Patent Application
20240181129
The presently disclosed subject matter is directed to guest-host supramolecular assembly of injectable hydrogel nanofibers for cell encapsulation.
The extracellular matrix (ECM) is a complex three-dimensional (3D) microenvironment that provides mechanical support, protection, and regulatory signals to embedded cells1,2. Hydrogels can mimic native ECM due to their ability to exhibit tissue-like properties, including viscoelastic mechanics and high water content comparable to the tissues they are intended to model and replace3-5.
While recreating ECM biophysical cues, such as fibrous architecture, has been a focus of the tissue engineering community in recent years30, there remains a need to develop injectable (shear-thinning and self-healing) or in situ gelling materials for both clinical (including drug delivery and tissue engineering with minimally invasive delivery) and additive manufacturing (3D-printing) applications. Clinically, injectable materials offer major advantages such as reduced patient discomfort and treatment cost34,35.
While these needs have been understood for some time, and efforts have been made to address the same, the current solutions fall far short of providing viable compositions suitable for mimicking ECM that are also patient friendly. Thus, there remains an unmet need for fibrous hydrogel systems amenable to minimally invasive delivery. The present disclosure provides such solutions, and demonstrates the superiority of the disclosed injectable fibrous hydrogels based on an analysis of the architecture of the fabricated hydrogel nanofibers, their rheological properties, and their ability to support sustained cell viability following injection.
This summary lists several embodiments of the presently disclosed subject matter, and in many cases lists variations and permutations of these embodiments. This summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently disclosed subject matter, whether listed in this summary or not. To avoid excessive repetition, this Summary does not list or suggest all possible combinations of such features.
In some embodiments, provided herein are injectable fibrous hydrogels, the injectable fibrous hydrogels comprising a guest macromer of a hyaluronic acid (HA) backbone and host macromer of a HA backbone, wherein the guest macromer comprises a HA electrospun hydrogel nanofiber functionalized with adamantane (Ad), wherein the host macromer comprises a HA electrospun hydrogel nanofiber functionalized with β-cyclodextrin (CD). In some embodiments, the HA of the guest macromer and the HA of the host macromer comprises a methacrylated HA (MeHA), wherein the MeHA is Ad-modified to form Ad-MeHA in the guest macromer, wherein the MeHA is CD-modified to form CD-MeHA in the host macromer. In some aspects, the methacrylated HA of the electrospun hydrogel nanofibers is covalently photocrosslinked in the presence of a photoinitiator via ultraviolet (UV) light-mediated radical polymerization. In some embodiments, the guest macromer and host macromer are both hydrophobic and form a stable supramolecular, yet reversible, guest-host interaction.
In some embodiments, the injectable fibrous hydrogel nanofibers are configured to imbibe water upon hydration rather than dissolving. In some aspects, the guest macromer and host macromer have a molar ratio ranging from about 1:1 to about 3:1, optionally about 2:1. In some embodiments, the guest macromers and host macromers of the hydrogel nanofibers associate via hydrophobic supramolecular interactions to form a mechanically robust 3D fibrous hydrogel configured for shear-thinning and self-healing post injection. In some aspects, the guest macromers and host macromers of the injectable fibrous hydrogel have an association constant (Ka) of about 1×104 M−1 to about 1×105 M−1, or at least about 1×105 M−1.
In some embodiments, the fibrous hydrogel is flowable through a needle at about 8 mL h−1 to about 20 mL h−1 (using a 16 to 22 gauge needle), optionally at about 12 mL h−1 (16 gauge needle), wherein the fibrous hydrogel is configured to transform to a stable hydrogel plug post injection. The injectable fibrous hydrogel can comprise a self-assembling guest-host fibrous hydrogel configured as a cell carrier for injectable tissue engineering. In some aspects, the injectable fibrous hydrogel can be configured to mimic an extra cellular matrix (ECM) upon injection, optionally wherein the injectable fibrous hydrogel is configured to form a hierarchical assembly upon injection to provide physical cues to cells at different length scales, mimicking the 3D cues provided by a native fibrous ECM. In some embodiments, the injectable fibrous hydrogel further comprises one or more ligands, optionally one or more cell adhesion peptides, optionally a cell adhesion peptide comprising arginylglycylaspartic acid (RGD), and other thiolated molecutles including peptide and fibronectin fragments, to permit integrin-mediated cell adhesion.
Also provided herein in some embodiments are injectable formulations comprising an injectable fibrous hydrogel. Such injectable formulations can further comprise one or more cells encapsulated in the fibrous hydrogel. Any suitable type of cell can be encapsulated, including for example but not limited to, hMSC, myoblasts (C2C12), fibroblasts (3T3), adipose-derived stem cells (ADSC), or pluripotent stem cells (PSC), all with varying applications. The one or more cells can have a post-injection survival rate of at least about 70%, optionally at least about 80%, optionally at least about 90%. In some embodiments, the injectable formulation can be configured for injection into a tissue of a subject, optionally a fibrous tissue, optionally a muscle, tendon, or ligament tissue. In some aspects, the fibrous hydrogel is flowable through a needle at about 8 mL h−1 to about 20 mL h−1 (using a 16 to 22 gauge needle), optionally at about 12 mL h−1 (16 gauge needle), wherein the fibrous hydrogel is configured to transform to a stable hydrogel plug post injection. In some aspects, the injectable formulation comprises one or more pharmaceutically acceptable carriers or excipients. In some embodiments, the injectable formulation comprises a storage modulus (G′) of about 6.6 kPa at 1% fibrous content at 10 Hz, and a G′ of about 9.2 kPa at 5% fibrous content at 10 Hz.
Provided herein in some embodiments are methods of forming a fibrous hydrogel nanofiber in a polymer solution, the method comprising methacrylating a hyaluronic acid (HA) backbone via methacrylate esterification with a primary hydroxyl group of a sodium HA to form methacrylated HA (MeHA); synthesizing adamantane (Ad)-modified MeHA (Ad-MeHA) and β-cyclodextrin (CD)-modified MeHA (CD-MeHA) by anhydrous coupling; electrospinning the fibrous hydrogel nanofiber in the polymer solution; and crosslinking the fibrous hydrogel nanofiber by exposure to ultraviolet (UV) light. In some embodiments, a degree of methacrylate modification of HA is controlled by an amount of a methacrylic anhydride introduced during the methacrylating step, optionally wherein the degree of methacrylate modification of HA is about 10% to about 40%, optionally about 20% to about 30%, optionally about 28%. In some aspects, Ad-MeHA is prepared using 1-adamantane acetic acid via di-tert-butyl bicarbonate (BOC2O)/4-dimethylaminopyridine (DMAP) esterification, wherein CD-MeHA is prepared using CD-HDA via (benzotriazol-1-yloxy) tris(dimethylamino) phosphonium hexafluorophosphate (BOP) amidation. In some embodiments, the electrospinning comprises a collection plate set-up using an applied voltage of about 9.5-10.5 kV (optionally ranging from about 7.5-12.5 kV), a distance from needle to collector of about 16 cm, a needle gauge of about 20, and a flow rate of about 0.4 mL h−1. In some aspects, crosslinking the fibrous hydrogel nanofiber with UV light comprises exposure to UV light at about 320-390 nm for about 10-15 minutes, optionally about 365 nm for about 15 minutes. In some embodiments, such methods further comprise repeatedly triturating the hydrogel fibers via needle extrusion to produce short fiber segments of a length of about 5 μm to about 20 μm, optionally about 12.7±5.0 μm.
Finally, provided herein in some embodiments are methods of treating a tissue of a subject, the methods comprising providing a subject to be treated and delivering to a tissue of the subject an injectable fibrous hydrogel as disclosed herein. In some embodiments treating the tissue can be a component of treating a wide range of musculoskeletal conditions or diseases, in addition to further tissue applications, e.g. brain, adipose, or skin tissues. In some aspects, the injectable fibrous hydrogel is administered to the tissue to be treated by injection. In some embodiments, the tissue to be treated is selected from a fibrous tissue, optionally a muscle, tendon, or ligament tissue. In some embodiments, the injectable fibrous hydrogel comprises one or more encapsulated cells (varying types of cells are suitable, as discussed further herein). In some aspects, the encapsulated cells have a higher viability post-injection when encapsulated in the fibrous hydrogel than when not encapsulated in the fibrous hydrogel, optionally a survivability rate of at least about 80%.
These and other objects are achieved in whole or in part by the presently disclosed subject matter. Objects of the presently disclosed subject matter having been stated above, other objects and advantages of the presently disclosed subject matter will become apparent to those skilled in the art after a study of the following description, Drawings and Examples.
The presently disclosed subject matter can be better understood by referring to the following figures. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the presently disclosed subject matter (often schematically). In the figures, like reference numerals designate corresponding parts throughout the different views. A further understanding of the presently disclosed subject matter can be obtained by reference to an embodiment set forth in the illustrations of the accompanying drawings. Although the illustrated embodiment is merely exemplary of systems for carrying out the presently disclosed subject matter, both the organization and method of operation of the presently disclosed subject matter, in general, together with further objectives and advantages thereof, may be more easily understood by reference to the drawings and the following description. The drawings are not intended to limit the scope of this presently disclosed subject matter, which is set forth with particularity in the claims as appended or as subsequently amended, but merely to clarify and exemplify the presently disclosed subject matter.
For a more complete understanding of the presently disclosed subject matter, reference is now made to the following drawings in which:
The presently disclosed subject matter now will be described more fully hereinafter, in which some, but not all embodiments of the presently disclosed subject matter are described. Indeed, the presently disclosed subject matter can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the presently disclosed subject matter.
While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.
All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art. While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.
In describing the presently disclosed subject matter, it will be understood that a number of techniques and steps are disclosed. Each of these has individual benefit and each can also be used in conjunction with one or more, or in some cases all, of the other disclosed techniques.
Accordingly, for the sake of clarity, this description will refrain from repeating every possible combination of the individual steps in an unnecessary fashion. Nevertheless, the specification and claims should be read with the understanding that such combinations are entirely within the scope of the invention and the claims.
Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.
Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.
As used herein, the term “about,” when referring to a value or to an amount of a composition, dose, sequence identity (e.g., when comparing two or more nucleotide or amino acid sequences), mass, weight, temperature, time, volume, concentration, percentage, etc., is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.
The term “comprising”, which is synonymous with “including” “containing” or “characterized by” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. “Comprising” is a term of art used in claim language which means that the named elements are essential, but other elements can be added and still form a construct within the scope of the claim.
As used herein, the phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. When the phrase “consists of” appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.
As used herein, the phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.
With respect to the terms “comprising”, “consisting of”, and “consisting essentially of”, where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms.
As used herein, the term “and/or” when used in the context of a listing of entities, refers to the entities being present singly or in combination. Thus, for example, the phrase “A, B, C, and/or D” includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D.
A “test” cell, tissue, sample, or subject is one being examined or treated.
A “compound”, as used herein, refers to any type of substance or agent that is commonly considered a drug, or a candidate for use as a drug, combinations, and mixtures of the above, as well as other non-limiting examples like polypeptides and antibodies.
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.
In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.
The term “ingredient” refers to any compound, whether of chemical or biological origin, that can be used the disclosed compositions and formulations. The terms “component”, “nutrient”, “supplement”, and ingredient” can be used interchangeably and are all meant to refer to such compounds.
The term “pharmaceutical composition” shall mean a composition comprising at least one active ingredient, whereby the composition is amenable to investigation for a specified, efficacious outcome in a mammal (for example, without limitation, a human). Those of ordinary skill in the art will understand and appreciate the techniques appropriate for determining whether an active ingredient has a desired efficacious outcome based upon the needs of the artisan.
As used herein, the term “pharmaceutically-acceptable carrier” means a chemical composition with which an appropriate compound or derivative can be combined and which, following the combination, can be used to administer the appropriate compound to a subject.
“Plurality” means at least two.
The term “subject,” as used herein, generally refers to a mammal. Typically, the subject is a human. However, the term embraces other species, e.g., pigs, mice, rats, dogs, cats, or other primates. In certain embodiments, the subject is an experimental subject such as a mouse or rat. The subject may be a male or female. The subject may be an infant, a toddler, a child, a young adult, an adult or a geriatric.
A subject under the care of a physician or other health care provider may be referred to as a “patient”.
A “subject” of diagnosis or treatment is an animal, including a human. It also includes pets and livestock.
As used herein, a “subject in need thereof” is a patient, animal, mammal, or human, who will benefit from the compositions, formulations and methods of the presently disclosed subject matter.
A “therapeutic” treatment is a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing or eliminating those signs.
A “therapeutically effective amount” of a compound is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered.
As used herein, the term “treating” may include prophylaxis of the specific injury, disease, disorder, or condition, or alleviation of the symptoms associated with a specific injury, disease, disorder, or condition and/or preventing or eliminating said symptoms. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease. “Treating” is used interchangeably with “treatment” herein.
The extracellular matrix (ECM) is a complex three-dimensional (3D) microenvironment that provides mechanical support, protection, and regulatory signals to embedded cells1,2. Hydrogels can mimic native ECM due to their ability to exhibit tissue-like properties, including viscoelastic mechanics and high water content comparable to the tissues they are intended to model and replace3,5. In the context of tissue engineering, there is growing appreciation for the importance of the fibrillar architecture of ECM and its role in providing biophysical cues that regulate cell behavior6-9. In particular, there is a need for robust 3D hydrogels that can mimic the fibrous networks present in many musculoskeletal tissues while allowing tuning of network biophysical and biochemical properties. A method of forming mechanically robust fibrous materials is electrospinning, a simple, scalable, and cost-effective biofabrication technique that produces nanofibers with tunable physicochemical properties. Electrospinning has also garnered interest owing to its ability to produce nanofibers.
While recreating ECM biophysical cues, such as fibrous architecture, has been a focus of the tissue engineering community in recent years30, there remains a need to develop injectable (shear-thinning and self-healing) or in situ gelling materials for both clinical (including drug delivery and tissue engineering with minimally invasive delivery) and additive manufacturing (3D-printing) applications. Clinically, injectable materials offer major advantages such as reduced patient discomfort and treatment cost34,35.
Guest-host (e.g., adamantane-β-cyclodextrin) supramolecular chemistries are considered herein for injectable hydrogel design due to their ability to support shear-thinning and self-healing behavior while also allowing careful tuning of viscoelastic mechanical properties through both primary and secondary crosslinking mechanisms. Guest-host assembled hydrogels can also be suitable cell carriers for injectable delivery since their non-Newtonian properties help protect cells from excessive shear during needle extrusion. However, achieving fibrillar topographies in mechanically robust injectable hydrogels remains challenging. While self-assembling peptides can serve as injectable nanofibrous hydrogels45, these materials are often limited by poor long-term mechanical properties5,8. Electrospun hydrogel materials, as described earlier, show robust mechanics and support biomimetic cellular behaviors. However, electrospun scaffolds typically require physical implantation and are not amenable to injectable delivery46,47.
To address the need for a fibrous hydrogel system amenable to minimally invasive delivery, disclosed herein are studies and results based on a unique combination of strategies in biomanufacturing, supramolecular chemistry, and tissue engineering to produce a mechanically robust injectable fibrous hydrogel capable of supporting cell encapsulation. The engineering design brings together hyaluronic acid (HA) electrospun hydrogel nanofibers, functionalized with either guest (adamantane, Ad) or host (β-cyclodextrin, CD) supramolecular moieties, that when joined together create a shear-thinning and self-healing hydrogel fiber network via guest-host complexation. HA was utilized for its amenability to functionalization with reactive groups enabling both photocrosslinking and guest-host assembly. In this work the architecture of the fabricated hydrogel nanofibers, their rheological properties, and their ability to support sustained cell viability following injection were assessed.
More particularly, as disclosed and discussed in the working Examples herein, the disclosed injectable fibrous hydrogels can comprise a guest macromer of a hyaluronic acid (HA) backbone and host macromer of a HA backbone, wherein the guest macromer comprises a HA electrospun hydrogel nanofiber functionalized with adamantane (Ad), wherein the host macromer comprises a HA electrospun hydrogel nanofiber functionalized with β-cyclodextrin (CD). The HA of the guest macromer and the HA of the host macromer can include a methacrylated HA (MeHA), wherein the MeHA is Ad-modified to form Ad-MeHA in the guest macromer, and the MeHA is CD-modified to form CD-MeHA in the host macromer. In some aspects, the methacrylated HA of the electrospun hydrogel nanofibers is covalently photocrosslinked in the presence of a photoinitiator via ultraviolet (UV) light-mediated radical polymerization. Notably, the guest macromer and host macromer are both hydrophobic and form a stable supramolecular, yet reversible, guest-host interaction.
The injectable fibrous hydrogel nanofibers can be configured to imbibe water upon hydration rather than dissolving. The guest macromer and host macromer can be optimized as needed and can in some aspects have a molar ratio ranging from about 1:1 to about 3:1, optionally about 2:1. In some embodiments, the guest macromers and host macromers of the hydrogel nanofibers associate via hydrophobic supramolecular interactions to form a mechanically robust 3D fibrous hydrogel. Advantageously, particularly as compared to existing hydrogels, the disclosed compositions are shear-thinning and self-healing post injection. In some aspects, the guest macromers and host macromers of the injectable fibrous hydrogel have an association constant (Ka) of about 1×104 M−1 to about 1×105 M−1, or at least about 1×105 M−1.
Importantly, the fibrous hydrogels disclosed herein are flowable through a needle at about 12 mL h−1 (via a 16 gauge needle). The disclosed fibrous hydrogels can transform to a stable hydrogel plug post injection. The injectable fibrous hydrogel can comprise a self-assembling guest-host fibrous hydrogel configured as a cell carrier for injectable tissue engineering. In some aspects, the injectable fibrous hydrogel can be configured to mimic an extra cellular matrix (ECM) upon injection, optionally wherein the injectable fibrous hydrogel is configured to form a hierarchical assembly upon injection to provide physical cues to cells at different length scales, mimicking the 3D cues provided by a native fibrous ECM. The injectable fibrous hydrogels can further include one or more ligands, optionally one or more cell adhesion peptides, optionally a cell adhesion peptide comprising arginylglycylaspartic acid (RGD), and other thiolated molecutles including peptide and fibronectin fragments, to permit integrin-mediated cell adhesion.
The disclosed hydrogels can also be incorporated into injectable formulations. Such injectable formulations can further comprise one or more cells encapsulated in the fibrous hydrogel. Any suitable type of cell can be encapsulated, including for example but not limited to, hMSC, myoblasts (C2C12), fibroblasts (3T3), adipose-derived stem cells (ADSC), or pluripotent stem cells (PSC), all with varying applications. The one or more cells can have a post-injection survival rate of at least about 70%, optionally at least about 80%, optionally at least about 90%. In some embodiments, the injectable formulation can be configured for injection into a tissue of a subject, optionally a fibrous tissue, optionally a muscle, tendon, or ligament tissue. In some aspects, the fibrous hydrogel is flowable through a needle at about 12 mL h−1 (16 gauge needle), wherein the fibrous hydrogel is configured to transform to a stable hydrogel plug post injection. In some aspects, the injectable formulation comprises one or more pharmaceutically acceptable carriers or excipients. In some embodiments, the injectable formulation comprises a storage modulus (G′) of about 6.6 kPa at 1% fibrous content at 10 Hz, and a G′ of about 9.2 kPa at 5% fibrous content at 10 Hz.
Provided herein, and as detailed further in the working Examples, are methods of forming a fibrous hydrogel nanofiber in a polymer solution, the method comprising methacrylating a hyaluronic acid (HA) backbone via methacrylate esterification with a primary hydroxyl group of a sodium HA to form methacrylated HA (MeHA); synthesizing adamantane (Ad)-modified MeHA (Ad-MeHA) and β-cyclodextrin (CD)-modified MeHA (CD-MeHA) by anhydrous coupling; electrospinning the fibrous hydrogel nanofiber in the polymer solution; and crosslinking the fibrous hydrogel nanofiber by exposure to ultraviolet (UV) light. In some embodiments, a degree of methacrylate modification of HA is controlled by an amount of a methacrylic anhydride introduced during the methacrylating step, optionally wherein the degree of methacrylate modification of HA is about 10% to about 40%, optionally about 20% to about 30%, optionally about 28%. In some aspects, Ad-MeHA is prepared using 1-adamantane acetic acid via di-tert-butyl bicarbonate (BOC2O)/4-dimethylaminopyridine (DMAP) esterification, wherein CD-MeHA is prepared using CD-HDA via (benzotriazol-1-yloxy) tris(dimethylamino) phosphonium hexafluorophosphate (BOP) amidation. In some embodiments, the electrospinning comprises a collection plate set-up using an applied voltage of about 9.5-10.5 kV, a distance from needle to collector of about 16 cm, a needle gauge of about 20, and a flow rate of about 0.4 mL h−1. In some aspects, crosslinking the fibrous hydrogel nanofiber with UV light comprises exposure to UV light at about 320-390 nm for about 10-15 minutes, optionally about 365 nm for about 15 minutes. In some embodiments, such methods further comprise repeatedly triturating the hydrogel fibers via needle extrusion to produce short fiber segments of a length of about 5 μm to about 20 μm, optionally about 12.7±5.0 μm.
Finally, provided herein are methods of treating a tissue of a subject, the methods comprising providing a subject to be treated and delivering to a tissue of the subject an injectable fibrous hydrogel as disclosed herein. In some embodiments treating the tissue can be a component of treating a wide range of musculoskeletal conditions or diseases, in addition to further tissue applications, e.g. brain, adipose, or skin tissues. In some aspects, the injectable fibrous hydrogel is administered to the tissue to be treated by injection. In some embodiments, the tissue to be treated is selected from a fibrous tissue, optionally a muscle, tendon, or ligament tissue. In some embodiments, the injectable fibrous hydrogel comprises one or more encapsulated cells. In some aspects, the encapsulated cells have a higher viability post-injection when encapsulated in the fibrous hydrogel than when not encapsulated in the fibrous hydrogel, optionally a survivability rate of at least about 80%.
The following examples are included to further illustrate various embodiments of the presently disclosed subject matter. However, those of ordinary skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the presently disclosed subject matter.
Materials. Sodium hyaluronate (sodium HA, 64 kDa) was purchased from Lifecore Biosciences. β-cyclodextrin (CD), hexamethylenediamine (HDA), ammonium chloride, and β-Toluenesulfonyl chloride were purchased from TCI America. Tetrabutylammonium hydroxide (TBA-OH) was purchased from Acros Organics. All other materials were purchased from Sigma-Aldrich.
Synthesis of β-CD-HDA. Synthesis of 6-(6-aminohexyl)amino-6-deoxy-β-cyclodextrin (β-CD-HDA) was performed as previously described34,42, first via synthesis of the intermediate 6-o-monotosyl-6-deoxy-β-cyclodextrin (CD-Tos) using β-cyclodextrin (β-CD) and p-toluenesulfonyl chloride (TosCl). Briefly, β-CD was suspended in water and cooled to 0° C. TosCl was dissolved in minimal acetonitrile and added dropwise. After stirring at room temperature for 2 h, sodium hydroxide was added dropwise. The reaction was stirred at room temperature for 30 min before the addition of solid ammonium chloride to obtain a pH of 8.5. The solution was cooled on ice and the precipitate collected. The CD-Tos product was washed first with cold deionized (DI) water and then cold acetone before being dried under vacuum. After product confirmation using 1H NMR (500 MHz Varian Inova 500), a round bottom flask was charged with the CD-Tos, HDA, and dimethyl formamide (DMF). The reaction vessel was purged with nitrogen and then the reaction was allowed to proceed with constant nitrogen flow at 80° C. for 18 h. The product was precipitated in cold acetone (5×50 mL/g CD-Tos), washed with cold diethyl ether (3×100 mL), and dried. The degree of modification was determined by 1H NMR.
Synthesis of Ad-MeHA and CD-MeHA. Methacrylate modification of the HA backbone was achieved via methacrylate esterification with the primary hydroxyl group of sodium HA at basic pH48,49. The degree of methacrylate modification was controlled by the amount of methacrylic anhydride introduced during synthesis and was determined to be 28% of the HA disaccharides via 1H NMR (
Electrospinning. Ad-MeHA, or CD-MeHA, was dissolved at 2% (w/v) in DI water along with 3.5% (w/v) poly(ethylene oxide) (PEO, 900 kDa) and 0.05% (w/v) Irgacure 2959 (I2959) for 24-48 h prior to electrospinning. The polymer solutions were electrospun using a Spraybase (Kildare, Ireland) collection plate set-up and using the following collection parameters: applied voltage: 9.5-10.5 kV, distance from needle to collector: 16 cm, needle gauge: 20, flow rate: 0.4 mL h−1. Hydrogel nanofibers were deposited onto foil covering the collector plate, placed into a container which was purged with nitrogen, and crosslinked with UV light (365 nm) for 15 minutes. Electrospinning parameters were chosen based on previous work with MeHA50 and CD-MeHA51.
Nanofiber imaging and morphological characterization. To measure the diameters of the Ad-MeHA and CD-MeHA fibers, samples were electrospun onto foil. After electrospinning, samples were photocrosslinked and analyzed in both dry and swollen states. Dry fibers were imaged using scanning electron microscopy (SEM, FEI Quanta 650) at a magnification of 10,000×. To visualize swollen fibers, a methacrylated rhodamine dye (MeRho, Polysciences, 2 mM) was incorporated prior to electrospinning. Rhodamine-labeled fibers were hydrated, and broken up via trituration through increasingly smaller needle gauges (16G-30G) before encapsulation in a 2% (w/v) MeHA hydrogel to facilitate image analysis. Fibers were encapsulated at 0.2% (w/v) for hydrated fiber diameter quantification and at 0.05% (w/v) for post-trituation fiber length quantification. The hydrated fibers were allowed to equilibrate within the MeHA hydrogels overnight in PBS before being imaged using confocal microscopy (Leica inverted confocal microscope, DMi8). Fiber diameters (n=300 fibers per group) and fiber lengths (n=90) were quantified from the resulting SEM (dry fibers) and confocal images (hydrated fibers) using ImageJ (NIH). The corresponding violin plots were made using R. The fiber mass swelling ratio QM for the Ad-MeHA and the CD-MeHA hydrogel fibers was calculated using the equation:
Hydrogel formulations. Collected guest Ad-MeHA and host CD-MeHA fibers were hydrated (0.1% w/v in DI water) overnight at 37° C. to remove PEO from the electrospinning process52, centrifuged, supernatant discarded, and then lyophilized. Once dry, the fibers were again hydrated at 0.1% w/v in DI water, allowed to swell at 37° C. for at least 2 hours, and then passed through needles of progressively smaller gauge sizes via trituration. Starting with a 16G needle and progressing up to a 30G needle, the fibers were passed through the needles until the fiber solution could easily pass through the needle. This process separated any adjoined fibers and resulted in a reproducible fiber suspension. Fiber density was calculated as the weight percentage of the dry fiber mass per volume solvent (w/v). When determining the volume, the dry fibers were first hydrated in a known volume of DI water and allowed to swell overnight. Hydrated fibers were then centrifuged to remove excess liquid. Finally, water was added back to the hydrated fibers to achieve the desired w/v density28. For mechanical testing, the complementary guest and host hydrogel fibers were gently mixed together directly on the rheology plate to create the mixed fibrous guest-host network. Non-fibrous MeHA and guest-host (Ad-MeHA/CD-MeHA) hydrogel groups were prepared as 3% (w/v) solutions and underwent covalent crosslinking (for mechanical stabilization) via photopolymerization in the presence of UV light (365 nm, 10 mW cm−2) and 1 mM lithium acylphosphinate (LAP) photoinitiator for 5 min. LAP was used as the photoinitiator for cell culture studies due to its ability to facilitate increased polymerization rates under cytocompatible longer wavelength (365-405 nm) light, even at lower intensities, when compared to 1295953. However, since dense electrospinning solutions take 1-2 days to homogenize (and since cells are not present in electrospinning), it is advantageous to use the less efficient photoinitiator 12959 to prevent solution gelation prior to electrospinning.
Rheology. All rheological measurements were performed on an Anton Paar MCR 302 rheometer with the plate temperature set at 37° C. The non-fibrous MeHA and guest-host hydrogels were characterized using a cone-plate geometry (25 mm diameter, 0.5°, 25 μm gap) immediately following photopolymerization while guest-host fibrous hydrogels were characterized using a parallel-plate geometry (25 mm diameter, 25 μm gap). Mechanical properties for the non-fibrous MeHA and guest-host hydrogel groups were tested using oscillatory frequency sweeps (0.1-10 Hz, 0.5% strain). Fibrous guest-host hydrogels mechanical properties were tested using oscillatory frequency sweeps (also 0.1-10 Hz, 0.5% strain), strain sweeps, and cyclic deformation tests alternating between 0.5% and 250% strain to assess shear-thinning and self-healing capabilities42.
Cell culture. Human mesenchymal stromal cells (hMSCs, Lonza) were used at passage 7 for all experiments. Culture media contained Gibco minimum essential medium (MEM-α) supplemented with 20 v/v % fetal bovine serum (Gibco) and 1 v/v % penicillin/streptomycin/amphotericin B (1000 U/mL, 1000 μg/mL, and 0.25 μg/mL final concentrations, respectively, Gibco). Prior to cell encapsulation, all materials were modified with thiolated Arginylglycylaspartic acid (RGD) peptide (Genscript) via Michael-type addition in 0.2 M triethanolamine at pH 8, dialyzed against DI water, and then lyophilized. The final RGD concentration was 1 mM for all hydrogel formulations used for cell culture. After materials were put into solution (non-fibrous MeHA and guest-host hydrogels) or hydrated (guest-host fibers), the materials were sterilized using germicidal UV irradiation for 3 h. Prior to the addition of cells, guest-host fibers were centrifuged briefly, with excess liquid aspirated under sterile conditions. To evaluate cell protection during injection under needle flow, hMSCs were added to the non-fibrous hydrogels or hydrogel fibers such that the final encapsulation density was 1×106 cells/mL. Prior to UV exposure, materials with cells were loaded into 1 mL tuberculin syringes and hydrogels were injected through a 16G needle at 12 mL h−1 using a syringe pump. The extruded material was used to make hydrogel plugs with identical volumes of 40 μL. All groups were subjected to 5 min UV polymerization post-injection to ensure that differences in cell viability were not due to UV cytotoxicity. For all cell culture experiments, media was replaced every 2-3 days.
Confocal imaging and analysis. Cell viability was assessed with a LIVE/DEAD assay kit (Invitrogen) by incubating hydrogels at 37° C. for 30 min with 2 mM calcein-AM and 4 mM ethidium homodimer-1 in PBS plus glucose (G-PBS, glucose 25 mM) followed by three PBS washes. Cells were imaged using a Leica DMi8 inverted confocal microscope and live/dead cells were quantified from 50 μm thick z-stacks (10 μm slices) using ImageJ's 3D object counter tool. Cell projected area, cell shape index (CSI), and aspect ratio were determined using ImageJ's measurement tool. CSI determines the circularity of the cell, where a line and a circle have CSI values of 0 and 1, respectively, and was calculated using the formula
where A is the cell projected area and P is the cell perimeter. Individual measurements were made for 60 cells from at least 6 hydrogels per experimental group.
Statistical Analysis. All experimental groups included at least 6 hydrogels for analysis of cell viability and shape. Cell viability and shape data were statistically analyzed using one-way analysis of variance (ANOVA) followed by Tukey's HSD post-hoc testing. These statistical analyses were conducted using GraphPad Prism 9.0 and R. P values <0.05 were considered statistically significant. For analysis of hydrogel rheological properties, 3 hydrogels were tested per group where the mean values are plotted. Bar graph heights correspond to the mean with standard deviation error bars and individual data points are included as scatter plots overlaying the bars. Tukey box plots of individual cell data show the second and third quartiles as boxes, the median as a line between the boxes, and error bars with the lower value of either 1.5 times the interquartile range or the maximum/minimum value. Data points outside this range are shown individually.
Guest-Host Hydrogel Fiber Design, Synthesis, and Quantification of Fiber Properties.
Disclosed herein is the design, synthesis, and characterization of a self-assembling guest-host fibrous hydrogel that can serve as a cell carrier for injectable tissue engineering applications. Hydrophobic Ad (guest) groups associate strongly with the hydrophobic core of CD (host) molecules resulting in a stable supramolecular, yet reversible, guest-host interaction (association constant, Ka=1×105 M−1) (
To create the electrospun hydrogel nanofibers capable of supramolecular assembly, Ad-MeHA and CD-MeHA (2% w/v) aqueous polymer solutions were mixed separately with poly(ethylene oxide) (PEO) and the photoinitiator Irgacure 2959 (I2959), and then electrospun to produce guest and host fiber populations (
Upon hydration, the hydrophilic nanofibers imbibe water, resulting in significant fiber swelling and increased diameter. The hydrated Ad-MeHA nanofibers swelled to an average diameter of 2.16±0.92 μm and hydrated CD-MeHA nanofibers swelled to an average diameter of 1.65±0.54 μm. SEM analysis of native fibrous ECMs has reported fibril diameters in the range of 75-400 nm57,58 with small type I collagen fibers in the 1-5 μm range59. Therefore, these CD-MeHA and Ad-MeHA hydrogel nanofibers are within the physiologically relevant range for fibrous ECM components15. In addition, both the distribution of hydrogel nanofiber diameters and the morphological change of the nanofibers following hydration (and the resulting PEO extraction) were observed. Calculated swelling ratios for the guest Ad-MeHA fibers and host CD-MeHA fibers were 93±15 and 49±4.8 respectively. These results correlate with the greater degree of swelling in the Ad-MeHA fibers seen in
Guest-Host-Assembled Fibers Show Mechanical Integrity as Well as Shear-Thinning and Self-Healing Character.
With the stable structure of the hydrated electrospun hydrogel nanofibers confirmed via confocal microscopy, mechanical properties of the fibrous hydrogels were then characterized via oscillatory shear rheology (
Biopolymer density can also be adjusted to tune mechanical properties. Fibrous hydrogel matrices formed with 5% fiber density (also 2:1 Ad:CD molar ratio) showed improved storage moduli over the 1% fiber density formulation (
Having demonstrated the guest-host assembly of the functionalized HA hydrogel fibers, efforts were next taken to assess their shear-thinning and self-healing capabilities. Strain sweep testing of the fibrous guest-host hydrogel showed loss moduli crossover at strains greater than 100%, highlighting the potential for injectability (
Studies using non-fibrous Ad-CD guest-host assembled hydrogels demonstrated the tunable mechanics and flow characteristics of the guest-host assembly by altering the guest-host pair ratio and density as well as network structure42,72,73. The fibrous hydrogel developed and disclosed herein displays analogous rheological behavior to non-fibrous guest-host hydrogels. However, a surprising advantage over the non-fibrous hydrogels is that as a result of the dynamic bonding interactions between fibers, the guest-host fibrous hydrogel is capable of shear-induced flow (injectability) and rapid recovery. Overall, rheological analysis of the guest-host fibrous hydrogel demonstrated properties of robust mechanical integrity, shear-thinning, and rapid recovery for stability post-injection. Indeed, the fibrous hydrogel scaffold was readily injectable, flowing easily through a needle (12 mL h−1, 16G) and recovered as a stable hydrogel plug.
Injected hMSCs Encapsulated in Fibrous Hydrogels are Viable and Show Increased Spreading Compared to Non-Fibrous Hydrogels.
Experiments were next designed to test the ability of the fibrous guest-host hydrogel to support sustained viability and spreading of encapsulated hMSCs following injection. hMSCs were chosen for these experiments due to their multipotential for differentiation toward cell types relevant for a broad range of fibrous tissues74 such as muscle, tendon, and ligament75. Protecting cells during injection and preserving high viability is one of the fundamental requirements for subsequent therapeutic application, but many injectable delivery vehicles suffer from poor cell survival76,77. Previous studies have attempted to address this issue using materials that leverage physical crosslinking since their compliant mechanical properties support non-uniform network deformation40,68,78. However, in these non-uniform matrices, mechanical stresses imposed on the material do not induce uniform strain fields allowing network deformations to dissipate stress8. Therefore, there remained a need to address this deficiency, which led to the design and discovery that the disclosed guest-host fibers could mitigate cell-damaging forces during injection due to the force dissipation capabilities of shear-thinning materials.
The post-injection viability and spreading of hMSCs when cultured in three hydrogel groups: non-fibrous MeHA, non-fibrous guest-host, and fibrous guest-host (
Similar to the initial cell viability, the live cell fractions observed on days 3 and 7 were largely the same (day 3: non-fibrous MeHA: 83±3% and guest-host: 83±3% versus the fibrous guest-host hydrogel: 82±6%), although on day 7 there was a statistically significant difference in viability between the non-fibrous MeHA hydrogel group compared to both guest-host hydrogel groups (non-fibrous: 86±2%, fibrous: 87±3%). Although hMSC viability was similar across all experimental groups, qualitative differences in cell shape/spreading observed at day 7 provided motivation to quantify hMSC shape metrics such as projected cell area, cell shape index (CSI, a measure of cell circularity), and aspect ratio (
Confocal imaging of hMSCs in fibrous networks showed direct hMSC interactions with fiber segments (
In summary, the present disclosure details the design and fabrication of an injectable fibrous hydrogel providing significant advantages over existing hydrogels, including for example, the capabilities of shear-thinning and self-healing under physiologic conditions. Using HA as a naturally-derived polymer, these materials offer unique advantages with respect to biocompatibility and mechanical integrity (G′=6.6±2.0 kPa) while also recapitulating the fibrous architecture of tissue. In addition to exhibiting shear-thinning and self-healing behavior, the guest-host fibrous hydrogel demonstrated injectability wherein encapsulated hMSCs were protected from membrane-disrupting shear forces during injection, resulting in sustained 3D hMSC viability (greater than 85%). Cells were also more spread and elongated in viscoelastic guest-host hydrogels compared to non-fibrous elastic hydrogel controls, with hMSCs showing significantly reduced CSI (circularity) in fibrous guest-host hydrogels compared to both non-fibrous MeHA and non-fibrous guest-host hydrogels. In summary, the injectable fibrous hydrogel platform introduced here offers the ability to broaden minimally invasive delivery to musculoskeletal tissue engineering applications requiring robust structural properties while also laying the foundation for future opportunities in 3D bioprinting and fundamental studies of cell-microenvironment interactions.
All references listed herein including but not limited to all patents, patent applications and publications thereof, scientific journal articles, and database entries (e.g., GENBANK® database entries and all annotations available therein) are incorporated herein by reference in their entireties to the extent that they supplement, explain, provide a background for, or teach methodology, techniques, and/or compositions employed herein.
It will be understood that various details of the presently disclosed subject matter may be changed without departing from the scope of the presently disclosed subject matter. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation.
This application is a U.S. National Phase Application of PCT International Patent Application No. PCT/US2022/016744, filed Feb. 17, 2022, incorporated by reference in its entirety, and which claims benefit of U.S. Provisional Patent Application Ser. No. 63/150,328, filed Feb. 17, 2021, the disclosure of which is herein incorporated by reference in its entirety.
This invention was made with government support under Grant Nos. R21AR075181 awarded by the National Institutes of Health and W81XWH-19-1-0157 awarded by the Department of Defense. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/016744 | 2/17/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63150328 | Feb 2021 | US |
