Fabrication of versatile dynamic hyaluronic acid-based hydrogels

Journal Pre-proof Fabrication of versatile dynamic hyaluronic acid-based hydrogels Wen Shi, Blake Hass, Mitchell A. Kuss, Haipeng Zhang, Sangjin Ryu, Dongze Zhang, Tieshi Li, Yu-long Li, Bin Duan

PII:

S0144-8617(19)31471-7

DOI:

https://doi.org/10.1016/j.carbpol.2019.115803

Reference:

CARP 115803

To appear in:

Carbohydrate Polymers

Received Date:

8 November 2019

Revised Date:

24 December 2019

Accepted Date:

27 December 2019

Please cite this article as: Shi W, Hass B, Kuss MA, Zhang H, Ryu S, Zhang D, Li T, Li Y-long, Duan B, Fabrication of versatile dynamic hyaluronic acid-based hydrogels, Carbohydrate Polymers (2020), doi: https://doi.org/10.1016/j.carbpol.2019.115803

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Fabrication of versatile dynamic hyaluronic acid-based hydrogels Wen Shia, b, Blake Hassc, Mitchell A. Kussa, b, Haipeng Zhangd, Sangjin Ryud, e, Dongze Zhangf, Tieshi Lig , Yu-long Lif, and Bin Duana, b, d, h* & Dick Holland Regenerative Medicine Program, University of Nebraska

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aMary

bDivision

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Medical Center, Omaha, NE, USA

of Cardiology, Department of Internal Medicine, University of Nebraska

of Medicine, University of Nebraska Medical Center, Omaha, NE, USA

dDepartment

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cCollege

of Mechanical Engineering, University of Nebraska-Lincoln, Lincoln, NE,

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USA e

Nebraska Center for Materials and Nanoscience, University of Nebraska-Lincoln, Lincoln,

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NE, USA f

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Medical Center, Omaha, NE, USA

Department of Emergency Medicine, University of Nebraska Medical Center, Omaha, NE,

USA

of Pediatrics, University of Nebraska Medical Center, Omaha, NE, USA

hDepartment

of Surgery, University of Nebraska Medical Center, Omaha, NE, USA

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gDepartment

*Corresponding Author: [email protected] ; Tel: +1 402 559 9637

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Highlights

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HA hydrogels formed by boronic ester dynamic covalent bond were facilely prepared.

Dynamic HA hydrogels possess good injectability and self-healing properties.

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Dynamic hydrogels can be used for reactive oxygen species responsive drug

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Dynamic hydrogels were biocompatible and protected cells from ROS induced

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delivery.

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damage.

Hydrogels can be used for 3D printed cell-laden scaffolds and “direct-in-

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gel”printing.

Abstract

In this study, an injectable and self-healing hydrogel based on the boronic ester dynamic covalent bond between phenylboronic acid modified hyaluronic acid (HA-PBA) and the commercially available poly (vinyl alcohol) (PVA) is prepared and should have multifunctions for biomedical applications. The hydrogels were rapidly formed under mild 2

conditions, and the rheological properties and in vitro degradation were systematically characterized. The HA-based hydrogels possessed good injectability and self-healing properties because of the dynamic bond. Moreover, due to the sensitivity of boronic ester to the biologically relevant concentration of hydrogen peroxide (H2O2), a major reactive oxygen species (ROS), the injectable hydrogel could be used as a H2O2/ROS responsive drug delivery system. The hydrogels supported good viability of encapsulated neural

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progenitor cells (NPC) and protected NPC from ROS induced damage in vitro when H2O2

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was present in the media. The dynamic hydrogels were further applied as bio-inks for 3D printing/bioprinting. Overall, this facilely prepared dynamic hydrogel based on HA-PBA

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and PVA may have many potential biomedical applications, including drug delivery, 3D

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culture of cells, and 3D bioprinting.

HA, hyaluronic acid;

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PBA, phenylboronic acid;

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Abbreviations

PVA, poly (vinyl alcohol);

ROS, reactive oxygen species; NPC, neural progenitor cells;

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GPGs, methacryloyl physical gels;

DMTMM, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride; DI, de-ionized;

PPBS, Dulbecco's phosphate-buffered saline; MWCO, molecular weight cut-off; ARS, Alizarin red S; 3

DOX, doxorubicin; RPMI, Roswell Park Memorial Institute medium; DMEM/F-12, Dulbecco's Modified Eagle medium: Nutrient Mixture F-12; FBS, fetal bovine serum; bFGF, basic fibroblast growth factor MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;

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LDH, lactate dehydrogenase;

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EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; pKa, ionization constant;

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CNS, central nervous system.

Keywords: Dynamic hydrogel, hyaluronic acid, self-healing, anti-oxidative, boronic

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acid ester, ROS responsive drug delivery, 3D printing

1. Introduction

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Injectable hydrogels can mimic human soft tissue and extracellular matrix (ECM), and they are promising platforms for drug and cell delivery and tissue engineering, as they allow minimal invasiveness and the filling of irregularly shaped lesion sites (Lee, 2018). Hyaluronic acid (HA) is an important component of ECM and can be used to prepare a variety of injectable hydrogels for tissue repair and regeneration (Highley, Prestwich, &

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Burdick, 2016). To form hydrogels, HA normally requires chemical modifications in the structures for crosslinking and the incorporation of different derivatives into HA polymer leads to distinct characteristics of HA hydrogels (Khunmanee, Jeong, & Park, 2017). HA hydrogels with diverse functions such as stimuli-responsiveness, adhesion and antibacterial are expected to aid their biomedical applications and improve the therapeutic responses and are gaining more attention (H. Chen et al., 2018; Patel & Dalwadi, 2013;

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Zhu, Li, Wang, Yu, & Wu, 2018).

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In terms of drug and cell delivery, the stimuli-responsive hydrogels are considered more efficient than non-responsive hydrogels (Oliva, Conde, Wang, & Artzi, 2017; Willner,

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2017). The stimuli-responsive hydrogels can respond to pathophysiological environmental

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change and shift the release kinetics for the delivery cargo under certain external stimuli, such as temperature, pH, light, and magnetic field (T. Fan, Li, Wu, Li, & Wu, 2011; Hu,

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Chen, Li, Zhou, & Cheng, 2017; Liu, Li, & Lam, 2018). Pathological microenvironments in many solid tumors and other diseases feature abundant reactive oxygen species (ROS),

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such as H2O2, which could be used as an efficient trigger for the drug delivery (Jin et al., 2019). The ROS responsive drug delivery systems have been extensively realized by many kinds of nanoparticles and prodrugs, which mainly rely on the incorporation of diverse ROS responsive linkers during nanoparticle and prodrug preparation steps (Ballance, Qin,

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Chung, Gillette, & Kong, 2019; Ye et al., 2019). However, ROS stimuli have rarely worked out in hydrogels (Ye et al., 2019). There are only a few reported ROS responsive hydrogels, and most of them require either multiple-step reactions or complicated reaction conditions for the hydrogel precursor synthesis (Ye et al., 2019). There is a great need to develop ROS

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responsive hydrogels that can be easily prepared and are applicable to drug and cell delivery. It is found that many of the current ROS responsive hydrogels are developed with innate cytoprotective abilities under oxidative stress (Dollinger, Gupta, Martin, & Duvall, 2017; Gupta et al., 2014; Q. Xu, He, Ren, Xiao, & Chen, 2016). Anti-oxidative hydrogels are capable of shielding the encapsulated cells from ROS related damage and can be a good

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platforms for cell transplantation, especially for the treatment of diseases involving high

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ROS, such as stroke, myocardial infarction, osteoarthritis, etc. (Crivelli et al., 2019; Hao et al., 2017; Lv et al., 2018; Santos, Sinha, & Lindner, 2018). The overall survival rate of

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transplanted cells can be improved after encapsulation in the anti-oxidative hydrogels and

therapeutic efficacy (Ye et al., 2019).

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delivery to the ROS rich environment, thereby improving the cell transplantation/

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Among all kinds of injectable hydrogels, greater attention is received by a special type of hydrogels called dynamic hydrogels, which can be formed through dynamic covalent

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bonds and present the self-healing ability due to the reversibility and equilibrium of dynamic bonds under certain conditions (Chakma & Konkolewicz, 2019). This hydrogel system shows many advantages compared to traditional, non-reversible covalent bond crosslinked hydrogels. The self-healing hydrogels were considered invulnerable to stress-

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induced crack formation, which should enhance their stability during implantation (Bastings et al., 2014; Burattini, Greenland, Chappell, Colquhoun, & Hayes, 2010; He, Fullenkamp, Rivera, & Messersmith, 2011). This unique characteristic of many types of dynamic hydrogels also allows the hydrogels to be injected through a fine needle due to the viscosity decrease under high shear stress (Gaffey et al., 2019; Hou, Wang, Park, Jin,

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& Ma, 2015). Such self-healing and injectable hydrogels allow the delivery of cargo (drugs and cells) directly to the target injection site, avoiding the immediate diffusion of the cargo that is found in traditional in situ formed hydrogels, which could improve the therapeutic effects and decrease toxicity to non-target tissues (Alarcin et al., 2018; Chen et al., 2017). Self-healing HA hydrogels have been prepared through host-guest interactions, imine bond, hydrazine bond, coordination bond, and others types of bonds (Chen et al., 2017; Y. Deng

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et al., 2017; Tian et al., 2018; L. L. Wang et al., 2018). However, there have been few

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reports on self-healing HA hydrogels that can respond to ROS (X. Xu et al., 2020).

Another important application of dynamic hydrogels is that they can also be used as bio-

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inks for 3D bio-printing (L. L. Wang et al., 2018). HA is an important component of many

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bio-inks, and Dr. Burdick’s group applied a dynamic HA hydrogel based on host-guest interaction to prepare bio-inks and successfully 3D printed stable scaffolds with multiple

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layers (Ouyang, Highley, Rodell, Sun, & Burdick, 2016). HA hydrogels based on a dynamic covalent hydrazone bond have been explored for 3D bioprinting and show

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promising effects (L. L. Wang et al., 2018). Given the high application potential of 3D bioprinting in tissue engineering, there is a need to develop novel and functional bio-inks, such as shear-thinning hydrogels, for the fabrication of miscellaneous scaffolds (Jammalamadaka & Tappa, 2018).

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In this work, we facilely prepared an injectable dynamic hydrogel based on phenylboronic

acid (PBA) grafted HA and commercially available poly (vinyl alcohol) (PVA). This hydrogel system is relatively stable, has good injectability, and is self-healing due to the formation of dynamic boronic ester bonds between phenylboronic acid groups and 1,3-diol groups (Marco-Dufort & Tibbitt, 2019). The dynamic HA based hydrogel has ROS

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responsiveness for drug delivery, as well as ROS scavenging properties. We demonstrated that the hydrogels were biocompatible toward encapsulated neural progenitor cells (NPC), and the ROS scavenging property protected the encapsulated NPC cells from ROS induced damage in vitro. We also applied the hydrogels as bio-inks for 3D bioprinting to fabricate multi-layer and cell-laden constructs. All of these results suggest that the multifunctional, injectable, and self-healing HA hydrogels are promising for a variety of biomedical

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applications, such as ROS responsive drug delivery, 3D culture, anti-oxidative protection,

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and 3D bioprinting.

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2. Materials and methods

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2.1 Synthesis of hyaluronic acid phenylboronic acid polymer conjugates (HA-PBA) The HA-PBA polymer conjugates were prepared by conjugating the 3-aminomethyl PBA

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(Alfar Aesar) to HA (290 kDa, Bloomage Biotech) using 4-(4,6-dimethoxy-1,3,5-triazin2-yl)-4-methyl-morpholinium chloride (DMTMM, TCI America) as the coupling agent

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(Fig. 1A). In a typical reaction, 100 mg of HA (0.25 mmol) were completely dissolved in 20 mL de-ionized (DI) water under constant stirring. Next, 47 mg PBA (0.25 mmol) and 80 mg DMTMM (0.25 mmol) were added to the HA solution separately. After all the agents were dissolved, the pH of the solution was adjusted to 6.5 using 1M HCl (Fisher

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Chemical) solution. In order to adjust the conjugation efficiency, another reaction was performed using 0.25 mmol HA, 0.25 mmol PBA, and 0.5 mmol DMTMM at the same pH. All reaction mixtures were stirred at room temperature for 72 h. After that, the mixtures were transferred to 6-8 kDa molecular weight cut-off (MWCO) dialysis bags (Spectrum) and dialyzed against DI water for at least 4 days at room temperature, with the water

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changed twice every day. The dialyzed solutions were freeze-dried using a benchtop lyophilizer (model FreeZone, Labconco) to obtain the HA-PBA polymer conjugates. The conjugates were stored in a desiccator at room temperature before use.

2.2 Proton nuclear magnetic resonance (1H NMR) analysis of HA-PBA conjugates 1

H NMR analysis was carried out on a 500 MHz Bruker NMR system and analyzed with

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Topspin 4.0 software. The polymer conjugates were dissolved in D2O at 5 mg/mL for NMR

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acquisition, and the chemical shifts were referred to the solvent peak of D2O (4.78 ppm at 25 °C). The degree of substitution was determined by the ratio of the integral of aromatic

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protons from the conjugated phenylboronic acid group (between 7.5~8 ppm, -C6H4) to the

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2.3 Alizarin red S complexation assay

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integral of the HA methyl proton peak (at 2.0 ppm, -CH3).

Alizarin red S (ARS) was prepared at 0.1 mM from a dilution of commercially available

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40 mM ARS solution (EMD Millipore) with PBS at pH 7.4. An HA-PBA solution was first prepared at 20 mM (referred to PBA amount) in 100 µl PBS at pH 7.4 and then mixed with 2 ml of ARS. The absorbance spectra of ARS with and without HA-PBA were collected by a microplate reader using wavelengths from 400 nm to 650 nm (SpectraMax M5,

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Molecular Devices).

2.4 Fabrication of injectable HA-PBA-PVA dynamic hydrogels Two different types of hydrogels were prepared by mixing 1.5 wt% or 2.5 wt% HA-PBA, dissolved in phosphate buffer (PBS, Calbiochem) at pH 7.4, with corresponding 1.5 wt%

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or 2.5 wt% PVA (13-23 kDa, Sigma), dissolved in PBS at pH 7.4, in a 3:1 volume ratio, respectively, at room temperature. An inversion test in an Eppendorf tube was conducted to verify the successful hydrogel formation. The gelation was caused by the boronic ester dynamic bond formation between the PBA groups in HA-PBA and 1,3-diol groups in PVA. The prepared hydrogels were referred to as HA-PBA-PVA 1.5% (prepared by using 1.5 wt% HA-PBA and 1.5 wt% PVA) and HA-PBA-PVA 2.5% (prepared by using 2.5 wt%

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HA-PBA and 2.5 wt% PVA). The gelation process was also evaluated using the same

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concentrations of HA-PBA and PVA dissolved in PBS at pH 5.5. However, hydrogels

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evaluated in all other studies were prepared in PBS at pH 7.4.

Figure 1. Scheme for the synthesis of the HA-PBA conjugate and the fabrication of the injectable hydrogel. The HA-PBA (hyaluronic acid-phenylboronic acid) polymer conjugate was prepared by conjugating the 3-aminomethyl PBA to HA (290 kDa), using DMTMM as the coupling agent. The HA-PBA hydrogel is prepared by mixing HA-PBA with PVA solutions with the same concentrations (13-23 kDa) at a 3:1 volume ratio. The 10

gelation is caused by the formation of dynamic covalent bonds (boronic ester). The resulting gel is self-healing and injectable through needles.

2.5 Rheological studies The rheological properties testing of the hydrogels was conducted at 25C and 37C by

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employing an AR1500ex rheometer (TA Instruments) using three different methods. In each study, around 0.5 ml of the hydrogel precursor solution was mixed in an Eppendorf

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tube, and the formed hydrogel was transferred to the geometry gap between the 25 mm parallel plate and the base plate (X. Deng et al., 2018). The oscillatory amplitude sweep

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method (γ = 0.1%-300%), with constant frequency of 2π rad/s (=1 Hz), was performed first

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to record the storage (G’) and loss moduli (G’’) changes and the critical strain region for two different hydrogels. Frequency sweeps from 0.1 to 10 Hz were performed at 10%

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constant strain. Then, to evaluate the shear-thinning behavior, the hydrogels were subjected to flow sweep experiments with a linearly ramped shear rate from 1 to 40 s–1, and the

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viscosity was measured under different shear rates. In the end, the self-healing characteristics of the hydrogel were determined by the alternate step strain sweep test with a fixed angular frequency (2π rad/s). Amplitude oscillatory strains were switched from a small strain (γ = 10 % for 1.5% hydrogel and 5% for 2.5% hydrogel) to a subsequent large

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strain (γ = 300%) with a 120 s strain interval, and 3 cycles were conducted.

2.6 Self-healing and injectability studies of HA-PBA-PVA hydrogel Each piece of colored hydrogel was prepared by mixing 30 µL of 2.5% HA-PBA, 10 µL of 2.5% PVA, and 2 µL of food dye solution (green and red). After gelation, two pieces of

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dye colored hydrogels were placed next to each other in a plastic dish. The dish was covered and kept in cell incubator for 10 min to evaluate the self-healing ability. After 10 min, the healed hydrogel was held up and pulled on both ends with tweezers for observation. For the injectability test, another 150 µL of 2.5 wt% HA-PBA, 50 µL of 2.5 wt% PVA, and 10 µL of doxorubicin (DOX) (10 mg/mL) were mixed all together to prepare the hydrogel for the injectability demonstration. After gelation, the hydrogel was loaded into

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a 1 mL syringe that was capped with a 21-gauge(G) needle to evaluate whether the

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hydrogel could be extruded through the needle into a plastic dish after manually pushing

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2.7 Degradation of HA-PBA-PVA hydrogels

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the syringe plunger.

The HA-PBA-PVA 1.5% hydrogels and HA-PBA-PVA 2.5% hydrogels were prepared by

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mixing 30 µL of HA-PBA and 10 µL of PVA for each gel, as previously mentioned. Each different hydrogel was incubated in a 1.5 mL centrifuge tube with 1 mL of PBS alone at

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pH 7.4 or with 1 mL of PBS at pH 7.4 containing 0.2 mM H2O2 (Fisher Scientific) at 37 °C. The weight of each hydrogel sample (Ws) was measured after removing excess PBS from the hydrogel surface and was recorded at each time point, with the initial weight set as W0. The remaining weight% was calculated using the equation: Ws/W0×100%. Each group had

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three replicates.

2.8 Cell culture and cytotoxicity study of HA-PBA polymer conjugates Both the breast cancer cell line MDA-MB-231 and a human induced pluripotent stem cell (iPS) derived NPC cell line (UTY1) were used to evaluate the cytotoxicity of HA-PBA

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polymer conjugates. The MDA-MB-231 cells were cultured in Roswell Park Memorial Institute medium (RPMI 1640, Gibco) supplemented with 10% fetal bovine serum (FBS, Sigma) and 1% Penicillin-Streptomycin (Invitrogen). The UTY1 cells were cultured in a mixture of 50% of Dulbecco's Modified Eagle medium: Nutrient Mixture F-12 (DMEM/F12) and 50% of neurobasal medium (Gibco) supplemented with 1% N2 (Gibco), 1% B27 (Gibco), 1% Penicillin-Streptomycin (Hyclone), and 20 ng/µL basic fibroblast growth

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factor (bFGF, Peprotech). A total number of 1 ×104 of MDA-MB-231 or 2 × 104 of UTY1

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cells were seeded in each well of a 48-well plate, and the cells were allowed to attach to the plate overnight at 37 °C in a humidified incubator containing 5% CO2. The plate was

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previously coated with a 1:100 dilution of growth factor reduced Matrigel (Corning) for 4

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hours before UTY1 cell seeding. The HA-PBA polymer conjugate, in a series of concentrations from 0.01 mg/mL to 1 mg/mL, was dissolved in corresponding culture

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media and added to each type of cells, followed by 48 h incubation. The control group contained no polymer conjugate. Each concentration and control group had 4 replicates.

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After that, the cytotoxicity was determined by the MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide, Sigma) assay (Ying Wang et al., 2018).

2.9 Live/dead assay of encapsulated cells inside hydrogels

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Both the MDA-MB-231 and UTY1 cells were expanded in the tissue culture plate in advance and re-suspended at 5×106/mL cell density in each culture media. For each hydrogel, a volume of 5 µL of cell suspension from each cell type was added to each 30 µL of HA-PBA and 10 µL of PVA at 1.5% and 2.5% (w/v) concentration, respectively, as previously mentioned. After gelation, each hydrogel was transferred to one well of a 96-

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well plate and 200 µL of cell culture media was added. The media was changed every two days. UTY1 cells encapsulated in growth factor reduced Matrigel (Corning) served as the control, and each one was prepared by adding 5 µL of UTY1 cell suspension at 5×106/mL into 40 µL of Matrigel. At each pre-determined time point, a live/dead assay was used to

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test the cell viability inside the hydrogel and Matrigel (Y. Wang et al., 2018).

2.10 DOX release from injectable hydrogels

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The drug release was evaluated in both types of hydrogels, i.e. HA-PBA-PVA 1.5% and HA-PBA-PVA 2.5%. Each of the DOX loaded hydrogels was prepared by mixing

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corresponding solutions of 30 µL HA-PBA with 10 µL PVA and 5 µL DOX (10 mg/mL).

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Hydrogels were individually incubated in 10 mL of PBS pH 7.4 alone or 10 mL of PBS at pH 7.4 with 0.1 mM H2O2 at 37°C for 2 days. At each time point, 100 µL of release buffer

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was withdrawn and stored at -20 °C before analysis. The amount of released DOX at each time point was determined by fluorescence using a microplate reader with an excitation

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wavelength set at 480 nm and an emission wavelength set at 550 nm. A DOX calibration curve with good linearity (R2=0.994) was generated by plotting the fluorescence emission value against the standard DOX concentration at 0, 1.25, 2.5, 5, and 10 µg/mL.

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2.11 ROS scavenging properties of hydrogel precursor The ROS scavenging property of the hydrogel precursor was determined by the pyrogallol autooxidation assay modified from reported methods (X. Li, 2012; Y. Zhu et al., 2018). Generally, 1.5% (w/v) HA-PBA hydrogel precursor solution or HA solution or PVA solution was prepared. After 30 µL of each solution was individually mixed with 120 µL

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Tris-HCl (50 mM, pH=7.5) in every well of a 96-well plate, 15 µL of pyrogallol (Alfar Aesar, 3 mM) water solution was subsequently added into the mixture, and the whole mixture was incubated in the dark for 20 min. In the control group, 30 µL of DI water was added instead of the precursor solution. Each group had 3 replicates. The absorbance change (∆Ab) during pyrogallol autooxidation of each sample from time zero to 20 min was measured by the microplate reader at 325 nm wavelength. The scavenging effect on

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ROS species was calculated using the following equation:

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Scavenging effect (%) = [ (∆Abcontrol- ∆Absample)/ ∆Abcontrol] ×100

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2.12 Anti-oxidative protection effect against H2O2 induced damage to NPC

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UTY1 cells were encapsulated in hydrogels and Matrigel, as described in Section 2.9. After 24 h incubation in the normal growth media, the media was replaced with fresh media

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containing 0.1 mM H2O2. The cells in the hydrogels and Matrigel were incubated in the H2O2 containing media for another 24 h, with each gel in one well of a 96-well plate. The

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live/dead assay was used to evaluate the cell viability in different gels after H2O2 treatment. Cells in the hydrogels and Matrigel were incubated in normal media for another 24 h to be used as the control.

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2.13 The lactate dehydrogenase assay (LDH) Aliquots (50 μL) of the media from the 1.5% hydrogels and Matrigel groups, with and without 24 h H2O2 (0.1 mM) treatment, were collected to determine the release of lactate dehydrogenase (LDH) using the CytoTox 96®NonRadioactive Cytotoxicity Assay kit

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(Promega), following the manufacturer’s protocol and the reported method (Ying Wang et al., 2018). The absorbance (OD) at 490 nm wavelength was recorded on a microplate reader.

2.14 ROS reactive fluorescent staining To preliminarily investigate the mechanism behind the anti-oxidative protection effect of developed hydrogels, the ROS/H2O2 reactive fluorescent probe 2’,7’-dicholorodihydro

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fluorescein diacetate (H2DCFDA, EMD Millipore) was applied to test the intracellular

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H2O2 levels. UTY1 cells encapsulated in hydrogels and Matrigel were incubated in medium containing 0.1 mM H2O2 for two hours and then washed with DPBS. The H2DCFDA probe

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was prepared as 5 mM stock solution in DMSO and diluted to 5 µM with DPBS instantly

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before use. Each hydrogel and Matrigel was then incubated in 5 µM probe solution for 20 min at 37°C before confocal microscope (Zeiss 880) observation. The intracellular

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2.15 Statistical analysis

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fluorescence was excited at 480 nm and emitted at 540 nm.

All quantitative data is expressed as the mean ± standard deviation (SD). Statistical analysis was performed using student t-tests in GraphPad Prism 7.0 Software. A pvalue < 0.05 was considered statistically significant. In all cases, * represents p<0.05, **

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represents p<0.01 and *** represents p<0.005.

3. Results

3.1 Synthesis and characterization of HA-PBA conjugates

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The HA-PBA conjugate was prepared by grafting the 3-aminomethyl PBA moiety to the HA backbone through an amide bond. The schematic synthesis of HA-PBA is shown in Fig. 1A. DMTMM was used as the coupling agent instead of 1-ethyl-3-(3-dimethylamino propyl) carbodiimide (EDC), as DMTMM showed improved conjugation efficiency in previous reports, and it is difficult to remove impurities after EDC reaction (D'Este, Eglin, & Alini, 2014; Gennari et al., 2017). The NMR analysis first confirmed the conjugation

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and revealed that about 34% of the carboxylic acid in the HA backbone was amidated by

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the PBA moiety (Fig. S1A). It was also found that increasing amount of DMTMM in the conjugation reaction raised the grafting ratio and resulted in over 50% of the PBA moiety

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being grafted in the HA backbone. DMTMM proved efficient for coupling PBA groups to

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HA. No significant change in the HA solubility was found after the PBA grafting. Based on NMR, the HA-PBA with a grafting ratio of 34% seemed to contain less impurities and

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was used for following studies.

The successful conjugation of the PBA moiety to the HA was also confirmed by the ARS

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assay, which has often been used to investigate the interaction between boronic acid and diols (Gennari et al., 2017). The original ARS solution in PBS at 7.4 pH showed red color; however, after the addition of the HA-PBA conjugate, the solution turned into a yellow color (Fig. S2A). From the visible light absorbance scanning study, the ARS solution

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showed a maximal absorbance peak at 520 nm, while the solution with HA-PBA showed a maximal absorbance peak at 470 nm (Fig. S2B). All of this data is consistent with reported findings, of which the complexation between phenylboronic acid and the catechol in ARS caused the absorbance spectra shift from red to yellow (Gennari et al., 2017; Negri & Deming, 2017). Those results indicated the successful conjugation of PBA on HA, and

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the resulting product maintained the ability of boronic ester bond formation after conjugation to the HA backbone.

3.2 Fabrication and characterization of injectable hydrogels The injectable hydrogel was prepared by mixing the HA-PBA solution and PVA solution together at pH 7.4. Rapid gelation (<15 s) occurred at room temperature due to the boronic

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ester dynamic bond formation (Fig. S1B) (Brooks & Sumerlin, 2016). It has been

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previously reported that alkaline conditions (pH 8~10) favored the boronic ester bond formation due to the ionization constant (pKa) of many PBA derivatives (Hong et al., 2018;

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Marco-Dufort & Tibbitt, 2019). For application purposes in pathophysiological conditions,

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we tried to evaluate the effect of neutral and acidic pH conditions on the gelation process. It was found that even at pH 5.5, the gelation still occurred easily (Fig. S1B), which might

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be explained by the significant lowering of the pKa of PBA in the HA backbone due to the negative charge on HA, as a previous study discussed (Tarus et al., 2014). No gelation time

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difference was found at two different pH levels. The results indicated that the hydrogel gelation could happen in a wide range of pH conditions (at least from pH 5.5 to 7.4 or even higher). As we were interested in the biomedical applications of the hydrogels, a pH of 7.4 was selected as our gelation pH.

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The rheological properties of the prepared hydrogels were determined by oscillatory amplitude sweep tests at 25C and 37C. The rheological properties are dependent on the hydrogel precursor concentration (Fig. 2A), with no significant difference found between the two temperatures. The storage modulus (G’) of the 1.5% and 2.5% hydrogels reached 235 ± 21 Pa and 1098 ± 81 Pa, respectively, and was higher than the loss modulus (G’’) at

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moderate strains. The intersection, or critical point, appeared at nearly 162% for the 1.5% hydrogel and 102% for the 2.5% hydrogel. When the strain was continually increased, the G’ value showed a dramatic decrease and was lower than the G’’ value, suggesting a collapse of the hydrogel network and a gel to sol state transition. The frequency sweep tests showed that both hydrogels maintained their elastic characteristic across the frequency range tested, with G’ consistently larger than G’’ (Fig. S3A).

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The injectability of the hydrogel was confirmed by the sharp decrease of the viscosity with

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the increase of an applied shear rate (Fig. 2B). The viscosity for the 1.5% and 2.5% hydrogels were 103.5 and 227.5 Pa·s, respectively, at the shear rate of 1 s-1and substantially

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declined to 7.2 at the rate of 40 s-1 for the 1.5% hydrogel and 19.5 Pa·s at the rate of 20 s-1

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for the 2.5% hydrogel.

The self-healing property of the hydrogel, caused by the dynamic boronic ester bond, was

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evaluated by the rheological recovery test (Fig. 2C). The recovery process was found to be consistent during the three alternating cycles of high and low strain. When the hydrogel

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was subjected to a high strain, the G’ value decreased from 250 Pa to 19 Pa for the 1.5% hydrogel and from 1155 Pa to 61 Pa for the 2.5% hydrogel. However, once the strain returned to the initial low strain, the value of G’ immediately recovered to 80% of the initial G’ value for the 1.5% hydrogel and to 72% of the initial G’ value for the 2.5% hydrogel.

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When a lag time (60 s) was added between the high and low strains (last cycle in the recovery test), the G’ value recovered to almost 100% of the initial G’ value for the 1.5% hydrogel and to 83% of the initial G’ value for the 2.5% hydrogel. To better prove the selfhealing property, a piece of 2.5% hydrogel was cut in half and then the two pieces were put together for healing for 10 min. We measured the storage modulus before cutting and

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after healing, and we found the storage modulus of the healed hydrogel almost recovered

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to the level before cutting (Fig. S3B).

Figure 2. Characterization of the rheological properties of HA-PBA-PVA hydrogels

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prepared at 1.5% and 2.5% precursor concentrations. A) Storage and loss moduli of two HA-PBA-PVA hydrogels; B) shear rate dependent viscosity change of HA-PBA-PVA hydrogels; C) storage and loss modulus changes with high recovery for G’ under

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alternating strains indicated the self-healing property of the hydrogels.

The hydrogels were relatively stable in PBS at 37 °C, with about 50% degradation in two weeks (Fig. 3C). On average, the HA-PBA-PVA 2.5% hydrogel degraded slightly slower than the HA-PBA-PVA 1.5% hydrogel, which indicated that the half-life of the hydrogels might be manipulated by changing the initial HA-PBA concentration. The influence of pH 20

on the stability of the hydrogel was also evaluated by incubating the 1.5% hydrogel in PBS at pH 5.5 (Fig. S2C). The result showed that such low pH did not significantly accelerate the degradation rate, and the hydrogel was relatively stable in slightly acidic conditions, which may preserve the hydrogel stability in the slightly acidic tumor microenvironment (W. Li & Sun, 2018). However, the stability of the hydrogels seemed very sensitive to H2O2, as the presence of H2O2 notably drove the degradation of the hydrogels (Fig. 3C).

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The mechanism behind this was probably involving a C-B bond breakage in the

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phenylboronate ester and a phenol product generation (Gennari et al., 2017), which would cause irreversible damage of the hydrogel cross-linking network. The HA-PBA-PVA 1.5%

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hydrogel was completely degraded within two days, and the HA-PBA-PVA 2.5% hydrogel

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degraded over 90% in three days in 0.2 mM H2O2 conditions. It was evident that the higher HA-PBA content caused the delay of the degradation degree. When the concentration of

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H2O2 was increased to 2 mM, the hydrogels could even be degraded within several hours. We also examined the self-healing ability of the hydrogel macroscopically. A red colored

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hydrogel was put in contact with another yellow colored hydrogel, allowing them to heal with each other at 37 °C for 10 min. It was found that the boundaries between the contacting hydrogels gradually became obscure. The two pieces of hydrogels were healed together as a single hydrogel and could be easily lifted by holding one end of the hydrogels. The

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pulling on both ends of the healed hydrogel did not cause any breakage. Those results, together with the rheology recovery test results, verified a good self-healing ability of the hydrogel.

The chemotherapeutic drug-DOX can be mixed with a hydrogel precursor, and a DOX loaded HA-PBA-PVA 2.5% hydrogel can be transferred into syringes and easily extruded

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out through 21G needles (Fig. 3B). We also proved that the HA-PBA-PVA 1.5% hydrogel could be easily extruded out through a very fine 31G needle (Video 1). All of the results showed good injectability of the dynamic hydrogels, which would facilitate their clinical

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application as a minimally invasive drug delivery system through catheters or needles.

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Figure 3. Self-healing and injectability characteristics of HA-PBA-PVA dynamic hydrogels (2.5%) and the swelling degradation profile of hydrogels (1.5% and 2.5%) in different solutions. A) Separate hydrogels could rapidly heal together after being put next to each other; B) hydrogels could be easily extruded from a syringe through 21G needles;

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C) degradation profile of hydrogels in PBS, with and without H2O2 (0.2 mM), revealed faster degradation in the presence of H2O2.

3.3 ROS responsive drug delivery from the injectable hydrogels To evaluate the injectable hydrogel as a drug delivery system, we first confirmed that the hydrogel itself did not affect the growth of mammalian cells by using a triple negative

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breast cancer cell line MDA-MB-231. The MTT study indicated that the polymer conjugate

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showed no toxicity toward the cells at up to 1 mg/mL (Fig. 4A). From the live and dead staining, cancer cells encapsulated within 1.5% and 2.5% hydrogels showed 94 ± 2% and

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88 ± 2% live cells at day 1, respectively, and 90 ± 2% and 85 ± 1% viability at day 5,

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respectively (Fig. 4B and 4C). Cancer cells grown in the 2.5% hydrogel had slightly lower viability than those on the 1.5% hydrogel (p< 0.01 at day 1 and p< 0.05 at day 5). This

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might be explained as a locally high concentration of PBA inside the hydrogel may not support the cancer cell growth as well as the relatively low concentration PBA. A previous

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study has also shown that free PBA molecules showed no toxicity to breast cancer cell 4T1 at up to 1 mg/ml, but they presented cytotoxicity at 10 mg/ml (Marasovic et al., 2017). However, from a drug delivery perspective, the hydrogel itself can be considered as

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biocompatible and as having no anti-cancer cell effects.

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Figure 4. Cytotoxicity of HA-PBA conjugate to MDA-MB-231 cells and live/dead assay

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of MDA-MB-231 encapsulated in two HA-PBA-PVA hydrogels. Scale bar=100 µm. A) MTT study of HA-PBA conjugate to MDA-MB-231 cells has shown no significant toxicity at up to 1 mg/ml; B&C) Live and dead assay of MDA-MB-231 cells encapsulated in HA-

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PBA-PVA hydrogels for at least 5 days indicated good biocompatibility.

The boronic ester-based hydrogel networks can be disrupted due to the sensitivity of

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boronic ester towards low pH, high glucose, and the existence of H2O2 (Guo et al., 2017; He et al., 2011; Marco-Dufort & Tibbitt, 2019; C. Wang et al., 2018; Yesilyurt et al., 2016). Even though the boronic ester-based hydrogels have been evaluated for pH or glucose responsive drug delivery, there are only a few studies regarding the H2O2 responsive drug delivery (Hong et al., 2018; C. Wang et al., 2018). We found that the release of DOX from the hydrogel followed a typical biphasic release profile, with an immediate, burst release 24

over a relatively short time period, followed by a sustained release over a relatively long period of time. H2O2, a major species of the ROS, can accelerate the chemotherapeutic drug release from the hydrogels (Fig. 5A and 5B), at a biologically relevant concentration (100 µM). DOX loaded hydrogels: HA-PBA-PVA 1.5% and 2.5% released 63.6 ± 1.8% and 53.4 ± 6.7% of total drugs, respectively, at the 2 h time point in PBS buffer at pH 7.4. In the presence of H2O2, the DOX release was increased to 74.2 ± 1.6% and 66.2 ± 0.6%

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for the 1.5% and 2.5% hydrogels, respectively. At the 4 h time point, there was 79.2 ± 6.8%

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and 75.1 ± 2.2% release from 1.5% and 2.5% hydrogel, respectively, in the absence of H2O2; however, 96.0 ± 0.2% and 85.8 ± 5.9% were released from 1.5% and 2.5% hydrogels,

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respectively, when H2O2 existed in the PBS. From 4 h to 48 h, the drug release from both

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hydrogels were relatively slow under normal conditions, and the total release ratio after 48 h was 82.4 ± 2.4% and 83.1 ± 6.4% for the 1.5% and 2.5% hydrogels, respectively.

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However, the total release ratio after 48 h reached 95.5 ± 0.6% and 98.5 ± 0.5% for the 1.5% and 2.5% hydrogel, when H2O2 was present. It can be concluded that the DOX release

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from the hydrogels was responding to H2O2. With an increased hydrogel precursor concentration, the time required for the “complete” drug release (> 95% loaded drug) also increased. When excess H2O2 was present, the boronate esters were easily degraded, which caused an enhanced erosion of the hydrogel network. We attribute the ROS/H2O2

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responsive drug release to the enhanced erosion, while without ROS/ H2O2, the drug release was dominated by the diffusion, which is consistent with a previously reported ROS responsive hydrogel based on boronate ester (C. Wang et al., 2018). As indicated in the literature (de Gracia Lux et al., 2012; Gennari et al., 2017), the oxidative cleavability of the boronate esters is due to the cleavage of sp2 carbon−boron bond and correspondingly

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increasing amounts of phenol product were produced. Furthermore, we also validated that

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the hydrogel would not affect the loaded drug efficacy, through in vitro cytotoxicity studies.

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Figure 5. H2O2 responsive drug release profile of DOX loaded hydrogels and the efficacy evaluation of released DOX from the hydrogel. A&B) The presence of H2O2 accelerated

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the DOX release from both the 1.5% and 2.5% hydrogels.

3.4 3D culture of NPC within hydrogels and anti-oxidative protection against H2O2 induced damage

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Injectable hydrogels can be used as a 3D culture scaffold of cells or as a cell delivery system to repair the central nervous system (CNS) (Tseng et al., 2015). HA is a majorly important ECM component (Bignami, Hosley, & Dahl, 1993). We presumed that our

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hydrogel should be biocompatible with NPC, given the substantial amount of HA in our hydrogel and the similar mechanical properties of our hydrogel to CNS tissues. We first evaluated the toxicity of the HA-PBA conjugate to NPC by an MTT study and found no significant difference in the NPC growth, even when the HA-PBA concentration was at 1 mg/mL (Fig. 6A). The viability of NPC within those two hydrogels was then evaluated by a live/dead assay. The results showed 90 ± 5% and 89 ± 5% live cells in the 1.5% and 2.5% 26

hydrogels, respectively, at day 1 after 3D culture and 88 ± 18% and 87 ± 2% in the 1.5% and 2.5% hydrogels, respectively, at day 4 (Fig. 6B). The NPC viability in our hydrogels is comparable to that in Matrigel, which is widely used for NPC 3D culture (Fig. 6B). Some dispersed NPC started aggregation from day 2, and most of them were aggregated and formed neurospheres of apparent size at day 4 in both hydrogels (Fig. 6C). Those results

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demonstrated good biocompatibility of the hydrogel to NPC.

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Figure 6. Cytotoxicity of HA-PBA conjugate to neural progenitor cells (UTY1) and live/dead assay of UTY1 encapsulated in two HA-PBA-PVA hydrogels compared with Matrigel. Scale bar (white)=100 µm; scale bar (black)=250 µm. A) MTT study of HA-PBA conjugate to UTY1 cells has shown no significant toxicity at up to 1 mg/ml; B &C) live and dead assay of UTY1 cells encapsulated in HA-PBA-PVA hydrogels for up to 4 days indicated good biocompatibility comparable to Matrigel. Both fluorescent images (C 27

middle panel) and bright field images (C bottom panel) revealed neurospheres were formed in the hydrogel starting at day 2, with more apparent size at day 4.

To prove the anti-oxidative property of the hydrogels, we performed several studies. First, the pyrogallol autooxidation assay was carried out in an alkaline buffer. During the autooxidation process, a colored compound called purpurogallin is formed, which has a

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prominent UV absorption at around 325 nm (Ramasarma et al., 2015). By adding the

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hydrogel precursor in the initiation of pyrogallol autooxidation, the scavenging of oxygen radicals by the hydrogel precursor would cause a decrease in the purpurogallin generation

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rate, as well as the UV absorbance at 325 nm. It was found that neither the HA nor PVA

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showed any scavenging effect, while the 1.5% hydrogel precursor showed a 45.8 ± 2.5% scavenging effect, and the 2.5% hydrogel precursor showed a 69.1 ± 3.7% scavenging

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effect (Fig. 7A). This indicated a satisfactory oxygen radical scavenging effect of our hydrogels. The scavenging effect seemed proportional to the concentration of the hydrogel

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precursor.

After confirming the ROS scavenging effect by chemical assay, we further implemented such anti-oxidative characteristic to protect the encapsulated cells from ROS damage. The encapsulated NPC within our HA-PBA-PVA hydrogels and Matrigel were exposed to

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H2O2, and the cell viability was determined and compared to those without H2O2 treatment. It is evident that NPC cultured in hydrogels were protected from ROS induced damage and showed comparable viability to the non-treatment group. In contrast, NPC cultured in Matrigel showed only 60 ± 3% live cells, which is significantly lower than the viability of the non-treatment group of 85 ± 1% live cells (Fig. 7B &C). Supernatants from the cultured

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media were collected, and the released LDH was measured (Fig. 7D). There is only a small amount of LDH increase in the hydrogel group after H2O2 treatment. However, we found a 2.5-time increase of LDH in the Matrigel group after H2O2 treatment. The released LDH in the Matrigel group with H2O2 is also 2 times higher than that in the 1.5% hydrogel group with H2O2. The H2DCFDA is a cell permeable and non-fluorescent agent (Oparka et al., 2016). In the cytosol and in the presence of H2O2, H2DCFDA is converted to a fluorescent

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product (DCF) and therefore has been used as a “H2O2 specific” probe (Gomes, Fernandes,

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& Lima, 2005). The preliminary staining study using this probe to evaluate the intracellular ROS/H2O2 of NPC cultured in Matrigel and dynamic hydrogels in the media containing

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H2O2 indicated a much higher H2O2 level (higher fluorescence intensity) in NPC cultured

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in Matrigel compared to dynamic hydrogels (Fig. 7E). This might explain the increased survival in dynamic hydrogels, as the anti-oxidative hydrogels could shield and scavenge

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the environmental H2O2 from cells. All of these results verified the anti-oxidative

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protection against ROS damage of 3D cultured cells in the hydrogels.

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Figure 7. Anti-oxidative ability of HA-PBA-PVA hydrogel precursors and the anti-ROS

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protection effect of the HA-PBA-PVA hydrogel for 3D cultured UTY1 cells in the presence of 0.1 mM H2O2. Scale bar=250 µm. A) Pyrogallol assay indicated significant ROS scavenging ability of the HA-PBA conjugate, which was absent in both HA and PVA; B&C) live and dead assays revealed an anti-oxidative protective effect of hydrogels to the encapsulated UTY1 cells, as compared to Matrigel, when exposed to H2O2 containing media for 24 h; D) LDH assay confirmed significantly more cell death in Matrigel, as 30

compared to that in the anti-oxidative hydrogel after H2O2 treatment; E) H2O2 specific probe H2DCFDA staining revealed higher intracellular H2O2 in cells cultured in Matrigel compared to cells cultured in HA-PBA-PVA hydrogels.

4. Discussion The rapid complexation between boronic acid and 1,2-diol or 1,3-diols leads to a dynamic

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boronic ester bond in aqueous solution under ambient conditions without any catalyst and

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has been studied and applied in many areas, including analyte sensors, disease diagnosis, and drug delivery (Brooks & Sumerlin, 2016; Chakma & Konkolewicz, 2019; Marco-

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Dufort & Tibbitt, 2019). Boronic acid compounds normally show low toxicity, and

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engineered hydrogel networks based on boronic ester have been reported (Marco-Dufort & Tibbitt, 2019). Boronic ester bond cross-linked hydrogels can be easily formed in an

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alkaline buffer, but very few studies have been reported to prepare dynamic hydrogels with H2O2 responsive properties. In this study, we developed multifunctional HA-PBA-PVA

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hydrogels that can form under neutral pH and respond to ROS. By employing the dynamic boronic ester bond, hydrogels are easily equipped with shearthinning and self-healing properties. However, many of the previously reported dynamic boronic ester hydrogels suffer from stability problems due to the weak binding strength

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between boronic acids and diols, particularly under physiological conditions, thereby limiting their applications (Marco-Dufort & Tibbitt, 2019). In this study, we designed a 3aminomethyl PBA grafted HA and mixed it with PVA solution to prepare the dynamic hydrogel. The HA-based hydrogel is advantageous, as HA has been exploited in many hydrogel systems and is highly biocompatible and supportive for a variety of cells, such as

31

breast cancer, glial, and neural cells due to their CD44 expression (Agrawal et al., 2018; Dzwonek & Wilczynski, 2015; Lam, Truong, & Segura, 2014). PVA is also biocompatible and has been used in diverse hydrogels for tissue engineering and drug delivery (Z. Wu, Kong, Liu, Sun, & Mi, 2018; Yuan et al., 2017). Our dynamic hydrogel preparation process only requires one simple amidation reaction before mixing, and a relatively stable, injectable, and self-healing hydrogel, with a half-life of ~14 days in PBS at pH 7.4, is

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obtained. Although other PBA derivatives have been tried to conjugate to HA before, such

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as the 2/3-aminophenylbornic acid group instead of 3-aminomethyl PBA in our case, which also resulted in dynamic hydrogels, they still suffered from stability issues. Limited

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applications have also been reported, with a focus on pH or glucose responsiveness (X.

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Deng et al., 2018; Figueiredo et al., 2019; Tarus et al., 2014). We have initially synthesized a 2-aminophenylboronic grafted HA with a close grafting ratio (~30%, Fig. S5) and mixed

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it with the PVA at 1.5 wt% at pH 7.4. Even though the hydrogel was formed like our system, the resulting hydrogel was unstable and easily dissociated in a PBS buffer within a few

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hours. We suspected this is probably why the reported HA-2-amionphenylboronic acidbased dynamic hydrogel would require another interpenetrating hydrogel system, like a Ca2+ cross-linked alginate to stabilize the network for 3D cell culture (X. Deng et al., 2018). It seems that 3-aminomethyl PBA is a better choice for HA based dynamic hydrogel.

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The ROS responsive drug delivery system can augment the therapeutic efficacy for ROS

rich tumors, such as prostate and breast tumors (D. Chen et al., 2018; C. Wang et al., 2018; Yu, Su, Chen, Chiang, & Lo, 2016). Some good reviews have summarized the advancement in ROS responsive nanoparticles for drug delivery purposes (Ballance et al., 2019; Saravanakumar, Kim, & Kim, 2017; Ye et al., 2019). Nanoparticles with boronic

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ester bonds have also been prepared and have shown enhanced drug release and therapeutic results in response to biologically relevant ROS (de Gracia Lux et al., 2012; Lv et al., 2018). Dr. Zhen Gu’s group reported the use of a boronic ester-based ROS responsive hydrogel for the programmed release of gemcitabine and a checkpoint inhibitor as combination therapy to fight cancer and achieved exciting results (C. Wang et al., 2018). Their hydrogel was formed in situ, which might cause drug diffusion to non-target tissues before gelation

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and is not as convenient as our shear-thinning hydrogel. On the contrary, therapeutic drugs

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can be first loaded into our hydrogel precursor, followed by the hydrogel formation, and finally, injection into the target site in the thixotropic gel, thereby avoiding the diffusion

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issue and giving an expected improvement to the therapeutic outcome. The innate anti-

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oxidative property of our hydrogel was proven to protect the encapsulated cells from environmental ROS and is an advantage for cell therapy. High ROS remains a great

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challenge to many cell-based therapies and transplantations (Arts, Gennaris, & Collet, 2015; Q. Xu et al., 2016). It was found that the H2O2 concentration in a brain ischemia site can

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reach up to 0.1~0.2 mM (Hyslop, Zhang, Pearson, & Phebus, 1995). Anti-oxidative hydrogels can increase of survival ratio of transplanted cells in ROS rich pathological environments and enhance the therapeutic efficacy (Q. Xu et al., 2016). Some of the reported anti-oxidative hydrogels are not easily injectable, which will hamper their

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translational application, and many others are more difficult to prepare, which is not costefficient (Gupta et al., 2014; Q. Xu et al., 2018). Our hydrogel could overcome those problems and become a good candidate for cell transplantation. Due to the stability limitation of boronic ester hydrogels, which retards their sustainable use in cell culture media, a limited number of studies have demonstrated their use for 3D

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cell culture (Marco-Dufort & Tibbitt, 2019). Hydrogels formed between two zwitterionic polymers with pendant benzoxaborole and catechol moieties only allowed the encapsulation and culture of cells for 24 h (Y. Chen et al., 2018). Dual networks/crosslinking were required for long term cell culture for up to 7 days (Tang et al., 2018). Although our developed hydrogel was also found to degrade faster in cell culture media than in PBS, the hydrogels still enabled the growth of 3D cultured cells for at least 4 days

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without the support of a secondary network, which is an advantage compared to many

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previous boronic ester hydrogels. Smithmyer et al. have demonstrated the application of boronic ester hydrogels for a combined culture of multiple cell types, as they could

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encapsulate different populations of cells within separate gels and then assemble them into

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a single microtissue, due to the self-healing properties (Smithmyer et al., 2018). Our hydrogel is also capable of such use, owing to its good self-healing properties, as shown in

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section 2.6. Not only single dispersed cells, but also different spheroids can be loaded into the hydrogel and delivered to positions of interest (Fig. S4E). It is anticipated that our

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hyaluronic acid dynamic hydrogel will be extremely useful for many in vitro and in vivo cell culture and delivery applications. 3D bioprinting is becoming an important tool in bio-fabrication and tissue regeneration (G. H. Wu & Hsu, 2015). Development of novel and multifunctional bio-inks is greatly

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encouraged in order to assemble more complicated tissue mimicking constructs and to investigate cell-cell interactions. Even though boronic ester based hydrogels have been suggested for potential use in 3D printing (Yinan Wang et al., 2016), we successfully fabricated cell-laden scaffolds for the first time using such type of dynamic hydrogel as bio-inks in 3D bioprinting (Fig. S4A-C, Video 2). The printing process showed minimal

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damage to the cells, and cells in the bio-inks showed good viability (85 ± 2% live cells) after extrusion and maintained an acceptable viability of 81 ± 2% live cells after 3-day culture (Fig. S4B). We also showed that it could be particularly useful for “direct-in-gel” printing (Fig. S4C &D) due to the self-healing properties. The dynamic boronic ester bonds from the ink gel break and reform new dynamic bonds with the corresponding groups from the support gel, making them heal together after “direct-in-gel” printing. Such process was

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so mild and fast that it would not affect the cell viability in the hydrogels. The distinct

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regions of the two gels/cells at the interface could be identified under confocal microscopy (Fig. S4C). The cancer cells (red) were localized in the area into which they were printed

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and were surrounded in a 3D region by a population of fibroblasts (green). Dr. Burdick’s

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group applied host-guest interaction-based dynamic HA-based hydrogels and successfully achieved the direct writing in self-healing hydrogels, which has opened many new

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opportunities in 3D printing, as the technique allowed the printing of complex hydrogel structures or open channels in the 3D space of another construct (Highley, Rodell, &

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Burdick, 2015). Hydrogels with shear-thinning and self-healing properties are pivotal to this approach. While it takes several chemical reactions to prepare the functional host-guest hydrogels, it only takes a one-step reaction to prepare our HA-based dynamic hydrogel. Our preliminary study confirmed that such “direct-in-gel” printing, using dynamic boronic

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ester hydrogels, can be used to incorporate multiple types of cells, such as fibroblasts and breast cancer cells, in defined regions of a 3D space, and it can be further used to investigate their interaction. A preliminary 3D printing study also showed that a more complex hydrogel structure (a circle) can be easily printed inside another support hydrogel (Fig. S4D), which is similar to a reported work using the host-guest hydrogel (Song, Highley,

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Rouff, & Burdick, 2018). In this work, they fabricated microchannels inside the support hydrogel by the direct-in-gel printing strategy and then washed away the ink gel and stabilized the support gel. After seeding endothelial cells in the microchannels, they found that the channel curvature had some influence on angiogenic sprouting, and increased sprouting is observed at curved locations. There are still several limitations of our developed dynamic hydrogels. First, based on

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the drug release profile, even though ROS responsive drug release was successfully

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realized by our hydrogel, DOX may not be an optimal choice for tumor in situ delivery and therapy using our system. This is because this drug showed high burst release, possibly due

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to a relatively large pore size and a lack of hydrophobic regions in the hydrogel network.

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The encapsulation of nanoparticles, microspheres, or liposomes in our hydrogel might be better options, given their relatively large size, and they might be released in a more

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sustained manner (Burade et al., 2017; Y. Wang et al., 2011). Second, like the problem associated with the host-guest HA-based hydrogel, the relaxation after extrusion of the

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hydrogel during 3D printing easily de-stabilized the scaffold structure, suggesting that a secondary crosslinking strategy compatible with our hydrogel system, e.g. thiol-maleimide click chemistry, should be incorporated to fabricate better constructs for future applications (W. Fan et al., 2016; Jansen, Negron-Pineiro, Galarza, & Peyton, 2018; Ouyang et al.,

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2016).

5. Conclusions In summary, we successfully and facilely synthesized dynamic HA-based hydrogels with self-healing and ROS-responsive properties that are relatively stable in physiologically

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relevant conditions. We further demonstrated that the injectable HA-based hydrogels can be applied for drug delivery and as anti-oxidative cell carriers. The storage modulus and in vitro degradation were tunable by changing the hydrogel precursor concentration. The dynamic hydrogel showed a rapid self-healing ability at a neutral pH due to the rapid dynamic exchange of the boronic ester bond. The ROS responsive DOX release was achieved due to the sensitivity of boronic ester to H2O2. In addition, the hydrogels are

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biocompatible and can be used in 3D culture of breast cancer cells and NPC. The anti-

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oxidative property of the hydrogels can shield the encapsulated cells from environmental ROS stress and prevent cell death. Furthermore, the hydrogels can be used as bio-inks in

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3D bioprinting. The implementation of the self-healing hydrogel as a support gel resulted

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in “direct-in-gel” printing, given the boronic ester bond can break fast and reform between the ink gel and the support gel. The injectable, self-healing, and ROS responsive dynamic

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HA-based hydrogel should be a promising candidate for drug/cell delivery, 3D culture, and

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3D bioprinting, and it will become a good platform for tissue engineering.

Acknowledgements

This work has been supported by Mary & Dick Holland Regenerative Medicine Program start-up grant, Mary & Dick Holland Regenerative Medicine Program pilot project grant,

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Nebraska Research Initiative Funding (B.D.), and University of Nebraska Collaboration Initiative Seed Grant (B.D. and Y.L). We would like to thank Dr. Peng Jiang at Rutgers University for providing the UTY1 cells and Dr. Sameer Mirza at the University of Nebraska Medical Center for providing the H16NF cells.

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Declaration of Interest None.

Supplementary Materials

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See attached.

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Supplementary videos (2) Video 1.

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Video 2.

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Reference

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Agrawal, S., Dwivedi, M., Ahmad, H., Chadchan, S. B., Arya, A., Sikandar, R., . . . Dwivedi, A. K. (2018). CD44 targeting hyaluronic acid coated lapatinib nanocrystals foster the efficacy against triple-negative breast cancer. Nanomedicine, 14(2), 327-337. Alarcin, E., Lee, T. Y., Karuthedom, S., Mohammadi, M., Brennan, M. A., Lee, D. H., . . . Jang, H. L. (2018). Injectable shear-thinning hydrogels for delivering osteogenic and angiogenic cells and growth factors. Biomater Sci, 6(6), 1604-1615. Arts, I. S., Gennaris, A., & Collet, J. F. (2015). Reducing systems protecting the bacterial cell envelope from oxidative damage. FEBS Lett, 589(14), 1559-1568. Ballance, W. C., Qin, E. C., Chung, H. J., Gillette, M. U., & Kong, H. (2019). Reactive oxygen species-responsive drug delivery systems for the treatment of neurodegenerative diseases. Biomaterials, 217, 119292. Bastings, M. M., Koudstaal, S., Kieltyka, R. E., Nakano, Y., Pape, A. C., Feyen, D. A., . . . Dankers, P. Y. (2014). A fast pH-switchable and self-healing supramolecular hydrogel carrier for guided, local catheter injection in the infarcted myocardium. Adv Healthc Mater, 3(1), 70-78. Bignami, A., Hosley, M., & Dahl, D. (1993). Hyaluronic acid and hyaluronic acid-binding proteins in brain extracellular matrix. Anat Embryol (Berl), 188(5), 419-433. Brooks, W. L. A., & Sumerlin, B. S. (2016). Synthesis and Applications of Boronic Acid-Containing Polymers: From Materials to Medicine. Chemical Reviews, 116(3), 1375-1397. Burade, V., Bhowmick, S., Maiti, K., Zalawadia, R., Ruan, H., & Thennati, R. (2017). Lipodox(R) (generic doxorubicin hydrochloride liposome injection): in vivo efficacy and bioequivalence versus Caelyx(R) (doxorubicin hydrochloride liposome injection) in human mammary carcinoma (MX-1) xenograft and syngeneic fibrosarcoma (WEHI 164) mouse models. BMC Cancer, 17(1), 405. 38

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Burattini, S., Greenland, B. W., Chappell, D., Colquhoun, H. M., & Hayes, W. (2010). Healable polymeric materials: a tutorial review. Chem Soc Rev, 39(6), 1973-1985. Chakma, P., & Konkolewicz, D. (2019). Dynamic Covalent Bonds in Polymeric Materials. Angew Chem Int Ed Engl. Chen, D., Zhang, G., Li, R., Guan, M., Wang, X., Zou, T., . . . Wan, L. J. (2018). Biodegradable, Hydrogen Peroxide, and Glutathione Dual Responsive Nanoparticles for Potential Programmable Paclitaxel Release. J Am Chem Soc, 140(24), 7373-7376. Chen, H., Cheng, J., Ran, L., Yu, K., Lu, B., Lan, G., . . . Lu, F. (2018). An injectable self-healing hydrogel with adhesive and antibacterial properties effectively promotes wound healing. Carbohydr Polym, 201, 522-531. Chen, M. H., Wang, L. L., Chung, J. J., Kim, Y. H., Atluri, P., & Burdick, J. A. (2017). Methods To Assess Shear-Thinning Hydrogels for Application As Injectable Biomaterials. ACS Biomater Sci Eng, 3(12), 3146-3160. Chen, Y., Diaz-Dussan, D., Wu, D., Wang, W., Peng, Y.-Y., Asha, A. B., . . . Narain, R. (2018). Bioinspired Self-Healing Hydrogel Based on Benzoxaborole-Catechol Dynamic Covalent Chemistry for 3D Cell Encapsulation. ACS Macro Letters, 7(8), 904-908. Crivelli, B., Bari, E., Perteghella, S., Catenacci, L., Sorrenti, M., Mocchi, M., . . . Torre, M. L. (2019). Silk fibroin nanoparticles for celecoxib and curcumin delivery: ROS-scavenging and antiinflammatory activities in an in vitro model of osteoarthritis. Eur J Pharm Biopharm, 137, 37-45. D'Este, M., Eglin, D., & Alini, M. (2014). A systematic analysis of DMTMM vs EDC/NHS for ligation of amines to hyaluronan in water. Carbohydr Polym, 108, 239-246. de Gracia Lux, C., Joshi-Barr, S., Nguyen, T., Mahmoud, E., Schopf, E., Fomina, N., & Almutairi, A. (2012). Biocompatible polymeric nanoparticles degrade and release cargo in response to biologically relevant levels of hydrogen peroxide. J Am Chem Soc, 134(38), 15758-15764. Deng, X., Attalla, R., Sadowski, L. P., Chen, M., Majcher, M. J., Urosev, I., . . . Hoare, T. (2018). Autonomously Self-Adhesive Hydrogels as Building Blocks for Additive Manufacturing. Biomacromolecules, 19(1), 62-70. Deng, Y., Ren, J., Chen, G., Li, G., Wu, X., Wang, G., . . . Li, J. (2017). Injectable in situ cross-linking chitosan-hyaluronic acid based hydrogels for abdominal tissue regeneration. Sci Rep, 7(1), 2699. Dollinger, B. R., Gupta, M. K., Martin, J. R., & Duvall, C. L. (2017). Reactive Oxygen Species Shielding Hydrogel for the Delivery of Adherent and Nonadherent Therapeutic Cell Types<sup>. Tissue Eng Part A, 23(19-20), 1120-1131. Dzwonek, J., & Wilczynski, G. M. (2015). CD44: molecular interactions, signaling and functions in the nervous system. Front Cell Neurosci, 9, 175. Fan, T., Li, M., Wu, X., Li, M., & Wu, Y. (2011). Preparation of thermoresponsive and pHsensitivity polymer magnetic hydrogel nanospheres as anticancer drug carriers. Colloids Surf B Biointerfaces, 88(2), 593-600. Fan, W., Shi, W., Zhang, W., Jia, Y., Zhou, Z., Brusnahan, S. K., & Garrison, J. C. (2016). Cathepsin S-cleavable, multi-block HPMA copolymers for improved SPECT/CT imaging of pancreatic cancer. Biomaterials, 103, 101-115. Figueiredo, T., Jing, J., Jeacomine, I., Olsson, J., Gerfaud, T., Boiteau, J. G., . . . Auzely-Velty, R. (2019). Injectable Self-Healing Hydrogels Based on Boronate Ester Formation between Hyaluronic Acid Partners Modified with Benzoxaborin Derivatives and Saccharides. Biomacromolecules. Gaffey, A. C., Chen, M. H., Trubelja, A., Venkataraman, C. M., Chen, C. W., Chung, J. J., . . . Atluri, P. (2019). Delivery of progenitor cells with injectable shear-thinning hydrogel maintains 39

Jo

ur na

lP

re

-p

ro

of

geometry and normalizes strain to stabilize cardiac function after ischemia. J Thorac Cardiovasc Surg, 157(4), 1479-1490. Gennari, A., Gujral, C., Hohn, E., Lallana, E., Cellesi, F., & Tirelli, N. (2017). Revisiting Boronate/Diol Complexation as a Double Stimulus-Responsive Bioconjugation. Bioconjug Chem, 28(5), 1391-1402. Gomes, A., Fernandes, E., & Lima, J. L. F. C. (2005). Fluorescence probes used for detection of reactive oxygen species. Journal of Biochemical and Biophysical Methods, 65(2), 45-80. Guo, R., Su, Q., Zhang, J., Dong, A., Lin, C., & Zhang, J. (2017). Facile Access to Multisensitive and Self-Healing Hydrogels with Reversible and Dynamic Boronic Ester and Disulfide Linkages. Biomacromolecules, 18(4), 1356-1364. Gupta, M. K., Martin, J. R., Werfel, T. A., Shen, T., Page, J. M., & Duvall, C. L. (2014). Cell protective, ABC triblock polymer-based thermoresponsive hydrogels with ROS-triggered degradation and drug release. J Am Chem Soc, 136(42), 14896-14902. Hao, T., Li, J., Yao, F., Dong, D., Wang, Y., Yang, B., & Wang, C. (2017). Injectable Fullerenol/Alginate Hydrogel for Suppression of Oxidative Stress Damage in Brown Adipose-Derived Stem Cells and Cardiac Repair. ACS Nano, 11(6), 5474-5488. He, L., Fullenkamp, D. E., Rivera, J. G., & Messersmith, P. B. (2011). pH responsive self-healing hydrogels formed by boronate-catechol complexation. Chem Commun (Camb), 47(26), 7497-7499. Highley, C. B., Prestwich, G. D., & Burdick, J. A. (2016). Recent advances in hyaluronic acid hydrogels for biomedical applications. Curr Opin Biotechnol, 40, 35-40. Highley, C. B., Rodell, C. B., & Burdick, J. A. (2015). Direct 3D Printing of Shear-Thinning Hydrogels into Self-Healing Hydrogels. Adv Mater, 27(34), 5075-5079. Hong, S. H., Kim, S., Park, J. P., Shin, M., Kim, K., Ryu, J. H., & Lee, H. (2018). Dynamic Bonds between Boronic Acid and Alginate: Hydrogels with Stretchable, Self-Healing, StimuliResponsive, Remoldable, and Adhesive Properties. Biomacromolecules, 19(6), 20532061. Hou, S., Wang, X., Park, S., Jin, X., & Ma, P. X. (2015). Rapid Self-Integrating, Injectable Hydrogel for Tissue Complex Regeneration. Adv Healthc Mater, 4(10), 1491-1495, 1423. Hu, J., Chen, Y., Li, Y., Zhou, Z., & Cheng, Y. (2017). A thermo-degradable hydrogel with lighttunable degradation and drug release. Biomaterials, 112, 133-140. Hyslop, P. A., Zhang, Z., Pearson, D. V., & Phebus, L. A. (1995). Measurement of striatal H2O2 by microdialysis following global forebrain ischemia and reperfusion in the rat: correlation with the cytotoxic potential of H2O2 in vitro. Brain Res, 671(2), 181-186. Jammalamadaka, U., & Tappa, K. (2018). Recent Advances in Biomaterials for 3D Printing and Tissue Engineering. J Funct Biomater, 9(1). Jansen, L. E., Negron-Pineiro, L. J., Galarza, S., & Peyton, S. R. (2018). Control of thiol-maleimide reaction kinetics in PEG hydrogel networks. Acta Biomater, 70, 120-128. Jin, H., Zhu, T., Huang, X., Sun, M., Li, H., Zhu, X., . . . Yan, D. (2019). ROS-responsive nanoparticles based on amphiphilic hyperbranched polyphosphoester for drug delivery: Light-triggered size-reducing and enhanced tumor penetration. Biomaterials, 211, 6880. Khunmanee, S., Jeong, Y., & Park, H. (2017). Crosslinking method of hyaluronic-based hydrogel for biomedical applications. J Tissue Eng, 8, 2041731417726464. Lam, J., Truong, N. F., & Segura, T. (2014). Design of cell-matrix interactions in hyaluronic acid hydrogel scaffolds. Acta Biomater, 10(4), 1571-1580. Lee, J. H. (2018). Injectable hydrogels delivering therapeutic agents for disease treatment and tissue engineering. Biomater Res, 22, 27. 40

Jo

ur na

lP

re

-p

ro

of

Li, W., & Sun, X. (2018). Recent Advances In Developing Novel Anti-Cancer Drugs Targeting Tumor Hypoxic and Acidic Microenvironments. Recent Pat Anticancer Drug Discov, 13(4), 455-468. Li, X. (2012). Improved Pyrogallol Autoxidation Method: A Reliable and Cheap SuperoxideScavenging Assay Suitable for All Antioxidants. Journal of Agricultural and Food Chemistry, 60(25), 6418-6424. Liu, Q., Li, H., & Lam, K. Y. (2018). Optimization of Deformable Magnetic-Sensitive HydrogelBased Targeting System in Suspension Fluid for Site-Specific Drug Delivery. Mol Pharm, 15(10), 4632-4642. Lv, W., Xu, J., Wang, X., Li, X., Xu, Q., & Xin, H. (2018). Bioengineered Boronic Ester Modified Dextran Polymer Nanoparticles as Reactive Oxygen Species Responsive Nanocarrier for Ischemic Stroke Treatment. ACS Nano, 12(6), 5417-5426. Marasovic, M., Ivankovic, S., Stojkovic, R., Djermic, D., Galic, B., & Milos, M. (2017). In vitro and in vivo antitumour effects of phenylboronic acid against mouse mammary adenocarcinoma 4T1 and squamous carcinoma SCCVII cells. J Enzyme Inhib Med Chem, 32(1), 1299-1304. Marco-Dufort, B., & Tibbitt, M. W. (2019). Design of moldable hydrogels for biomedical applications using dynamic covalent boronic esters. Materials Today Chemistry, 12, 1633. Negri, G. E., & Deming, T. J. (2017). Protein Complexation and pH Dependent Release Using Boronic Acid Containing PEG-Polypeptide Copolymers. Macromol Biosci, 17(1). Oliva, N., Conde, J., Wang, K., & Artzi, N. (2017). Designing Hydrogels for On-Demand Therapy. Acc Chem Res, 50(4), 669-679. Oparka, M., Walczak, J., Malinska, D., van Oppen, L. M. P. E., Szczepanowska, J., Koopman, W. J. H., & Wieckowski, M. R. (2016). Quantifying ROS levels using CM-H2DCFDA and HyPer. Methods, 109, 3-11. Ouyang, L., Highley, C. B., Rodell, C. B., Sun, W., & Burdick, J. A. (2016). 3D Printing of ShearThinning Hyaluronic Acid Hydrogels with Secondary Cross-Linking. ACS Biomaterials Science & Engineering, 2(10), 1743-1751. Patel, G. C., & Dalwadi, C. A. (2013). Recent patents on stimuli responsive hydrogel drug delivery system. Recent Pat Drug Deliv Formul, 7(3), 206-215. Ramasarma, T., Rao, A. V. S., Devi, M. M., Omkumar, R. V., Bhagyashree, K. S., & Bhat, S. V. (2015). New insights of superoxide dismutase inhibition of pyrogallol autoxidation. Molecular and Cellular Biochemistry, 400(1), 277-285. Santos, A. L., Sinha, S., & Lindner, A. B. (2018). The Good, the Bad, and the Ugly of ROS: New Insights on Aging and Aging-Related Diseases from Eukaryotic and Prokaryotic Model Organisms. Oxid Med Cell Longev, 2018, 1941285. Saravanakumar, G., Kim, J., & Kim, W. J. (2017). Reactive-Oxygen-Species-Responsive Drug Delivery Systems: Promises and Challenges. Adv Sci (Weinh), 4(1), 1600124. Smithmyer, M. E., Deng, C. C., Cassel, S. E., LeValley, P. J., Sumerlin, B. S., & Kloxin, A. M. (2018). Self-Healing Boronic Acid-Based Hydrogels for 3D Co-cultures. ACS Macro Letters, 7(9), 1105-1110. Song, K. H., Highley, C. B., Rouff, A., & Burdick, J. A. (2018). Complex 3D-Printed Microchannels within Cell-Degradable Hydrogels. Advanced Functional Materials, 28(31), 1801331. Tang, S., Ma, H., Tu, H.-C., Wang, H.-R., Lin, P.-C., & Anseth, K. S. (2018). Adaptable Fast Relaxing Boronate-Based Hydrogels for Probing Cell–Matrix Interactions. Advanced Science, 5(9), 1800638.

41

Jo

ur na

lP

re

-p

ro

of

Tarus, D., Hachet, E., Messager, L., Catargi, B., Ravaine, V., & Auzély-Velty, R. (2014). Readily Prepared Dynamic Hydrogels by Combining Phenyl Boronic Acid- and Maltose-Modified Anionic Polysaccharides at Neutral pH. Macromolecular Rapid Communications, 35(24), 2089-2095. Tian, R., Qiu, X., Yuan, P., Lei, K., Wang, L., Bai, Y., . . . Chen, X. (2018). Fabrication of Self-Healing Hydrogels with On-Demand Antimicrobial Activity and Sustained Biomolecule Release for Infected Skin Regeneration. ACS Appl Mater Interfaces, 10(20), 17018-17027. Tseng, T. C., Tao, L., Hsieh, F. Y., Wei, Y., Chiu, I. M., & Hsu, S. H. (2015). An Injectable, SelfHealing Hydrogel to Repair the Central Nervous System. Adv Mater, 27(23), 3518-3524. Wang, C., Wang, J., Zhang, X., Yu, S., Wen, D., Hu, Q., . . . Gu, Z. (2018). In situ formed reactive oxygen species-responsive scaffold with gemcitabine and checkpoint inhibitor for combination therapy. Sci Transl Med, 10(429). Wang, L. L., Highley, C. B., Yeh, Y. C., Galarraga, J. H., Uman, S., & Burdick, J. A. (2018). Threedimensional extrusion bioprinting of single- and double-network hydrogels containing dynamic covalent crosslinks. J Biomed Mater Res A, 106(4), 865-875. Wang, Y., Li, L., Kotsuchibashi, Y., Vshyvenko, S., Liu, Y., Hall, D., . . . Narain, R. (2016). SelfHealing and Injectable Shear Thinning Hydrogels Based on Dynamic Oxaborole-Diol Covalent Cross-Linking. ACS Biomaterials Science & Engineering, 2(12), 2315-2323. Wang, Y., Mirza, S., Wu, S., Zeng, J., Shi, W., Band, H., . . . Duan, B. (2018). 3D hydrogel breast cancer models for studying the effects of hypoxia on epithelial to mesenchymal transition. Oncotarget, 9(63), 32191-32203. Wang, Y., Shi, W., Kuss, M., Mirza, S., Qi, D., Krasnoslobodtsev, A., . . . Duan, B. (2018). 3D Bioprinting of Breast Cancer Models for Drug Resistance Study. ACS Biomaterials Science & Engineering, 4(12), 4401-4411. Wang, Y., Wei, Y. T., Zu, Z. H., Ju, R. K., Guo, M. Y., Wang, X. M., . . . Cui, F. Z. (2011). Combination of hyaluronic acid hydrogel scaffold and PLGA microspheres for supporting survival of neural stem cells. Pharm Res, 28(6), 1406-1414. Willner, I. (2017). Stimuli-Controlled Hydrogels and Their Applications. Acc Chem Res, 50(4), 657658. Wu, G. H., & Hsu, S. H. (2015). Review: Polymeric-Based 3D Printing for Tissue Engineering. J Med Biol Eng, 35(3), 285-292. Wu, Z., Kong, B., Liu, R., Sun, W., & Mi, S. (2018). Engineering of Corneal Tissue through an Aligned PVA/Collagen Composite Nanofibrous Electrospun Scaffold. Nanomaterials (Basel), 8(2). Xu, Q., He, C., Ren, K., Xiao, C., & Chen, X. (2016). Thermosensitive Polypeptide Hydrogels as a Platform for ROS-Triggered Cargo Release with Innate Cytoprotective Ability under Oxidative Stress. Adv Healthc Mater, 5(15), 1979-1990. Xu, Q., Venet, M., Wang, W., Creagh-Flynn, J., Wang, X., Li, X., . . . Wang, W. (2018). Versatile Hyperbranched Poly(beta-hydrazide ester) Macromers as Injectable Antioxidative Hydrogels. ACS Appl Mater Interfaces, 10(46), 39494-39504. Xu, X., Zeng, Z., Huang, Z., Sun, Y., Huang, Y., Chen, J., . . . Zhao, C. (2020). Near-infrared lighttriggered degradable hyaluronic acid hydrogel for on-demand drug release and combined chemo-photodynamic therapy. Carbohydrate Polymers, 229, 115394. Ye, H., Zhou, Y., Liu, X., Chen, Y., Duan, S., Zhu, R., . . . Yin, L. (2019). Recent Advances on Reactive Oxygen Species-Responsive Delivery and Diagnosis System. Biomacromolecules, 20(7), 2441-2463.

42

Jo

ur na

lP

re

-p

ro

of

Yesilyurt, V., Webber, M. J., Appel, E. A., Godwin, C., Langer, R., & Anderson, D. G. (2016). Injectable Self-Healing Glucose-Responsive Hydrogels with pH-Regulated Mechanical Properties. Adv Mater, 28(1), 86-91. Yu, L. Y., Su, G. M., Chen, C. K., Chiang, Y. T., & Lo, C. L. (2016). Specific Cancer Cytosolic Drug Delivery Triggered by Reactive Oxygen Species-Responsive Micelles. Biomacromolecules, 17(9), 3040-3047. Yuan, F., Ma, M., Lu, L., Pan, Z., Zhou, W., Cai, J., . . . Yin, F. (2017). Preparation and properties of polyvinyl alcohol (PVA) and hydroxylapatite (HA) hydrogels for cartilage tissue engineering. Cell Mol Biol (Noisy-le-grand), 63(5), 32-35. Zhu, J., Li, F., Wang, X., Yu, J., & Wu, D. (2018). Hyaluronic Acid and Polyethylene Glycol Hybrid Hydrogel Encapsulating Nanogel with Hemostasis and Sustainable Antibacterial Property for Wound Healing. ACS Appl Mater Interfaces, 10(16), 13304-13316. Zhu, Y., Matsumura, Y., Velayutham, M., Foley, L. M., Hitchens, T. K., & Wagner, W. R. (2018). Reactive oxygen species scavenging with a biodegradable, thermally responsive hydrogel compatible with soft tissue injection. Biomaterials, 177, 98-112.

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FIGURE CAPTIONS (7)

Figure 1. Scheme for the synthesis of the HA-PBA conjugate and the fabrication of the injectable hydrogel. The HA-PBA (hyaluronic acid-phenylboronic acid) polymer conjugate was prepared by conjugating the 3-aminomethyl PBA to HA (290 kDa) using DMTMM as the coupling agent. The HA-PBA hydrogel is prepared by mixing HA-PBA

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with PVA solutions with the same concentrations (13-23 kDa) at a 3:1 volume ratio. The

resulting gel is self-healing and injectable through needles.

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gelation is caused by the formation of dynamic covalent bonds (boronic ester). The

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Figure 2. Characterization of the rheological properties of HA-PBA-PVA hydrogels

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prepared at 1.5% and 2.5% precursor concentrations. A) Storage and loss moduli of two HA-PBA-PVA hydrogels; B) shear rate dependent viscosity change of HA-PBA-PVA

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hydrogels; C) storage and loss modulus changes with high recovery for G’ under alternating strains indicated the self-healing property of the hydrogels.

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Figure 3. Self-healing and injectability characteristics of HA-PBA-PVA dynamic hydrogels (2.5%), and the swelling degradation profile of hydrogels (1.5% and 2.5%) in different solutions. A) Separate hydrogels could rapidly heal together after being put next to each other; B) hydrogels could be easily extruded from a syringe through 21G needles;

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C) degradation profile of hydrogels in PBS, with and without H2O2 (0.2 mM), revealed faster degradation in the presence of H2O2. Figure 4. Cytotoxicity of HA-PBA conjugate to MDA-MB-231 cells and live/dead assay of MDA-MB-231 encapsulated in two HA-PBA-PVA hydrogels. Scale bar=100 µm. A) MTT study of HA-PBA conjugate to MDA-MB-231 cells has shown no significant toxicity

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at up to 1 mg/ml; B&C) Live and dead assay of MDA-MB-231 cells encapsulated in HAPBA-PVA hydrogels for at least 5 days indicated good biocompatibility. Figure 5. H2O2 responsive drug release profile of DOX loaded hydrogels and the efficacy evaluation of released DOX from the hydrogel. A&B) The presence of H2O2 accelerated the DOX release from both the 1.5% and 2.5% hydrogels. Figure 6. Cytotoxicity of HA-PBA conjugate to neural progenitor cells (UTY1) and

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live/dead assay of UTY1 encapsulated in two HA-PBA-PVA hydrogels compared with

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Matrigel. Scale bar (white)=100 µm; scale bar (black)=250 µm. A) MTT study of HA-PBA conjugate to UTY1 cells has shown no significant toxicity at up to 1 mg/ml; B&C) live and

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dead assay of UTY1 cells encapsulated in HA-PBA-PVA hydrogels for up to 4 days

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indicated good biocompatibility comparable to Matrigel. Both fluorescent images (C middle panel) and bright field images (C bottom panel) revealed neurosphere was formed

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in the hydrogel starting at day 2 with more apparent size at day 4. Figure 7. Anti-oxidative ability of HA-PBA-PVA hydrogel precursors and the anti-ROS

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protection effect of the HA-PBA-PVA hydrogel for 3D cultured UTY1 cells in the presence of 0.1 mM H2O2. Scale bar=250 µm. A) Pyrogallol assay indicated significant ROS scavenging ability of the HA-PBA conjugate, which was absent in both HA and PVA; B&C) live and dead assay revealed an anti-oxidative protective effect of hydrogels to the

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encapsulated UTY1 cells, as compared to Matrigel, when exposed to H2O2 containing media for 24 h; D) LDH assay confirmed significantly more cell death in Matrigel, as compared to that in the anti-oxidative hydrogel after H2O2 treatment; E) H2O2 specific probe H2DCFDA staining revealed higher intracellular H2O2 in cells cultured in Matrigel compared to cells cultured in HA-PBA-PVA hydrogels.

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