Patent Application
20240366784
Systemic drug administration is often hampered by biodistribution challenges, including rapid renal clearance, low or no tissue distribution specificity, and poor aqueous solubility of many small molecule drugs. Local delivery strategies mitigate these issues by minimizing drug exposure in off-target tissues and extending the window of therapeutically active drug concentrations at the site of action. Hydrogels therefore continue to evolve as tailorable platforms for therapeutic delivery that can support tissue regeneration, alter cell or tissue responses, or present appropriate therapeutic cargo. Such cargo may include pharmacologic drugs, exosomes, or cells intended to support processes of immune modulation and tissue repair. In particular, small molecule drugs are attractive payloads due to their ease of synthesis and ability for optimization; however, their local delivery from hydrogels is often challenging due to their rapid diffusive release and non-specific cellular biodistribution.
Thus, there is a need in the art for hydrogel compositions and methods of use thereof for local and sustained release of therapeutic cargo (e.g., small molecules). The disclosure addresses this need.
In one aspect, the disclosure provides a hydrogel composition. In certain embodiments, the hydrogel composition comprises a nanoparticle comprising a plurality of macrocycles, wherein each of the macrocycles are covalently linked, either directly or indirectly, by at least one nanoparticle linker. In certain embodiments, the hydrogel composition comprises a functionalized polymer comprising a hydrophilic polymer core substituted with a plurality of hydrophobic substituents, wherein at least a portion of the hydrophobic substituents are independently non-covalently associated with one of the macrocycles of the nanoparticle. In certain embodiments, the hydrogel composition comprises at least one therapeutic agent, wherein the at least one therapeutic agent is non-covalently associated with a macrocycle of the nanoparticle.
In another aspect, the disclosure provides a method for treating, preventing, and/or ameliorating an inflammatory disease or disorder in a subject, the method comprising administering to the subject the hydrogel composition of the disclosure.
In another aspect, the disclosure provides a method for delivering a therapeutic agent to a macrophage in a subject, the method comprising administering to the subject the hydrogel composition of the disclosure.
In another aspect, the disclosure provides a method for reducing or inhibiting pro-inflammatory (M1-like) behavior of a macrophage in a subject, the method comprising administering to the subject the hydrogel composition of the disclosure.
In another aspect, the disclosure provides a method for promoting pro-healing (M2-like) behavior of a macrophage in a subject, the method comprising administering to the subject the hydrogel composition of the disclosure.
The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments of the present application.
Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.
Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.
In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. The statement “at least one of A and B” or “at least one of A or B” has the same meaning as “A, B, or A and B.” In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.
In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.
Hydrogels are conventionally formed ex vivo via covalent crosslinking, necessitating invasive implantation of the resulting solid material. In contrast, injectable hydrogels may be formed through varying methods to enable minimally invasive delivery, including for the local presentation of encapsulated therapeutic cargo. One such approach is in situ hydrogel formation after the injection of liquid precursors, accomplished by mechanisms such as radical polymerization, addition crosslinking, or environmental stimuli. However, the kinetics of hydrogel formation often complicate the delivery process. Rapid gelation risks clogging the delivery device, whereas slow gelation results in material dispersion throughout the tissue after injection.
As an alternative to in situ hydrogel formation, some approaches capitalize on the use of dynamic crosslinks, such as dynamic covalent chemistries, engineered biomolecular interactions, or guest-host chemistries. Dynamic crosslinking allows hydrogels to be formed ex vivo and then extruded during injection by shear-thinning processes, as the dynamic bonds temporarily break in response to shear stress and rapidly bind again. These dynamic material behaviors ultimately allow for injectable delivery that mitigates the risks for invasive implantation, while also increasing material retention at the target site, including precious therapeutic cargo.
As an extension of these self-assembling hydrogel systems, hydrogels may also be formed from discrete micro- or nano-structural units instead of polymeric building blocks alone. Such non-homogenous structures uniquely allow for the structural subunits to take on discrete functions and the unique behavior of the bulk hydrogel formed. For example, granular hydrogels have emerged as a unique class of injectable materials, assembled through inter-particle crosslinking reactions or particle jamming. Discrete microgel components within these systems may have tunable behavior to control drug delivery. Other systems have leveraged polymer-nanoparticle interactions to aid in the self-assembly of injectable hydrogels. The inclusion of discrete nanoparticles within these structures has been leveraged to endow polymer-nanoparticle hydrogels with distinct therapeutic, diagnostic, and physical properties. While such polymer-nanoparticle hydrogels open new avenues for local therapeutic delivery, their use as a means to provide controlled release of nanoparticles to naturally target highly phagocytic innate immune cells remains unexplored.
Such local and cell-specific delivery is particularly important for immune modulation. Specifically, the systemic administration of immunosuppressive drugs places patients at an increased risk of infection, which has hampered their clinical acceptance. Despite these challenges, there remains a growing interest in the use of immunomodulatory drugs to combat inflammatory disease and promote tissue healing.
Macrophages (MF) are crucial regulators of the tissue-immune microenvironment, and unsolicited MF-derived inflammation commonly underlies failed tissue healing processes and an array of chronic diseases, making them an attractive therapeutic target. While these cells exist across an array of phenotypes, they are often broadly characterized as pro-inflammatory (‘M1-like’) or pro-healing (‘M2-like’), particularly in the context of tissue repair. MF phenotypes are highly plastic, subject to modulation by external stimuli such as pharmaceuticals that arrest inflammatory behavior and promote a reparative M2-like phenotype for injury resolution. While immunosuppressants are widely available and used in clinical practice, few M2-polarizing drugs have been reported and necessitate high dosing. Immunoregenerative medicine therefore remains limited by a lack of knowledge regarding which drugs or drug classes can potently promote reparatory MF phenotypes.
Small molecule drugs may be of use towards the goal of modulating MF phenotype and are amenable to delivery by guest-host interactions. Guest-host interactions are a subset of supramolecular associations, characterized by the transient complexation of a macrocyclic host with a small molecule guest. In the case of many macrocycles, and β-cyclodextrin (CD) in particular, complexation is driven by hydrophobic interactions that enable the inclusion of a wide variety of guest molecules. It is therefore a versatile and common excipient in pharmaceutical formulations on market, used to enhance drug solubility and bioavailability. CD is also widely used in biomaterial applications, including in molecular imaging probes, surface coatings, and polymeric drug carriers for affinity-based delivery.
The development of cyclodextrin nanoparticles (CDNPs) that possess a high drug loading capacity and inherent capacity for MF-targeted therapy have been previously described in the literature (U.S. Patent Publication No. US2020/0338011, which is hereby incorporated herein by reference in its entirety). The CDNPs are formed only from CD crosslinked by L-lysine, creating a dense network of the host macrocycle that perpetuates a high drug loading capacity. Due to the saccharide-based structure of CD, it is readily internalized by MF, likely via recognition by cell surface receptors that include scavenger receptor A1 (SR-A1) and mannose receptor (MRC1). The drug loading capacity and MF avidity have been leveraged for systemically administered MF-targeted cancer immunotherapy, and similar MF-targeting strategies are highly effective in a range of applications. However, the delivery of these and other therapeutic nanocarriers is typically accomplished via systemic administration; their local and sustained delivery has not previously been reported.
In one aspect, the disclosure relates to the development of an injectable hydrogel platform for the local administration of small molecule drugs that uniquely leverages guest-host interactions for injectable hydrogel assembly and cell-targeted nanoparticle therapy to directly address the need for location-specific modulation of MF behavior. Through supramolecular assembly by guest-host interactions, CDNPs were crosslinked with adamantane-modified hyaluronic acid (Ad-HA) to yield an injectable hydrogel. The same guest-host interactions also serve as a mechanism to retain immunomodulatory therapeutics. The drug of choice was selected by scrutiny of a targeted library of 45 small molecules drugs that spanned a variety of drug classes with reported immunomodulatory capacity. Through a two-step drug screening process and subsequent in-depth transcriptional analysis of both human and murine MF, celastrol was identified as a potent modulator of MF phenotype that suppresses M1-like and promotes M2-like behavior. Celastrol was included within the nanoparticle core by guest-host interaction prior to hydrogel formation. The resulting therapeutic hydrogels degraded by surface erosion over the course of greater than one month, continually releasing drug-loaded nanoparticles that were uptaken by MF and arrested their pro-inflammatory response. The approach represents a promising strategy to achieve functional re-orientation of the local immune microenvironment by the continual release of drug-loaded nanoparticles from the hierarchical hydrogels formed.
The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range.
The term “alkenyl” as used herein refers to straight and branched chain and cyclic alkyl groups as defined herein, except that at least one double bond exists between two carbon atoms. Thus, alkenyl groups have from 2 to 40 carbon atoms, or 2 to about 20 carbon atoms, or 2 to 12 carbon atoms or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to vinyl, —CH═C═CH2, —CH═CH(CH3), —CH═C(CH3)2, —C(CH3)═CH2, —C(CH3)═CH(CH3), —C(CH2CH3)═CH2, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl among others.
The term “alkoxy” as used herein refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can include about 1 to about 12, about 1 to about 20, or about 1 to about 40 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms. For example, an allyloxy group or a methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structure are substituted therewith.
The term “alkynyl” as used herein refers to straight and branched chain alkyl groups, except that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have from 2 to 40 carbon atoms, 2 to about 20 carbon atoms, or from 2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to —C═CH, —C≡C(CH3), —C═C(CH2CH3), —CH2C═CH, —CH2C≡C(CH3), and —CH2C≡C(CH2CH3) among others.
The term “alkyl” as used herein refers to straight chain and branched alkyl groups and cycloalkyl groups having from 1 to 40 carbon atoms, 1 to about 20 carbon atoms, 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms. Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. As used herein, the term “alkyl” encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.
A disease or disorder is “ameliorated” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.
The term “amine” as used herein refers to primary, secondary, and tertiary amines having, e.g., the formula N(group)3 wherein each group can independently be H or non-H, such as alkyl, aryl, and the like. Amines include but are not limited to R—NH2, for example, alkylamines, arylamines, alkylarylamines; R2NH wherein each R is independently selected, such as dialkylamines, diarylamines, aralkylamines, heterocyclylamines and the like; and R3N wherein each R is independently selected, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like. The term “amine” also includes ammonium ions as used herein.
The term “aryl” as used herein refers to cyclic aromatic hydrocarbon groups that do not contain heteroatoms in the ring. Thus aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, aryl groups contain about 6 to about 14 carbons in the ring portions of the groups. Aryl groups can be unsubstituted or substituted, as defined herein. Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, a phenyl group substituted at any one or more of 2-, 3-, 4-, 5-, or 6-positions of the phenyl ring, or a naphthyl group substituted at any one or more of 2- to 8-positions thereof. Use of the term “aryl” in combination with another term (e.g., iodide, chloride, boronic acid, and magnesium halide, inter alia), indicates that the aryl group is substituted at one or more positions with the substituent defined by the term used in the combination. For example, an aryl chloride indicates that the aryl is substituted with at least one chloride.
The term “cycloalkyl” as used herein refers to cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. Cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined herein. Representative substituted cycloalkyl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4-2,5- or 2,6-disubstituted cyclohexyl groups or mono-, di- or tri-substituted norbornyl or cycloheptyl groups, which can be substituted with, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. The term “cycloalkenyl” alone or in combination denotes a cyclic alkenyl group.
The term “cycloalkylene” or “cycloalkylenyl” as used herein refers to a bivalent saturated cycloalkyl radical
In certain embodiments, the term may be regarded as a product of removal of two hydrogen atoms from the corresponding cycloalkane (e.g., cyclobutyl) by removal of two hydrogen atoms from the same
different
carbon atoms.
A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.
As used herein, the term “derived from” refers to a compound or moiety that is structurally identical in most respects to the compound to which it refers. In some embodiments, the compound that the moiety is derived from was used as a reagent or intermediate in the synthesis of the compound that is substituted with the moiety. In some embodiments, the moiety only differs structurally from the compound it is derived from at the portion of the moiety that links to the remainder of the molecule that the moiety substitutes. As used herein, a “derivative” of a particular compound or moiety encompasses compounds and moieties that are derived from the particular compound. For example, 2-hydroxypropyl-α-cyclodextrin is derived from α-cyclodextrin.
The terms “halo,” “halogen,” or “halide” group, as used herein, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
The term “haloalkyl” group, as used herein, includes mono-halo alkyl groups, poly-halo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkyl include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, 1,3-dibromo-3,3-difluoropropyl, perfluorobutyl, and the like.
The term “heteroaryl” as used herein refers to aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S; for instance, heteroaryl rings can have 5 to about 8-12 ring members. A heteroaryl group is a variety of a heterocyclyl group that possesses an aromatic electronic structure. A heteroaryl group designated as a C2-heteroaryl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C4-heteroaryl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups can be unsubstituted, or can be substituted with groups as is discussed herein. Representative substituted heteroaryl groups can be substituted one or more times with groups such as those listed herein.
Additional examples of aryl and heteroaryl groups include but are not limited to phenyl, biphenyl, indenyl, naphthyl (1-naphthyl, 2-naphthyl), N-hydroxytetrazolyl, N-hydroxytriazolyl, N-hydroxyimidazolyl, anthracenyl (1-anthracenyl, 2-anthracenyl, 3-anthracenyl), thiophenyl (2-thienyl, 3-thienyl), furyl (2-furyl, 3-furyl), indolyl, oxadiazolyl, isoxazolyl, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydryl, acridinyl, thiazolyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl), imidazolyl (1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl), triazolyl (1,2,3-triazol-1-yl, 1,2,3-triazol-2-yl 1,2,3-triazol-4-yl, 1,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4-oxazolyl, 5-oxazolyl), thiazolyl (2-thiazolyl, 4-thiazolyl, 5-thiazolyl), pyridyl (2-pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl), pyrazinyl, pyridazinyl (3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl), quinolyl (2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6-quinolyl, 7-quinolyl, 8-quinolyl), isoquinolyl (1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5-isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), benzo[b]furanyl (2-benzo[b]furanyl, 3-benzo[b]furanyl, 4-benzo[b]furanyl, 5-benzo[b]furanyl, 6-benzo[b]furanyl, 7-benzo[b]furanyl), 2,3-dihydro-benzo[b]furanyl (2-(2,3-dihydro-benzo[b]furanyl), 3-(2,3-dihydro-benzo[b]furanyl), 4-(2,3-dihydro-benzo[b]furanyl), 5-(2,3-dihydro-benzo[b]furanyl), 6-(2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro-benzo[b]furanyl), benzo[b]thiophenyl (2-benzo[b]thiophenyl, 3-benzo[b]thiophenyl, 4-benzo[b]thiophenyl, 5-benzo[b]thiophenyl, 6-benzo[b]thiophenyl, 7-benzo[b]thiophenyl), 2,3-dihydro-benzo[b]thiophenyl, (2-(2,3-dihydro-benzo[b]thiophenyl), 3-(2,3-dihydro-benzo[b]thiophenyl), 4-(2,3-dihydro-benzo[b]thiophenyl), 5-(2,3-dihydro-benzo[b]thiophenyl), 6-(2,3-dihydro-benzo[b]thiophenyl), 7-(2,3-dihydro-benzo[b]thiophenyl), indolyl (1-indolyl, 2-indolyl, 3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl), indazole (1-indazolyl, 3-indazolyl, 4-indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl), benzimidazolyl (1-benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl, 6-benzimidazolyl, 7-benzimidazolyl, 8-benzimidazolyl), benzoxazolyl (1-benzoxazolyl, 2-benzoxazolyl), benzothiazolyl (1-benzothiazolyl, 2-benzothiazolyl, 4-benzothiazolyl, 5-benzothiazolyl, 6-benzothiazolyl, 7-benzothiazolyl), carbazolyl (1-carbazolyl, 2-carbazolyl, 3-carbazolyl, 4-carbazolyl), 5H-dibenz[b,f]azepine (5H-dibenz[b,f]azepin-1-yl, 5H-dibenz[b,f]azepine-2-yl, 5H-dibenz[b,f]azepine-3-yl, 5H-dibenz[b,f]azepine-4-yl, 5H-dibenz[b,f]azepine-5-yl), 10,11-dihydro-5H-dibenz[b,f]azepine (10,11-dihydro-5H-dibenz[b,f]azepine-1-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-2-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-3-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-4-yl, 10,11-dihydro-5H-dibenz[b,f]azepine-5-yl), and the like.
The term “heteroarylalkyl” as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined herein.
The term “heteroarylene” or “heteroarylenyl” as used herein refers to a bivalent heteroaryl radical (e.g., 2,4-pyridylene). In certain embodiments, the term may be regarded as a divalent radical formed by the removal of two hydrogen atoms from one or more rings of a heteroaryl moiety, wherein the hydrogen atoms may be removed from the same or different rings, preferably the same ring.
The term “heterocycloalkyl” as used herein refers to an aliphatic, partially unsaturated or fully saturated, 3- to 14-membered ring system, including single rings of 3 to 8 atoms and bi- and tricyclic ring systems where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. A heterocycloalkyl can include one to four heteroatoms independently selected from oxygen, nitrogen, and sulfur, wherein a nitrogen and sulfur heteroatom optionally can be oxidized and a nitrogen heteroatom can be optionally substituted. Representative heterocycloalkyl groups include, but are not limited, to the following exemplary groups: pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl. The term heterocycloalkyl group can also be a C2 heterocycloalkyl, C2-C3 heterocycloalkyl, C2-C4 heterocycloalkyl, C2-C5 heterocycloalkyl, C2-C6 heterocycloalkyl, C2-C7 heterocycloalkyl, C2-C8 heterocycloalkyl, C2-C9 heterocycloalkyl, C2-C10 heterocycloalkyl, C2-C11 heterocycloalkyl, and the like, up to and including a C2-145 heterocycloalkyl. For example, a C2 heterocycloalkyl comprises a group which has two carbon atoms and at least one heteroatom, including, but not limited to, aziridinyl, diazetidinyl, oxiranyl, thiiranyl, and the like. Alternatively, for example, a C5 heterocycloalkyl comprises a group which has five carbon atoms and at least one heteroatom, including, but not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, diazepanyl, and the like. It is understood that a heterocycloalkyl group may be bound either through a heteroatom in the ring, where chemically possible, or one of carbons comprising the heterocycloalkyl ring. The heterocycloalkyl group can be substituted or unsubstituted.
The term “heterocycloalkylene” or “heterocycloalkylenyl” as used herein refers to a bivalent saturated cycloalkyl radical
In certain embodiments, the term may be regarded as a product of removal of two hydrogen atoms from the corresponding heterocycloalkane (e.g., piperidine) by removal of two hydrogen atoms from the same
different
carbon atom(s) and/or heteroatom(s).
The term “independently selected from” as used herein refers to referenced groups being the same, different, or a mixture thereof, unless the context clearly indicates otherwise. Thus, under this definition, the phrase “X1, X2, and X3 are independently selected from noble gases” would include the scenario where, for example, X1, X2, and X3 are all the same, where X1, X2, and X3 are all different, where X1 and X2 are the same but X3 is different, and other analogous permutations.
The term “linker” as used herein refers to an organic moiety that connects two parts of a compound (e.g., two macrocycles). The linker can be, in non-limiting examples, a direct bond, a single atom (e.g., —O—), a peptide, or a substituted or unsubstituted alkylene or heteroalkylene moiety (e.g., polyethylene glycol). One skilled in the art would be apprised of the common linkers suitable for use herein and methods of preparation thereof.
As used herein, the term “macrocycle” refers to any compound or chemical group that is a cyclic group comprising a minimum of 12 ring members (e.g., 12 or more contiguous atoms that form a ring), wherein the cyclic group is capable of binding a compound (e.g., a therapeutic agent, e.g., an anticancer agent) by means of intermolecular forces that, under certain conditions, last greater than 1 second (e.g., greater than 2 seconds, 4 seconds, 10 seconds, 60 seconds 1 minute, 2 minutes, 5 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 1 day, 3 days, 1 week, 2 weeks, 1 month, 2 months, 6 months, 1 year, 2 years, 5 years, or 10 years). Example classes of host macrocycles include cyclodextrins, pillar[n]arenes, calix[n]arenes, and cucurbit[n]urils. Included in each of the foregoing classes are macrocycles derived from any members of that class through, for example, chemical derivatization. For example, “cyclodextrin” encompasses α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin, as well as any chemically derivatized versions of the same including, but not limited to, 2-hydroxypropyl-α-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin 2-hydroxypropyl-7-cyclodextrin, methyl-α-cyclodextrin, methyl-β-cyclodextrin, methyl-γ-cyclodextrin, a cyclodextrin sulfobutylether, a cyclodextrin thioether, a cyanoethylated cyclodextrin, a succinyl-cyclodextrin, or an aminated cyclodextrin.
The terms “patient,” “subject,” or “individual” are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In a non-limiting embodiment, the patient, subject or individual is a human.
The term “polymer” may include, according to some embodiments, any molecule comprising repeating structural units connected to each other, typically, by covalent chemical bonds. The term “polymer” may include, according to some embodiments, a homopolymer (which is a polymer derived from one monomer species), a copolymer (which is a polymer derived from two (or more) monomeric species) or a combination thereof. A polymer, as referred to herein, may include a mixture of polymers. A polymer, as referred to herein, may include linear and/branched polymers which consist of a single main chain with one or more polymeric side chains.
The term “substituted” as used herein in conjunction with a molecule or an organic group as defined herein refers to the state in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms. The term “functional group” or “substituent” as used herein refers to a group that can be or is substituted onto a molecule or onto an organic group. Examples of substituents or functional groups include, but are not limited to, a halogen (e.g., F, Cl, Br, and I); an oxygen atom in groups such as hydroxy groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, hydroxyamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other heteroatoms in various other groups. Non-limiting examples of substituents that can be bonded to a substituted carbon (or other) atom include F, Cl, Br, I, OR, OC(O)N(R)2, CN, NO, NO2, ONO2, azido, CF3, OCF3, R, O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2, (CH2)0-2N(R)C(O)R, (CH2)0-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(═NH)N(R)2, C(O)N(OR)R, and C(═NOR)R, wherein R can be hydrogen or a carbon-based moiety; for example, R can be hydrogen, (C1-C100) hydrocarbyl, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl; or wherein two R groups bonded to a nitrogen atom or to adjacent nitrogen atoms can together with the nitrogen atom or atoms form a heterocyclyl.
A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.
The terms “treat,” “treating” and “treatment,” as used herein, means reducing the frequency or severity with which symptoms of a disease or condition are experienced by a subject by virtue of administering an agent or compound to the subject.
In one aspect, the disclosure provides a hydrogel composition. In certain embodiments, the hydrogel composition comprises a nanoparticle comprising a plurality of macrocycles, wherein each of the macrocycles is independently covalently linked, either directly or indirectly, by at least one independently selected nanoparticle linker. In certain embodiments, the hydrogel composition comprises a functionalized polymer comprising a hydrophilic polymer core substituted with a plurality of independently selected hydrophobic substituents, wherein at least a portion of the hydrophobic substituents are independently non-covalently associated with one of the macrocycles of the nanoparticle. In certain embodiments, the hydrogel composition comprises at least one therapeutic agent, wherein the at least one therapeutic agent is non-covalently associated with a macrocycle of the nanoparticle.
In certain embodiments, the nanoparticle linker comprises a moiety of Formula (I):
wherein:
In certain embodiments, L1a comprises —C(═O)—. In certain embodiments L1a comprises —(CH2)—. In certain embodiments, L1a comprises —(CH2)2—. In certain embodiments L1a comprises —(CH2)—. In certain embodiments, L1a comprises —(CH2)3—. In certain embodiments L1a comprises —(CH2)—. In certain embodiments, L1a comprises —(CH2)4—. In certain embodiments, L1a comprises
In certain embodiments, L1b comprises —C(═O)—. In certain embodiments L1b comprises —(CH2)—. In certain embodiments, L1b comprises —(CH2)2—. In certain embodiments L1b comprises —(CH2)—. In certain embodiments, L1b comprises —(CH2)3—. In certain embodiments L1b comprises —(CH2)—. In certain embodiments, L1b comprises —(CH2)4—. In certain embodiments, L1b is
In certain embodiments, X1a is —NH—. In certain embodiments, X1b is —NH—.
In certain embodiments, L2 comprises —[CH(C(═O)OH)]—. In certain embodiments L2 comprises —(CH2)—. In certain embodiments, L2 comprises —(CH2)2—. In certain embodiments L2 comprises —(CH2)—. In certain embodiments, L2 comprises —(CH2)3—. In certain embodiments L2 comprises —(CH2)—. In certain embodiments, L2 comprises —(CH2)4—. In certain embodiments, L2 is
In certain embodiments, the nanoparticle linker is
In certain embodiments, at least one macrocycle is a cyclodextrin. In certain embodiments, at least one macrocycle is a pillar[n]arene. In certain embodiments, at least one macrocycle is a calix[n]arene. In certain embodiments, at least one macrocycle is a cucurbit[n]uril.
In certain embodiments, the cyclodextrin is an α-cyclodextrin. In certain embodiments, the cyclodextrin is a β-cyclodextrin. In certain embodiments, the cyclodextrin is a 7-cyclodextrin.
In certain embodiments, the cyclodextrin is a 2-hydroxypropyl-α-cyclodextrin. In certain embodiments, the cyclodextrin is a 2-hydroxypropyl-β-cyclodextrin. In certain embodiments, the cyclodextrin is a 2-hydroxypropyl-7-cyclodextrin. In certain embodiments, the cyclodextrin is a methyl-α-cyclodextrin. In certain embodiments, the cyclodextrin is a methyl-β-cyclodextrin. In certain embodiments, the cyclodextrin is a methyl-γ-cyclodextrin. In certain embodiments, the cyclodextrin is a cyclodextrin sulfobutylether. In certain embodiments, the cyclodextrin is a cyclodextrin thioether. In certain embodiments, the cyclodextrin is a cyanoethylated cyclodextrin. In certain embodiments, the cyclodextrin is a succinyl-cyclodextrin. In certain embodiments, the cyclodextrin is an aminated cyclodextrin.
In certain embodiments, the nanoparticle linker is covalently conjugated to a primary hydroxyl of the cyclodextrin.
In certain embodiments, the hydrophilic polymer core is a hyaluronic acid derivative. In certain embodiments, the hydrophilic polymer core is a dextran derivative. In certain embodiments, the hydrophilic polymer core is a chitosan derivative. In certain embodiments, the hydrophilic polymer core is a fucoidan derivative. In certain embodiments, the hydrophilic polymer core is an alginate derivative. In certain embodiments, the hydrophilic polymer core is a cellulose derivative. In certain embodiments, the hydrophilic polymer core is a collagen derivative. In certain embodiments, the hydrophilic polymer core is a poly(ethylene glycol) derivative. In certain embodiments, the hydrophilic polymer core is a poly(hydroxyethyl acrylate) derivative. In certain embodiments, the hydrophilic polymer core is a poly(hydroxyethyl methacrylate) derivative. In certain embodiments, the hydrophilic polymer core is a poly(N-isopropylacrylamide) derivative. In certain embodiments, the hydrophilic polymer core is a poly(glycolic acid). In certain embodiments, the hydrophilic polymer core is a poly(lactic acid) derivative. In certain embodiments, the hydrophilic polymer core is a poly(lactic acid-glycolic acid) derivative. In certain embodiments, the hydrophilic polymer core is an oligo(poly(ethylene glycol)fumarate) derivative. In certain embodiments, the hydrophilic polymer core is a poly(vinyl alcohol) derivative. In certain embodiments, the hydrophilic polymer core is a poly(vinyl acid) derivative.
In certain embodiments, the hydrophobic substituent comprises a linear, branched, cyclic, or polycyclic C6-C20 hydrocarbon, C6-C20 aryl or alkylaryl, hetero or alkylaromatic hydrocarbon moieties. Additional hydrophobic substituents contemplated for use herein are described in the literature, for example U.S. Pat. No. 9,827,321, which is hereby incorporated herein by reference in its entirety. See also Bioconjugate Chem. 2015, 26, 2279-2289; and Coord. 2022, 454:214352.
In certain embodiments, the macrocycle comprises an α-cyclodextrin and the hydrophobic substituent comprises hexyl. In certain embodiments, the macrocycle comprises an α-cyclodextrin and the hydrophobic substituent comprises octyl. In certain embodiments, the macrocycle comprises an α-cyclodextrin and the hydrophobic substituent comprises polyethylene oxide. In certain embodiments, the macrocycle comprises an α-cyclodextrin and the hydrophobic substituent comprises ferrocenyl. In certain embodiments, the macrocycle comprises an α-cyclodextrin and the hydrophobic substituent comprises azobenzyl.
In certain embodiments, the macrocycle comprises a β-cyclodextrin and the hydrophobic substituent comprises adamantyl. In certain embodiments, the macrocycle comprises a 3-cyclodextrin and the hydrophobic substituent comprises cyclohexyl. In certain embodiments, the macrocycle comprises a β-cyclodextrin and the hydrophobic substituent comprises benzyl. In certain embodiments, the macrocycle comprises an β-cyclodextrin and the hydrophobic substituent comprises ferrocenyl. In certain embodiments, the macrocycle comprises an β-cyclodextrin and the hydrophobic substituent comprises azobenzyl.
In certain embodiments, the macrocycle comprises a γ-cyclodextrin and the hydrophobic substituent comprises cyclodecyl. In certain embodiments, the macrocycle comprises an γ-cyclodextrin and the hydrophobic substituent comprises ferrocenyl. In certain embodiments, the macrocycle comprises an γ-cyclodextrin and the hydrophobic substituent comprises azobenzyl.
In certain embodiments, the macrocycle comprises a cucurbit[6]uril and the hydrophobic moiety comprises a polyamine. In certain embodiments, the polyamine is a hexanediamine. In certain embodiments, the polyamine is spermine.
In certain embodiments, the hydrophobic substituent is independently adamantyl.
In certain embodiments, the functionalized polymer is a compound of Formula (II):
wherein:
In certain embodiments, the compound of Formula (II) is a compound of Formula (IIa):
In certain embodiments, each occurrence of R2a, R2b, R2c, R2d, and R2e is independently selected from the group consisting of H, C(═O)RI, C(═O)ORI, C(═O)N(RI)(RII), optionally substituted C1-C6 alkyl, optionally substituted C3-C8 cycloalkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C8 heterocycloalkyl, optionally substituted C6-C10 aryl, and optionally substituted C2-C10 heteroaryl.
In certain embodiments, R2f is H. In certain embodiments, R2f is R3.
In certain embodiments, R2a is H. In certain embodiments, R2b is H. In certain embodiments, R2c is H. In certain embodiments, R2d is H. In certain embodiments, R2e is H. In certain embodiments, R2e is C(═O)Me.
In certain embodiments, Y is —NH—.
In certain embodiments, Z1 is —CH2—.
In certain embodiments, Z2 is —C(═O)CH2—.
In certain embodiments, R3 is
In certain embodiments, m is an integer selected from the group consisting of about 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or about 1000.
In certain embodiments, n and m have a ratio of about 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, or about 1:2.
In certain embodiments, the therapeutic agent is a small molecule therapeutic agent.
In certain embodiments, the therapeutic agent is an immunomodulatory and/or anti-inflammatory agent.
In certain embodiments, the anti-inflammatory agent is at least one selected from the group consisting of NSAIDs, steroids, glucocorticoids, statins, PPAR inhibitors, PDE4 inhibitors, resolvins, antibiotics, quinones, ROS-scavengers, or naturally-derived compounds, and the like.
In certain embodiments, the anti-inflammatory agent is celastrol. In certain embodiments, the anti-inflammatory agent is piclamilast. In certain embodiments, the anti-inflammatory agent is ciglitazone. In certain embodiments, the anti-inflammatory agent is celecoxib. In certain embodiments, the anti-inflammatory agent is betamethasone. In certain embodiments, the anti-inflammatory agent is pravastatin. In certain embodiments, the anti-inflammatory agent is aspirin. In certain embodiments, the anti-inflammatory agent is ketorolac. In certain embodiments, the anti-inflammatory agent is lornoxicam. In certain embodiments, the anti-inflammatory agent is cortisone. In certain embodiments, the anti-inflammatory agent is pioglitazone. In certain embodiments, the anti-inflammatory agent is prednisolone. In certain embodiments, the anti-inflammatory agent is triamcinolone. In certain embodiments, the anti-inflammatory agent is methylprednisolone. In certain embodiments, the anti-inflammatory agent is desoximetasone. In certain embodiments, the anti-inflammatory agent is dexamethasone. In certain embodiments, the anti-inflammatory agent is fluvastatin. In certain embodiments, the anti-inflammatory agent is lovastatin. In certain embodiments, the anti-inflammatory agent is simvastatin. In certain embodiments, the anti-inflammatory agent is atorvastatin. In certain embodiments, the anti-inflammatory agent is rosuvastatin. In certain embodiments, the anti-inflammatory agent is gemfibrozil. In certain embodiments, the anti-inflammatory agent is troglitazone. In certain embodiments, the anti-inflammatory agent is rolipram. In certain embodiments, the anti-inflammatory agent is fenofibrate. In certain embodiments, the anti-inflammatory agent is rosiglitazone. In certain embodiments, the anti-inflammatory agent is apremilast. In certain embodiments, the anti-inflammatory agent is cilomilast. In certain embodiments, the anti-inflammatory agent is crisoborole. In certain embodiments, the anti-inflammatory agent is roflumilast. In certain embodiments, the anti-inflammatory agent is iloprost. In certain embodiments, the anti-inflammatory agent is colchicine. In certain embodiments, the anti-inflammatory agent is quercetin. In certain embodiments, the anti-inflammatory agent is JSH-23. In certain embodiments, the anti-inflammatory agent is tuftsin. In certain embodiments, the anti-inflammatory agent is resolving D1. In certain embodiments, the anti-inflammatory agent is sivelestat. In certain embodiments, the anti-inflammatory agent is doxycycline. In certain embodiments, the anti-inflammatory agent is methotrexate. In certain embodiments, the anti-inflammatory agent is vortioxetine.
In certain embodiments, the therapeutic agent is a pro-inflammatory agent.
In certain embodiments, the pro-inflammatory agent is at least one selected from the group consisting of tyrosine kinase inhibitors, CSF1R inhibitors, mTOR inhibitors, or direct immune agonists that include agonists of toll-like receptors, NOD-like receptors, STING, or other pattern-recognition receptors, and the like.
In certain embodiments, the pro-inflammatory agent is GW2580. In certain embodiments, the pro-inflammatory agent is CEP32496. In certain embodiments, the pro-inflammatory agent is BLZ945. In certain embodiments, the pro-inflammatory agent is OSI930. In certain embodiments, the pro-inflammatory agent is PLX3397. In certain embodiments, the pro-inflammatory agent is dasatinib. In certain embodiments, the pro-inflammatory agent is sunitinib. In certain embodiments, the pro-inflammatory agent is ABT869. In certain embodiments, the pro-inflammatory agent is imatinib. In certain embodiments, the pro-inflammatory agent is foretinib. In certain embodiments, the pro-inflammatory agent is XL228. In certain embodiments, the pro-inflammatory agent is gefitinib. In certain embodiments, the pro-inflammatory agent is PD0325901. In certain embodiments, the pro-inflammatory agent is trametinib. In certain embodiments, the pro-inflammatory agent is bentamapimod. In certain embodiments, the pro-inflammatory agent is dabrafenib. In certain embodiments, the pro-inflammatory agent is vemurafenib. In certain embodiments, the pro-inflammatory agent is crizotinib. In certain embodiments, the pro-inflammatory agent is UNC2025. In certain embodiments, the pro-inflammatory agent is indoximod. In certain embodiments, the pro-inflammatory agent is celecoxib. In certain embodiments, the pro-inflammatory agent is rapamycin. In certain embodiments, the pro-inflammatory agent is NIK12192. In certain embodiments, the pro-inflammatory agent is trichostatin A. In certain embodiments, the pro-inflammatory agent is IBET151. In certain embodiments, the pro-inflammatory agent is TMP195. In certain embodiments, the pro-inflammatory agent is BYL719. In certain embodiments, the pro-inflammatory agent is GDC0941. In certain embodiments, the pro-inflammatory agent is BKM120. In certain embodiments, the pro-inflammatory agent is imiquimod. In certain embodiments, the pro-inflammatory agent is gardiquimod. In certain embodiments, the pro-inflammatory agent is resiquimod (R848). In certain embodiments, the pro-inflammatory agent is motolimod. In certain embodiments, the pro-inflammatory agent is GS9620.
In certain embodiments, the therapeutic agent is at least one selected from the group consisting of JAK inhibitors, sphingosine-1-phosphate receptor modulators, cannabinoid receptor ligands, COX inhibitors, ACE inhibitors, Chalcone derivatives, Indole derivatives, Pyrazole derivatives, Pyrazolo[3,4-d]pyrimidine derivatives, Thiazolidinone derivatives, Triazol derivatives, Oxazolidinone derivatives, Imidazole derivatives, Pyrrol derivatives, or Furan derivatives and the like.
In certain embodiments, the nanoparticle has a diameter of about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or about 100 nm. In certain embodiments, the nanoparticle has a diameter of about 80 nm.
In certain embodiments, the nanoparticle comprises β-cyclodextrin succinate crosslinked with lysine. In certain embodiments, the β-cyclodextrin succinate and lysine have a ratio of about 1:1.
In certain embodiments, the functionalized polymer has a degree of monomer functionalization ranging from about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50%. In certain embodiments, the functionalized polymer has a degree of monomer functionalization of about 10%. In certain embodiments, the functionalized polymer has a degree of monomer functionalization of about 18%. In certain embodiments, the functionalized polymer has a degree of monomer functionalization of about 43%. In certain embodiments, the term “degree of functionalization” refers to the degree to which each monomer of the functionalized polymer is substituted with a hydrophobic moiety. For example, in certain embodiments, wherein the hydrophobic moiety is adamantyl and the hydrophilic polymer core is hyaluronic acid, the “degree of functionalization” refers to the amount of disaccharide monomers which are adamantyl-substituted as a percentage of the total number of disaccharide monomers in the hyaluronic acid polysaccharide.
In certain embodiments, the functionalized polymer comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or about 15% (w/v) of the hydrogel composition. In certain embodiments, the functionalized polymer comprises about 2.5% (w/v) of the hydrogel composition. In certain embodiments, the functionalized polymer comprises about 5.0% (w/v) of the hydrogel composition. In certain embodiments, the functionalized polymer comprises about 7.5% (w/v) of the hydrogel composition. In certain embodiments, the functionalized polymer comprises about about 10.0% (w/v) of the hydrogel composition.
In certain embodiments, the nanoparticle and the functionalized polymer have a ratio in the hydrogel composition of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or about 1:10 (nanoparticle:functionalized polymer). In certain embodiments, the nanoparticle and the functionalized polymer have a ratio in the hydrogel composition of about 0.5:1.0 (nanoparticle:functionalized polymer). In certain embodiments, the nanoparticle and the functionalized polymer have a ratio in the hydrogel composition of about 1:0:1.0 (nanoparticle:functionalized polymer). In certain embodiments, the nanoparticle and the functionalized polymer have a ratio in the hydrogel composition of about 1.5:1.0 (nanoparticle:functionalized polymer).
In certain embodiments, the therapeutic agent has a concentration in the hydrogel composition of about 5 μM.
In another aspect, the disclosure provides a method for treating, preventing, and/or ameliorating a disease or disorder in a subject. In certain embodiments, the method comprises administering to the subject the hydrogel composition of the disclosure.
In certain embodiments, the disease or disorder is an inflammatory disease or disorder. In certain embodiments, the disease or disorder is a focal tissue injury. In certain embodiments, the disease or disorder is an autoimmune disease and/or associated tissue dysfunction. In certain embodiments, the disease or disorder is cancer. In certain embodiments, the disease or disorder is or relates to a cytokine storm. In certain embodiments, the disease or disorder is sepsis.
In certain embodiments, the inflammatory disease or disorder is selected from the group consisting of cardiovascular disease, diabetic wounds, osteoarthritis, inflammatory bowel disease, and colitis. In certain embodiments, the inflammatory disease or disorder is a cardiovascular disease. In certain embodiments, the cardiovascular disease is atherosclerosis. In certain embodiments, the cardiovascular disease is myocarditis. In certain embodiments, the cardiovascular disease is endocarditis.
In certain embodiments, the cardiovascular disease is at least one selected from the group consisting of atherosclerosis, myocarditis, endocarditis, myocardial infarction, heart failure, stroke, aneurism, aortic dissection, peripheral arterial disease, congenital defects, and valvular disease.
In certain embodiments, the focal tissue injury is at least one selected from the group consisting of acute kidney injury, spinal cord injury, traumatic brain injury, muscle loss, and bone fracture
In certain embodiments, the chronic autoimmune disease and associated tissue dysfunction is at least one selected from the group consisting of multiple sclerosis, systemic lupus, rheumatoid arthritis, type 1 or type 2 diabetes, scleroderma, and psoriasis.
In certain embodiments, the cancer is at least one selected from the group consisting of hematologic cancer, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, and skin cancer.
In certain embodiments, the hydrogel composition is locally administered to an inflamed tissue or organ. In certain embodiments, the inflamed tissue or organ is the heart.
In another aspect, the disclosure provides a method for delivering a therapeutic agent to a macrophage in a subject. In certain embodiments, the method comprises administering to the subject the hydrogel composition of the disclosure.
In another aspect, the disclosure provides a method for reducing or inhibiting pro-inflammatory (M1-like) behavior of a macrophage in a subject. In certain embodiments, the method comprises administering to the subject the hydrogel composition of the disclosure.
In another aspect, the disclosure provides a method for promoting pro-healing (M2-like) behavior of a macrophage in a subject. In certain embodiments, the method comprises administering to the subject the hydrogel composition of the disclosure.
In certain embodiments, the subject is a mammal. In certain embodiments, the mammal is a human.
Various embodiments of the present application can be better understood by reference to the following Examples which are offered by way of illustration. The scope of the present application is not limited to the Examples given herein.
Unless otherwise indicated, solvents and general reagents were purchased from Sigma-Aldrich or TCI America and used without additional purification. Pharmaceutical drugs were obtained from Selleckchem, MedChemExpress, or Cayman Chemical Company and prepared at stock concentrations of 100 mM in dimethyl sulfoxide (DMSO). Cell culture reagents were purchased from VWR, unless otherwise stated.
Cyclodextrin nanoparticles (CDNPs) were prepared by methods as previously reported in the literature. Briefly, succinyl-β-cyclodextrin (1.0-3% w/v, 1.0 eq. succinylated groups), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 5-12.5 eq. to succinylate), and N-hydroxysuccinimide (NHS, 0.5 eq. to EDC) were dissolved in MES buffer (50 mM, pH 6.0). The reaction was stirred (30 min, RT) prior to the dropwise addition of L-lysine (0.5-4 eq. to succinylate) and overnight crosslinking. The product was recovered by addition of 100 μL brine and precipitation from a tenfold excess of anhydrous ethanol on ice. Following immediate re-dissolution in water, the product was purified by size-exclusion chromatography (SEC; PD-10, Fisher). Saccharide positive fractions were identified by spotting on a silica gel TLC plate, developing with 5% H2SO4 in ethanol, and then heating the plate. Positive fractions were concentrated by centrifugal filtration (10 kDa MWCO, Amicon), washed repeatedly with water, and lyophilized until dry. The final CDNP products were re-dissolved at 20% w/v in MilliQ water and stored at −20° C. until later use. Particle size was determined by dynamic light scattering (DLS; Zetasizer, Malvern) in triplicate at a concentration of 5 mg/mL in phosphate buffered saline (PBS). For scanning electron microscopy (SEM), samples were prepared at 200 μg/mL in DI water, lyophilized in a thin layer on conductive scaffolding, attached to SEM stubs using double sided carbon tape, and sputter coated prior to imaging (Zeiss, Supra 50VP).
Hyaluronic acid (HA; MW=82 kDa or 337 kDa; Lifecore Biomedical) was modified by pendant addition of 1-adamantane acetic acid (Ad), similar to previous reports. HA (5 g) was dissolved in DI water at 2% w/v, exchanged against Dowex 50W resin (15 g), neutralized by tetrabutylammonium hydroxide (TBA) to a final pH of 7.02-7.05, and lyophilized to yield HA-TBA (
Hydrogels were prepared from stock solutions of CDNP (20% w/v) and the denoted Ad-HA polymer (82 kDa Ad-HA (Low), 337 kDa Ad-HA (Low), and 82 kDa Ad-HA (High)) prepared in PBS. Hydrogels were formed by pipette mixing of the two separate solutions, followed by vortexing, manual stirring, and sonication to ensure homogenization with brief centrifugation to remove entrapped air bubbles. Hydrogel formulations were varied by adjusting the initial Ad-HA polymer concentration (2.5-10% w/v), the volumetric ratio of Ad-HA to CDNP (8:1-1:4), and anneal time (0-14 days) following mixing.
Characterization was performed on a TA Instruments Discovery HR20 rheometer fitted with a cone and plate geometry (20 mm diameter, 1° cone angle, 27 μm gap). Temperature was maintained at 25° C. through use of a Peltier plate stage. Properties of hydrogel samples were examined by oscillatory time sweeps at varying frequencies (0.1, 1, and 10 Hz; 1% strain), oscillatory frequency sweeps (0.01 Hz to 100 Hz; 1% strain), oscillatory strain sweeps (1% to 500% strain; 10 Hz), and continuous flow experiments with the shear rate linearly ramped from 0.005 to 50 s−1. Recovery experiments were performed using oscillatory time sweeps at 500% strain with recovery at 1% strain.
Cells were maintained under standard culture conditions (37° C., 5% CO2) in the indicated medium which was replenished every two days. The murine MF cell lines, RAW 264.7 (ATCC) and RAW-Blue™ (InvivoGen), were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with heat-inactivated fetal bovine serum (FBS), and 1% penicillin-streptomycin (Pen-Strep). RAW-Blue™ media was additionally supplemented by 100 μg/ml Normocin with the addition of 100 μg/ml of Zeocin every other passage to maintain selection pressure, as recommended by the manufacturer. Cells were passaged at 70% confluency. Primary MF were derived from murine and human tissues. Bone marrow derived macrophages (BMDMs) were isolated and derived according to standard protocols. Marrow was extracted from the surgically resected femur and tibia of male C57BL/6 mice, dissociated, and filtered using a 40 μm strainer. Red blood cells were lysed with ammonium chloride, and recovered cells were plated at 2×106 cells/well in 24 well plates and maintained in Iscove's Modification of DMEM (IMDM) supplemented with 10% heat-inactivated FBS, 1% Pen-Strep, and 10 ng/mL recombinant mouse macrophage colony-stimulating factor (M-CSF; PeproTech). Primary human monocytes were isolated from peripheral blood (New York Blood Center) from a single healthy human donor via density centrifugation as previously described in the literature. Harvested monocytes were cultured for 5 days on non-tissue culture-treated well plates in RPMI-1640 media supplemented with 10% heat-inactivated human serum, 1% Pen-Strep, and 20 ng/mL recombinant human M-CSF (PeproTech).
The process of drug selection leveraged a unique two-step drug screen that first identified potent M1-inhibitors using a high-throughput reporter cell assay followed by secondary identification of M2-promoting drugs in cell lines. Furthermore, a rigorous approach for validating drug activity was pursued, including in depth transcriptional analysis of primary murine and human cells to ensure cross-species drug activity. For initial drug screens, RAW-Blue™ cells were plated at 1×105 cells/well in 384 well plates. To induce polarization towards an M1-like phenotype, media was replaced after 24 h including zymosan (100 μg/mL). Drug dosing was performed concurrently, spanning concentrations from 100 μM to 0.03 μM in half-log dilutions; n=4 per group. At 24 h after drug treatment, secreted embryonic alkaline phosphatase (SEAP) reporter activity was quantified using QUANTI-Blue™ Solution following the manufacturer's protocol. Absorbance was read at 620 nm (BioTek Instruments, Synergy H1) and is presented following normalization to zymosan-treated controls.
For secondary identification of M2-promoting drugs, transcriptional analysis using qPCR was performed. RAW264.7 cells were plated at 1×106 cells/well in 24 well plates. After 24 h, media was replaced, and IL-4 (10 ng/mL, PeproTech) or zymosan (100 μg/mL) were included to serve as internal controls for M2-like and M1-like phenotypes, respectively. Treatment groups were concurrently activated via zymosan and treated with 1 μM drug concentrations, n=3 per group. After 24 hrs, cells were lysed by freezing, RNA was extracted (RNEasy Mini Kit; Qiagen), and cDNA synthesis was performed (High-Capacity cDNA Reverse Transcription Kit; Fisher). Samples were subject to qPCR using Taqman Fast Advanced Master Mix and probes (Fisher) for analysis of hprt (Mm01545399_m1), nos2 (Mm00440502_m1), il12b (Mm01288989_m1), il6 (Mm00446190_m1), mrc1 (Mm00485148_m1), il10 (Mm01288386_m1), and arg1 (Mm00475988_m1). Data is expressed as a fold change in gene expression using the ΔΔCt method, relative to the hprt and zymosan-treated controls.
For in depth transcriptional profiling of best drug candidates (piclamilast and celastrol), primary murine and human cells were subject to nanoString analysis. BMDMs were differentiated in 24 well plates as described. Zymosan (100 μg/mL) and IL-4 (10 ng/mL) treatments were again included as internal references for M1-like and M2-like MF phenotypes, respectively. Cells were treated with 1 μM of the prescribed compounds for 24 hrs, n=3 per group. Following RNA extraction, nanoString multiplex gene expression analysis was performed using 100 ng of extracted RNA and a custom-designed panel of 91 genes (Table 1), which relate primarily to murine MF phenotypes, as well as angiogenesis and fibrosis. Transcriptional analysis was similarly performed for human MF. Following differentiation from peripheral blood monocytes as described, cells were stimulated with lipopolysaccharide (LPS, 100 ng/mL). After 24 hrs, celastrol was added (1-10 μM, 24 hrs). RNA was isolated (RNAqueous-Micro Total RNA Isolation Kit, Fisher) and nanoString was performed with a custom-designed panel of 70 genes related primarily to MF phenotypes, angiogenesis, and fibrosis (Table 2). For both murine and human datasets, data was normalized to internal positive and negative controls using the nSolver 4.0 software and subsequently normalized to housekeeping genes (geometric mean of hprt and tbp for mouse, gapdh and tbp for human), as recommended by the manufacturer. Data is presented for all genes expressed above background as raw gene counts or as the Z-score of log-transformed data relative to M1-like controls or row means as indicated.
To examine potential cytotoxicity of drug and hydrogel components, RAW-Blue™ cells were plated at 5×103 cells/well in 96 well plates. Media was replaced at 24 h containing either drug (100 μM to 0.03 μM in half-log dilutions) or polymeric components (Ad-HA or CDNP, 5 to 0.04% w/v in five-fold dilutions) of interest. After 24 h, metabolic activity was assessed by PrestoBlue™ (Fisher) following the manufacturer's protocol, n=3 per treatment group, Absorbance was recorded at 570 nm, background subtracted from cell-free control wells, and normalized to untreated controls.
Surface plasmon resonance (Nicoya, OpenSPR) was used to quantify binding affinity between CD and celastrol. The instrument was primed with running buffer (0.5% v/v DMSO in PBS), a high sensitivity carboxyl sensor was installed, and both channels were cleaned with 10 mM HCl at 150 μL/min. The surface of the sensor was activated via injection of 200 μL of 0.1 M EDC/NHS in DI water in both channels at 20 μL/min. The ligand, aminated β-cyclodextrin, prepared as previously described, was dissolved in sodium acetate buffer (10 mM, pH 6, 1.2 mg/mL) and immobilized on the surface in channel 2 at 20 μL/min. Residual succinyl esters were deactivated by ethanolamine. The analyte, celastrol, was dissolved in running buffer and injected at concentrations of 62.5 μM, 125 μM, and 250 μM. Between tests, the injection port was rinsed with 1 mL of running buffer. Curves were analyzed in GraphPad Prism 8 via the association kinetics model.
To determine the rate at which hydrogels were eroded, established methods of one-dimensional hydrogel degradation were used. Assays were performed in custom made acrylic erosion wells, having a hydrogel chamber (4.3 mm diameter, 7 mm depth) overlaid with a supernatant chamber (1.6 cm diameter, 10 mm depth). Hydrogels (n=4 per group, 30 μL each) were deposited in the hydrogel chamber, wells were centrifuged to provide an even hydrogel surface, and the hydrogel was covered with 1 mL of PBS. Samples were incubated at 37° C., and the supernatant was collected at regular intervals with replacement by fresh buffer. At the endpoint, hydrogels were degraded in 2 mg/mL hyaluronidase (Sigma) for complete sample recovery. Quantification of polymeric content in release buffer was performed via uronic acid assay. For each supernatant sample, 50 μL of the sample was combined with ice-cold sulfuric acid containing sodium tetraborate decahydrate (1 mL, 19 mg/mL). Samples were incubated at 100° C. for 10 mins and cooled on ice prior to addition of carbazole (30 μL, 1.25 mg/mL in ethanol). Samples were briefly vortexed, incubated at 100° C. (15 min), and cooled on ice prior to recording absorbance at 525 nm (Thermo, Spectronic BioMate 3). Data is presented as the cumulative erosion over time, normalized to total sample recovery after enzymatic degradation.
For release studies, hydrogels were prepared including celastrol (5 mM). Celastrol was loaded into CDNPs by overnight mixing in PBS prior to the addition of Ad-HA. Hydrogels (n=3 per group) were prepared and loaded into erosion wells as previously described, with 1 mL of RAW-Blue™ cell media used as the supernatant which was subsequently collected with replacement at set time points over 14 days. Samples were stored at −20° C. until analysis of bioactivity in RAW-Blue™ cells, which were plated at 25×103 cells/well in 96 well plates. After 24 h, media was replaced with unconditioned media (control) or conditioned media from the release studies, supplemented with zymosan (100 μg/mL) as an inflammatory stimulus. Inflammatory activity was detected at 24 h after treatment using QUANTI-Blue™ Solution, following the manufacturer's protocol as described elsewhere herein.
For in vitro imaging of nanoparticle uptake, CDNPs were dissolved at 50 mg/mL in carbonate buffer (0.1 M, pH 8.5) and fluorescently labelled with Alexa Flour 555 NHS (10 μg/mL, Fisher). The reaction proceeded for 2 h at RT in the dark prior to product recovery by centrifugal filtration (10 kDa MWCO, Amicon). The product was repeatedly washed with water to remove unbound dye and lyophilized.
To determine if CDNPs released from hydrogels were uptaken by MF, hydrogels were prepared from the fluorescently labeled nanoparticle (CDNP-AF555) with or without celastrol inclusion (5 mM) and loaded into erosion wells as previously described (n=3 per group), with 1 mL of RAW-Blue™ cell media used as the supernatant which was collected after 48 hours. Conditioned media was transferred to RAW-Blue™ cells, seeded 24 hours prior at 15×103 cells/well in a 96 well glass bottom plate. After 24 h, cells were washed by PBS, fixed with 4% paraformaldehyde (15 min, 37° C.), and stained for cell membrane (5 μg/mL AlexaFluor 488 wheat germ agglutinin, Fisher) and nuclei (NucBlue™, Fisher) for 15 min at room temperature. Plates were washed by PBS prior to imaging (Leica, DMI 6000B). CDNP-AF555 uptake was assessed in ImageJ, quantified as the integrated fluorescence density.
Data presented are mean±standard deviation (SD), unless otherwise indicated.
Statistical analysis was performed using GraphPad Prism v9.3.1 and determined by analysis of variance (ANOVA), using repeated measurements where appropriate in conjunction with post-hoc Tukey's honest significant difference test. Normality was assessed by Shapiro-Wilk test. For two-way ANOVA, the Geisser-Greenhouse's epsilon correction was applied. Significance was determined at p<0.05.
Cyclodextrins are versatile macrocyclic hosts, used in the pharmaceutical industry as excipients to improve drug solubility and bioavailability, a use which has been meaningfully extended through the development of nano- and bulk-materials that leverage this host capacity for drug retention, including for macrophage (MF)-targeted therapies. In biomaterial applications, the transient guest-host complexes formed between CD and hydrophobic guest molecules is also a common means of forming dynamically crosslinked hydrogels. For this purpose, adamantane (Ad) is frequently used, as its size readily complements that of CD's hydrophobic cavity, contributing to formation of a one-to-one inclusion complex with relatively high affinity (Keq=105 M). Here, CD's versatile host capacity is leveraged to develop a nanotherapeutic drug delivery vehicle which is dynamically crosslinked by a host-modified polymer.
To prepare cyclodextrin nanoparticles (CDNPs) of varying size, particles were formed through EDC/NHS mediated amidation between succinyl-β-cyclodextrin and L-lysine (
Further increase in the particle size was not observed, as increased substrate or catalyst concentrations resulted in formation of a solid hydrogel during the reaction. Thus, the largest available CDNPs were pursued for hydrogel development; nanoparticles formed with 3.3% w/v CD, a 0.5:1 lysine feed ratio, and a 12.5:1 EDC feed ratio reliably produced CDNPs with a diameter of 80 nm, as confirmed by dynamic light scattering (DLS,
Development a shear-thinning and self-healing hydrogel was sought by leveraging the high affinity supramolecular interaction of CD at the nanoparticle surface with Ad. Here, hyaluronic acid (HA, MW=82 kDa or 337 kDa) was exchanged against Dowex-100 resin and neutralized by tetrabutylammonium hydroxide to yield HA-TBA (
The guest-polymers prepared serve as a crosslinker between host (CDNP) particles to yield polymer-nanoparticle hydrogels that self-assemble through guest-host crosslink formation (
The viscoelastic properties of the hydrogels was further investigated via oscillatory shear rheology. As the number of host sites accessible for guest-polymer binding at the nanoparticle surface is indeterminate, the ratio of guest to host components was empirically investigated (
Across these datasets, it is apparent that crosslink density is critical to stable gel formation as increased polymer modification yielded increased hydrogel moduli when molecular weight remained constant. Consistent with these observations, hydrogel moduli were likewise dependent on hydrogel concentration (
In sum, these studies demonstrate a method of tuning the mechanical properties of associative polymer-nanoparticle hydrogels and highlight the dominant role of dynamic bond density as a critical factor in stable hydrogel formation. More specifically, these studies revealed that 82 kDa Ad-HA (High) affords the most robust hydrogel formation and does so at polymer-nanoparticle ratios that are beneficial for subsequent drug loading.
Hydrogels formed through dynamic bonds assume varying structural integrity under strained conditions. As the bonds are continually broken by the application of sufficient external force, bond disassembly enables fluid-like flow and shear-thinning hydrogel injection. Continuous flow experiments (
In sum, the hydrogels exhibit fluid-like flow under high strain for ease of injection (shear-thinning). When the strain is removed, the hydrogels rapidly recover such as to allow for retention within the tissue. As a result, hydrogels could be pre-formed within a syringe and easily injected as liquids that rapidly re-solidify (
Identification of small molecule drugs capable of modulating MF phenotype were next sought for application in the context of tissue injury and inflammatory disease. Specifically, identification of a candidate that could suppress the damaging M1-like MF phenotype following injury and promote the reparatory M2-like MF phenotype for injury resolution was pursued. Therefore, a two-step screening process was developed that first identified pharmacological inhibitors of M1-like transcription using RAW-Blue™ cells, which are a readily available and inexpensive reporter cell line that incorporates a SEAP reporter construct downstream of both AP-1 and NF-κB promoter regions, and thus reports pro-inflammatory activation. The cell line is frequently used to evaluate both agonists and antagonists of M1-like polarization. While some approaches have been successful in identifying compounds for MF re-polarization such as through morphological analysis, applications have focused on redirection towards an M1 and not an M2 state. Currently, few approaches exist to directly assay for M2-like polarization. In the second step of the screening process, transcriptional analysis, via qPCR and multiplex analysis (nanoString), was used to identify compounds that promote canonical M2-like MF gene expression.
Initial drug screens were approached through an unbiased evaluation that compared dozens of drugs across multiple drug classes as such direct comparison is currently lacking from literature. Suppression of M1-like activation was therefore examined for a targeted library of 45 small molecule drug candidates that span a diverse set of specific drugs and drug classes that have established immunosuppressive or immunomodulatory capacity, including glucocorticoids, statins, and PPAR inhibitors among others. For primary evaluation in RAW-Blue™ cells, cells were stimulated by zymosan, a toll-like receptor 2 (TLR2) agonist that mimics sterile inflammation, and concurrently treated with the drugs of interest. HDAC inhibitors examined had no effect and were excluded from the data presented and subsequent studies. Other drug classes had varying degrees of efficacy (
After initial inhibitory screening, the effects of six selected drugs (i.e., celecoxib, betamethasone, pravastatin, ciglitazone, piclamilast, and celastrol) on MF phenotype were further scrutinized by transcriptional analysis in zymosan-activated RAW264.7 MF in order to determine if these drug candidates further promote M2-activation. Canonical markers of both M1-like (nos2, i16, il12b) and M2-like (mrc1, arg1, il10) phenotypes were included (
Further in-depth transcriptional analysis of the two drugs candidates was performed by multiplex gene expression analysis (nanoString) in zymosan-activated bone marrow derived MF (BMDMs) treated with piclamilast and celastrol. The analysis measured gene expression across 91-genes that represent multiple M1-like and M2-like markers, as well as genes associated with ECM regulation, fibroblast activity, angiogenesis, and immune signaling pathways (Table 1).
Cluster analysis and corresponding dendograms (
While results in cell lines and primary murine cells are promising, there exist nuanced differences between the murine and human inflammatory programs that may hinder translation and warrant early investigation. To ensure drug activity across species, transcriptional analysis was further performed in LPS-activated MF derived from peripheral blood mononuclear cells (PBMCs) using a panel of human genes (Table 2). The choice of LPS instead of zymosan as a pro-inflammatory stimulus was made to allow comparison to other studies of human MF, which more commonly use LPS. Subsequent to LPS activation, cells were treated with varying concentrations of celastrol (1-10 μM). Both cluster analysis (
While celastrol has moderate oral bioavailability which can be enhanced by formulation, it displays significant sex-dependences in systemic bioavailability and such administration is associated with hepatotoxicity, hematopoietic system toxicity, nephrotoxicity, and undesirable biodistribution. Moreover, local biomaterial-delivery strategies afford an opportunity to reduce clearance rates, prolong release, concentrate drug dose at the site of action, and enhance cell-specific delivery. For these reasons, examination of its potential as a suitable cargo for CDNP encapsulation and delivery from the polymer-nanoparticle hydrogels is described herein (
Supramolecularly assembled hydrogels typically degrade via surface erosion, with dependence on network structure and composition. It was therefore anticipated that nanoparticle release from the hydrogels would be both a means of hydrogel degradation and a potential method of cell-targeted delivery, owing to the rapid uptake of CDNPs by phagocytic immune cells that occurs both in vitro and in vivo. Hydrogel erosion was assessed over a six week period at 37° C., spanning a range of hydrogel compositions. Across these formulations, hydrogels typically exhibited slow erosion over the course of greater than one month, with 82 kDa Ad-HA (High) degrading slower than less effectively crosslinked network architectures (
Celastrol-loaded hydrogel formulations were next prepared by simple mixing of celastrol (Cel) and CDNP under aqueous conditions and the resulting drug-loaded nanoparticle (CDNP-Cel) were used directly in hydrogel formation. Importantly, drug incorporation did not alter the rheological properties (
Next, the ability of CDNP-Cel to be released from the hydrogels by erosion and subsequently uptaken my MF was assessed. For this purpose, CDNPs were fluorescently labeled by Alexa Fluor555 (CDNP-AF555). Both drug loaded and unloaded CDNP-AF555 nanoparticles were formulated in 82 kDa Ad-HA (High) hydrogels that were subsequently allowed to erode in media. RAW264.7 cells were treated with the conditioned media prior to imaging and quantification (
The final objective was to investigate the ability of the drug-laden hydrogels to provide bioactive concentrations of CDNP-Cel release, capable of modulating cell phenotype over time in vitro (
Overall, these findings demonstrate the release of CDNP-Cel from the hydrogels is a promising strategy for MF-targeted drug delivery that can bias cell phenotype over the course of weeks. This timespan coincides well with the established timeline of exuberant inflammation that frequently underlies failed tissue repair programs, particularly in organ injury, and is therefore a promising immunoregenerative strategy towards such applications.
The following exemplary embodiments are provided, the numbering of which is not to be construed as designating levels of importance:
Embodiment 1 provides a hydrogel composition comprising:
Embodiment 2 provides the hydrogel composition of Embodiment 1, wherein the least one nanoparticle linker independently comprises a moiety of Formula (I):
wherein:
Embodiment 3 provides the hydrogel composition of Embodiment 2, wherein L1a and L1b are each independently
Embodiment 4 provides the hydrogel composition of Embodiment 2 or 3, wherein X1a and X1b are each independently —NH—.
Embodiment 5 provides the hydrogel composition of any one of Embodiments 2-4, wherein L2 is
Embodiment 6 provides the hydrogel composition of any one of Embodiments 2-5, wherein the nanoparticle linker is
Embodiment 7 provides the hydrogel composition of any one of Embodiments 1-6, wherein each of the plurality of macrocycles is independently selected from the group consisting of a cyclodextrin, a pillar[n]arene, a calix[n]arene, and a cucurbit[n]uril.
Embodiment 8 provides the hydrogel composition of Embodiment 7, wherein each cyclodextrin is independently selected from the group consisting of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.
Embodiment 9 provides the hydrogel composition of Embodiment 7, wherein each cyclodextrin is independently selected from the group consisting of 2-hydroxypropyl-α-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, 2-hydroxypropyl-γ-cyclodextrin, methyl-α-cyclodextrin, methyl-β-cyclodextrin, methyl-γ-cyclodextrin, a cyclodextrin sulfobutylether, a cyclodextrin thioether, a cyanoethylated cyclodextrin, a succinyl-cyclodextrin, and an aminated cyclodextrin.
Embodiment 10 provides the hydrogel composition of Embodiment 7, wherein each nanoparticle linker is covalently conjugated to a primary hydroxyl of the cyclodextrin.
Embodiment 11 provides the hydrogel composition of any one of Embodiments 1-10, wherein the hydrophilic polymer core is selected from the group consisting of a hyaluronic acid derivative, a dextran derivative, a chitosan derivative, a fucoidan derivative, an alginate derivative, a cellulose derivative, a collagen derivative, a poly(ethylene glycol) derivative, a poly(hydroxyethyl acrylate) derivative, a poly(hydroxyethyl methacrylate) derivative, a poly(N-isopropylacrylamide) derivative, a poly(glycolic acid), a poly(lactic acid) derivative, a poly(lactic acid-glycolic acid) derivative, a oligo(poly(ethylene glycol)fumarate) derivative, a poly(vinyl alcohol) derivative, and a poly(vinyl acid) derivative.
Embodiment 12 provides the hydrogel composition of any one of Embodiments 1-11, wherein each hydrophobic substituent is independently selected from the group consisting of adamantyl, cyclohexyl, benzyl, azobenzyl, and ferrocenyl.
Embodiment 13 provides the hydrogel composition of any one of Embodiments 1-12, wherein the functionalized polymer is a compound of Formula (II):
wherein:
Embodiment 14 provides the hydrogel composition of Embodiment 13, wherein the compound of Formula (II) is a compound of Formula (IIa):
Embodiment 15 provides the hydrogel composition of Embodiment 13 or 14, wherein each of the following apply:
Embodiment 16 provides the hydrogel composition of any one of Embodiments 13-15, wherein each occurrence of R2a, R2b, R2c, and R2d is independently H.
Embodiment 17 provides the hydrogel composition of any one of Embodiments 13-16, wherein each occurrence of R2e is independently C(═O)Me.
Embodiment 18 provides the hydrogel composition of any one of Embodiments 13-17, wherein each occurrence of Y is independently —NH—.
Embodiment 19 provides the hydrogel composition of any one of Embodiments 13-18, wherein each occurrence of Z1 is independently —CH2—.
Embodiment 20 provides the hydrogel composition of any one of Embodiments 13-19, wherein each occurrence of Z2 is —C(═O)CH2—.
Embodiment 21 provides the hydrogel composition of any one of Embodiments 13-20, wherein each occurrence of R3 is independently
Embodiment 22 provides the hydrogel composition of any one of Embodiments 13-21, wherein m is an integer ranging from 200 to 1000.
Embodiment 23 provides the hydrogel composition of any one of Embodiments 13-22, wherein n and m have a ratio of about 1:10 to about 1:2 (n:m).
Embodiment 24 provides the hydrogel composition of any one of Embodiments 1-23, wherein the therapeutic agent is a small molecule therapeutic agent.
Embodiment 25 provides the hydrogel composition of any one of Embodiments 1-24, wherein the therapeutic agent is selected from the group consisting of an anti-inflammatory agent and a pro-inflammatory agent.
Embodiment 26 provides the hydrogel composition of Embodiment 25, wherein the anti-inflammatory agent is selected from the group consisting of celastrol, piclamilast, ciglitazone, celecoxib, betamethasone, pravastatin, aspirin, ketorolac, lornoxicam, cortisone, pioglitazone, prednisolone, triamcinolone, methylprednisolone, desoximetasone, dexamethasone, Fluvastatin, lovastatin, simvastatin, atorvastatin, rosuvastatin, gemfibrozil, troglitazone, rolipram, fenofibrate, rosiglitazone, apremilast, cilomilast, crisoborole, roflumilast, iloprost, colchicine, quercetin, JSH-23, tuftsin, resolving D1, sivelestat, doxycycline, methotrexate, and vortioxetine.
Embodiment 27 provides the hydrogel composition of Embodiment 25, wherein the pro-inflammatory agent is selected from the group consisting of GW2580, CEP32496, BLZ945, OSI930, PLX3397, dasatinib, sunitinib, ABT869, imatinib, foretinib, XL228, gefitinib, PD0325901, trametinib, bentamapimod, dabrafenib, vemurafinib, crizotinib, UNC2025, indoximod, celecoxib, rapamycin, NIK12192, trichostatin A, IBET151, TMP195, BYL719, GDC0941, BKM120, imiquimod, gardiquimod, resiquimod (R848), motolimod, and GS9620.
Embodiment 28 provides the hydrogel composition of any one of Embodiments 1-27, wherein the nanoparticle has a diameter ranging from about 50 nm to about 100 nm, optionally wherein the nanoparticle has a diameter of about 80 nm.
Embodiment 29 provides the hydrogel composition of any one of Embodiments 1-28, wherein the nanoparticle comprises β-cyclodextrin succinate crosslinked with lysine, optionally wherein the β-cyclodextrin succinate and lysine have a ratio of about 1:1.
Embodiment 30 provides the hydrogel composition of any one of Embodiments 1-29, wherein the functionalized polymer has a degree of monomer functionalization ranging from about 5% to about 50%, optionally wherein the functionalized polymer has a degree of monomer functionalization selected from the group consisting of about 10%, about 18%, and about 43%.
Embodiment 31 provides the hydrogel composition of any one of Embodiments 1-30, wherein the functionalized polymer comprises about 1% (w/v) to about 15% (w/v) of the hydrogel composition, optionally wherein the functionalized polymer comprises about 2.5% (w/v), about 5.0% (w/v), 7.5% (w/v), or about 10.0% (w/v) of the hydrogel composition.
Embodiment 32 provides the hydrogel composition of any one of Embodiments 1-31, wherein the nanoparticle and the functionalized polymer have a ratio in the hydrogel composition ranging from about 10:1 to about 1:10 (nanoparticle:functionalized polymer), optionally wherein the nanoparticle and the functionalized polymer have a ratio in the hydrogel composition of about 0.5:1.0, about 1.0:1.0, or about 1.5:1 (nanoparticle:functionalized polymer).
Embodiment 33 provides the hydrogel composition of any one of Embodiments 1-32, wherein the therapeutic agent has a concentration in the hydrogel composition of about 5 μM.
Embodiment 34 provides a method for treating, preventing, and/or ameliorating a disease or disorder in a subject, the method comprising administering to the subject the hydrogel composition of any one of Embodiments 1-33.
Embodiment 35 provides the method of Embodiment 34, wherein the disease or disorder is at least one selected from the group consisting of an inflammatory disease or disorder, a focal tissue injury, an autoimmune disease and/or associated tissue dysfunction, cancer, cytokine storm, and sepsis.
Embodiment 36 provides the method of Embodiment 35, wherein the inflammatory disease or disorder is selected from the group consisting of cardiovascular disease, diabetic wounds, osteoarthritis, inflammatory bowel disease, and colitis.
Embodiment 37 provides the method of Embodiment 36, wherein the cardiovascular disease is at least one selected from the group consisting of atherosclerosis, myocarditis, endocarditis, myocardial infarction, heart failure, stroke, aneurism, aortic dissection, peripheral arterial disease, congenital defects, and valvular disease.
Embodiment 38 provides the method of any one of Embodiments 34-37, wherein the hydrogel composition is locally administered to an inflamed tissue or organ, optionally wherein the inflamed tissue or organ is the heart.
Embodiment 39 provides a method for delivering a therapeutic agent to a macrophage in a subject, the method comprising administering to the subject the hydrogel composition of any one of Embodiments 1-33.
Embodiment 40 provides a method for reducing or inhibiting pro-inflammatory (M1-like) behavior of a macrophage in a subject, the method comprising administering to the subject the hydrogel composition of any one of Embodiments 1-33.
Embodiment 41 provides a method for promoting pro-healing (M2-like) behavior of a macrophage in a subject, the method comprising administering to the subject the hydrogel composition of any one of Embodiments 1-33.
The terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments of the present application. Thus, it should be understood that although the present application describes specific embodiments and optional features, modification and variation of the compositions, methods, and concepts herein disclosed may be resorted to by those of ordinary skill in the art, and that such modifications and variations are considered to be within the scope of embodiments of the present application.
This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/463,575, filed May 3, 2023, which is incorporated herein by reference in its entirety.
This invention was made with government support under 5R35GM147184-02 awarded by National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63463575 | May 2023 | US |
