable as a triplet (also called a triplet of triplets) at 2.25 ppm in the 1H NMR spectrum (Figure 11). The amine - terminal groups derived from ethylenediamine of mucic acid are 1 not directly observable by 1H NMR. However, analysis of the monomer's NMR spectrum and the HMBC NMR spectrum of cMAP allowed an assignment to be made from the methylene group adjacent to the amine - functional group as a triplet at 2.85 ppm. Furthermore, the TNBSA assay for primary amines was positive, and thus it was confirmed that cMAP has terminal primary amines as terminal groups. Finally, there was sufficient hydrolysis of the methyl ester or a small amount of carboxylic acid as a terminal group present as an impurity in the starting DMS. The methylene group adjacent to the carboxylic acid is 1 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), 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 end groups in PEG such as succinimidyl propionate (SPA) or succinimidyl valerate (SVA). cMAP was reacted with di-SPA-PEG or mPEG-SVA resulting copolymers or triblock polymers having PEG lengths of 2, 3.4, or 5 kD, respectively.

[0151] Since a significant amount of diamine-terminated polymer chains were present in the cMAP mixture, reaction with di-SPA-PEG (Figure 2) resulted in cMAP-PEG copolymers having a broad size distribution (from diblock cMAP-PEG copolymers just slightly larger than 10 kD to cMAP-PEG-cMAP triblock polymers terminated with methyl esters or carboxylic acids in cMAPs, to polymers over 100 kD in length; the size distributions reported in Table 6-7 are from polymer yields obtained by fractionating the crude polymer through successively smaller molecular weight cut-off centrifugal spin filters).

[0152] [Table 6]

[0153] [Table 7]

[0154] Such large molecular weight polymers can impose substantial toxicity in vivo, so to synthesize well-defined polymers of appropriate length, cMAP-PEG-cMAP triblock polymer species were separated from the copolymers using this fractionation method. Other triblock polymers of 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 also exist and are separated from the desired triblock by fractionation.

[0156] Example 6.2. Molecular weight of the polymer by GPC. Gel permeation chromatography was used to characterize the molecular weight of cMAP. 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. For the concentration of cMAP, dn / dc, the differential refractive index was determined (Table 8) and used to measure the molecular weight. The average molecular weight of 9 batches of cMAP was around 6 kD and had a polydispersity index (PDI) of less than 1.1 (Table 8). The results from individual batches can be found in Table 9.

[0157] [Table 8]

[0158] [Table 9]

[0159] Using the same method, the 5k cMAP-PEG copolymer had a larger size distribution with a PDI of 1.4 and Mw of 42kD and Mn of 29kD (Table 8). The 5k mPEG-cMAP-PEGm triblock was approximately 21kD and had a PDI of less than 1.1 (Table 8). Further, the results of the 3.4kD PEGcMAP-PEG copolymer and 2kD PEG mPEG-cMAP-PEGm triblock, as well as the cMAP-PEG-cMAP triblock derived from the fraction of the cMAP-PEG copolymer, are all reported in Table 10.

[0160]

Table 10

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

[0162] Example 7. Nanoparticle Formulations and Properties.

[0163] Example 7.1. Formulations. 5-Nitrophenylboronic acid-PEGm (5-nPBA-PEGm) contains a boronic acid group that enables one end of this 5 kD PEG to bind to adjacent diol groups on the mucic acid of cMAP at pH above 6.8, resulting in the steric stabilization of siRNA containing NPs as shown in Figure 15. Various NP formulations using cMAP, cMAP-PEG copolymers, and mPEG-cMAP-PEGm triblock polymers with or without additional 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).

[0164] In contrast to cMAP alone, the cMAP-PEG copolymers and mPEG-cMAP-PEGm triblock polymers were able to form stable particles without additional 5-nPBA-PEGm. However, the pure cMAP-PEG-cMAP triblock polymer separated from the cMAP-PEG copolymer was unable to form stable siRNA-containing NPs without the addition of 5-nPBA-PEGm, presumably because it did not contain sufficient PEG for adequate shielding (also called masking) and steric stabilization of the NPs (Table 11 and Figures 18 - 20).

[0165] [Table 11]

[0166] The cMAP-PEG copolymer and the mPEG-cMAP-PEGm triblock polymer formed stable NPs in PBS. To test whether the additional 5-nPBA-PEGm provides even greater steric stability to the NPs when tested in vivo, formulations with additional 5-nPBA-PEGm were 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 were 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: 10 mM phosphate buffered at pH 7.4 when 5-nPBA-PEGm binds to the adjacent diol on cMAP; and 1 mM KCl at pH 5.5 when 5-nPBA-PEGm dissociates from the diol of mucic acid. The cMAP-siRNA NPs with 5-nPBA-mPEG had a slightly negative zeta potential of -3 mV in phosphate buffer at pH 7.4 when 5-nPBA-mPEG was present on the NPs. However, when these NPs were placed in 1 mM KCl at pH 5.5, the zeta potential was approximately +1 mV. These results were consistent with the boronic acid binding to the diol with 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. A similar effect was observed with cMAP-PEG copolymers and mPEG-cMAP-PEGm triblock polymers with and without 5-nPBA-PEGm (Table 12).

[0170] Example 7.4. Nanoparticle Stoichiometry. The amounts of cMAP and copolymer bound to NPs are shown in Table 13. For all three polymers (cMAP, cMAP-PEG copolymer, and mPEG-cMAP-PEGm triblock polymer), 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 found to bind to the NPs for cMAP-PEG copolymer and mPEG-cMAP-PEGm triblock polymer NP formulations. When the particles were formulated at a 3+ / - charge ratio, taking into account the excess cationic polymer present and that the effective NP charge ratio is 1+ / - , this meant that there was less than one PEG per diol present on the NPs. As shown above in the data regarding siRNA encapsulation, substantially all of the siRNA was encapsulated in the NPs (Figure 12).

[0171] [Table 13]

[0172] Example 8. In Vivo Pharmacokinetic Studies in Mice.

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

[0174] NP composed of cMAP polymer and siRNA mixed at a 3+ / - charge ratio and stabilized with 5-nPBA-PEGm was tested because this NP formulation was 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). Since CALAA-01 used an inclusion complex for the interaction between CDP and adamantane-PEG (AD-PEG), there was a possibility that AD-PEG detached from the NP during circulation and the NP lost its stability. Others synthesized AD2-PEG and showed that this compound had a higher ability to stabilize CDP-based NPs than AD-PEG in the original CALAA-01 formulation. This is due to the enhanced binding of two adamantanes per PEG (to two CDs), resulting in a further steric stabilization during circulation (Figure 24A). Together with this cMAP boronic acid system, the interaction between the PEG compound and the polymer ends successfully through the boronic acid ester formed from the boronic acid and diol on the polymer, with ca. 30% of the PEG bound to the NP. Since only 1 / 3 of the cMAP used to formulate the NP bound to the particles (Table 3), this was roughly equivalent to one PEG per diol. The boronic acid-diol interaction was unexpectedly stronger than the inclusion complex between adamantine and cyclodextrin. As a result, 5-nPBA-PEGm remained attached to cMAP longer than AD-PEG to CDP and was able to bring about improvements in terms of even higher steric stability and circulation time.

[0175] 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 PEG loops that shield the NP core. Additional 5-nPBA-mPEG can be used for further stabilization of the NPs. The zeta potential switching from negative at pH 7.4 to positive at pH 5.5 indicated that 5-nPBA-PEGm was able to interact with the cMAP-PEG copolymer in the NP formulation by measuring the filtered amount of excess PEG in addition to the 20% PEG bound to the particles. NPs formulated with the cMAP-PEG copolymer did not provide a significantly longer circulation time beyond the cMAP:5-nPBA-PEGm-based NPs regardless of whether 5-nPBA-PEGm was added (Figure 24B).

[0176] NPs formed using the mPEG-cMAP-PEGm triblock form 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 (the other formulations were below the limit of detection by 60 minutes). Similar results were observed with this formulation in nude mice (Figure 25). These 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. Addition of 5-nPBA-PEGm to the triblock polymer-siRNA NPs did not result in an improvement in circulation time (Figure 24B). Furthermore, an 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 by spin filtering the formulation through a 30kD MWCO membrane (Figure 24C). It was difficult to remove all of the excess polymer using this purification method. These results suggested that polymers not contained 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 addition of the 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 had two PEGs per cMAP unit and might have been 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 in the CDP-adamantane system. Some of the 5-nPBA-PEGm had to remain on the NPs during circulation.

[0178] To provide further evidence that the NPs remained 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 by a fluorophore-labeled siRNA with a Typhoon imager. Results from these experiments showed that the siRNA and the fluorescently labeled siRNA remained intact in the NPs while circulating in vivo.

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[0180] As will be understood by those skilled in the art, numerous modifications and variations of the present disclosure are possible in light of these teachings, and all of them are considered herein. For example, 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 and the cited prior art documents that complement the features of the present disclosure. Similarly, any of the substances, features, or articles described 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 herein by reference in their entirety for all purposes. In addition to the references already mentioned, the present disclosure relates to the subject matter of 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 of chemical substances, applications, and methods of making and using the block copolymers described therein.

Claims

1. One or more of the following structural units of formula (I), formula (II), or formula (III): 【Chemical 1】 comprising; 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, a polymer comprising alternating charged and uncharged segments.

2. The polymer of claim 1, wherein A includes polyethylene glycol and a suitable linking group.

3. The polymer of claim 1, wherein the polyalkylene glycol has a nominal weight in the range of about 500 daltons to about 50,000 daltons.

4. The polymer of claim 1, wherein B is a cationic charged segment containing at least one polyhydroxy sugar linkage including at least a pair of adjacent diols.

5. The polymer of claim 1, wherein B includes at least one repeating subunit having the structure of formula (IV): 【Chemical 2】 including.

6. The polymer of claim 1, wherein B further includes at least one repeating subunit having the structure of formula (V): 【Chemical Formula 3】 including, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

7. The polymer of claim 1, wherein B includes at least one repeating subunit containing cMAP, and the subunit structure is represented by formula (VI): 【Chemical 4】 represented as; wherein, m is, independently of each occurrence, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 4 - 6; and n is, independently of each occurrence, 1, 2, 3, 4, or 5. the polymer of claim 1.

8. Described by the structure of formula (VII): 【Chemical Formula 5】 wherein, chain A is and 【Chemical Formula 6】 chain B is and 【Chemical Formula 7】 cMAP is and 【Chemical 8】 p and q are sufficient to provide a number average molecular weight in the range of about 500 Da to about 50,000 Da, independently, for the subunits containing cMAP and PEG; m is, independently of each occurrence, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 4 - 6; n and r are, independently of each occurrence, 0, 1, 2, 3, 4, or 5; and

9. Described by the structure of formula (VII): X 1 and X 2 is, independently of their presence, C 1-6 alkyl, optionally substituted by -OH, -COOH, -C(=O)O(alkyl), -C(=O)O(aryl), -NH 2 , -NH(alkyl), -N(alkyl) 2 The polymer according to claim 1, which is substituted by or is a salt or protected analog thereof.

9. Described by the structure of formula (VII): 【Chemical Formula 9】 wherein, cMAP is and 【Chemical 10】 chain B is and 【Chemical 11】 chain C is and 【Chemical 12】 the terminal group D is: and 【Chemical 13】 is p and q are each independently sufficient to provide a number average molecular weight in the range of from about 500 Da to about 50,000 Da for the sub-units containing cMAP and PEG; m is each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 4 - 6 if possible; n and r are each independently 0, 1, 2, 3, 4, or 5; z is equal to 1 or greater than it and at most 10; and X 2 is, independently of their presence, C 1-6 alkyl, optionally substituted by -OH, -COOH, -C(=O)O(alkyl), -C(=O)O(aryl), -NH 2 , -NH(alkyl), -N(alkyl) 2 or a salt or protected analog thereof; and X 3 is -NH 2 , -COOH, -C(=O)O(alkyl), or a salt or protected analog thereof, the polymer of claim 1.

10. The structure of formula (IX): 【Chemical Formula 14】 as described by wherein the terminal group D is: 【Chemical Formula 15】 ; cMAP is 【Chemical 16】 ; the chain C is 【Chemical 17】 ; p and q are each independently sufficient to provide a number average molecular weight in the range of from about 500 Da to about 50,000 Da for the sub-units containing cMAP and PEG; m is each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 4 - 6 if possible; n and r are each independently 0, 1, 2, 3, 4, or 5; z is equal to 1 or greater than it and at most 10, and X 3 and X 4 are, independently of their presence, -NH 2 , -COOH, -C(=O)O(alkyl), or salts or protected analogs thereof the polymer of claim 1.

11. The polymer of claim 6, wherein m is 4, 5, or 6.

12. The polymer of claim 7, wherein n is 1.

13. The polymer of claim 7, wherein r is 2, 3, or 4.

14. The polymer of claim 8, wherein p is sufficient to provide a number average molecular weight for the sub-unit containing cMAP in the range of from about 5 kDa to about 15 kDa, from about 6 kDa to about 14 kDa, 7 kDa to about 13 kDa, from about 8 kDa to about 12 kDa, 9 kDa to about 11 kDa, or about 10 kDa.

15. The polymer of claim 8, wherein q is sufficient to provide a number average molecular weight for the sub-unit containing PEG in the range of from about 500 Da to about 50 kDa, from about 1 kDa to about 40 kDa, 5 kDa to about 30 kDa, or from about 5 kDa to about 20 kDa.

16. The polymer of claim 1 and a second boronic acid-containing polymer comprising the structure of formula (X) 【Chemical 18】 wherein n is 0, 1, 2, 3, or 4, preferably 1 if possible; The polymer and the second boronic acid-containing polymer are reversibly connected to each other by a borate condensation linkage between a boronic acid moiety of formula (IX) and at least one pair of adjacent diols of polyhydroxy linkage of formula (I), (II), or (III), and X 3 is at the distal end of this connection; R A is nitro; s is 20 - 1200; L is a linking group between the phenyl ring and the polyethylene oxide linkage; and

17. 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 and is a polymer complex, or a salt or protected analog thereof.

18. 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)-, the polymer composite of claim 16. ​ The polymer complex of claim 17, wherein L is -NH-C(=O)-, -C(=O)-NH-, -OC(=O)-, or -C(=O)-O-.

19. Nanoparticles comprising the polymer of claim 1.

20. Nanoparticles comprising the polymer complex of claim 16.

21. The nanoparticles of claim 19 or 20, wherein the nanoparticles are substantially spherical and have a cross-sectional dimension in the range of about 20 nm to about 300 nm.

22. A plurality of nanoparticles of claim 19 or 20.

23. The plurality of nanoparticles of claim 20, wherein the plurality of nanoparticles are substantially monodisperse and exhibit a standard deviation in the cross-sectional dimension between nanoparticles of less than 20%, 30%, 40%, 50%, or 60% as measured by cryogenic transmission electron microscopy (cryo-TEM).

24. Nanoparticles comprising the polymer of claim 1 or the polymer complex of claim 16, further comprising an encapsulated biological substance.

25. The nanoparticles of claim 24, wherein the biological substance is covalently bound to the polymer or polymer complex.

26. The nanoparticles of claim 24, wherein the biological substance is a polynucleotide or a small molecule therapeutic agent.

27. The nanoparticles of claim 24, wherein the biological substance is a polynucleotide that is an RNA molecule.

28. The nanoparticles of claim 27, wherein the RNA molecule is an siRNA molecule.

29. The nanoparticles of claim 20, further conjugated to a target-directed ligand, wherein the conjugation occurs through a condensation linkage between the distal end of the boronic acid-containing polymer and the target-directed ligand.

30. The nanoparticles of claim 29, wherein a single target-directed ligand is conjugated to each polymer.

31. The nanoparticles of claim 29, wherein a plurality of target-directed ligands are conjugated to each polymer.

32. A pharmaceutical composition comprising a biologically active agent, the polymer of claim 1 or the polymer complex of claim 16, and a pharmaceutically acceptable carrier or excipient.

33. A pharmaceutical composition comprising a biologically active agent, the nanoparticles of claim 19, and a pharmaceutically acceptable carrier or excipient.

34. A method comprising administering the nanoparticles of claim 24 to a patient, wherein the bioavailability of the biological substance is improved compared to administration of the biological substance itself.

35. A method for preparing the polymer of claim 1, 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.

36. The method of claim 35, wherein at least one polyhydroxy linkage comprises mucic acid and at least one linking group is an amide.

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