Anionic citrate-based biomaterials

Abstract

This disclosure provides anionic citrate-based compositions for use in regeneration applications. The disclosed compositions may be in the form of scaffolds and generally comprise (i) a citrate component, (ii) a diol, and (iii) an anionic moiety. The anionic moiety may comprise one or more sulfates, sulfonates, phosphates, phosphonates, sulfamates, or carboxylic acids. The sulfate and / or sulfonate-containing moieties may be conjugated with sulfur trioxide complexes by amidation, esterification, and / or substitution reactions. Peptides may be conjugated on the surface of the composition.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application was filed on June 7, 2023, and is assigned Ser. No. 63 / 471,567, and claims the benefit of priority of a U.S. Provisional Patent Application entitled “Anionic Citrate - Based Biomaterials”. The entire contents of the prior U.S. Provisional Patent Application are incorporated herein by reference.

[0002] Background 1. Technical Field This disclosure refers to anionic citrate - based compositions with controlled binding and release of proteins for regenerative engineering applications.

Background Art

[0003] 2. Background Art Despite the ability of bone to regenerate after injury, delayed healing or nonunion (pseudarthrosis) in orthopedics is a significant clinical problem (Chen C. H., Hsu E. L., Stupp S. I. Supramolecular self - assembling peptides to deliver bone morphogenetic proteins for skeletal regeneration. Bone. Vol. 141, December 2020, 115565). Among the 8 million annual fracture repair cases in the United States, more than 10% of cases result in nonunion, and it has been reported that the rate is very high in smokers, diabetics, and osteoporotic patients (Buza J. A. 3rd; Einhorn T. Bone healing in 2016. Clinical Cases Mineral and Bone Metabolism 2016, 13(2), 101 - 105).

[0004] Autologous bone grafting from the iliac crest is considered the highest standard of clinical practice for osteoplasia in the repair and fusion of large bone defects (Dohzono S., Imai Y., Nakamura H., Wakitani S., Takaoka K. Successful spinal fusion by E. coli-derived BMP-2-adsorbed porous beta-TCP granules: a pilot study. Clinical Orthopaedics and Related Research. December 2009, 467(12), 3206~3212). However, site deformation, pain, infection, and limited supply are the main drawbacks associated with harvesting iliac crest grafts (Arrington ED, Smith WJ, Chambers HG, Bucknell AL, Davino NA Complications of iliac crest bone graft harvesting. Clinical Orthopaedics and Related Research. 1996, 329, 300~309). These limitations, combined with advances in bone biology, are leading researchers to move towards alternative approaches using bioactive molecules and materials. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] For example, bone morphogenetic proteins (BMPs) are naturally occurring, bioactive molecules that can form bone ectopically (Urist MR Bone - formation by Autoinduction. Science. 1965, 150(3698), 893~899). A cure rate of 92% has been reported for BMP treatment in patients with nonunion (Zhou YQ, Tu HL, Duan YJ, Chen X. Comparison of bone morphogenetic protein and autologous grafting in the treatment of limb long bone nonunion: a systematic review and meta-analysis. Journey of Orthopedic Surgery and Research. 2020, 15(1), 288). However, side effects such as inflammatory responses, soft tissue hematomas, bone cysts, ectopic osteogenesis, cancer, retrograde ejaculation, infection, and nerve root damage have been associated with BMP treatment (El Bialy I., Jiskoot W., Reza Nejadnik M. Formulation, Delivery and Stability of Bone Morphogenetic Proteins for Effective Bone Regeneration. Pharmaceutical Research. 2017, 34(6), 1152~1170). It has been widely reported that the main cause of these adverse reactions is the administration of high doses of BMP (Mroz TE, Wang JC, Hashimoto R., Norvell DC Complications related to Osteobiologics use in spine surgery a systematic review. Spine. 2010, 35(9), S86~S104.).For example, Medtronic's INFUSE bone graft kit utilizes 1.5 mg / ml of BMP-2, which is more than 1 million times the physiological concentration during normal bone repair conditions (Gamradt SC, Lieberman JR Genetic modification of stem cells to enhance bone repair. Annals Biomedical Engineering. 2004, 32(1), 136~147).

[0006] The efficacy of BMP and some of the reported side effects have been shown to be controllable through improvements in its delivery system. Current BMP delivery systems are collagen-based due to collagen's biocompatibility and its ability to be processed into powders, films, gels, fibers, and absorbent sponges (Lee SH, Shin H. Matrices and scaffolds for delivery of bioactive molecules in bone and cartilage tissue engineering. Advanced Drug Delivery Review. 2007, 59(4~5), 339~359.). However, in vivo studies have shown that due to rapid initial release, only 5% of BMP remains in the collagen sponge after two weeks (Geiger M., Li RH, Friess W. Collagen sponges for bone regeneration with rhBMP-2. Advanced Drug Delivery Review. 2003, 55(12), 1613~1629).

[0007] To address this limitation in collagen-based BMP delivery, much research has focused on designing delivery systems for biomaterials that improve protein binding capacity and reduce the required dose of BMP-2. Many studies have attempted to reduce the dose of BMP-2 and decrease its release rate by binding heparin to its carrier. Heparin is a highly sulfurized glycosaminoglycan, is electrostatically charged, and induces electrostatic interactions with growth factors such as BMP-2, forming polyelectrolyte complexes (Terauchi M., Tamura A., Tonegawa A., Yamaguchi S., Yoda T., Yui N. Polyelectrolyte complexes between polycarboxylates and BMP-2 for enhancing osteogenic differentiation: effect of chemical structure of polycarboxylates. Polymers. 2019, 11(8), 1327~1327). However, the main drawback of using heparin to induce growth factors is its anticoagulant effect, which can increase the risk of complications during surgery and healing (Terauchi et al.).

[0008] For at least the reasons stated above, there is a demand for biocompatible compositions with improved protein binding and controlled protein release capabilities for regenerative engineering applications, such as the replacement of damaged tissue. [Means for solving the problem]

[0009] In accordance with this disclosure, highly advantageous anionic citrate compositions can be produced and used to control the binding and release of proteins through polyelectrolyte complexes.

[0010] In exemplary embodiments, the disclosed compositions comprise (i) a citrate component, (ii) a polyol, and (iii) an anionic moiety. The citrate component may comprise one or more citric acid, citrate salts, or esters of citric acid. The polyol may comprise one or more diols, such as butanediol, hexanediol, octanediol, or polyethylene glycol. Other exemplary polyols intended in accordance with this disclosure include one or more glycerol, β-glycerol phosphate, or xylitol.

[0011] The anionic portion may comprise one or more sulfates, sulfonates, phosphates, phosphonates, sulfamates, or carboxylic acids. In certain exemplary embodiments, the sulfate and / or sulfonate-containing portion may further comprise one or more hydroxyls, amines, or carboxylic acids. In other exemplary embodiments, the sulfate and / or sulfonate-containing portion may comprise one or more taurine, isethionic acid, or sodium 2-carboxyethane-1-sulfonate.

[0012] In some exemplary embodiments, the sulfate and / or sulfonate-containing portion may be conjugated with a sulfur trioxide complex via amidation, esterification, and / or substitution reactions. In more specific examples, the amidation reaction may be facilitated by 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride (EDC).

[0013] In some embodiments, the portion containing the phosphate and phosphonate may include one or more hydroxyls, amines, or carboxylic acids. In more specific examples, the portion containing the phosphate and phosphonate may include 3-(phosphonooxy)propanoic acid or 2-hydroxyethyl phosphate. In other exemplary embodiments, the anionic portion may further include one or more alkenes, alkynes, halogens, borates, or azides.

[0014] In exemplary embodiments, the composition may further comprise a peptide conjugated on the surface of the composition. In some embodiments, the peptide is a protein-binding peptide. In other embodiments, the peptide is a protein-mimicking peptide.

[0015] In exemplary embodiments, the composition may further comprise granular inorganic material. The granular inorganic material may comprise one or more hydroxyapatite, tricalcium phosphate, biphasic calcium phosphate, or bioglass. In some embodiments, the granular inorganic material is present in an amount of 10 to 60% by weight of the composition. In certain embodiments, the granular inorganic material is microsized or nanosized. In some exemplary embodiments, the granular inorganic material is rod-shaped. In other exemplary embodiments, the granular inorganic material is fibrous.

[0016] In some exemplary embodiments, the scaffold may be formed in part from the disclosed composition, wherein the scaffold is biodegradable. In some embodiments, the scaffold is 50-90% porous. In exemplary embodiments, the scaffold contains a gradient porous structure.

[0017] In some exemplary embodiments, the composition is bound to a protein through electrostatic interactions. In some embodiments, the protein is one or more of the following: bone morphogenetic proteins (BMPs), basic fibroblast growth factor (bFGF), transforming growth factor β (TGF-β1), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and / or epidermal growth factor (EGF). In more specific examples, the BMP may include BMP-2, BMP-4, or BMP-7.

[0018] Additional properties, functions, and advantages of the disclosed citrate-based compositions may become apparent from the following description of exemplary embodiments. [Brief explanation of the drawing]

[0019] The accompanying drawings are provided for assistance to those skilled in the art in the preparation and use of the disclosed citrate-based compositions.

[0020] [Figure 1] Figure 1 is a schematic diagram of the chemical structure of POXC-IA as described herein. [Figure 2] Figure 2 is a schematic diagram of the chemical structure of POXC-IA and its associated 1H-NMR spectrum, demonstrating the successful addition of IA as described herein. [Figure 3] Figure 3 is a schematic diagram of sulfate / sulfonate conjugation on the surface of citrate-based biomaterials, showing the use of free carboxylic acids (conjugation "A") or hydroxyl groups (conjugation "B"). [Figure 4-1]Figure 4 includes four panels. Panel A shows a poly(octamethylene-xylitol citrate) (POXC) scaffold modified such that its surface presents sulfate and / or sulfonate groups. Panel B shows a high-resolution X-ray photoelectron spectroscopy (XPS) spectrum of S2p. Panel C shows the sulfate / sulfonate content obtained from the integration of the high-resolution XPS spectra of S2p and C1s (this is defined as the ratio of the content of S atoms and C atoms, and the C atom content for POXC-IA-Ta was corrected by subtracting the C atoms derived from taurine). Panel D shows the surface zeta potential of scaffolds of INFUSE collagen (Medtronic plc, Minneapolis, MN), POXC, POXC-IA-Ta, and POXC-IA-SO3Na at different pHs. [Figure 4-2] Figure 4 includes four panels. Panel A shows a poly(octamethylene-xylitol citrate) (POXC) scaffold modified such that its surface presents sulfate and / or sulfonate groups. Panel B shows a high-resolution X-ray photoelectron spectroscopy (XPS) spectrum of S2p. Panel C shows the sulfate / sulfonate content obtained from the integration of the high-resolution XPS spectra of S2p and C1s (this is defined as the ratio of the content of S atoms and C atoms, and the C atom content for POXC-IA-Ta was corrected by subtracting the C atoms derived from taurine). Panel D shows the surface zeta potential of scaffolds of INFUSE collagen (Medtronic plc, Minneapolis, MN), POXC, POXC-IA-Ta, and POXC-IA-SO3Na at different pHs. [Figure 5] Figure 5 is a plot showing the cumulative rhBMP-2 release from an INFUSE collagen scaffold and a POXC scaffold modified with sulfate / sulfonate. **Mode for Carrying Out the Invention**

[0021] Description of Exemplary Embodiments As described above, the present disclosure provides highly advantageous anionic citrate-based compositions for the control of protein binding and release. In certain embodiments, the present disclosure provides compositions comprising (i) a citrate component, (ii) a polyol, and (iii) an anionic moiety.

[0022] Citric acid is an inexpensive, non-toxic, and naturally occurring metabolite that is related to bone anatomy and physiology through the control of apatite nanocrystal growth and cellular energy production. Furthermore, citrate-based biomaterials present useful chemical functional groups for bulk chemistry modification and surface conjugation chemistry. In addition to the chemical properties of citrate, citrate-based biomaterials provide unique elastomeric properties that can aid in the development of native tissue during regeneration. As used herein, the terms “citrate-based composition” and “citric-based biomaterial” are generally interchangeable and refer to polymer compounds generated by reacting citric acid with diol monomers to produce polymers having a backbone containing hydrolyzable ester bonds.

[0023] Citrate-based biomaterials are rich in the bulk chemistry of carboxyl and hydroxyl, which can be modified with anionic moieties through monomer addition or surface modification. In exemplary embodiments, the anionic moiety can include one or more of sulfate, sulfonate, phosphate, phosphonate, sulfamate, or carboxylic acid. For example, monomers containing sulfate and sulfonate (including, but not limited to, taurine or its salt form and isethionic acid or its salt form) can react with the free carboxylic acid groups of citric acid through esterification or amidation reactions.

[0024] For example, the addition of sodium isethionate (IA) to a mixture of citric acid, xylitol, and 1,8-octanediol yields poly(octamethylene-xylitol citrate) (POXC) with sodium isethionate attached, forming POXC-IA (this structure is shown in Figure 1). Proton nuclear magnetic resonance of the polymer precursor ( 1 The 1H NMR characterization demonstrates the successful introduction of the sulfonate moiety into citrate polymers, such as POXC-IA (see Figure 2). As shown in Figure 2, characteristic peaks originating from the isethionic acid moiety are located at 4.22–4.16 ppm and 2.70–2.62 ppm.

[0025] In other exemplary embodiments, sulfates and / or sulfonates may be conjugated onto the surface of citrate-based biomaterials using free carboxylic acids (see Figure 3: Conjugation "A") and / or hydroxyl groups (see Figure 3: Conjugation "B").

[0026] Figure 4, comprising four panels (Figures A-D), illustrates exemplary embodiments of modified citrate-based polymer scaffolds having sulfate and / or sulfonate-containing portions. Specifically, panel A shows the surface of a poly(octamethylene-xylitol citrate) (POXC) scaffold modified to present sulfate and / or sulfonate. Panel B shows the high-resolution X-ray photoelectron spectroscopy (XPS) spectrum of S2p. Panel C shows the sulfate / sulfonate content obtained from the integration of the high-resolution XPS spectra of S2p and C1s, which is defined as the ratio of S and C atom content. The C atom content for POXC-IA-Ta was corrected by subtracting C atoms from taurine. Panel D shows the surface zeta potentials of INFUSE collagen, POXC, POXC-IA-Ta, and POXC-IA-SO3Na scaffolds at different pH levels.

[0027] To produce a porous polymer scaffold, a polymer precursor of POXC or POXC-IA was mixed with sodium chloride and cured at 80°C for 7 days. The salt was leached with deionized water. The resulting POXC-IA scaffold may be immersed in a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS) / taurine solution and shaken at room temperature for 24 hours to form a scaffold having both isethionic acid (IA) and sodium taurine salt (Ta), as shown in panel A of Figure 4, POXC-IA-Ta. The success of surface conjugation was confirmed by X-ray photoelectron spectroscopy (XPS), which showed an increase in sulfur peak intensity (see panels B and C of Figure 4). As shown in panel B of Figure 4, the S2p peak centered at approximately 168 eV corresponds to the CSO bond. The sulfonate content can be defined as the ratio of sulfur to carbon (S / C) content obtained from the integration of high-resolution XPS spectra of S2p and C1s. As shown in panel C of Figure 4, the S / C ratio increases from 0 in the POXC scaffold to 0.00785 in POXC-IA and to 0.00954 in POXC-IA-Ta.

[0028] As another example, surface modification of POXC-IA scaffolds can be achieved by immersion in a sulfur trioxide triethylamine complex in DMF for 24 hours at 35°C. The substitution reaction on the pendant hydroxyl group leads to a scaffold having both isethionic and sulfate groups, such as POXC-IA-SO3Na shown in panel A of Figure 4. As shown in panels B and C of Figure 4, XPS characterization confirms the success of the surface modification, with a significant increase in sulfate and sulfonate content. As shown in panel B of Figure 4, POXC-IA-SO3Na exhibits a strong S2p peak centered at approximately 168 eV. As shown in panel C of Figure 4, the S / C ratio in POXC-IA-SO3Na has increased to 0.250.

[0029] In exemplary embodiments, the sulfate / sulfonate content can be adjusted by the amount of sulfiding agent and / or the reaction temperature. The sulfate / sulfonate content efficiently modulates the surface charge of these citrate polymer scaffolds. As shown in panel D of Figure 4, the surface zeta potential increases with increasing sulfate / sulfonate content at pH 4–10. It is noteworthy that the surface zeta potential of POXC scaffolds is higher than that of commercially available INFUSE collagen sponges (Medtronic plc, Minneapolis, MN).

[0030] To demonstrate the protein-binding and protein-releasing capabilities of sulfate and / or sulfonate-modified citrate materials, recombinant human BMP-2 (rhBMP-2) was used as a model protein in experimental studies. Specifically, 1 μg of rhBMP-2 was packed into INFUSE collagen scaffolds, POXC scaffolds, and sulfurized / sulfonated POXC scaffolds modified to varying degrees of sulfurization / sulfonation. Subsequently, cumulative rhBMP-2 release was measured on days 1, 2, 3, 7, 14, and 21 using the BMP-2 ELISA kit (R&D Systems, Minneapolis, MN).

[0031] As shown in Figure 5, rhBMP-2 release from the INFUSE collagen scaffold resulted in a rapid increase of 71.17 ± 5.16% after 24 hours. After 7 days, 99.14 ± 5.72% had been released from the INFUSE collagen scaffold. In contrast, rhBMP-2 release from the POXC scaffold and modified POXC scaffold was slow and controlled by the degree of sulfidation / sulfonation. The measured results are consistent with the surface zeta potential, confirming that surface charge plays a crucial role in controlling rhBMP-2 binding and release.

[0032] For example, as shown in Figure 5, after 24 hours, rhBMP-2 release was 27.02 ± 8.26%, 13.42 ± 2.01%, and 6.33 ± 2.29% for the POXC, POXC-IA-Ta (S / C = 0.00954), and POXC-IA-SO3Na (S / C = 0.0250) scaffolds, respectively. The slow and stable rhBMP-2 release continued after 28 days, with only 59.74 ± 11.71% of rhBMP-2 released from POXC, only 32.22 ± 4.26% from the POXC-IA-Ta scaffold, and only 16.85 ± 2.97% from the POXC-IA-SO3Na scaffold.

[0033] As shown in Figure 5, unmodified POXC scaffolds can also sequester BMP-2 more efficiently than the absorbent collagen sponges used in INFUSE.

[0034] In some exemplary embodiments, compositions disclosed for protein binding for regenerative engineering comprise (i) a citrate component and (ii) a polyol. Experimental data presented herein demonstrate that release can be modulated by the addition of other load electroactive groups, such as sulfates / sulfonates.

[0035] Based on the experimental results presented herein, those skilled in the art can expect to obtain similar results using other anions, for example, monomers containing phosphates, phosphonates, sulfamates, and carboxylates, but not limited to those listed above. This is due to the ability of these anions to increase the surface zeta potential in a manner similar to that of sulfates and sulfonates presented herein. The BMP-2 release data shows a direct correlation with the zeta potential on the scaffold surface. Finally, those skilled in the art can also expect that other heparin-binding growth factors, such as bFGF, TGF-β1, VEGF, PDGF, and EGF, will also be retained by the chemical properties presented above.

[0036] This disclosure is described with reference to exemplary embodiments and examples, but is not limited to such exemplary embodiments / examples.

Claims

1. (i) Citrate components, (ii) polyols, and (iii) Anionic portion A composition containing the following:

2. The composition according to claim 1, wherein the citrate component comprises one or more citric acid, citrate salts, or esters of citric acid.

3. The composition according to claim 1, wherein the polyol comprises a diol.

4. The composition according to claim 3, wherein the diol comprises one or more butanediol, hexanediol, octanediol, or polyethyleneglycerol.

5. The composition according to claim 1, wherein the polyol comprises one or more of glycerol, β-glycerol phosphate, or xylitol.

6. The composition according to claim 1, wherein the anionic portion comprises one or more sulfates, sulfonates, phosphates, phosphonates, sulfamates, or carboxylic acids.

7. The composition according to claim 6, wherein the portion containing sulfate and / or sulfonate further comprises one or more hydroxyls, amines, or carboxylic acids.

8. The composition according to claim 6, wherein the portion containing sulfate and / or sulfonate comprises one or more of taurine, isethionic acid, or sodium 2-carboxyethane-1-sulfonate.

9. The composition according to claim 6, wherein the portion containing sulfate and / or sulfonate is conjugated with a sulfur trioxide complex via an amidation reaction, esterification, and / or substitution reaction.

10. The composition according to claim 9, wherein the amidation reaction is accelerated by 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride (EDC).

11. The composition according to claim 6, wherein the portion containing the phosphate and phosphonate comprises one or more hydroxyls, amines, or carboxylic acids.

12. The composition according to claim 11, wherein the portion containing the phosphate and phosphonate comprises 3-(phosphonooxy)propanoic acid or 2-hydroxyethyl phosphate.

13. The composition according to claim 6, wherein the anionic portion further comprises one or more alkenes, alkynes, halogens, borates, or azides.

14. The composition according to claim 1, wherein the peptide is conjugated on the surface of the composition.

15. The composition according to claim 14, wherein the peptide is a protein-binding peptide.

16. The composition according to claim 14, wherein the peptide is a protein-mimetic peptide.

17. The composition according to claim 1, wherein the composition further comprises a granular inorganic material.

18. The composition according to claim 17, wherein the granular inorganic material comprises one or more of hydroxyapatite, tricalcium phosphate, biphasic calcium phosphate, or bioglass.

19. The composition according to claim 17, wherein the granular inorganic material is present in an amount of 10 to 60% by weight of the composition.

20. The composition according to claim 17, wherein the granular inorganic material is micro-sized or nano-sized.

21. The composition according to claim 17, wherein the granular inorganic material is formed into a rod shape.

22. The composition according to claim 17, wherein the granular inorganic material is fibrous.

23. The composition according to claim 1, wherein the citrate component and the polyol react to form a polymer.

24. A scaffold formed in part from the composition described in claim 1, wherein the scaffold is biodegradable.

25. The composition according to claim 22, wherein the scaffold is 50-90% porous.

26. The composition according to claim 22, wherein the scaffold contains a gradient porous structure.

27. The composition according to claim 1, wherein the composition is bound to a protein through electrostatic interactions.

28. The composition according to claim 27, wherein the protein is one or more of bone morphogenetic protein (BMP), basic fibroblast growth factor (bFGF), transforming growth factor β (TGF-β1), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and / or epidermal growth factor (EGF).

29. The composition according to claim 28, wherein the BMP comprises one or more of BMP-2, BMP-4, or BMP-7.

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