molecules Article
Development of Gallic Acid-Modified Hydrogels Using Interpenetrating Chitosan Network and Evaluation of Their Antioxidant Activity Byungman Kang 1,† , Temmy Pegarro Vales 2,3,† Ho-Joong Kim 2, * 1 2 3 4 5
* †
ID
, Byoung-Ki Cho 4 , Jong-Ki Kim 5, * and
Nuclear Chemistry Research Division, Korea Atomic Energy Research Institute, 989-111 Daedeok-daero, Yuseong-gu, Daejeon 34057, Korea;
[email protected] Department of Chemistry, Chosun University, Gwangju 61452, Korea;
[email protected] Department of Natural Sciences, Caraga State University, Butuan City 8600, Philippines Department of Chemistry, Dankook University, 119, Dandae-ro, Chungnam 31116, Korea;
[email protected] Department of Biomedical Engineering, School of Medicine, Catholic University of Daegu, Daegu 42472, Korea Correspondence:
[email protected] (J.-K.K.);
[email protected] (H.-J.K.) These authors equally contributed to this work.
Received: 14 October 2017; Accepted: 13 November 2017; Published: 15 November 2017
Abstract: In this work, antioxidant hydrogels were prepared by the construction of an interpenetrating chitosan network and functionalization with gallic acid. The poly(2-hydroxyethyl methacrylate) p(HEMA)-based hydrogels were first synthesized and subsequently surface-modified with an interpenetrating polymer network (IPN) structure prepared with methacrylamide chitosan via free radical polymerization. The resulting chitosan-IPN hydrogels were surface-functionalized with gallic acid through an amide coupling reaction, which afforded the antioxidant hydrogels. Notably, gallic-acid-modified hydrogels based on a longer chitosan backbone exhibited superior antioxidant activity than their counterpart with a shorter chitosan moiety; this correlated to the amount of gallic acid attached to the chitosan backbone. Moreover, the surface contact angles of the chitosan-modified hydrogels decreased, indicating that surface functionalization of the hydrogels with chitosan-IPN increased the wettability because of the presence of the hydrophilic chitosan network chain. Our study indicates that chitosan-IPN hydrogels may facilitate the development of applications in biomedical devices and ophthalmic materials. Keywords: chitosan; IPN; hydrogels; antioxidant activity; gallic acid
1. Introduction Biomaterials with unprecedented levels of structural organization and extraordinary properties have been sought for many years. One emerging material for the design and synthesis of functional biomaterials is hydrogels. Hydrogels are robust, three-dimensional cross-linked hydrophilic polymeric materials, which are capable of retaining large amounts of water and biological fluids [1–3]. More than 50 years after their discovery, poly(2-hydroxyethyl methacrylate) p(HEMA)-based hydrogels remain a prominent and relevant member of the hydrogel family. Owing to their intrinsic high biocompatibility, good mechanical properties, and excellent swelling behavior, p(HEMA)-based hydrogels are employed as biomedical materials, such as in drug delivery systems [4–6], contact lenses [7], dental adhesives [8], and carrier materials for wound healing [9,10]. In the past few decades, there has been a shift toward multi-component hydrogels. Some strategies for the preparation of multi-component hydrogels
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include thiol-ene/yne click reactions [11,12], native chemical ligation [13,14], oxime chemistry [15], chemical ligation [13,14], oxime chemistry [15], and interpenetrating polymer networks (IPNs) [16]. and interpenetrating polymer networks (IPNs) [16]. In particular, an IPN is an intriguing system In particular, an IPN is an intriguing system comprising cross-linked polymers, with at least one comprising cross-linked polymers, with at least one being synthesized and/or cross-linked within being synthesized and/or cross-linked within the immediate presence of the other. In an IPN system, thethe immediate presence of the other. In an IPN system, the polymer networks are physically polymer networks are physically interlocked and entangled on the molecular scale without interlocked and ondifferent the molecular without bonds between different covalent bonds entangled between the types of scale polymer chainscovalent [17–19]. Furthermore, the the fabrication types of polymer [17–19]. Furthermore, the fabrication ofwhere hydrogels via IPNs is typically of hydrogels viachains IPNs is typically simple and straightforward, pre-polymerized hydrogelssimple are and straightforward, where pre-polymerized hydrogels are submerged into a solution monomers submerged into a solution of monomers in the presence of a polymerization initiator. of The resulting in thedouble presence of a polymerization initiator. The resulting double network structure generally network structure generally produces an advanced multicomponent hybrid system,produces which an often advanced multicomponent system, which often exhibits significantly improved component exhibits significantly hybrid improved component polymer properties [20–22] and synergistic polymer properties [20–22] and synergistic properties of the constituent polymers [23].properties of the constituent polymers [23]. In In recent years, advances ininpolymer recent years, advances polymerscience scienceand andbiotechnology biotechnologyhave havefacilitated facilitated the the production production of of biomaterials with excellent bioactivities. Particularly, antioxidants haveconsiderable gained considerable biomaterials with excellent bioactivities. Particularly, antioxidants have gained attention in attentionapplications in biomedical applications becausetoof ability to act ashydrogen-donating reducing agents, hydrogenbiomedical because of their ability acttheir as reducing agents, antioxidants, donating and single[24]. oxygen Anof important class is free radical antioxidants, scavengers, free andradical singlescavengers, oxygen quenchers An quenchers important[24]. class antioxidants of antioxidants is have polyphenols, which have a distinguished ability to undergo reactions. oxidation/reduction polyphenols, which a distinguished ability to undergo oxidation/reduction For instance, reactions. For instance, gallic acid and caffeic acid play key roles in the defense mechanism gallic acid and caffeic acid play key roles in the defense mechanism against free radicals andagainst reactive free radicals reactive oxygen species by chain breaking the free chain reaction oxygen speciesand (ROS) by breaking the free(ROS) radical reaction viaradical the hydroxyl groupsvia onthe their hydroxyl groups on their aromatic rings [25,26]. However, the use of bare antioxidants in the aromatic rings [25,26]. However, the use of bare antioxidants in the pharmaceutical, biomedical, and pharmaceutical, biomedical, and food industries has faced various challenges, such as volatilization, food industries has faced various challenges, such as volatilization, instability, and oxidation under instability, and oxidation under ambient oxygen [27,28]. Hence, antioxidants have been functionalized ambient oxygen [27,28]. Hence, antioxidants have been functionalized into biopolymers and inorganic into biopolymers and inorganic materials to address the aforementioned impediments. Moreover, by materials to address the aforementioned impediments. Moreover, by using the advantages of each using the advantages of each constituent, antioxidant–biopolymer conjugates could be employed as constituent, antioxidant–biopolymer conjugates could be employed as new food additives, in packing, new food additives, in packing, and as biomedical materials. Several studies have reported on the and as biomedical materials. Several studies have on the and functionalization of biomacromolecules functionalization of biomacromolecules suchreported as chitosan its derivatives with phenolic such as chitosan and its derivatives with phenolic compounds extracted from plants, such[29–32]. as gallic compounds extracted from plants, such as gallic acid, caffeic acid, tannic acid, and catechin acid, caffeic acid, tannic acid, and catechin [29–32]. Furthermore, the antioxidant curcumin has been Furthermore, the antioxidant curcumin has been incorporated into bandages and collagen matrices incorporated into bandages and collagen matrices to promote wound healing [33]. to promote wound healing [33].
Figure 1. (a) Schematic representation for development of chitosan-interpenetrating polymer network Figure 1. (a) Schematic representation for development of chitosan-interpenetrating polymer network (IPN) hydrogels functionalized with polyphenols; (b) Photograph of the fabricated antioxidant hydrogel. (IPN) hydrogels functionalized with polyphenols; (b) Photograph of the fabricated antioxidant hydrogel.
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Recently, we demonstrated the potential of IPN structures consisting of succinyl chitosan polymers and spiropyran as photochromic [34]. The nucleophilic amino groups of of chitosan Recently, we demonstratedhydrogels the potential of IPN structures consisting succinylpolymers chitosan within theand IPNspiropyran structure facilitated modifications and [34]. conjugations with various functional molecules. polymers as photochromic hydrogels The nucleophilic amino groups of chitosan In the present paper, we report a synthetic strategy for the preparation of antioxidant hydrogels using polymers within the IPN structure facilitated modifications and conjugations with various functional amolecules. simple method by constructing an IPN architecture based on methacrylamide chitosan (MC) and an In the present paper, we report a synthetic strategy for the preparation of antioxidant antioxidant polyphenol, gallic acid (Figure 1). Initially, p(HEMA)-based hydrogels synthesized and hydrogels using a simple method by constructing an IPN architecture basedwere on methacrylamide achitosan chitosan-IPN was constructed using intermolecularly cross-linked chitosan chains and p(HEMA) (MC) and an antioxidant polyphenol, gallic acid (Figure 1). Initially, p(HEMA)-based networks. By means of amide reactions, chitosan-IPN hydrogels were further surfacehydrogels were synthesized and coupling a chitosan-IPN was constructed using intermolecularly cross-linked functionalized with gallic acid, which significantly improved the antioxidant activity of the chitosan chains and p(HEMA) networks. By means of amide coupling reactions, chitosan-IPN hydrogels. The radical scavenging efficiency of the fabricated antioxidant hydrogels was investigated hydrogels were further surface-functionalized with gallic acid, which significantly improved the in two model assaysofemploying 2,2-diphenyl-1-picrylhydrazyl (DPPH) of and antioxidant activity the hydrogels. The radical scavenging efficiency the3-ethylbenzothiazolinefabricated antioxidant 6-sulfonic acid (ABTS) free radicals. hydrogels was investigated in two model assays employing 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) free radicals. 2. Results and Discussion 2. Results and Discussion Antioxidant hydrogels were prepared according to the synthetic route illustrated in Figure 1. Initially, p(HEMA)-based hydrogels were synthesized viathe free radical polymerization HEMA Antioxidant hydrogels were prepared according to synthetic route illustratedwith in Figure 1. monomers using ethylenehydrogels glycol dimethacrylate (EGDMA) a cross-linking agent with and Initially, p(HEMA)-based were synthesized via freeas radical polymerization azobisisobutyronitrile (AIBN) as the glycol initiator. The resulting p(HEMA)-based hydrogels were surfaceHEMA monomers using ethylene dimethacrylate (EGDMA) as a cross-linking agent and modified with an IPN structure cross-linked chains. Chitosan is a natural polysaccharide azobisisobutyronitrile (AIBN)using as the initiator. chitosan The resulting p(HEMA)-based hydrogels were that has been usedwith in various applications because of its biodegradability, surface-modified an IPNbiomedical structure using cross-linked chitosan chains. Chitosan nontoxicity, is a natural and biocompatibility. However, chitosan hasbiomedical limited solubility in both water of and organic polysaccharide that has been used in various applications because its common biodegradability, solvents because of extensive intramolecular and intermolecular hydrogen in the and βnontoxicity, and biocompatibility. However, chitosan has limited solubility inbonding both water andαcommon conformations [35,36]. Although chitosans with an acetylation degree in the range ofbonding 40–60% in and a organic solvents because of extensive intramolecular and intermolecular hydrogen the medium molecular weight are Although soluble atchitosans physiological pH values [37], theyinmust be chemically α- and β-conformations [35,36]. with an acetylation degree the range of 40–60% modified to improve the solubility insoluble neutralat aqueous media pH andvalues common organic solvents and to be and a medium molecular weight are physiological [37], they must be chemically processed into IPN hydrogels. Herein, chitosan polymers were chemically modified by introducing modified to improve the solubility in neutral aqueous media and common organic solvents and to be methacrylate the chitosan N-position of the were primary amine modified groups inbythe chitosan processed intofunctionalities IPN hydrogels.onto Herein, polymers chemically introducing backbone, yielding a methacrylamide derivative of primary chitosan amine (MC).groups MCs with methacrylate functionalities onto the N-position of the in thedifferent chitosanmolecular backbone, weights (MWs) were synthesized using 100–300 (MC). kDa and 600–800 kDa chitosan, for lowand(MWs) highyielding a methacrylamide derivative of chitosan MCs with different molecular weights 1H-NMR spectroscopic measurements (Figure 2) revealed the degrees of MC, synthesized respectively.using were 100–300 kDa and 600–800 kDa chitosan, for low- and high-MC, respectively. 1methacrylation, which were found to be(Figure about 59.30% and 37.78% for lowand high-MC, respectively. H-NMR spectroscopic measurements 2) revealed the degrees of methacrylation, which were The degree of methacrylation was calculated previouslyThe reported [38] by found to be about 59.30% and 37.78% for low- andaccording high-MC,to respectively. degree literature of methacrylation comparing the according integratedtoarea of the H2–H6 peaks at 2.8–4.0 ppm to that ofthe theintegrated methylene peaks at was calculated previously reported literature [38] by comparing area of the 5.35 and 5.65 ppm. H2–H6 peaks at 2.8–4.0 ppm to that of the methylene peaks at 5.35 and 5.65 ppm.
1 1 Figure spectra of (a)oflow-methacrylamide chitosan (MC) gallic (GA); and (b) highFigure 2. 2. H-NMR H-NMR spectra (a) low-methacrylamide chitosan (MC)acid gallic acid (GA); and MC-GA. (b) high-MC-GA.
Next, Next, chitosan-IPN chitosan-IPN hydrogels hydrogels (low-MC-H (low-MC-H and and high-MC-H high-MC-H based based on on low-MC low-MC and and high-MC, high-MC, respectively) were constructed by loading MC into p(HEMA) hydrogels, followed by the crossrespectively) were constructed by loading MC into p(HEMA) hydrogels, followed by the cross-linking linking of MC via radical polymerization across the methacryl carbon–carbon double bond using
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of MC via radical polymerization across the methacryl carbon–carbon double bond using ammonium persulfate and sodium metabisulfite (SMBS) as polymerization initiators. The fabricated ammonium(APS) persulfate (APS) and sodium metabisulfite (SMBS) as polymerization initiators. The chitosan-IPN hydrogels were surface-functionalized by amide coupling reactions of antioxidant gallic fabricated chitosan-IPN hydrogels were surface-functionalized by amide coupling reactions of acid (GA) togallic the chitosan network, which resulted in which two antioxidant low-MC-GA and antioxidant acid (GA) to the chitosan network, resulted inhydrogels, two antioxidant hydrogels, high-MC-GA and based on low- and based high-MC-H, respectively. After the cross-linking reaction, the yields of low-MC-GA high-MC-GA on lowand high-MC-H, respectively. After the cross-linking surface modification chitosans were estimated to be ~61.6% ~73.8%to forbelowand high-MC-H, reaction, the yields ofwith surface modification with chitosans wereand estimated ~61.6% and ~73.8% respectively. The amountsrespectively. of conjugated MC corresponded to about 0.123 and 0.148 gtofor low-0.123 and for low- and high-MC-H, The amounts of conjugated MC corresponded about high-MC-H, respectively, for 1 g of the corresponding This was hydrogels. obtained simply by and 0.148 g for low- and high-MC-H, respectively, for 1 ghydrogels. of the corresponding This was measuringsimply the amount of MC bound the cross-linking reaction it with the initial obtained by measuring the after amount of MC bound after and the comparing cross-linking reaction and amount of MC. comparing it with the initial amount of MC. To quantify the amount amount of of GA GA attached attached to to the the chitosan-IPN chitosan-IPN hydrogels, hydrogels, UV/Vis UV/Vis absorption onon GA were taken. TheThe method relies only on measurements using using aastandard standardcalibration calibrationcurve curvebased based GA were taken. method relies only the use of the free GA as the standard compound, and the results are given as moles of GA per surface on the use of the free GA as the standard compound, and the results are given as moles of GA per area of hydrogel. Employing Beer’s Law regression at 293 nm, the quantities of GA per hydrogel surface area of hydrogel. Employing Beer’s Law regression at 293 nm, the quantities of GAwere per estimatedwere to beestimated ~0.019 µmol low-MC-GA 0.160 µmol high-MC-GA (Figure 3), which were hydrogel to befor ~0.019 μmol for and low-MC-GA andfor 0.160 μmol for high-MC-GA (Figure 3), calculated the total surface area surface of hydrogels with a size ofwith 10.0a×size 10.0ofmm a thickness which werefrom calculated from the total area of hydrogels 10.0and × 10.0 mm andof a 0.24 mm. As in Table 1, a much higher1,quantity attached antioxidant residuesantioxidant was found thickness of illustrated 0.24 mm. As illustrated in Table a muchofhigher quantity of attached in the hydrogels withina longer MC polymer to those withcompared a shorter MC. The longer residues was found the hydrogels with compared a longer MC polymer to those with achitosan shorter seemsThe to produce a more seems accessible site to GA for the amide coupling reaction as amide compared to its MC. longer chitosan to produce a more accessible site to GA for the coupling shorter counterpart, that thecounterpart, amounts of incorporated chitosans are of nearly same regardless of reaction as compareddespite to its shorter despite that the amounts incorporated chitosans the nearly length same of chitosans [16,34]. are regardless of the length of chitosans [16,34]. Absorbance
0.3
Absorbance
0.25 0.2
1.5
y = 3.5127x + 0.0046 R² = 0.9995
1 0.5 0 0
0.15
0.1 0.2 0.3 Con. of Gallic Acid, mM
0.4
High-MC-GA
0.1
Low-MC-GA
0.05
Low-MC High-MC
0 285
335
385
435
Wavelength, nm
Figure 3. Absorbance prepared methacrylamide methacrylamide chitosan-interpenetrating chitosan-interpenetrating polymer Figure 3. Absorbance spectra spectra of of the the prepared polymer network with gallic gallic acid. acid. network (IPN) (IPN) hydrogels hydrogels functionalized functionalized with Table hydrogels. Table 1. 1. Characteristics Characteristics of of prepared prepared antioxidant antioxidant hydrogels. Contact MW of Chitosan Amounts of Attached Polyphenols Hydrogels MW of Chitosan Amounts ofper Attached Polyphenols a Angle (°) b (◦ ) b (kDa) Hydrogel (μmol) Hydrogels Contact Angle (kDa) per Hydrogel (µmol) a c p(HEMA) — — 73.2 ± 1.9 c p(HEMA) — 100–300 — — 73.2 ±±2.9 1.9 Low-MC-H 68.8 Low-MC-H 100–300 — 68.8 ± 2.9 High-MC-H 600–800 — 60.5 ± 12.3 High-MC-H 600–800 — ± 0.0028 60.5 12.3 Low-MC-GA 100–300 0.019 69.9 ± ± 4.1 Low-MC-GA 100–300 0.019 ± 0.0028 69.9 ± 4.1 High-MC-GA 600–800 0.160 ± 0.0536 66.4 ± 5.0 High-MC-GA 600–800 0.160 ± 0.0536 66.4 ± 5.0 a Data are means ± SD (n = 3); b Data are means ± SD (n = 4), c p(HEMA) is pristine p(HEMA)-based hydrogel. a b c Data are means ± SD (n = 3); Data are means ± SD (n = 4), p(HEMA) is pristine p(HEMA)-based hydrogel.
Contact angle measurements were carried out to investigate the surface properties of the prepared Contacthydrogels angle measurements werein carried to investigate thesurface surfacemodification properties of of thep(HEMA) prepared antioxidant [39]. As shown Figureout 4 and Table 1, the antioxidant hydrogels [39]. As shown in Figure 4 and Table 1, the surface modification of p(HEMA) hydrogels with cross-linked chitosan-IPN structures resulted in a decrease in the water contact angle, hydrogels enhanced with cross-linked chitosan-IPN structures resulted inexhibited a decrease in theangles water of contact indicating surface wettability. Lowand high-MC-H contact aboutangle, 68.8° and 60.5°, respectively. These values represent decreases of about 4.4° and 12.7° for the hydrogels, respectively, relative to the value of 73.2° for the unmodified control. The observed decrease in the
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indicating enhanced surface wettability. Low- and high-MC-H exhibited contact angles of about 68.8◦ and 60.5◦ , respectively. These values represent decreases of about 4.4◦ and 12.7◦ for the hydrogels, Molecules 2017, 22, 1976 5 of 11 respectively, relative to the value of 73.2◦ for the unmodified control. The observed decrease in the hydrogel to to thethe relatively hydrophilic chitosan-IPN, which enhanced the hydrogelcontact contactangle anglewas wasattributed attributed relatively hydrophilic chitosan-IPN, which enhanced surface-hydrophilicity of the prepared hydrogels. Moreover, the hydrogel surface-modified with longer the surface-hydrophilicity of the prepared hydrogels. Moreover, the hydrogel surface-modified with chitosan networks showedshowed higher wettability compared to its shorter counterpart, becausebecause a longera longer chitosan networks higher wettability compared to its shorter counterpart, chitosan covered the hydrogel surface with its hydrophilic glucosamine units more so than longer chitosan covered the hydrogel surface with its hydrophilic glucosamine units more shorter so than chains. However, the water contact angle of hydrogels functionalized with GA increased by about shorter chains. However, the water contact angle of hydrogels functionalized with GA increased by ◦ and 5.9◦ for low- and high-MC-GA, respectively, relative to GA-unfunctionalized chitosan-IPN 1.1 about 1.1° and 5.9° for low- and high-MC-GA, respectively, relative to GA-unfunctionalized chitosanhydrogels, demonstrating that thethat relatively hydrophobic GA slightly the surface the wettability IPN hydrogels, demonstrating the relatively hydrophobic GAdecreased slightly decreased surface of the prepared hydrogels. Furthermore, the values depicted in Table 1 were in fairly good accordance wettability of the prepared hydrogels. Furthermore, the values depicted in Table 1 were in fairly good with the contact reported in the literature [40].literature Ketelson[40]. et al.Ketelson have reported that commercially accordance withangles the contact angles reported in the et al. have reported that ◦ [40]. available contact lenses exhibited contact angles of 30–105 commercially available contact lenses exhibited contact angles of 30–105° [40]. (b)
(a)
69.9°
68.8° (d)
(c)
60.5°
66.4°
Figure 4. Contact angles of nanopure water droplets (4.5 μL) on (a) low-methacrylamide chitosan (MC) Figure 4. Contact angles of nanopure water droplets (4.5 µL) on (a) low-methacrylamide chitosan (MC) hydrogel; (b) low-MC-gallic acid (GA) hydrogel; (c) high-MC hydrogel; and (d) high-MC-GA hydrogel. hydrogel; (b) low-MC-gallic acid (GA) hydrogel; (c) high-MC hydrogel; and (d) high-MC-GA hydrogel.
The antioxidant properties of the fabricated chitosan-IPN hydrogels were assessed using DPPH antioxidant propertiesassays. of the fabricated assessed using DPPH and The ABTS radical scavenging Herein, thechitosan-IPN antioxidant hydrogels efficiencieswere of the prepared hydrogels and ABTS radical scavenging assays. Herein, the antioxidant efficiencies of the prepared hydrogels were investigated using ascorbic acid as a positive control. In the DPPH assay, the antioxidant activity were investigated ascorbic aciddecolorization as a positive control. In the DPPH antioxidant activitya is determined by using the extent of the of the DPPH radical.assay, The the DPPH radical shows is determined by the extent of the decolorization of the DPPH radical. The DPPH radical strong absorption maximum at 517 nm and its color changes from purple to colorless followedshows by the aformation strong absorption 517 nm and its the color changes from purple tofrom colorless followed by the of stable maximum hydrazine at (DPPH-H) upon absorption of hydrogen an antioxidant. Thus, formation of stable hydrazine (DPPH-H) proportional upon the absorption of hydrogen from an antioxidant. Thus, the antioxidant effect is stoichiometrically to the decrease in the UV absorption at 517 nm. In the antioxidant effect is stoichiometrically proportional to the decrease in the UV absorption at 517 nm. contrast, the ABTS assay is based on the reduction of the generated blue/green ABTS•+ species with In the ABTS of assay is based onatthe of thein generated ABTS•scavenging + species thecontrast, percent inhibition the absorbance 734reduction nm. As shown Figures 5blue/green and 6, the radical with the of percent inhibition of the absorbance at 734 nm.reaction As shown inDPPH Figures 5 and 6, radicals. the radical abilities the prepared hydrogels were evaluated upon with and ABTS As scavenging abilities of the prepared hydrogels were evaluated upon reaction with DPPH ABTSa expected, the polyphenol-free hydrogels did not exhibit any radical scavenging abilities. and Notably, radicals. As expected, the polyphenol-free hydrogels did notscavenging exhibit anyabilities radical scavenging abilities. remarkable improvement in the DPPH and ABTS radical by the polyphenolNotably, a remarkable improvement in the DPPH and ABTS radical scavenging abilities by the modified hydrogels was observed. polyphenol-modified hydrogels was observed. In the DPPH and ABTS assays, moderate antioxidant abilities were observed for low-MC-GA, In the DPPH39.40% and ABTS assays, of moderate antioxidant were observed for which inhibited and 38.25% the DPPH and ABTSabilities radicals, respectively. On low-MC-GA, the contrary, which inhibited 39.40% and 38.25% of the DPPH and ABTS radicals, respectively. On the exhibited contrary, strong antioxidant activities were observed for the hydrogels with longer chitosan chains, which strong antioxidant activities were observed for the hydrogels with longer chitosan chains, which exhibited a 74.65% and 95.79% inhibition of DPPH and ABTS radicals, respectively, while the positive control, aascorbic 74.65%acid, and exhibited 95.79% inhibition DPPH and ABTS radicals, respectively, while the positive control, a 93.65% of and 95.31% inhibition against DPPH and ABTS radicals, respectively. ascorbic acid, exhibited a 93.65% and 95.31% inhibition against DPPH and ABTS radicals, respectively. The results suggest that hydrogels based on MC species with a higher MW exhibited stronger The results suggest that hydrogels based MC species with a higher to MW stronger antioxidant effects than those with shorter MC on moieties. This was attributed theexhibited potent antioxidant antioxidant effects than those with shorter MC moieties. This was attributed to the potent antioxidant residues being attached to the longer MC-based hydrogels. Generally, polyphenols possessing an residues beingarrangement, attached to for the example, longer MC-based hydrogels. o-diphenolic a catechol structure, Generally, can donatepolyphenols a hydrogenpossessing radical to scavenge DPPH and ABTS free radicals. The resulting phenolic radical is stabilized by resonance, whereby the radical is delocalized across the aromatic ring and is further oxidized to form the fully conjugated o-dione structure, o-quinone. Moreover, the additional hydroxyl group in GA enhances its antioxidant activity, as the added hydroxyl group adjacent to the o-dihydroxyl phenolic structure forms an intramolecular hydrogen bond in the o-position during the radical scavenging reaction,
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an o-diphenolic arrangement, for example, a catechol structure, can donate a hydrogen radical to scavenge DPPH and ABTS free radicals. The resulting phenolic radical is stabilized by resonance, whereby the radical is delocalized across the aromatic ring and is further oxidized to form the fully conjugated o-dione structure, o-quinone. Moreover, the additional hydroxyl group in GA enhances Molecules 2017, 22, 22, 1976 1976 of 11 11 its antioxidant activity, as the added hydroxyl group adjacent to the o-dihydroxyl phenolic structure Molecules 2017, 66 of forms an intramolecular hydrogen bond in the o-position during the radical scavenging reaction, which provides additional stability to the phenoxy radical owing to its hydrogen-donating capacity. which which provides providesadditional additionalstability stabilityto tothe thephenoxy phenoxyradical radicalowing owingto toits itshydrogen-donating hydrogen-donatingcapacity. capacity. Several studies have reported the enhanced antioxidant activity of tri-hydroxyl derivatives in thethe oSeveral studies have reported the enhanced antioxidant activity of tri-hydroxyl derivatives in oSeveral studies have reported the enhanced antioxidant activity of tri-hydroxyl derivatives the in position, such as catechin gallate ester and GA, because of the hydrogen-donating capacity of the position, such asas catechin gallate o-position, such catechin gallateester esterand andGA, GA,because becauseofofthe thehydrogen-donating hydrogen-donatingcapacity capacityof of the the third hydroxyl group to the phenoxy radical [24,41,42]. third third hydroxyl hydroxyl group group to to the the phenoxy phenoxy radical radical [24,41,42]. [24,41,42].
DPPH DPPH Inhibition, Inhibition, % %
100 100 80 80 60 60 40 40 20 20 00 11
22
33
44
55
66
Figure 5. Radical Radical scavenging capacity of the prepared antioxidant hydrogels against 2,2-diphenyl-1Figure 5. Radicalscavenging scavengingcapacity capacityof ofthe theprepared preparedantioxidant antioxidanthydrogels hydrogelsagainst against2,2-diphenyl-12,2-diphenyl-1Figure picrylhydrazyl (DPPH) free radicals. The amount of ascorbic acid was 0.85 μmol. Legend: poly(2picrylhydrazyl(DPPH) (DPPH)free free radicals. radicals.The Theamount amountof ofascorbic ascorbicacid acidwas was0.85 0.85μmol. µmol.Legend: Legend:111===poly(2poly(2picrylhydrazyl hydroxyethyl methacrylate) (p(HEMA)); low-methacrylamide chitosan hydrogel (MC-H); hydroxyethyl methacrylate) methacrylate) (p(HEMA)); (p(HEMA)); 222 === low-methacrylamide low-methacrylamide chitosan chitosan hydrogel hydrogel (MC-H); (MC-H); hydroxyethyl high-MC-H; low-MC-gallic acid (GA); high-MC-GA; ascorbic acid. high-MC-H;444===low-MC-gallic low-MC-gallicacid acid(GA); (GA);555===high-MC-GA; high-MC-GA;666===ascorbic ascorbicacid. acid. 333===high-MC-H;
ABTS ABTS Inhibition, Inhibition, % %
100 100
80 80
60 60
40 40
20 20
00 11
22
33
44
55
66
Figure 6. Radical scavenging capacity of of the the prepared antioxidant hydrogels against 3Figure scavenging capacity of the prepared antioxidant hydrogels against 3Figure 6. 6. Radical Radical scavenging capacity prepared antioxidant hydrogels against ethylbenzothiazoline-6-sulfonic acid (ABTS) free radicals. The amount of ascorbic acid was 0.85 μmol. ethylbenzothiazoline-6-sulfonic acid 3-ethylbenzothiazoline-6-sulfonic acid(ABTS) (ABTS)free freeradicals. radicals.The Theamount amountofofascorbic ascorbicacid acidwas was0.85 0.85μmol. µmol. Legend: poly(2-hydroxyethyl methacrylate) (p(HEMA)); low-methacrylamide chitosan hydrogel Legend: Legend:111===poly(2-hydroxyethyl poly(2-hydroxyethylmethacrylate) methacrylate)(p(HEMA)); (p(HEMA));222===low-methacrylamide low-methacrylamidechitosan chitosanhydrogel hydrogel (MC-H); high-MC-H; low-MC-gallic acid (GA); high-MC-GA; ascorbic acid. (MC-H); (MC-H); 333 ===high-MC-H; high-MC-H;444===low-MC-gallic low-MC-gallicacid acid(GA); (GA);555===high-MC-GA; high-MC-GA;666===ascorbic ascorbicacid. acid.
3. Materials Materials and and Methods Methods 3. 3.1. Chemicals Chemicals 3.1. HEMA, EGDMA, EGDMA, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride hydrochloride (EDC-HCl), (EDC-HCl), HEMA, N-hydroxysuccinimide (NHS), (NHS), GA, GA, APS, APS, SMBS, SMBS, methacrylic methacrylic anhydride, anhydride, DPPH, DPPH, and and ABTS ABTS were were N-hydroxysuccinimide purchased from from Sigma Sigma Aldrich Aldrich (St. (St. Louis, Louis, MO, MO, USA). USA). AIBN AIBN was was purchased purchased from from Junsei Junsei (Tokyo, (Tokyo, purchased Japan), while while chitosan chitosan (100–300 (100–300 kDa kDa and and 600–800 600–800 kDa) kDa) was was acquired acquired from from Acros Acros Organics Organics (Geel, (Geel, Japan), Belgium). The The degree degree of of deacetylation deacetylation was was provided provided by by the the supplier supplier and and was was found found to to be be ≥90%. ≥90%. Belgium).
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3. Materials and Methods 3.1. Chemicals HEMA, EGDMA, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC-HCl), N-hydroxysuccinimide (NHS), GA, APS, SMBS, methacrylic anhydride, DPPH, and ABTS were purchased from Sigma Aldrich (St. Louis, MO, USA). AIBN was purchased from Junsei (Tokyo, Japan), while chitosan (100–300 kDa and 600–800 kDa) was acquired from Acros Organics (Geel, Belgium). The degree of deacetylation was provided by the supplier and was found to be ≥90%. Deuterium oxide was purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). 3.2. Synthesis of HEMA-Based Hydrogels A HEMA monomer was initially purified using vacuum distillation prior to polymerization. Briefly, EGDMA (0.04 g) and AIBN (0.04 g) were dissolved in HEMA (9.92 g). The resulting solution was mixed for 30 min, injected into a square mold comprising two glass plates internally covered with a polypropylene sheet and separated by a 0.20 mm wide Teflon frame, and was heated at 90 ◦ C for 5 h to allow for polymerization to take place. The samples were then removed from the molds and subjected to extensive dialysis. They were then placed in 400 mL of de-ionized water (changed three times daily), for 2 days, to remove any unreacted monomer and initiators. Subsequently, square hydrogels (10 mm × 10 mm × 0.24 mm) were cut from the square mold, immersed in boiling water for 15 min, and dried at 40 ◦ C overnight. 3.3. Preparation of MC and Analysis of Degree of Methacrylation MC was synthesized according to previously reported literature [43]. Chitosan of varying MW (Mw of 100,000–300,000 Da and 600,000–800,000 Da) was separately dissolved in 2 wt % acetic acid overnight at room temperature (RT) to constitute a 3 wt % solution of chitosan in distilled water. Methacrylic anhydride was added to the chitosan solutions at a 0.44 methacrylic anhydride/glucosamine molar ratio. The resulting mixture was stirred at RT for 3 h before being subjected to extensive dialysis against distilled water for 2 days with at least three to four changes of distilled water a day. The mixture was freeze-dried and stored at −20 ◦ C until use. The degree of methacrylation of chitosan was determined using 1 H-NMR spectroscopic measurements [35]. An appropriate amount of MC was dissolved in D2 O to constitute a ~0.5% (w/v) MC solution. The degree of methacrylation was then calculated by comparing the integrated area of H2–H6 peaks at 2.8–4.0 ppm to that of the methylene peaks at 5.35 and 5.65 ppm. The 1 H-NMR spectra were recorded using JNM-AL300 (JEOL, Tokyo, Japan). 3.4. Synthesis of MC-IPN Functionalized with GA An appropriate amount of previously freeze-dried MC was dissolved to reconstitute a 2 wt % solution in distilled water. Then, previously prepared p(HEMA)-based hydrogels were immersed in the MC solution at RT. After 24 h, the p(HEMA)-based hydrogels were washed with distilled water and immersed in 10 mL of distilled water followed by the addition of polymerization initiators, APS and SMBS. The mixture was allowed to sit for 2 h to allow for the cross-linking reaction to proceed completely. The yield of the surface modification was calculated from Equation (1). To remove any unreacted cross-linking agents, MC-IPN hydrogels were washed with phosphate buffer saline (PBS; pH 7.4) for 3 days with at least four to five changes of buffer each day. Subsequently, the functionalization of MC-IPN hydrogels with GA was then performed. This was done by submerging the MC-IPN hydrogels in 20 mL of distilled water, followed by the addition of EDC-HCl, NHS and GA. The mixture was allowed to sit for 24 h at RT. The mixture was immersed in distilled water for 2 days to completely remove any unreacted chemicals prior to characterization. % Yield = {(Weight of dried IPN Hydrogel − Weight of p(HEMA) hydrogel)/Weight of MC)} × 100
(1)
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3.5. UV-Vis Absorption Measurements The absorption spectra of the hydrogels were measured at a wavelength range of 285–750 nm with a Shimadzu, UV-1650PC (Shimadzu, Tokyo, Japan) spectrophotometer. The measurements for each sample were repeated four times, and the results were averaged. 3.6. Contact Angle Measurements A drop of nanopure water (4.5 µL) was positioned on the hydrogel surface. Contact angles were then measured using a DSA100 instrument (Krüss GmbH, Hamburg, Germany). The measurements for each sample were taken four times, and the results were averaged. 3.7. DPPH Radical-Scavenging Assay of the MC-IPN Hydrogels A method described by Brand–Williams and modified by Miliauskas [44] was used in determining the DPPH radical-scavenging capacity of the prepared MC-IPN hydrogels. 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 sample was shaken for 30 min at 37 ◦ C in the dark. The reaction of the DPPH radical was estimated by measuring the absorption at 517 nm against ethanol as a blank in the spectrophotometer. The percentage of the DPPH• scavenging inhibition capacity was calculated from Equation (2): % Inhibition = {1 − (Absorbance of sample/Absorbance of control)} × 100
(2)
3.8. ABTS Radical-Scavenging Assay of the MC-IPN Hydrogels The ABTS radical-scavenging capacity of each sample was determined according to the modified method described by Arnao et al. [45]. ABTS radical cations (ABTS•+) were produced by adding 7 mM ABTS solution and 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 h 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 ◦ C, in the dark. The reaction of the ABTS•+ species was estimated by measuring the absorption at 734 nm against methanol as a blank. The percentage scavenging inhibition capacity of ABTS•+ was calculated using Equation (2). 4. Conclusions We have prepared antioxidant p(HEMA)-based hydrogels using a chitosan-based IPN structure and surface immobilization with GA. We have successfully synthesized polymerizable MCs and applied them to the construction of chitosan-based IPN structures on p(HEMA) hydrogels. Remarkably, the IPN synthesis was carried out in an aqueous solution without an additional cross-linker, which makes this approach more facile and practical than those previously reported using chitosan-based IPN structures. Further covalent modifications with GA on the chitosan backbone yielded antioxidant chitosan-IPN hydrogels. Superior antioxidant effects were observed by the hydrogels with longer chitosan species, as more antioxidant residues were attached to the longer chitosan chains. The surface wettability of the prepared antioxidant hydrogels was enhanced in the presence of the relatively hydrophilic chitosan-IPN structure but was slightly decreased upon conjugation with GA. The results described herein support the feasibility of chitosan-IPN hydrogels as versatile platforms for the development of ophthalmic materials and functional biomaterials with intrinsic bioactivities and biocompatibility.
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Acknowledgments: This work was performed with financial support from the Industrial Materials Fundamental Technology Development Program (10052981, Development of Smart Contact Lens Materials for Glaucoma Therapy and IOP Measurements), which is funded by the Ministry of Trade, Industry and Energy of Korea. Author Contributions: Ho-Joong Kim and Jong-Ki Kim conceived and designed the experiments; Temmy Pegarro Vales performed the experiments; Ho-Joong Kim, Byoung-Ki Cho, and Temmy Pegarro Vales analyzed the data; Ho-Joong Kim and Byungman Kang contributed reagents/materials/analysis tools; Temmy Pegarro Vales and Byungman Kang wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors. © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
