Adhesive Catechol-Conjugated Hyaluronic Acid for Biomedical Applications: A Mini Review

: Recently, catechol-containing polymers have been extensively developed as promising materials for surgical tissue adhesives, wound dressing, drug delivery depots, and tissue engineering scaffolds. Catechol conjugation to the polymer backbone provides adhesive properties to the tissue and does not signiﬁcantly affect the intrinsic properties of the polymers. An example of a catecholic polymer is catechol-conjugated hyaluronic acid. In general, hyaluronic acid shows excellent biocompatibility and biodegradability; thus, it is used in various medical applications. However, hyaluronic acid alone has poor mechanical and tissue adhesion properties. Catechol modiﬁcation considerably increases the mechanical and underwater adhesive properties of hyaluronic acid, while maintaining its biocompatibility and biodegradability and enabling its use in several biomedical applications. In this review, we brieﬂy describe the synthesis and characteristics of catechol-modiﬁed hyaluronic acid, with a speciﬁc focus on catechol-involving reactions. Finally, we discuss the basic concepts and therapeutic effects of catechol-conjugated hyaluronic acid for biomedical applications. including involving bone, cartilage, salivary gland, and


Introduction
Hyaluronic acid (HA) is a naturally occurring polysaccharide composed of D-glucuronic acid and N-acetyl-D-glucosamine, which is used in various biomedical applications, such as wound healing, viscosupplementation for wrinkle fillers, post-surgical antiadhesive barriers, drug delivery carriers, and tissue engineering scaffolds [1][2][3][4][5][6][7][8][9][10][11][12][13]. HA is abundantly found in connective tissues and fluids, particularly in articular cartilage and synovial fluids [1][2][3][4][5]. The roles of HA in biological functions are diverse, including the hydration of the extracellular matrix, the regulation of tissue homeostasis, receptor-mediated regulation of cell proliferation, migration, and differentiation, and the lubrication of cartilage and of the eyes [14,15]. For instance, HA in synovial fluids is responsible for the lubrication of cartilage in the synovium owing to its viscoelasticity and biocompatibility with synovial fluids [16]. In addition, HA is fully degraded by hyaluronidase in the body [17]. As a result, HA is widely used for drug delivery depots and tissue engineering scaffolds. However, there are difficulties in using unmodified HA in various medical settings due to its poor mechanical properties. Therefore, chemical modifications of the amine, thiol, and catechol functional groups on the HA backbone can address this drawback.
In this review, we briefly describe the methodologies used for the preparation and characterization of HA-catechol. In addition, we focus on the biomedical uses of HAcatechol, which are categorized into the following four subsections: antifouling, wound healing, drug/gene delivery carriers, and tissue engineering scaffolds. Furthermore, we discuss the current challenges and opportunities for technologies using HA-catechol, which require further fundamental research, and its use in translational medicine.

Methodologies for the Synthesis and Characterization of Hyaluronic Acid-Catechol
Carbodiimide chemistry is commonly used to prepare HA-catechol . It involves the formation of an amide bond between the carboxylic group of HA and the primary amine of dopamine ( Figure 1a). The important reaction conditions for HA-catechol synthesis include maintaining the pH between 4 and 6 to prevent oxidation of the catechol groups [37]. The reaction solution turns black at a pH above 6, indicating the oxidation of the catechol groups. The degree of catechol substitution (DS) in HA is 10-35%, identified using standard carbodiimide chemistry. Similar approaches have been used to prepare catechol-conjugated negatively charged polymers (i.e., alginate-catechol [82,83], heparincatechol [84], and polyacrylic acid-catechol [85]). In a previous study, a Schiff's base reaction between aldehyde-modified HA and dopamine was used to prepare HA-catechol [86]. HA was first oxidized using sodium periodate to prepare dialdehyde-functionalized HA. The formation of the Schiff base was achieved by the reaction between dialdehyde groups in oxidized HA and the primary amine groups in dopamine. The DS (25-45%) obtained using the Schiff base reaction was higher than that obtained using the carbodiimide coupling reaction (~10%), probably due to the dialdehyde groups of the oxidized HA [86].

Biocompatibility
High levels of HA are present in tissues and cartilage, particularly in those of the musculoskeletal system. In addition, HA is widely used in medical applications mainly due to its biocompatibility. The conjugation of molecules to HA does not significantly affect its biocompatibility. For instance, injectable tyramine-conjugated HA hydrogels prepared by the oxidative crosslinking of phenolic groups maintain high cell viability (>95%) of human mesenchymal stem cells (hMSCs) for up to 14 days [87]. In addition, the viability of MSCs in an HA hydrogel crosslinked with polyethylene glycol (HA-PEG gel) was ~100% when 10 6 cells mL −1 of MSCs were treated with the hydrogel for three days [88].
Similarly, HA-catechol is also non-cytotoxic and biocompatible. Treatment of cells at a density of 1.0 × 10 6 cells/100 μL with a pre-gelation HA-catechol solution preserves cell viability (~100%) [46]. Treatment with extracts of HA-catechol hydrogels maintains ~83% viability in L929 cells compared to that obtained using fresh media. In addition, the viability of human hepatocytes (hHEPs) encapsulated in HA-catechol hydrogels prepared by the addition of NaIO4 was ~70.9%, which is far higher than that observed using HAmethacrylate hydrogels (~52.2%) on day 14 of treatment [46,89]. Furthermore, HA-catechol hydrogels incorporated with basic fibroblast growth factor exhibit improved hHEPs cell viability to ~81.4% after 14 days [37]. The viability of human neural stem cells (hNSCs) in an HA-catechol hydrogel after seven days was approximately 70%, which is two-fold that observed using an HA-methacrylate hydrogel (less than 30%).

Tissue Adhesive Properties
HA has anti-adhesive properties; therefore, HA-based physical barriers have been developed to prevent post-surgical adhesion. However, the conjugation of catechol groups to HA significantly increases the adhesiveness of HA-catechol to tissues. As mentioned, the oxidized catechol groups (i.e., catechol quinone) can covalently react with the

Biocompatibility
High levels of HA are present in tissues and cartilage, particularly in those of the musculoskeletal system. In addition, HA is widely used in medical applications mainly due to its biocompatibility. The conjugation of molecules to HA does not significantly affect its biocompatibility. For instance, injectable tyramine-conjugated HA hydrogels prepared by the oxidative crosslinking of phenolic groups maintain high cell viability (>95%) of human mesenchymal stem cells (hMSCs) for up to 14 days [87]. In addition, the viability of MSCs in an HA hydrogel crosslinked with polyethylene glycol (HA-PEG gel) was~100% when 10 6 cells mL −1 of MSCs were treated with the hydrogel for three days [88].
Similarly, HA-catechol is also non-cytotoxic and biocompatible. Treatment of cells at a density of 1.0 × 10 6 cells/100 µL with a pre-gelation HA-catechol solution preserves cell viability (~100%) [46]. Treatment with extracts of HA-catechol hydrogels maintains~83% viability in L929 cells compared to that obtained using fresh media. In addition, the viability of human hepatocytes (hHEPs) encapsulated in HA-catechol hydrogels prepared by the addition of NaIO 4 was~70.9%, which is far higher than that observed using HAmethacrylate hydrogels (~52.2%) on day 14 of treatment [46,89]. Furthermore, HA-catechol hydrogels incorporated with basic fibroblast growth factor exhibit improved hHEPs cell viability to~81.4% after 14 days [37]. The viability of human neural stem cells (hNSCs) in an HA-catechol hydrogel after seven days was approximately 70%, which is two-fold that observed using an HA-methacrylate hydrogel (less than 30%).

Tissue Adhesive Properties
HA has anti-adhesive properties; therefore, HA-based physical barriers have been developed to prevent post-surgical adhesion. However, the conjugation of catechol groups to HA significantly increases the adhesiveness of HA-catechol to tissues. As mentioned, the oxidized catechol groups (i.e., catechol quinone) can covalently react with the amine or thiol groups that are abundantly found in native tissues, including proteoglycan structures, resulting in superior tissue adhesive properties. The tissue adhesive properties of HAcatechol are commonly measured with a lap shear test using animal skin, such as chicken or porcine skin (Figure 2a). In addition, post-crosslinking of HA-catechol adhesives is carried out prior to its use on skin or dermal tissues to improve tissues' adhesion properties in general. The adhesive stress withstood by joint chicken skins in the presence of HAcatechol hydrogels is approximately 10 kPa, which is far greater than that bore when using methacrylated HA hydrogels (Figure 2b) [52]. In addition, tissue adhesive properties are improved by the addition of an oxidative intermolecular crosslink agent (NaIO 4 ) and by an increase of DS in HA-catechol. The maximum adhesive strength is 90.0 ± 6.7 kPa when using HA-catechol with high DS (0.45) at a molar ratio of 1:2 (catechol/NaIO 4 ). Moreover, the addition of Fe 3+ ions to the hydrogels increases the adhesive stress of HAcatechol. The detachment stress of hydrogels containing HA-catechol/Fe 3+ complexes is 14.8 ± 0.9 kPa, while that of an HA-catechol solution is 0.9 ± 0.5 kPa, measured using mouse subcutaneous tissues [47]. HA-catechol-based composite materials also show excellent adhesive properties. For instance, HA-catechol and Pluronic F127 (16 wt%) composite hydrogels show excellent stability with approximately 10-fold greater adhesive strength (~7.2 kPa) than HA-catechol alone (~1.7 kPa) [48]. HA-catechol/PVA (Polyvinyl alcohol) hydrogel composite films can be used as mucoadhesive films for the treatment of oral candidiasis [49]. HA-catechol/PVA hydrogels have a high detachment force of approximately 0.23 N, which was measured using a bovine buccal membrane. HA-catechol/PLL (poly-L-lysine) hydrogels enzymatically crosslinked with horseradish peroxidase (HRP) with H 2 O 2 showed a significant improvement in adhesive strength, from 20.8 kPa to 33.8 kPa [39]. Therefore, HA-catechol is useful for long-term tissue adhesion and may have great potential for use in biomedical applications. Furthermore, the tissue adhesiveness of HA-catechol may prevent the malposition or movement of the HA-based materials on the targeted tissue surfaces.

Biomedical Applications
HA-catechol is widely used in biomedical applications owing to its biocompatibility, adhesiveness, and anti-inflammatory properties. In this section, we describe the various biomedical uses of HA-catechol, focusing on its physicochemical properties and catecholinvolving chemical reactions with tissues and/or biomacromolecules.

Antifouling Materials
HA is a representative antifouling material mainly due to its hydrophilicity and its unique moisture retention and lubrication abilities. The catechol moiety of HA-catechol can form a thin film on substrates (Figure 4a). These HA-catechol coatings on Au surfaces enhance hydrophilicity (11.9 • of water contact angle) compared to that measured on bare Au surfaces (81.6 • ) [40]. In addition, HA-catechol-coated Au substrates have extremely low protein-adsorption levels (<5 ng/cm 2 ) for single proteins (i.e., bovine serum albumin, lysozymes, and β-lactoglobulin). Polyimide (PI), Au, poly(methyl methacrylate) (PMMA), polytetrafluoroethylene (PTFE), and polyurethane (PU) are coated with HA-catechol to prepare antifouling substrates [60]. All substrates coated with HA-catechol exhibit improved hydrophilicity of the substrates (less than 50 • of water contact angle) compared to the unmodified substrates, as shown in Figure 4b. The adsorption of bovine serum albumin on HA-catechol-coated substrates was dramatically reduced to 22-46% compared to that on unmodified surfaces (Figure 4c). Therefore, HA-catechol is an excellent coating agent for the antifouling treatment of biomedical devices.

Antifouling Materials
HA is a representative antifouling material mainly due to its hydrophilicity and its unique moisture retention and lubrication abilities. The catechol moiety of HA-catechol can form a thin film on substrates (Figure 4a). These HA-catechol coatings on Au surfaces enhance hydrophilicity (11.9° of water contact angle) compared to that measured on bare Au surfaces (81.6°) [40]. In addition, HA-catechol-coated Au substrates have extremely low protein-adsorption levels (<5 ng/cm 2 ) for single proteins (i.e., bovine serum albumin, lysozymes, and β-lactoglobulin). Polyimide (PI), Au, poly(methyl methacrylate) (PMMA), polytetrafluoroethylene (PTFE), and polyurethane (PU) are coated with HA-catechol to prepare antifouling substrates [60]. All substrates coated with HA-catechol exhibit improved hydrophilicity of the substrates (less than 50° of water contact angle) compared to the unmodified substrates, as shown in Figure 4b. The adsorption of bovine serum albumin on HA-catechol-coated substrates was dramatically reduced to 22-46% compared to that on unmodified surfaces (Figure 4c). Therefore, HA-catechol is an excellent coating agent for the antifouling treatment of biomedical devices.

Wound-Healing Materials
HA-catechol materials have been investigated as wound-healing materials because reactive oxygen species can be scavenged by catechol groups. The incorporation of antiinflammatory drugs into HA-catechol hydrogels significantly improves wound closure and healing. For instance, arginine derivatives have been incorporated into HA-catechol hydrogels, as shown in Figure 5a [62]. To confirm the antioxidant properties, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radical (OH) scavenging experiments are generally used. An arginine/HA-catechol composite hydrogel showed strong antioxidant activity with maximum OH and DPPH free-radical scavenging rates of 41.1% and 67.4%, respectively. In addition, complete wound closure and healing could be achieved using these hydrogels after 21 days (Figure 5b). HA-catechol conjugated with chitosan is also used as a wound-healing material [57]. Wound healing can be improved by increasing the concentrations of H 2 O 2 and HRP. HA-catechol/chitosan composite hydrogels with the addition of H 2 O 2 (0.05%, v/v) and HRP (0.1 mg/mL) showed complete wound healing nine days post-surgery, which depicts a wound closure effect similar to that obtained using surgical suturing, indicating that these hydrogels could replace surgical sutures to achieve wound closure. In addition, a mild acute inflammatory response was observed for six days after the implantation of HA-catechol/chitosan hydrogels, and inflammation receded on the ninth day after surgery. This is similar to the biocompatibility of surgical sutures and enzymatically (HRP) crosslinked hydrogels.

Wound-Healing Materials
HA-catechol materials have been investigated as wound-healing materials because reactive oxygen species can be scavenged by catechol groups. The incorporation of antiinflammatory drugs into HA-catechol hydrogels significantly improves wound closure and healing. For instance, arginine derivatives have been incorporated into HA-catechol hydrogels, as shown in Figure 5a [62]. To confirm the antioxidant properties, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radical (OH) scavenging experiments are generally used. An arginine/HA-catechol composite hydrogel showed strong antioxidant activity with maximum OH and DPPH free-radical scavenging rates of 41.1% and 67.4%, respectively. In addition, complete wound closure and healing could be achieved using these hydrogels after 21 days (Figure 5b). HA-catechol conjugated with chitosan is also used as a wound-healing material [57]. Wound healing can be improved by increasing the concentrations of H2O2 and HRP. HA-catechol/chitosan composite hydrogels with the addition of H2O2 (0.05%, v/v) and HRP (0.1 mg/mL) showed complete wound healing nine days post-surgery, which depicts a wound closure effect similar to that obtained using surgical suturing, indicating that these hydrogels could replace surgical sutures to achieve wound closure. In addition, a mild acute inflammatory response was observed for six days after the implantation of HA-catechol/chitosan hydrogels, and inflammation receded on the ninth day after surgery. This is similar to the biocompatibility of surgical sutures and enzymatically (HRP) crosslinked hydrogels.

Drug/Gene Delivery Carriers
A challenge in cancer treatment is targeting anticancer drug delivery to increase the efficiency of drugs. Because HA interacts with CD44, overexpressed in cancer cells, HA-based hydrogels as drug/gene delivery carriers have been developed for anticancer drug therapy. The catechol groups in HA can act as coating and crosslinking moieties to achieve sustained drug release. For instance, HA-catechol/chitosan composite nanoparticles containing the anticancer drug doxorubicin (DOX), with a high loading capacity of 250 µg/mg, exhibited a controlled release of DOX, in comparison to the administration of free DOX [63]. In addition, the nanoparticles showed mucoadhesive properties; over 60% of the nanoparticles remained on the buccal mucosa after washing with artificial saliva, while only 10% remained when dextran was used as a control. In addition, these nanoparticles showed enhanced cellular uptake and accumulation in HN22 cells. Furthermore, treatment using DOX nanoparticles led to extensive apoptosis (22% of the apoptotic rate), which was higher than that observed using free DOX (16%). HA-catechol hydrogels containing amine-functionalized graphene oxide (GO) are prepared by covalent reactions involving the catechol groups of HA and the amine groups of GO [53]. DOX is noncovalently bound to GO in hydrogels by hydrophobic interactions and π-π stacking. In addition, controlled DOX release profiles have been obtained using HA-catechol/GO hydrogels. HA-catechol hollow nanoparticles have also been developed to deliver anticancer drugs. Silica nanoparticles are coated with HA-catechol, and the inner silica particles are removed to fabricate hollow HA-catechol particles to load anticancer drugs, such as DOX [65]. The hollow HA-catechol particles showed drug-loading efficiency (~20 wt%) with different drug-release profiles between pH 5.5 and 7.4. At pH 5.5, 67% of DOX was released from the particles within 8 h, while 25% of DOX release occurred at pH 7.4 after 60 h. Considering the pH of the physiological conditions and the cancer microenvironment, these particles can effectively lead to cancer cell death after pH-triggered DOX release. In addition, HA-catechol-coated Au nanorods have been fabricated for cancer therapy via the interaction of catechol and Au [66]. DOX was covalently conjugated to HA-catechol, which has an acid-labile hydrazone linkage. The release of DOX from the HA-catecholcoated Au nanorods was controlled by two stimuli, namely, pH and NIR (near-infrared) light, and was found to be successful in inhibiting the growth of breast cancer cells.
HA-catechol-based gene delivery has been performed using calcium phosphate (CaP)containing plasmid DNA or siRNA [45]. HA-catechol has been used as a binder for CaP and cancer-targeting materials. Stabilized CaP/siRNA/HA-catechol nanoparticles are prepared by the simple mixing of solutions and involve specific interactions between calcium and catechol. The nanoparticles deliver siRNA to cancer cells via endocytosis. In the HT29-luc tumor xenograft model, the nanoparticles showed no sign of hepatic toxicity after systemic administration and successfully inhibited cancer growth through the gene silencing effect of siRNA. In addition, CAP nanoparticles stabilized by HA-catechol have been designed to enhance the transfection of hMSCs in a gene delivery system, as shown in Figure 6 [41]. The CAP nanoparticles stabilized by HA-catechol showed efficient gene delivery of both pBMP-2 as a plasmid DNA and mir148b as a microRNA to stem cells via CD44-specific receptor-mediated endocytosis.

Tissue Engineering Scaffolds
In general, chemical modification of HA-based materials is required for tissue engineering, mainly due to the poor mechanical properties of unmodified HA under physiological conditions. The conjugation of catechol to HA increases its mechanical properties with excellent stability against hyaluronidase. HA-catechol is especially useful to promote osteogenesis and angiogenesis. Vascular endothelial growth factor (VEGF) was previously immobilized on a Ti surface using HA-catechol [50]. The VEGF-immobilized Ti substrate (60 ng/cm 2 ) sustained osteoblast proliferation (70,000 osteoblasts/cm 2 ) after seven days of incubation. In addition, the alkaline phosphatase activity (0.7 µM/mg) of VEGF-immobilized Ti substrate was greater than that of bare Ti surfaces (0.3 µM/mg).
Moreover, the VEGF-immobilized Ti substrate using HA-catechol not only allowed osteogenesis for bone formation, but also inhibited bacterial adhesion. In addition, HA-catechol can form thin films with chitosan on a Ti-Nb-Zr alloy (TNZ) [67]. HA-catechol-and chitosan-coated TNZ surfaces improve the proliferation of osteoblast cells, with approximately 35,000 osteoblasts/cm 2 after seven days, which is comparable with the proliferation levels achieved with bare TNZ (30,000 osteoblasts/cm 2 ). The modification of Ti surfaces with HA-catechol can be used to enhance osteogenesis and angiogenesis. HA-catechol has been used for salivary gland tissue engineering (Figure 7). A polycarbonate (PC) membrane coated with HA-catechol showed enhanced adhesion of embryonic submandibular glands (eSMGs) (5.2-fold increase of for the number of adherent eSMGs) compared to both bare and HA-coated membranes [44]. In addition, branching morphogenesis, including average bud counts, was enhanced by HA-catechol coatings mimicking an HA-rich environment. HA-catechol composite hydrogels with carbon nanotubes (CNTs) and/or polypyrrole (PPy) have been developed for hNSC engineering [54]. The catechol groups bound to CNTs via hydrophobic and π-π interactions promote the polymerization of and. CNT and/or PPy in hydrogels contribute to the improvement of electroconductivity to promote the differentiation of human fetal NSCs and human induced pluripotent stem cell-derived neural progenitor cells. In addition, upregulated calcium channel expression and activation of depolarization have been observed in human stem cells using HA-catechol/CNT hydrogels, and intracellular calcium influx is also enhanced by the stimulation of electrical signals. Therefore, HA-catechol can be used in various tissue-engineering applications, including those involving bone, cartilage, salivary gland, and neural tissue regeneration.

Tissue Engineering Scaffolds
In general, chemical modification of HA-based materials is required for tissue engineering, mainly due to the poor mechanical properties of unmodified HA under physiological conditions. The conjugation of catechol to HA increases its mechanical properties with excellent stability against hyaluronidase. HA-catechol is especially useful to pro- and activation of depolarization have been observed in human stem cells using HA-catechol/CNT hydrogels, and intracellular calcium influx is also enhanced by the stimulation of electrical signals. Therefore, HA-catechol can be used in various tissue-engineering applications, including those involving bone, cartilage, salivary gland, and neural tissue regeneration. Figure 7. Schematic illustration of mesenchymal HA-mimicking HA-catechol platform for salivary gland tissue engineering. Reprinted with permission from [44]. Copyright © 2020 American Chemical Society.

Conclusions
In this review, we have described the synthetic and characteristic methodologies, general properties, and various biomedical applications of HA-catechol, including antifouling, tissue adhesives, wound healing, drug/gene delivery, and tissue engineering. Catechol conjugation onto HA backbones significantly enhances HA mechanical and tissue adhesive properties due to catechol-involving reactions, allowing various biomedical applications. In addition, the intrinsic properties of HA, such as biocompatibility and biodegradability, are still conserved after the conjugation of the catechol groups. We expect that HA-catechol will be exploited as an adhesive and biocompatible material in many clinical settings, opening the doors to translational researches to enhance therapies.

Conclusions
In this review, we have described the synthetic and characteristic methodologies, general properties, and various biomedical applications of HA-catechol, including antifouling, tissue adhesives, wound healing, drug/gene delivery, and tissue engineering. Catechol conjugation onto HA backbones significantly enhances HA mechanical and tissue adhesive properties due to catechol-involving reactions, allowing various biomedical applications. In addition, the intrinsic properties of HA, such as biocompatibility and biodegradability, are still conserved after the conjugation of the catechol groups. We expect that HA-catechol will be exploited as an adhesive and biocompatible material in many clinical settings, opening the doors to translational researches to enhance therapies.