Journal Search Engine
Search Advanced Search Adode Reader(link)
Download PDF Export Citaion korean bibliography PMC previewer
ISSN : 1229-6457(Print)
ISSN : 2466-040X(Online)
The Korean Journal of Vision Science Vol.20 No.4 pp.513-521

Antioxidant Activity of Dopamine-Modified Hydrogels Containing Cross-linked Hyaluronic Acid

Geun-Chang Ryu1),Jeong Hee Hwang2),Cheol-Woo Lee1),*
Dept. of Optometry and Optic science of Dongshin University, Naju1)
Dept. of Optometry, Gangdong University, Eumseong ChungBuk2)
Address reprint request to Cheol-Woo Lee Dept. of Optometry and Optic science of Dongshin University, Naju TEL: +82-61-330-3550
November 27, 2018 December 17, 2018 December 17, 2018


Purpose :

In this study, we made dopamine-functionalized hydrogels containing a cross-linked hyaluronic acid (HA) network and investigated their antioxidant activities.

Methods :

In the first step, we made poly hydroxyethyl methacrylate(p(HEMA))-based hydrogels post-modified with an interpenetrating polymer network(IPN) structure composed of HA polymers and a p(HEMA) network. The subsequent functionalization with dopamine via an amide coupling reaction resulted in the antioxidant hydrogels. Their antioxidant activities were evaluated using 2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) and 2,2-diphenyl-1-picrylhydrazyl radical scavenging assays.

Results :

The dopamine-modified hydrogels exhibited significant antioxidant activities, when compared to unmodified control. The presence of the HA–IPN structure improved the surface wettability of the hydrogel while dopamine-conjugated IPN hydrogel did not demonstrate the significant difference compared to hydrogel control. Dopamine-modified hydrogels exhibited high transmittance (>88%).

Conclusion :

The results demonstrate that the development of antioxidant hydrogels based on dopamine-conjugated HA–IPN structures may help develop ophthalmic and biomedical materials.

도파민이 적용된 히알루론산 가교 하이드로겔의 항산화 활성

동신대학교 안경광학과, 나주1)
강동대학교 안경광학과, 충북 음성2)
    Dongshin University

    Ⅰ. 서 론

    Polyphenols are secondary metabolites of plants and are generally involved in the defense against ultraviolet radiation or aggression by pathogens.1,2) In the last decade, there has been significant interest in the potential health benefits of dietary plant polyphenols as antioxidants. Polyphenols act as singlet oxygen quenchers, free radical scavengers, peroxides and other reactive oxygen species inactivators, and metal ion chelators, and thus, they can prevent human aging and other related diseases/ disorders caused by oxidative stress and free- radical damage.3-10) However, the use of polyphenols in the pharmaceutical, biomedical, and food industries is limited owing to their thermal instability, volatility, and oxidizability.9,10) To address the abovementioned issues, biocompatible materials possessing antioxidant polyphenols have emerged as potent therapeutic agents owing to their potential applications in the biomedical and pharmaceutical research areas. For instance, quercetin, a naturally occurring plant polyphenol from the flavonoid group, has been entrapped in a silicabased inorganic material by a sol–gel route and it represents a useful strategy for preventing the onset of peri-implant diseases in the field of dentistry.11) The intrinsically antioxidant quercetin-entrapped silica-based materials could be used in dentistry as components in glass ionomer cement and in medicine as replacements for bone implants.12) Mesoporous silica nanoparticles were covalently coated with an antioxidant polyphenol, namely, caffeic acid or rutin, to diminish the impact of the oxidative stress induced after transfection into cells.13) Organic polymers such as gelatin, chitosan, and polypropylene have been functionalized with gallic acid and caffeic acid.14-16) Furthermore, hydrogels, proven to be promising materials for biomedical applications owing to their hydrated tissue-like structures, were modified with gallic acid-conjugated chitosan networks and exhibited strong antioxidant activity, according to the length of the chitosan chain.8)

    Recently, we have prepared protein-resistant hydrogel lenses surface-functionalized with polysaccharidebased interpenetrating polymer network (IPN) structures consisting of hyaluronic acid and polyethyleneimine chains.17) The hyaluronic acid within IPNs have many carboxylic acid groups for further conjugations with various functional molecules. As an extension of our previous work, we report a new approach to the preparation of dopamine-modified poly(hydroxyethyl methacrylate) p(HEMA)-based hydrogels containing cross-linked hyaluronic acid (HA) and investigated their antioxidant activities. Methacrylated hyaluronic acid (MeHA) polymers were synthesized and interpenetrated into pre-formed p(HEMA)-based hydrogels, and the subsequent radical polymerization between the MeHA chains resulted in HA–interpenetrating polymer network (IPN) hydrogels consisting of HA networks and cross-linked p(HEMA). The introduction of dopamine via amide coupling with the carboxyl groups of HA–IPNs resulted in the antioxidant hydrogels. Their antioxidant activities were evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’ -Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging assays. The changes in the surface wettability of the hydrogel were also investigated.

    Ⅱ. Methods

    1. Chemicals and equipments

    Sodium hyaluronate (NaHA) (10 kDa), 2, 2- dimethoxy-2-phenylacetophenone, ammonium persulfate (APS), 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2’-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were obtained from Sigma-Aldrich. 2-hydroxyethyl methacrylate (HEMA) was acquired from Junsei (Japan). Ethylene glycol dimethacrylate (EGDMA), 1-ethyl-3-(3-dimethylaminopropyl)- carbodiimide hydrochloric acid (EDC.HCl), triethylamine (TEA) and sodium metabisulfite (SMBS) were purchased from Daejung. N-hydroxysuccinimide (NHS) was procured from Fluka. Deuterium oxide was purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Methacrylic anhydride was purchased from Alfa Aesar. Hydrochloric acid, sodium hydroxide, dimethylformamide (DMF), tetrahydrofuran (THF), chloroform and other reagents were analytical grade and used without further purification. Water contact angle measurements were performed on a DSA100 instrument (Krüss GmbH, Hamburg, Germany). Hitachi 5300 spectrophotometer (Japan) was used to measure the absorbance of the hydrogels (in triplicate).

    2. Synthesis of methacrylated hyaluronic acid (MeHA)

    Methacrylated hyaluronic acid was synthesized according to previously reported procedure.18) 1 g of NaHA was dissolved in distilled water to make 1 wt% and the solution was cooled in an ice bath. 0.6 mL of methacrylic anhydride was added to the solution. With 5M NaOH, the pH of the solution was adjusted to 7.5-8.5. Then it was dialyzed against distilled water for 3 days, freezedried and stored until use. 1H NMR spectroscopy was used to determine the degree of methacrylation of methacrylated hyaluronic acid (MeHA).

    3. Synthesis of Dopamine-functionalized IPN Hydrogel

    HEMA monomer was purified by vacuum distillation prior to use. EGDMA (0.04 g) and a very small amount of initiator were dissolved in HEMA (9.92 g). The solution was sonicated for 30 min and transferred to a square mold made of two glass plates covered internally with polypropylene sheet. The molds were exposed to UV light for 30 min and heated at 80℃ for at least 4 hrs. Then the samples were removed from the molds and soaked in distilled water for 2 days to remove any unreacted reagents. After drying at 40℃ overnight, they were immersed in 1.3 w/v% solution of methacrylated hyaluronic acid in distilled water for 24 hours. After washing with distilled water, they were immersed in a 10-mL solution of APS (5 mg) and SMBS (5 mg) for 24 hrs. The hydrogels were washed with distilled water and were immersed in distilled H2O with EDC, NHS, TEA and dopamine HCl for 24 hrs. Finally, they were washed with distilled water for 2 days to remove any unreacted chemicals prior to characterization and to allow swelling to facilitate cutting. The resulting IPN hydrogels were cut into 5×5 mm samples for antioxidant capacity determination and 1×1 cm samples for absorbance and contact angle measurements.

    4. Contact Angle Measurements

    Static contact angle measurements were made using DSA100 instrument (Krüss GmbH, Hamburg, Germany). A drop of 5 μL Milli-Q water was placed onto the surface of hydrogels and contact angles were recorded by analyzing frames captured using an optical camera. Recorded frames were evaluated by ADVANCE software to calculate contact angles. Six measurements were done for each hydrogel and the mean value was reported.

    5. DPPH Radical-Scavenging Assay of the MeHA-IPN Hydrogels

    The DPPH radical-scavenging assay described by Brand-Williams and modified by Miliauskas was used in determining the DPPH radical- scavenging capacity of the prepared MC-IPN hydrogels.19) The test samples were compared to a known antioxidant, ascorbic acid (1000 ppm). Briefly, DPPH· solution (0.2 mM in ethanol) was mixed with the hydrogel samples. The reaction mixture was shaken for 30 min at 37℃ in the dark. The reaction of the DPPH·was estimated by measuring the adsorption at 517 nm against ethanol. The percentage of the DPPH· radical inhibition was calculated from Equation 1.

     Equation 1: %Inhibition = {1-1-(Absorbance of sample/ Absorbance of control)} × 100

    6. ABTS Radical-Scavenging Assay of the HA-IPN Hydrogels

    The ABTS radical-scavenging assay was based on the modified method described by Arnao, et al.20) ABTS radical cations (ABTS·+) were produced from the addition of 7 mM ABTS solution to 2.4 mM potassium persulfate solution. The diluted ABTS·+ solution was then prepared by mixing the two solutions in equal quantities and allowing them to react for 24 hrs at RT in the dark. The solution was then diluted with methanol to obtain an absorbance range of 0.7-1 ± 0.02 units at 734 nm. Hydrogel samples were added to the diluted ABTS·+ solution and incubated for 30 min, at 37℃ in the dark. The reaction of the ABTS·+ species was estimated by measuring the absorption at 734 nm against methanol. The percentage radical inhibition of ABTS·+ was calculated from Equation 1.

    7. Statistical Analyses

    All data are expressed as means ± standard deviations (SD) (n=3).

    Ⅲ. Results and Discussion

    The preparation of the antioxidant hydrogels is outlined in Fig. 1. First, the p(HEMA) network was prepared by radical copolymerization of HEMA and a cross linker, EGDMA. MeHA was loaded and interpenetrated within the p(HEMA) network and the subsequent intra/intermolecular cross-linking polymerization between the MeHA chains produced HA–IPN-modified hydrogels. Finally, dopamine was conjugated onto the HA–IPN hydrogels via amide coupling between the amine group of dopamine and the carboxyl unit of HA polymer, resulting in the antioxidant hydrogel (HA–IPN–DA). MeHA was synthesized according to a previously reported procedure.17) MeHA was synthesized through the esterification reaction of hyaluronic acid with methacrylic anhydride and its degree of methacrylation (DM) was determined using 1H-NMR spectroscopy (Fig. 2). DM was calculated to be approximately 4% based on digital integration of the methacrylate proton signal at 5.8 ppm or 6.2 ppm (1H) relative to the sugar ring of hyaluronic acid (10H) at 3.3 - 4.0 ppm.

    One of the most important properties required in hydrogels is optical transparency, especially in soft contact lenses. Herein, optical transmittance of the prepared hydrogels was measured at a wavelength range of 400–700 nm and reported as % transmittance. The MeHA IPN-DA hydrogel exhibited high transmittance (>90%, Fig. 3). The quantification of the incorporated dopamine onto the hydrogels was performed by UV/Vis spectroscopy, as shown in Fig. 4. Using Beer’s Law regression at 288 nm based on the calibration curve of the dopamine solution having a concentration range of 0–0.5 mM, the amount of dopamine on each surface of the hydrogel was calculated to be 0.525 ± 0.109 μmol/cm2.

    The surface wettability of hydrogels is an important parameter for their practical biomedical application as the hydrophobic substrate increases the protein adsorption in physiological conditions. The surface wettability of the hydrogel was investigated by measuring the water contact angle. As shown in Fig. 5, the presence of the HA–IPN structure reduced the contact angle of the hydrogel, which is indicative of the increment in surface wettability. The contact angles of the HA-containing hydrogels were observed to be approximately 71.2° and 78.8° for HA–IPN and HA–IPN–DA hydrogels, respectively. These values represent decreases of approximately 8.0° and 0.4°, respectively, relative to the value of 79.2° for the unmodified control. This result suggests that surface enrichment with hydrophilic HA–IPN structures may provide enhanced surface-hydrophilicity onto the p(HEMA)-based hydrogels. The water contact angle of the dopamine-HA– IPN hydrogel, demonstrating that a considerable number of hydrophilic carboxylate ions within the HA polymers were replaced with relatively hydrophobic dopamine molecules. Thus, surface wettability between dopamine-conjugated IPN hydrogel and p(HEMA) hydrogel did not demonstrate the significant difference.

    The antioxidant activities of the hydrogels were evaluated using DPPH and ABTS radical scavenging assays. Both assays are based on the theory that antioxidants are hydrogen donors and use the extent of decolorization of the radical solution to determine the radical scavenging capacity. Reduction by antioxidant causes the radicals to lose absorption at certain wavelengths (734 nm for ABTS and 515 nm for DPPH). As expected, the dopaminefree hydrogels did not exhibit significant radical scavenging ability, while the dopamine-modified ones exhibited a remarkable improvement in the DPPH and ABTS radical scavenging abilities. As shown in Fig. 6, the unmodified and HA–IPNmodified hydrogels inhibited 2.34% and 4.01% of the ABTS radicals, respectively. On the contrary, significant antioxidant activities were observed for the dopamine- modified hydrogel, which exhibited 22.79% inhibition against ABTS radicals. In the DPPH assay, the dopamine-free unmodified and HA–IPN hydrogels eliminated 4.23 % and 2.56% of the DPPH radicals, respectively, while the dopaminefunctionalized hydrogels inhibited 11.02% of them (Fig. 7). This result is attributed to the potent antioxidant residues being attached to the surface of hydrogels. Polyphenols such as dopamine, possessing an o-diphenolic arrangement, can donate a hydrogen radical to free radicals. The resulting phenolic radical stabilizes by delocalizing the radical electron across the aromatic ring and further oxidation converts the phenolic radical of dopamine to o-quinone.

    Some HA-IPN hydrogels have been reported.17,21) However, their functions were the improved protein resistance and surface wettability of hydrogels. In this study, we could provide an antioxidant activity to HA-IPN hydrogel by incorporation with polyphenols such as dopamine.

    Ⅳ. Conclusions

    In this study, we prepared dopamine-modified hydrogels containing HA-networks and investigated their antioxidant activities. First, p(HEMA) hydrogels were synthesized and surface- functionalized with HA–IPN structures consisting of cross-linked HA polymers and p(HEMA) networks. The subsequent conjugation of dopamine to the HA–IPN structures by an amide coupling reaction resulted in the antioxidant hydrogels. The presence of HA–IPN structures improved the surface hydrophilicity of p(HEMA) hydrogels relative to that of the unmodified ones. The dopamine-modified hydrogels exhibited strong antioxidant activities, which were evaluated by radical scavenging capacity against ABTS and DPPH radicals. The results described herein provide a useful strategy for the development of ophthalmologic and biomedical devices.


    This work was supported by the 2018 research year research grant of the Dongshin University.



    Schematic representation of the preparation of the dopamine-modified HA–IPN hydrogel. The inset photo shows the prepared hydrogel


    1H NMR spectrum of MeHA in D2O. The peaks at 5.6 and 6.0 ppm are due to the methacrylate groups, while the peaks at 3.3- 4.0 ppm are due to the sugar ring of HA.


    Transmission spectra of the antioxidant hydrogels


    Absorbance spectra of the MeHA IPN and dopamine-functionalized MeHA IPN hydrogels. Inset image displays the calibration curve obtained using five varying concentrations of dopamine in aqueous solution.


    Contact angles of water droplets on the (a) p(HEMA), (b) HA–IPN hydrogel, and (c) HA –IPN–DA hydrogel.


    Scavenging effect of the prepared antioxidant hydrogels on ABTS radical.


    a) Scavenging effect and b) raw data of the prepared antioxidant hydrogels on DPPH radical.



    1. Sardesai NP, Andreescu D et al.: Electroanalytical evaluation of antioxidant activity of cerium oxide nanoparticles by nanoparticle collisions at microelectrodes. J Am Chem Soc. 135(45), 16770- 16773, 2013.
    2. Lee HL, Choi CW et al.: Antimetastatic Activity of Gallic Acid-conjugated Chitosan against Pulmonary Metastasis of Colon CarcinomaCells. Bull Korean Chem Soc. 39(1), 90-96, 2018.
    3. Deligiannakis Y, Sotiriou GA et al.: Antioxidant and Antiradical SiO2 Nanoparticles Covalently Functionalized with Gallic Acid. ACS Appl Mater Interfaces 4(12), 6609–6617, 2012.
    4. Alam MS, Lee DU: Quantum‐Chemical Studies to Approach the Antioxidant Mechanism of Nonphenolic Hydrazone Schiff Base Analogs: Synthesis, Molecular Structure, Hirshfeld and Density Functional Theory Analyses. Bull KoreanChem Soc. 36(2), 682-691, 2015.
    5. Astete CE, Dolliver D et al.: Antioxidant Poly (lactic-co-glycolic) Acid Nanoparticles Made with α-Tocopherol–Ascorbic Acid Surfactant. ACS Nano. 5(12), 9913-9325, 2011.
    6. Lee SS, Song W et al.: Antioxidant Properties of Cerium Oxide Nanocrystals as a Function of Nanocrystal Diameter and Surface Coating. ACS Nano. 7(11), 9693-9703, 2013.
    7. Kanazawa K, Sakakibara H: High Content of Dopamine, a Strong Antioxidant, in Cavendish Banana. J Agric Food Chem. 48(3), 844-848, 2000.
    8. Kim B, Kang B et al.: Polyphenol-functionalized hydrogels using an interpenetrating chitosannetwork and investigation of their antioxidant activity. Macromolecular Res. 26(1), 35-39, 2018
    9. Scoponi M, Cimmino S et al.: Photo-stabilisation mechanism under natural weathering and accelerated photo-oxidative conditions of LDPE films foragricultural applications. Polymer 41(22), 7969-7980, 2000.
    10. Giannakopoulos E, Christoforidis KC et al.: Influence of Pb(II) on the radical properties of humic substances and model compounds. JPhys Chem A. 109(10), 2223-2232, 2005.
    11. Catauro M, Papale F et al.: Silica/quercetin sol–gel hybrids as antioxidant dental implant materials. Sci Technol Adv Mater. 16(3), 035001, 2015.
    12. Catauro M, Bollino F et al.: Biocompatibility improvement of titanium implants by coating with hybrid materials synthesized by sol–gel technique. J Biomed Mater Res A. 102(12), 4473-4479, 2014.
    13. Ebabe Elle R, Rahmani S et al.: Functionalized Mesoporous Silica Nanoparticle with Antioxidants as a New Carrier That Generates Lower OxidativeStress Impact on Cells. Mol Pharm. 13(8), 2647-2660, 2016.
    14. Pasanphan W, Buettner GR et al.: Chitosan conjugated with deoxycholic acid and gallic acid: A novel biopolymer‐based additive antioxidant for polyethylene. Appl Polym Sci. 109(1), 38-46, 2008.
    15. Cho YS, Kim SK et al.: Preparation, characterization, and antioxidant properties of gallic acidgrafted- chitosans. Carbohydr Polym. 83(4), 1617-1622, 2011.
    16. Cirillo G, Kraemer K et al.: Biological Activity of a Gallic Acid−Gelatin Conjugate. Biomacromolecules11(12), 3309-3315, 2010.
    17. Kim HJ, Ryu GC et al.: Hydrogel lenses functionalized with polysaccharide for reduction of protein adsorption.Macromolecular Res. 23(1), 74-78, 2015.
    18. Gramlich WM, Holloway JL et al.: Transdermal gelation of methacrylated macromers with nearinfraredlight and gold nanorods. Nanotechnology 25(1), 014004, 2014.
    19. Brand-Williams W, Cuvelier ME et al.: Use of a free radical method to evaluate antioxidant activity. LWT-Food Sci Technol. 28(1), 25-30, 1995.
    20. Arnao MB, Cano A et al.: The hydrophilic and lipophilic contribution to total antioxidant activity. Food Chem. 73(2), 239-244, 2001.
    21. Van BM, Jones L et al.: Hyaluronic acid containing hydrogels for the reduction of protein adsorption. Biomaterials. 29(7), 780-789, 2008.