Hyaluronic Acid-tyramine Hydrogels Research Articles

Enzyme-mediated fabrication of nanocomposite hydrogel microneedles for tunable mechanical strength and controllable transdermal efficiency

Microneedles (MNs) are increasingly used in transdermal drug delivery due to high bioavailability, simple operation, and improved patient compliance. However, further clinical applications are hindered by unsatisfactory mechanical strength and uncontrolled drug release. Herein, an enzyme-mediated approach is reported for the fabrication of nanocomposite hydrogel-based MNs with tunable mechanical strength and controllable transdermal efficiency. As a proof-of-concept, tetrakis(1-methyl-4-pyridinio)porphyrin (TMPyP) was chosen as a model drug for photodynamic therapy of melanoma. TMPyP-loaded PLGA nanoparticles (NP/TMPyP) served as an inner phase of MNs for controlled release of photosensitizers, and enzyme-mediated hyaluronic acid-tyramine (HAT) hydrogels served as an external phase for optimizing the mechanical strength of MNs. By changing the concentration of HRP and H2O2, three types of MNs were fabricated for transdermal delivery of TMPyP, which demonstrated different cross-linking densities and various mechanical strength. Among the three MNs, the HAT-Medium@NP/TMPyP-MN with a medium mechanical strength exhibited the highest values of transdermal efficiency in vitro and the longest retention time in vivo. As compared to pure TMPyP and TMPyP-loaded nanoparticles, the HAT-Medium@NP/TMPyP-MN demonstrated higher anticancer efficacy in both melanoma A375 cells and a xenografted tumor mouse model. Therefore, the enzyme-mediated nanocomposite hydrogel MNs show great promise in the transdermal delivery of therapeutic drugs with enhanced performance. Statement of SignificanceThis study reports an enzyme-mediated approach for the fabrication of photodynamically-active microneedles (HAT@NP/TMPyP-MNs) with tunable mechanical strength and controllable transdermal efficiency. On one hand, HAT hydrogels that bear different cross-linking densities, facilitate tunable mechanical strength and optimized transdermal performances of MNs; on the other hand, NP/TMPyP and HAT network contribute to sustained release of photosensitizers. Comparing to other formulation (i.e., NP/TMPyP or TMPyP), the HAT-Medium@NP/TMPyP-MN exhibited excelling anticancer efficacy in photodynamic therapy in vitro and in vivo. We believe that the combination of enzyme-mediated polymeric cross-linking and slow-releasing nano-vehicles in a single nanocomposite platform provides a versatile approach for the fabrication of MNs with enhanced therapeutic efficacy, which holds great promise in the transdermal delivery of various therapeutic drugs in future.

Optimization of hyaluronic acid-tyramine/silk-fibroin composite hydrogels for cartilage tissue engineering and delivery of anti-inflammatory and anabolic drugs

Injury of articular cartilage leads to an imbalance in tissue homeostasis, and due to the poor self-healing capacity of cartilage the affected tissue often exhibits osteoarthritic changes. In recent years, injectable and highly tunable composite hydrogels for cartilage tissue engineering and drug delivery have been introduced as a desirable alternative to invasive treatments. In this study, we aimed to formulate injectable hydrogels for drug delivery and cartilage tissue engineering by combining different concentrations of hyaluronic acid-tyramine (HA-Tyr) with regenerated silk-fibroin (SF) solutions. Upon enzymatic crosslinking, the gelation and mechanical properties were characterized over time. To evaluate the effect of the hydrogel compositions and properties on extracellular matrix (ECM) deposition, bovine chondrocytes were embedded in enzymatically crosslinked HA-Tyr/SF composites (in further work abbreviated as HA/SF) or HA-Tyr hydrogels. We demonstrated that all hydrogel formulations were cytocompatible and could promote the expression of cartilage matrix proteins allowing chondrocytes to produce ECM, while the most prominent chondrogenic effects were observed in hydrogels with HA20/SF80 polymeric ratios. Unconfined mechanical testing showed that the compressive modulus for HA20/SF80 chondrocyte-laden constructs was increased almost 10-fold over 28 days of culture in chondrogenic medium which confirmed the superior production of ECM in this hydrogel compared to other hydrogels in this study. Furthermore, in hydrogels loaded with anabolic and anti-inflammatory drugs, HA20/SF80 hydrogel showed the longest and the most sustained release profile over time which is desirable for the long treatment duration typically necessary for osteoarthritic joints. In conclusion, HA20/SF80 hydrogel was successfully established as a suitable injectable biomaterial for cartilage tissue engineering and drug delivery applications.

Enzymatically Crosslinkable Hyaluronic Acid-Gelatin Hybrid Hydrogels as Potential Bioinks for Tissue Regeneration

Hydrogels that mimic the composition and properties of the extracellular matrix have attracted great attention as potential polymeric scaffolds for tissue engineering applications. In this study, an injectable hydrogel composed of hyaluronic acid and gelatin was developed via the oxidative coupling reaction using horseradish peroxidase (HRP). The hydrogel was prepared by mixing two phenol-conjugated polymer solutions, hyaluronic acid-tyramine (HA-TA) and gelatin-hydroxyphenyl propionic acid (GH), in the presence of HRP and hydrogen peroxide (H202). The gelation rate and mechanical properties of composite hydrogel were controlled by adjusting the HRP and H202 concentrations, respectively Compared to the pure HA-TA hydrogels, the stability and cellular behaviors of composite hydrogel improved significantly In addition, the injectable hydrogel system showed good performance in 3D printing with high cell viability after one day of printing. The results suggest that enzymatically crosslinked HA-TA and GH hybrid (HA-TA/GH) composite of HA-TA and GH hydrogel has the potential as a material for tissue engineering and 3D printing biofabrication.

In situ formed collagen-hyaluronic acid hydrogel as biomimetic dressing for promoting spontaneous wound healing

Wound dressing is distinctly important to wound healing, because it can not only protect wound from external disturbance, but also provide an ideal environment for wound closure. However, most of wound dressings need additional active ingredients to assist the repair process. In order to develop new dressings that can present spontaneous healing activity, herein, an injectable hydrogel consisted of collagen I and hyaluronic acid has been designed to mimic extracellular matrix for vascular cells growing and wound closure. The preparation of hydrogel (COL-HA) was realized through in situ coupling of phenol moieties of collagen I-hydroxybenzoic acid (COL-P) and hyaluronic-acid-tyramine (HA-Tyr) through horseradish peroxidase (HRP). The physical structure and properties were characterized, and the biological performances were analyzed. COL-HA hydrogel presented porous structure that contributed to the exchange of gas, medium and nutrition. Human microvascular endothelial cells (HMEC) and fibroblasts (COS-7) cultured within this hydrogel showed significant proliferation behaviors. More importantly, a certain level of vascular endothelial growth factor (VEGF) was observed in HMEC cultured hydrogel, which led to the possibility of vascular regeneration. For the full-thickness wound, the healing ratio and validity of wound treated with COL-HA hydrogel were higher than commercial drug and individual COL-P hydrogel, HA-Tyr hydrogel groups, since collagen and hyaluronic acid made joint efforts to improve wound repair.

An injectable platelet lysate-hyaluronic acid hydrogel supports cellular activities and induces chondrogenesis of encapsulated mesenchymal stem cells

Developing scaffolds that can provide cells and biological cues simultaneously in the defect site is of interest in tissue engineering field. In this study, platelet lysate (PL) as an autologous and inexpensive source of growth factors was incorporated into a cell-laden injectable hyaluronic acid-tyramine (HA-TA) hydrogel. Subsequently, the effect of platelet lysate on cell attachment, viability and differentiation of human mesenchymal stem cell (hMSCs) toward chondrocytes was investigated. HA-TA conjugates having a degree of substitution of 20 TA moieties per 100 disaccharide units were prepared and crosslinked in the presence of horseradish peroxidase and low concentrations of hydrogen peroxide. The storage moduli of the gels ranged from 500 to 2000 Pa and increased with increasing polymer concentration. In contrast to a retained round shape of the cells when using pure HA-TA hydrogel, the hMSCs attached and spread out in PL enriched matrix. The enrichment of hMSCs laden HA-TA hydrogels with PL induced a cartilage like extra cellular matrix deposition in vitro. The hMSCs increasingly deposited collagen type II and proteoglycans over time. The deposition of the new extracellular matrix (ECM) is simultaneous with gel degradation and resulted ultimately in the formation of a tough dense matrix. These findings demonstrate the potential of injectable HA-TA-PL hydrogel as a cell delivery system for cartilage regeneration. Statement of SignificanceCartilage tissue has limited ability to self-repair because of its avascular nature. To have an efficient cartilage tissue regeneration, we combined platelet lysate (PL), as an autologous and inexpensive source of growth factors, with an injectable hyaluronic acid tyramine (HA-TA) hydrogel scaffold. Platelet lysate had a vital role in supporting human mesenchymal stem cells (hMSCs) activities, like cell attachment, viability and proliferation in the 3D hydrogel structure. Also, the hMSCs encapsulated HA-TA induced hyaline cartilage generation when placed in chondrogenic differentiation medium. This study introduces a new system for cartilage tissue engineering, which can be injected in a minimally invasive manner and is rich with patient’s own growth factors and biological cues.

Hyaluronidase-incorporated hyaluronic acid–tyramine hydrogels for the sustained release of trastuzumab

We developed an injectable hydrogel system for the sustained release of protein drugs that incorporated both protein drugs and hyaluronidase. Trastuzumab and hyaluronidase were incorporated in hydrogels composed of hyaluronic acid–tyramine (HA–Tyr) conjugates through the enzymatic crosslinking utilizing hydrogen peroxide (H2O2) and horseradish peroxidase (HRP). Through electrostatic interactions with the HA, trastuzumab was retained in the hydrogel to minimize its burst release. Hyaluronidase was incorporated in the hydrogel to release trastuzumab from the hydrogels. The hydrogels were degraded and showed sustained release of trastuzumab in phosphate buffer over four weeks in vitro. Both the rates of drug release and gel degradation were controlled by the concentration of hyaluronidase. Trastuzumab released from the hydrogels inhibited the proliferation of BT-474 cells in vitro. In an animal model, the single subcutaneous injection of a mixture solution of HA–Tyr conjugates, H2O2, HRP, trastuzumab and hyaluronidase inhibited tumor growth significantly, whereas injection of trastuzumab alone at the same dose failed to do so. Compared to trastuzumab alone, the hyaluronidase-incorporated HA–Tyr hydrogels improved the pharmacokinetic profile of trastuzumab in the plasma of mice. Furthermore, they were fully degraded over two weeks, and the formation of fibrous capsules was not observed in mice.

Enzyme-mediated hyaluronic acid–tyramine hydrogels for the propagation of human embryonic stem cells in 3D

The propagation of human embryonic stem cells (hESCs) in three-dimensional (3D) scaffolds facilitates the cell expansion process and supplies pluripotent cells of high quality for broad-spectrum applications in regenerative medicine. Herein, we report an enzyme-mediated hyaluronic acid–tyramine (HA–Tyr) hydrogel that encapsulated and propagated hESCs in 3D. HA–Tyr hydrogels were formed by crosslinking the tyramine moieties with horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). By changing the HRP and H2O2 concentration, we prepared HA–Tyr hydrogels of different mechanical strength and studied the self-renewal properties of hESCs in these scaffolds. We observed that both the chemical composition and mechanical strength of substrates were important factors affecting cell proliferation and pluripotency. The HA–Tyr hydrogel with a compressive modulus of ∼350Pa supported the proliferation of hESCs at the pluripotent state in both mTeSR1 medium and mouse embryonic fibroblast (MEF)-conditioned medium. Immunohistochemical analyses revealed that hESCs proliferated well and formed spheroid structures in 3D, without undergoing apoptosis. The hESCs cultured in HA–Tyr hydrogels showed high expression of CD44 and pluripotency markers. These cells exhibited the capability to form cell derivatives of all three embryonic germ layers in vitro and in vivo. In addition, the genetic integrity of the hESCs was unaffected in the 3D cultivation system. Statement of SignificanceThe scope of this study is to provide a stable 3D cultivation system for the expansion of human embryonic stem cells (hESCs) towards clinical applications. We report an enzyme mediated hyaluronic acid–tyramine (HA–Tyr) hydrogel that encapsulated and propagated hESCs in 3D. Unlike other HA-based photo-crosslinked hydrogel systems reported, we investigated the effects of mechanical strength of hydrogels on the self-renewal properties of hESCs in 3D. Then, we characterized hESCs cultured in hydrogels with lower mechanical strength that best supported the self-renewal of hESCs. Hence, we demonstrated a reliable approach for the controlled propagation of hESCs in 3D. We believe that such an approach would facilitate the development of stem cell-based therapy towards clinical applications.

Liposomal delivery of horseradish peroxidase for thermally triggered injectable hyaluronic acid-tyramine hydrogel scaffolds.

A thermoresponsive injectable hydrogel scaffold for tissue engineering has been developed, whereby the scaffold was injected as a liquid at room temperature, and gelled at the target site in response to the change in body temperature. Our approach involved suspending thermoresponsive liposomes, which encapsulated horseradish peroxidase (HRP), in a hyaluronic acid-tyramine (HA-Tyr) conjugate and hydrogen peroxide (H2O2) solution. At room temperature, HRP was separated from the HA-Tyr conjugate by the lipid membrane, and hence the precursor solution remained as a liquid and was injectable. Upon injection and exposure to body temperature, the lipids experienced a phase transition, which significantly increased the membrane permeability and led to the release of HRP and the oxidative coupling of Tyr moieties with H2O2, forming a crosslinked hydrogel scaffold. It was shown in this study that the precursor solution remained as a liquid on the order of hours at 20 °C, yet gelation could be induced within minutes by heating to 37 °C. Furthermore, it was shown that HRP release could be controlled by various material and processing parameters, and that the gelation rate could be adjusted to meet various clinical needs by adjusting HRP encapsulation, liposome concentration and HA-Tyr concentration.

Enzymatic conjugation of a bioactive peptide into an injectable hyaluronic acid–tyramine hydrogel system to promote the formation of functional vasculature

In this study, one-step enzyme-mediated preparation of a multi-functional injectable hyaluronic-acid-based hydrogel system is reported. Hydrogel was formed through the in situ coupling of phenol moieties by horseradish peroxidase (HRP) and hydrogen peroxide (H2O2), and bioactive peptides were simultaneously conjugated into the hydrogel during the gel formation process. The preparation of this multi-functional hydrogel was made possible by synthesizing peptides containing phenols which could couple with the phenol moieties of hyaluronic-acid–tyramine (HA–Tyr) during the HRP-mediated crosslinking reaction. Preliminary studies demonstrated that two phenol moieties per molecule resulted in a consistently high degree of conjugation into the HA–Tyr hydrogel network, unlike the one modified with one phenol moiety per molecule. Therefore, an Arg–Gly–Asp (RGD) peptide bearing two phenol moieties (phenol2–poly(ethylene glycol)–RGD) was designed for conjugation to endow the HA–Tyr hydrogel with adhesion signals and enhance its bioactivities. Human umbilical vein endothelial cells (HUVECs) cultured on or within the RGD-modified hydrogels showed significantly different adhesion behavior, from non-adherence on the HA–Tyr hydrogel to strong adhesion on hydrogels modified with phenol2–poly(ethylene glycol)–RGD. This altered cell adhesion behavior led to improved cell proliferation, migration and formation of capillary-like network in the hydrogel in vitro. More importantly, when HUVECs and human fibroblasts (HFF1) were encapsulated together in the RGD-modified HA–Tyr hydrogel, functional vasculature was observed inside the cell-laden gel after 2weeks in the subcutaneous tissue. Taken together, the in situ conjugation of phenol2–poly(ethylene glycol)–RGD into HA–Tyr hydrogel system, coupled with the ease of incorporating cells, offers a simple and effective means to introduce biological signals for preparation of multi-functional injectable hydrogels for tissue engineering application.

Injectable hyaluronic acid-tyramine hydrogels incorporating interferon-α2a for liver cancer therapy

We report an injectable hydrogel system that incorporates interferon-α2a (IFN-α2a) for liver cancer therapy. IFN-α2a was incorporated in hydrogels composed of hyaluronic acid-tyramine (HA-Tyr) conjugates through the oxidative coupling of Tyr moieties with hydrogen peroxide (H2O2) and horseradish peroxidase (HRP). IFN-α2a-incorporated HA-Tyr hydrogels of varying stiffness were formed by changing the H2O2 concentration. The incorporation of IFN-α2a did not affect the rheological properties of the hydrogels. The activity of IFN-α2a was furthermore well-maintained in the hydrogels with lower stiffness. Through the caspase-3/7 pathway in vitro, IFN-α2a released from HA-Tyr hydrogels inhibited the proliferation of liver cancer cells and induced apoptosis. In the study of the pharmacokinetics, a higher concentration of IFN-α2a was shown in the plasma of mice treated with IFN-α2a-incorporated hydrogels after 4h post injection, with a much higher amount of IFN-α2a delivered at the tumor tissue comparing to that of injecting an IFN-α2a solution. The tumor regression study revealed that IFN-α2a-incorporated HA-Tyr hydrogels effectively inhibited tumor growth, while the injection of an IFN-α2a solution did not demonstrate antitumor efficacy. Histological studies confirmed that tumor tissues in mice treated with IFN-α2a-incorporated HA-Tyr hydrogels showed lower cell density, with more apoptotic and less proliferating cells compared with tissues treated with an IFN-α2a solution. In addition, the IFN-α2a-incorporated hydrogel treatment greatly inhibited the angiogenesis of tumor tissues.

Modulation of mesenchymal stem cell chondrogenesis in a tunable hyaluronic acid hydrogel microenvironment

An injectable and biodegradable hydrogel system comprising hyaluronic acid-tyramine (HA-Tyr) conjugates can safely undergo covalent cross-linking in vivo by the addition of small amounts of peroxidase and hydrogen peroxide (H2O2), with the independent tuning of the gelation rate and degree of cross-linking. Such hydrogel networks with tunable mechanical and degradation properties may provide the additional level of control needed to enhance chondrogenesis and overall cartilage tissue formation in vitro and in vivo. In this study, HA-Tyr hydrogels were explored as biomimetic matrices for caprine mesenchymal stem cells (MSCs) in cartilage tissue engineering. The compressive modulus, equilibrium swelling and degradation rate could be controlled by varying the concentration of H2O2 as the oxidant in the oxidative coupling reaction. Cellular condensation reflected by the increase in effective number density of rounded cells in lacunae was greater in softer hydrogel matrices with lower cross-linking that displayed enhanced scaffold contracture. Conversely, within higher cross-linked matrices, cells adopted a more elongated morphology, with a reduced degree of cellular condensation. Furthermore, the degree of hydrogel cross-linking also modulated matrix biosynthesis and cartilage tissue histogenesis. Lower cross-linked matrix enhanced chondrogenesis with increases in the percentage of cells with chondrocytic morphology; biosynthetic rates of glycosaminoglycan and type II collagen; and hyaline cartilage tissue formation. With increasing cross-linking degree and matrix stiffness, a shift in MSC differentiation toward fibrous phenotypes with the formation of fibrocartilage and fibrous tissues was observed. These findings suggest that the tunable three-dimensional microenvironment of the HA-Tyr hydrogels modulates cellular condensation during chondrogenesis and has a dramatic impact on spatial organization of cells, matrix biosynthesis, and overall cartilage tissue histogenesis.

An injectable hyaluronic acid–tyramine hydrogel system for protein delivery

Previously, we reported the independent tuning of mechanical strength (crosslinking density) and gelation rate of an injectable hydrogel system composed of hyaluronic acid–tyramine (HA–Tyr) conjugates. The hydrogels were formed through the oxidative coupling of tyramines which was catalyzed by hydrogen peroxide (H 2O 2) and horseradish peroxidase (HRP). Herein, we studied the encapsulation and release of model proteins using the HA–Tyr hydrogel. It was shown that the rapid gelation achieved by an optimal concentration of HRP could effectively encapsulate the proteins within the hydrogel network and thus prevented the undesired leakage of proteins into the surrounding tissues after injection. Hydrogels with different mechanical strengths were formed by changing the concentration of H 2O 2 while maintaining the rapid gelation rate. The mechanical strength of the hydrogel controlled the release rate of proteins: stiff hydrogels released proteins slower compared to weak hydrogels. In phosphate buffer saline, α-amylase (negatively charged) was released sustainably from the hydrogel. Conversely, the release of lysozyme (positively charged) discontinued after the fourth hour due to electrostatic interactions with HA. In the presence of hyaluronidase, lysozymes were released continuously and completely from the hydrogel due to degradation of the hydrogel network. The activities of the released proteins were mostly retained which suggested that the HA–Tyr hydrogel is a suitable injectable and biodegradable system for the delivery of therapeutic proteins.

An injectable enzymatically crosslinked hyaluronic acid- hydrogel system with independent tuning of mechanical strength and gelation rate.

In this study, we propose an enzymatically crosslinked hyaluronic acid (HA) hydrogel with tunable mechanical strength and gelation rate as a novel injectable system. The hydrogel composed of HA-tyramine conjugate (HA-Tyr) was formed using the oxidative coupling of tyramine moieties catalyzed by hydrogen peroxide (H2O2) and horseradish peroxidase (HRP). The mechanical strength of the HA-Tyr hydrogel was tuned solely by the H2O2 amount without affecting the gelation rate. The hydrogels formed more rapidly with increasing HRP concentration and the gelation time ranged from 1 s to 20 min. A faster gelling system yielded more localized gel formation than a slower gelling one at the site where it was administered through subcutaneous injection. Studies on the swelling ratio and scanning electron microscopy images of the hydrogel structure further demonstrated that the crosslinking density was controlled by the concentration of H2O2 used. The mechanical strength of HA-Tyr hydrogels strongly affected the degradation rate in the presence of hyaluronidase in vitro; hydrogels degraded more slowly with increasing mechanical strength of the hydrogel. The independently tunable mechanical strength and gelation rate achieved by this enzymatically formed HA-Tyr hydrogel system will provide great advantages to a wide range of applications of injectable hydrogels, such as drug delivery and tissue regeneration.

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