Subscriber access provided by UNIV OF LOUISIANA
Surfaces, Interfaces, and Applications
Effective suppression of oxidative stress on living cells in hydrogel particles containing a physically immobilized WS2 radical scavenger Ji Eun Kim, DaBin Yim, Sang Woo Han, Jin Nam, Jong-Ho Kim, and Jin Woong Kim ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 01 May 2019 Downloaded from http://pubs.acs.org on May 1, 2019
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Applied Materials & Interfaces
Effective Suppression of Oxidative Stress on Living Cells in Hydrogel Particles Containing a Physically Immobilized WS2 Radical Scavenger Ji Eun Kim,† DaBin Yim,‡ Sang Woo Han,† Jin Nam,§ Jong-Ho Kim,‡,* Jin Woong Kim†,∥,* † Department ‡ Department
of Bionano Technology, Hanyang University, Ansan, 15588, Republic of Korea of Chemical Engineering, Hanyang University, Ansan 15588, Republic of
Korea § Amore-Pacific Co. R&D Center, Yongin, 17074, Republic of Korea. ∥ Department of Chemical and Molecular Engineering, Hanyang University, Ansan, 15588, Republic of Korea
Abstract We report on a tungsten disulfide (WS2) nanosheets-immobilized hydrogel system that can inhibit oxidative stress on living cells. First, we fabricated a highly stable suspension of WS2 nanosheets as a radical scavenger by enveloping them with the amphiphilic poly(caprolactone)-b-poly(ethylene oxide) copolymer (PCL-b-PEO) during in situ liquid exfoliation in an aqueous medium. After the PCL-b-PEO-enveloped WS2 nanosheets were embedded in three types of hydrogel systems, including, carrageenan gum/locust bean gum bulk hydrogels, physically crosslinked alginate microparticles, covalently crosslinked PEG hydrogel microparticles, they retained their characteristic optical properties. Intriguingly, the WS2 nanosheet-immobilized hydrogel particles exhibited sustainable radical scavenging performance without any deterioration in the original activity of the WS2 nanosheets, even after repeated use. This implies that hydrogen atoms dissociated from the chalcogen of the WS2 nanosheets effectively scavenged free radicals through the hydrogel mesh. Because of this unique behavior, the coexistence of the WS2 nanosheets with living cells in the hydrogel matrix improved cell viability up to 40%, which demonstrates that the WS2 nanosheets can suppress oxidative stress on living cells. Keywords WS2 nanosheets, hydrogel particles, radical scavenger, cell encapsulation, oxidative stress 1 ACS Paragon Plus Environment
ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 20
1. Introduction Hydrogels are widely used biomaterials because they can resemble the extracellular environment of biological tissues, thus allowing their application in a variety of bioindustries.15
Hydrogels are constructed by cross-linking natural, synthetic, or both hydrophilic polymers
so that they possess highly hydrated three-dimensional (3D) gel networks. These 3D gel networks can hold 1000 times more water than the same volume of dried hydrogel due mainly to their excellent affinity to water and structurally stable chain architecture.6-9 In particular, given their mesh structure, which is on the tunable molecular length scale, sustainable bioprocesses can be achieved either by allowing inflow of an aqueous continuous phase or by blocking interference, which allows the design of a biocompatible environment-friendly solid support capable of encapsulating living cells or bioactives.10-14 Also, because the dimension of hydrogel mesh can be easily manipulated by applying external stimuli such as heat, pH, ionic strength, light, and biomolecules, their phase characteristics can be intelligently controlled. Recent studies have shown that the integration of nanomaterials into hydrogel matrices enables the development of mechanically stable tissue scaffolds,15-18 bioadhesives,19-21 smart drug delivery systems,22-25 and advanced diagnostic technology.26-28 These technological advances are possible because the nanomaterials exhibit their own functions through the hydrogel mesh, which is sufficiently hydrated with water.20,
29
However, when a hydrogel
contains living cells, some nanomaterials often damage them, which lower the cell viability.30, 31
Therefore, the internal environment of the hydrogel in these systems must be improved so
that the living cells can perform their normal functions, even in the presence of nanomaterial. Living-cell activity can be further enhanced with appropriate antioxidant capability that prevents reactive oxygen species.32, 33 This suggests that the development of a hydrogel system with antioxidant power will provide an excellent environment for living cells. However, general organic antioxidants cannot be used because they do not retain long-term antioxidant capability in the hydrogel. This lack of retention is due to the short life-span of the antioxidants in water.34 Consequently, if we can develop a hydrogel system that can sustain antioxidant activity under hydrated conditions, we will be able to further diversify its biophysical functions. Herein, we present a transition metal dichalcogenide (TMD) radical scavenger-hybridized hydrogel microparticle (HMP) system that is capable of continuous and stable radical scavenging performance. Recently, TMDs have received attention due to their large surface area and high light absorbance in various biological applications such as photothermal therapy, bioimaging elements, and cellular mimic actuator.28,35,36 In this study, we used WS2 nanosheets 2 ACS Paragon Plus Environment
Page 3 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Applied Materials & Interfaces
as an option of TMD nanosheets. The essence of our approach is to physically immobilize the WS2 nanosheets, which can exhibit prolonged radical scavenging irrespective of the environmental stimuli, using a defect-mediated one-step hydrogen atom transfer in water.37 Because the WS2 nanosheets are much bigger than conventional hydrogel meshes, they can be physically fixed in the hydrogel phase. Accordingly, we fabricated a stable suspension of WS2 nanosheets by enveloping them with a monolayer of the amphiphilic diblock copolymer poly(caprolactone)-b-poly(ethylene oxide) (PCL-b-PEO) using a previously reported protocol.38 After embedding the in situ exfoliated WS2 nanosheets into the HMPs by either physical or chemical cross-linking, we assessed whether the WS2 nanosheets within the hydrogel phase still show the same optical properties. We also observed whether the WS2 nanosheets in the hydrogels exhibit the same level of antioxidant activity as that of the nanosheets alone. Finally, using WS2 nanosheet-immobilized HMPs together with L929 fibroblasts and then HaCaT keratinocytes, we attempted to show how the WS2 nanosheet radical scavenger affects the oxidative stress on living cells.
2. Experimental section 2.1. Materials Bulk tungsten disulfide (WS2) powder, sodium alginate (alginic acid sodium salt from brown algae, guluronic acid content 65–70%), calcium chloride solution (CaCl2, 1 M in H2O), poly(ethylene glycol) diacrylate (PEGDA, Mn = 700 gmol-1), N,N-methylenebisacrylamide (MBA), 2-hydroxy-2-methylpropiophenone (Darocur), olive oil, and 2,2′-azino-bis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Poly(ε-caprolactone)-b-poly(ethylene glycol) (PCL-b-PEO) (Mn = 7000 gmol-1) was generously provided by Mizon Co. (Seoul, Korea). CaCl2 powder, ethanol, and isopropanol were purchased from Daejung Chemical and Metal Co. (Shiheung-city, Korea). Carrageenan gum (CG), locust bean gum (LBG), ethylenediaminetetraacetic acid (EDTA), 1,3butanediol (1,3-BG), and cetyl PEG/PPG-10/1 dimethicone (Abil EM 90, Evonik Industries, Essen, Germany) were used as received. HyClone Dulbecco’s modified Eagle’s medium (DMEM) and Roswell Park Memorial Institute (RPMI) 1640 medium were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS), penicillin, and phosphate-buffered saline (PBS) solution were purchased from Gibco (Gaithersburg, MD,
3 ACS Paragon Plus Environment
ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
USA). All chemicals used in this study were reagent grade and were used without further purification. Deionized double-distilled water was used for all experiments. 2.2. Fabrication of aqueous suspension of WS2 nanosheets A stable aqueous suspension of WS2 nanosheets was produced using a previously reported protocol.38 Bulk WS2 (2 mmol) and PCL-b-PEO (40 mg) were added to water (20 mL). A tip sonicator sonicated the mixture in an ice bath for 1 h at 20 W. The WS2 suspension was centrifuged at 2000 rpm for 1 h, and then the supernatant was centrifuged at 10,000 rpm for 1 h. After dispersing the sediment in water (10 mL) again, the suspension was centrifuged at 3500 rpm for 1 h to obtain a supernatant. The lateral size of the WS2 nanosheets was in the range of 50–150 nm. High-resolution TEM images showed the trigonal prismatic arrangement of WS2 nanosheets with clear lattice fringe of ~2.7 Å. Finally, water was added to the recovered contents to adjust the concentration of the master dispersion. 2.3. Immobilization of WS2 nanosheets in HMPs To prepare the WS2 nanosheet-immobilized calcium alginate (CA) HMPs (WS2-CAHMPs), sodium alginate was dissolved in the suspension of WS2 nanosheets (1 w/v %). The suspension then was dropped into the CaCl2 solution (2 w/v %) via a syringe pump (Pump 11 Elite, Harvard Apparatus, Holliston, MA, USA) at a flow rate of 100 μL min−1 under gentle agitation for 30 min. The WS2-CAHMPs were fully washed with water. For comparison, we also made a WS2 nanosheet-immobilized CG/LBG hydrogel sheet (WS2-CG/LBGHG). First, we dissolved EDTA (0.02 w/v %) in the suspension of WS2 nanosheets (5 mL) at 80 C. Then, CG (1.3 w/v %) and LBG (0.2 w/v %) in 1,3-BG (1 mL) were mixed into the suspension. After sonication for 5 min, the mixture was poured into a petri dish and cooled to 25 C. The WS2-CG/LBGHG was washed three times with water. Monodisperse WS2 nanosheet-immobilized PEGDA HMPs (WS2-PEGDAHMPs) were produced using a capillary-based microfluidic device (see Supporting Information). Controlled injection of two immiscible fluids (a dispersion fluid and an outer fluid) produced monodisperse water-in-oil (W/O) PEGDA precursor emulsion drops in the microcapillary device. Typically, the outer fluid was a mixture of olive oil and Abil EM 90 (2 wt%). The dispersion fluid contained PEGDA (2 wt%), WS2 nanosheets (variable), MBA (0.2 wt%), and Darocur (0.1 wt%) as the photoinitiator. The flow rate of each fluid was controlled precisely with syringe pumps (Pump 11 Elite). The droplets collected in the continuous phase were chemically cross-linked via irradiation with 365-nm UV light (JHC1-051S-V2, A&D Co., 4 ACS Paragon Plus Environment
Page 4 of 20
Page 5 of 20 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Applied Materials & Interfaces
Tokyo, Japan) for 1 min. To remove the olive oil and other additives, the collected WS2PEGDAHMPs
were washed completely by repeated centrifugation at 3500 rpm with extra
isopropanol. The rinsed WS2-PEGDAHMPs were then redispersed in water. 2.4. Cell encapsulation in hydrogel matrix Sodium alginate filtered by a sterilized syringe filter was used to encapsulate mouse NCTC clone 929 cells (L929, Korean Cell Line Bank, Seoul, Korea) at a cell population concentration of 2.5 × 105 cells/mL. The concentration of WS2 nanosheets in the solution was controlled to 100 μM. The sodium alginate/WS2 nanosheets/L929 mixture was added dropwise into a 96well plate, with each well containing 100 mL of CaCl2 (20 mM) and left to incubate at 37 C for 15 min. After incubation, the samples were washed three times in nonsupplemented RPMI. The cells were grown and maintained in 100 mM high-glucose RPMI. All cultures were maintained at 37 C and 5% CO2. The medium was changed three times weekly. In the case of encapsulating HaCaT keratinocytes, they were cultured in high-glucose DMEM and then immobilized following the same process used for encapsulation of L929 cells. 2.5. ABTS radical scavenging assay Radical scavenging activity was measured using a modified ABTS radical scavenging assay.39 ABTS radicals were generated by mixing 7 mM ABTS with 2.45 mM potassium persulfate and then incubating the mixture at 25 C in the dark for 12 h. The radical solution was diluted with ethanol to an absorbance of
