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Fabrication of schizophyllan hydrogel via orthogonal thiol-ene photopolymerization
Accepted Manuscript Title: Fabrication of schizophyllan hydrogel via orthogonal thiol-ene photopolymerization Authors: Sora Lee, Hyunhyuk Tae, Chang Seok Ki PII: DOI: Reference:
S0144-8617(17)30298-9 http://dx.doi.org/doi:10.1016/j.carbpol.2017.03.042 CARP 12131
To appear in: Received date: Revised date: Accepted date:
13-1-2017 28-2-2017 12-3-2017
Please cite this article as: Lee, Sora., Tae, Hyunhyuk., & Ki, Chang Seok., Fabrication of schizophyllan hydrogel via orthogonal thiol-ene photopolymerization.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2017.03.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Fabrication of schizophyllan hydrogel via orthogonal thiol-ene photopolymerization
Sora Lee, Hyunhyuk Tae, Chang Seok Ki*
Department of Biosystems and Biomaterials Science and Engineering, Seoul National University, Seoul 08826, Republic of Korea
Sora Lee and Hyunhyuk Tae equally contributed to this study as co-first authors.
*To whom correspondence should be sent: Chang Seok Ki, Ph D Assistant Professor Department of Biosystems and Biomaterials Science and Engineering, Seoul National University, Seoul 08826, Republic of Korea Email: [email protected]
Highlights
SPG and PEG/SPG hydrogel fabrication via orthogonal thiol-ene reaction.
The properties of SPG-based hydrogel could be modulated by controlling components.
Mechanical enhancement of hydrogel has been achieved by dual mode crosslinking.
1
Abstract Schizophyllan (SPG) is a water soluble β-glucan, which is obtained from Schizophyllum commune. SPG has been widely explored with their unique properties such as triple helical structure, immune-modulation, and anti-tumoral activity. In this study, we tried to fabricate SPG hydrogel via thiol-ene photopolymerization. SPG-norbornene and SPG-thiol were synthesized from ultrasonicated SPG. Two types of SPG hydrogels (SPG and PEG/SPG hybrid) were formed via thiol-ene photo-click reaction. By controlling the SPG content and stoichiometric balance of norbornene and thiol groups, swelling ratio and shear elastic modulus of SPG hydrogel could be controlled in the range of 20-60 and 0.5-10 kPa, respectively. For PEG/SPG hybrid hydrogel, we found that the triple-helical structure of SPG played a role in physical network formation with thiol-ene cross-linking. The degradability of SPG hydrogel could be also controlled by varying formulation. Therefore, such highly tunable SPG hydrogels would be utilized for various applications.
Key words: schizophyllan, hydrogel, thiol-ene reaction, photopolymerization
1. Introduction Hydrogel is composed of three-dimensional polymer network retaining a large amount of water, resulting in unique physical and mechanical properties. For centuries, hydrogels have 2
been utilized in a wide variety of industries such as food, pharmaceutics, and cosmetics. Recently, soft tissue-like property of hydrogels elicits great potential in biomedical applications (e.g., tissue culture matrix, wound-care material) (Hoffman, 2012; Peppas, Hilt, Khademhosseini, & Langer, 2006). In general, hydrogel can be formed with various hydrophilic polymers by crosslinking (Hennink & van Nostrum, 2012). Polymer type for hydrogel fabrication is one of key factors which determine the hydrogel property and roughly categorized into synthetic and natural polymers (Drury & Mooney, 2003). Among abundant natural polymers, polysaccharides are especially advantageous in hydrogel fabrication for their abundance, low toxicity, structural diversity, and chemical functionality. In addition, they allow various chemical modifications, resulting in versatile manipulation of hydrogel property (Van Vlierberghe, Dubruel, & Schacht, 2011). Hence, for decades, hydrogels formed with various polysaccharides (e.g., hyaluronic acid (Gramlich, Kim, & Burdick, 2013), carrageenan (Popa, Gomes, & Reis, 2011), xanthan (Bueno, Bentini, Catalani, & Petri, 2013), chitosan (Mirahmadi, Tafazzoli-Shadpour, Shokrgozar, & Bonakdar, 2013), dextran (Corrente, Amara, Pacelli, Paolicelli, & Casadei, 2013)) have been extensively explored in biomedical field. β-glucan commonly refers to a family of glucopyranose polysaccharides containing 1,3-β-glycosidic linkages with 1,6-branch structure of varying degrees. β-glucans can be obtained from a wide variety of sources such as fungi, bacteria, algae, lichen, and some plants (Lehtovaara & Gu, 2011). Compared with other polysaccharides, the most distinctive feature of β-glucan is the triple-helix of which three β-glucan chains are self-assembled by hydrogen bonds. The triple-helical structure of β-glucan can be dissociated by changing solvent types or pH condition, resulting in a single-helix (Miyoshi, Uezu, Sakurai, & Shinkai, 2004). Such a structural uniqueness of β-glucan is very important in processing as well as its utilizations. 3
Meanwhile, tremendous studies have dedicated to evaluation of bioactivity of β-glucan. For centuries, herbal medicines which contain β-glucan have been clinically used and recent efforts have unveiled the multi-bioactivity of β-glucan (e.g., immune-stimulation, anti-inflammatory, anti-microbial, and anti-tumoral activities) (Chen & Seviour, 2007). One of key mechanisms that β-glucan exhibits bioactivity is triggered by phagocytic action of macrophage (Chan, Chan, & Sze, 2009). Such an activation mechanism is a product of long-term evolution of immune system against bacterial or fungal pathogens. For decades, β-glucan has been widely utilized as food additive, nutrition factor, and direct therapeutic material for enhancing immune response (Divyasri, Gunasekar, Benny, & Ponnusami, 2014). For β-glucan-based hydrogel fabrication, a considerable number of both physical and chemical cross-linking techniques have been proposed. Physically cross-linked β-glucan hydrogels can be easily fabricated by controlling temperature or pH of β-glucan solution. For example, linear β-glucan solution forms a reversible ‘low-set’ gel between 55-80 °C and it becomes sol by cooling. At a higher temperature range of 85-145 °C, an irreversible ‘high-set’ gel can be also formed (Maeda, Saito, Masada, Misaki, & Harada, 1967; Zhang, Nishinari, Williams, Foster, & Norton, 2002). Alternatively, β-glucan hydrogel can be prepared by neutralizing the alkali β-glucan solution (Kanzawa, Harada, Koreeda, & Harada, 1987). Such a hydrogel is easily changed to sol-state by increasing pH, as disrupting hydrogen bond. However, the conventional physical cross-linking methods are not physiologically compatible. Such βglucan hydrogels are therefore not suitable in biological uses (e.g., delivery vehicles for biomolecules or cells) (Kim et al., 2000). In recent years, carboxymethylated β-glucan has been cross-linked via ionic cross-linking and semi-interpenetrating hydrogel of β-glucan and other synthetic polymers has been reported (Aalaie, Rahmatpour, & Vasheghani‐ Farahani, 2009; 4
Corrente et al., 2009). However, such cross-linking techniques were somehow sensitive to external conditions (e.g., pH, ionic strength, temperature). Meanwhile, chemical cross-linking methods have been explored in β-glucan hydrogel fabrication. It can form more stable network compared with physically constructed β-glucan network. Xiao et al. (2009) and Itagati et al. (2016) formed β-glucan hydrogels with epichlorohydrin and hexamethylenediisocyanate, respectively. Without chemical cross-linkers, β-glucan hydrogel could be also fabricated by irradiation of gamma-ray. Gamma-ray of high energy might produce chemically functional groups on β-glucan, resulting in cross-links (Lim et al., 2015). Alternatively, β-glucan was oxidized and cross-linked with gelatin via imine linkage formation. This gelation could be quickly completed at a narrow pH range (Fang, Takahashi, & Nishinari, 2005). Recently, Qi et al. (2017) developed a salecan-based hydrogel by grafting 2-acrylamido-2-methyl-1propanesulfonic acid (AMPS) onto salecan chains using N,N’-methylenebis(acrylamide) as a cross-linker. Although such chemical cross-linking techniques are relatively handy to control cross-linking density and gelation kinetics, potential toxicity of cross-linking agent or crosslinking condition should be considered as drawback (Nussinovitch, 2010). In this study, we report β-glucan hydrogel fabrication with a kind of branched βglucans, schizophyllan (SPG) via thiol-norbornene photopolymerization. SPG is one of water soluble β-glucans obtained from Schizophyllum commune and contains a 1,3-β-linked backbone with single 1,6-β-linked glucose side chains at every third residue (Zhang, Kong, Fang, Nishinari, & Phillips, 2013). The thiol-ene photopolymerization is a radical mediated step-growth reaction via orthogonal cross-linking between thiol and norbornene groups, resulting in rapid gelation as well as ideal network formation. Besides, the reaction causes low cytotoxicity in cell encapsulation and less damage to bioactive molecules during gelation process due to less radical 5
generation compared with other radical polymerizations (Lin, Ki, & Shih, 2015; Mũnoz, Shih, & Lin, 2014). For the thiol-ene photopolymerization, SPG-derivatives (i.e., SPG-norbornene, SPGthiol) were synthesized. Next, SPG and PEG/SPG hybrid hydrogels were fabricated and their physical properties, degradability, and applicability as a protein drug carrier as well as a cell culture matrix were evaluated.
2. Materials and methods 2.1. Materials Schizophyllum commune lysate was purchased from CoSeedBioPharm (Korea). The crude SPG was obtained by precipitating with isopropanol after filtering the lysate, followed by vacuum drying. The SPG flake was re-dissolved in 0.1 M sodium hydroxide solution and vigorously stirred at 80 °C for 2 h. The alkali treated SPG was filtered and neutralized with diluted hydrochloric acid, followed by precipitation and thorough washing with isopropanol. Finally, the precipitate was washed again with chloroform to remove residual lipids and proteins. To reduce molecular weight of SPG, the refined SPG was re-dissolved in de-ionized water at 10 mg/mL. 30 mL SPG solution was transferred in to a 50 mL conical tube and ultrasonicated by an ultra-sonic processor at 130 W (VCX-130, SONICS). The ultrasonication time was controlled by measuring the shear viscosity change of SPG solution using a rheometer (Hakke MARSIII, ThermoScientific). 4-arm poly(ethylene oxide) (PEG4OH, 20 kDa), isopropanol, and 5norbornene-2-methylamine were obtained from JenKem Technology, Samchun Chemical, and Tokyo Chemical Industry, respectively. All the other chemicals were purchased from SigmaAldrich and used without further purification. 6
2.2. Synthesis For synthesis of carboxymethyl-schizophyllan (SPG-COOH), ultrasonicated SPG was dissolved in de-ionized water at 20 mg/mL and sodium hydroxide (24 eq. of pyranose of SPG) was added to activate hydroxyl groups of SPG, followed by addition of isopropanol of equal volume. Then, chloroacetic acid (14 eq. of pyranose of SPG) was drop-wisely added and the mixture was subsequently stirred at 60 °C for 3 h. To quench the reaction, de-ionized water of 2fold volume was added and dialyzed using a cellulose acetate tube (MWCO = 12-14 kDa) for 3 days, followed by lyophilization. The degree of substitution (DS) was determined by ASTM D1439-94 standard method (ASTM, 1994). Schizophyllan-norbornene (SPG-NB) was synthesized via EDC chemistry with 5-norbornene-2-methylamine (NB-amine). SPG-COOH was dissolved in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (0.1 M, pH 6) at 16 mg/mL and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) (1 eq. of pyranose of SPG) was added. Then, 5-norbornene-2-methylamine (2 eq.) was added and pH of the mixture solution was adjusted around 6 with hydrochloric acid. The solution was stirred for 18 h, followed by dialysis against de-ionized water at pH 6 for 3 days and lyophilization. Schizophyllan-thiol (SPG-SH) was synthesized according to the previous SPG-NB synthesis protocol with slight modification (Fig. 1). After dialysis, mercaptoethanol was added for reducing di-sulfide bond and pH of the solution was adjusted to 8 with sodium hydroxide, followed by stirring for 30 min. Then, the solution was re-dialyzed against de-ionized water at pH 5 for 3 days and subsequently lyophilized. The amount of SPG-SH thiol groups was determined by Ellman’s assay. PEG-ester linked-tetra-norbornene (PEG4NB) was synthesized by reacting 4-arm PEG-OH with 57
norbornene-2-carboxylic acid (Fairbanks et al., 2009; Ryu et al., 2016) and lithium arylphosphinate (LAP) were synthesized according to published protocols (Fairbanks, Schwartz, Bowman, & Anseth, 2009). 2.3. SPG hydrogel formation Fig. 2 presents schematics for SPG hydrogel formations via thiol-ene photo-click reaction. For SPG hydrogels, SPG-NB was dissolved in phosphate-buffered saline (PBS) at a desired concentration (i.e., 3, 5, 8 wt%) with 1 mM LAP and dithiothreitol (DTT). The concentration of DTT was varied in the range of 2-20 mM according to the concentration of SPG-NB in prepolymer solutions. For PEG/SPG hydrogel, the precursor solutions were prepared with SPG-SH of varying concentration (0-3 wt%), 4 wt% PEG4NB, and DTT in the presence of 1 mM LAP. DTT was used as a co-crosslinker to balance the stoichiometric ratio between norbornene and thiol moieties ([NB] = [SH]) (Fig. 2C). The precursor solution was injected into the gap between glass slides separated by 1 mm-thick spacers and subsequently exposed to ultraviolet (UV) light (5 mW/cm2, 365 nm) for 2 min to initiate thiol-ene photopolymerization. Then, formed hydrogel slabs were collected and immediately immersed in pH 7.4 PBS. 2.4. Nuclear magnetic resonance (NMR) and Fourier transform infrared (FT-IR) Spectroscopies For NMR analysis, intact and ultrasonicated SPGs were dissolved in deuterated dimethyl sulfoxide at 40 mg/mL for analysis. For SPG derivatives, each sample was dissolved in D2O at 40 mg/mL. 13C NMR spectra were obtained by using a 600 MHz NMR spectrometer (AVANCE 600, Bruker). To obtain FT-IR spectra, lyophilized intact SPG and SPG derivative samples were powdered and analyzed by an attenuated total reflectance Fourier transforminfrared (ATR FT-IR) spectrometer (Nicolet 6700, ThermoScientific). FT-IR spectra were 8
obtained with 32 scans at 4 cm-1 resolution.
2.5. Gel filtration chromatography (GFC) Molecular weights of SPG samples were measured by a high performance liquid chromatography (HPLC) (Ultimate 3000, Dionex) with sodium azide 0.1 M solution as an eluent through a serialized column of Waters Ultrahydrogel 120, 500, and 1000. Each SPG sample was dissolved in de-ionized water at 10 mg/mL and filtered with a 0.2 μm pore syringe filter. The flow rate was 1 mL/min and eluted SPG concentration was measured by a refractive index detector. The number and weight average molecular weights were determined based on a standard curve obtained with pullulan standards via software for GFC (Chromeleon 6.8 Extention-pak).
2.6. Rheometry To determine ultrasonication time, shear viscosity change of SPG solution (10 mg/mL) was measured using a MARSIII rheometer (ThermoScientific, rotational ramp CR (controlled rate) mode with continuous type, gap size: 0.1 mm) with a parallel-plate geometry at room temperature. Shear viscosity of SPG solution at a specific shear rate (416 s-1) was measured at different ultrasonication time. To observe gelation behavior, in situ photorheometry was conducted using a MARSIII rheometer (ThermoScientific, oscillation time-sweep mode with 5% strain, 1 Hz frequency, gap size: 80 μm) with a UV cure cell at room temperature. The prepolymer solution (50 μL) was placed on a quartz plate in the UV cure cell and irradiated with 9
UV light (Omnicure S1000, 5 mW/cm2, 365 nm) via a liquid light guide. UV light was shined 60 s after the onset of rheometrical measurements. Gel point (i.e., crossover time) was defined as the time when the elastic modulus (G′) surpassed the loss modulus (G″). For shear modulus measurement, 1 mm-thick SPG and PEG/SPG hydrogel slabs were fabricated and swollen in pH 7.4 PBS at 37 °C for 24 h. Then, circular gel discs (8 mm in diameter) were punched out from the gel slabs using a biopsy punch. Gel moduli were measured by performing oscillatory rheometry in strain-sweep mode (0.1-5%) with 0.8 mm gap-size using a parallel plate geometry (diameter = 8 mm). The average G′ value of each sample was obtained from the linear viscoelastic region (LVR) (Fig. S1) (Ki, Shih, & Lin, 2013).
2.7. Gel fraction and swelling ratio measurements To measure gel fraction and mass swelling ratio, 1 mm-thick circular hydrogels (50 μL) were formed and immersed in pH 7.4 PBS at 37 °C for 24 h until reaching equilibrium swelling. The swollen weight (WSwollen) of hydrogel was measured. Next, hydrogels were immersed in deionized water for 24 h to remove uncross-linked components (i.e., sol fraction) as well as salts. The hydrogels were then vacuum dried and weighed (WDry). The gel fraction and the mass swelling ratio were obtained by the following equations. Gel fraction = WDry / Theoretical weight of solid components Swelling ratio = WSwollen / WDry
(1) (2)
2.8. Degradability test 10
SPG hydrogels were formed with variable contents (i.e., 3, 5, 8 wt%) of SPG in prepolymer solutions. The DTT concentrations were 5, 8, and 13 mM for 3, 5, and 8 wt% SPG, respectively. For PEG/SPG hydrogels, the concentration of PEG was fixed at 4 wt% and SPGSH concentration was varied (i.e., 0, 1, 3 wt%). The formed hydrogels were incubated in pH 7.4 PBS at 37 °C for 30 days. PBS was replaced every 2 days. The shear elastic modulus (G′) change of the incubated hydrogel was tracked. The degradation rate constant (k′) was calculated from exponential curve fitting to G′ plots by the following equation (Metters, Bowman, & Anseth, 2000). ln G′/G′0 = - k′ t
(3)
where G′0 is the shear elastic modulus on day 1 and t is the incubation time.
2.9. Bovine serum albumin release assay Bovine serum albumin (BSA, Sigma Aldrich) was added in the precursor solutions of different concentrations (i.e., 3% and 5% SPG-NB) at 0.5 wt%. Then, the precursor solutions containing BSA were shined by UV light. Then, BSA-contained hydrogels were subsequently immersed in 2 mL of pH 7.4 PBS, followed by incubation at 37 °C for 78 h. The amount of released BSA from SPG hydrogel was quantified by Bradford assay (BioRad) using a microplate reader (Synergy HT, BioTek Instruments).
2.10. Cell encapsulation 11
Adenocarcinoma human alveolar basal epithelial cells (A549) were kept in high glucose Dulbecco's modified Eagle's medium (DMEM) (Corning) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco) and 1% (v/v) antibiotic–antimycotic solution (Gibco) at 5% CO2 and 37 °C. Before cell encapsulation, precursor solution was filtered using a 0.2 μm pore syringe filter for sterilization. A549 cells were trypsinized with 0.25% trypsin–EDTA solution and suspended in the precursor solution at 2 × 106 cell/mL. The cell suspended precursor solution of 20 μL was transferred into a 1 mL syringe mold with a cut-open tip and exposed to UV light (5 mW/cm2, 365 nm) for 2 min. Cell-laden hydrogels were incubated in culture media at 5% CO2 and 37 °C. Metabolic activity was measured by CellTiter-Blue assay (Promega). A cell-laden hydrogel was incubated in 500 μL of 1× reagent medium for 4 h. Then, 200 μL medium was transferred into a black 96-well plate and fluorescence was measured using the microplate reader (Ex/Em: 540/590 nm). Cell survival was evaluated by Live/Dead fluorescence staining. Cellladen hydrogels were washed with PBS 1-h post-encapsulation and stained with 1 μM calcein AM (AnaSpec Inc.) and 4 μM ethidium homodimer-1 (AnaSpec Inc.) for 1 h. The stained cell images were acquired by a fluorescence microscope (BX51, Olympus) and encapsulated cell morphology was observed using a phase-contrast microscope (CKX41, Olympus).
3. Results and discussion 3.1. Characterization of ultrasonicated SPG and SPG derivatives The intact SPG solution showed extremely high viscosity even at a low concentration due to its high molecular weight (891 kDa, Table S1), resulting in difficulty in subsequent processes. In order to decrease the solution viscosity, depolymerization process was therefore 12
conducted by ultrasonication prior to SPG derivative synthesis. The shear viscosity of SPG solution dramatically decreased with treatment time until 40 min. However, the viscosity did not change over 40 min (Fig. S2). After ultrasonication, the depolymerized SPG was characterized by GFC and NMR spectroscopy to confirm its structural change. As a result, it turned out that the molecular weight of SPG decreased to 338 kDa (Table S1) without distinct structural change (an identical 13C NMR spectrum of intact and ultrasonicated SPG (Fig. 3)) (Münzberg, Rau, & Wagner, 1995). Hence, the ultrasonic treatment time was fixed at 40 min and the resulted SPG was utilized in all the later synthesis processes. To synthesize both SPG-NB and SPG-SH, we first conducted carboxymethylation on the ultrasonicated SPG, resulting in SPG-COOH as an intermediate product (Fig. 1). Then, NBamine and cysteamine were immobilized to SPG-COOH via EDC chemistry for SPG-NB and SPG-SH, respectively. Fig. 3 shows 13C NMR spectra of SPG and SPG derivatives. In SPGCOOH spectrum, a newly formed peak at 178 ppm is attributed to carbonyl group (Mohan et al., 2015), indicating that carboxylic group was successfully immobilized to SPG. Its DS value determined by titration method was about 0.28. For SPG-NB, characteristic peaks of norbornene group appeared at 132 and 137.9 ppm with a peak at 173 ppm attributed to carbonyl group of newly formed amide bond. In addition, multiple peaks at 48.9, 43.7, 41.4, 40.4, and 29.2 ppm also indicate norbornene group (Dinda, Venu, Sarma, & Shunmugam, 2014). These peaks were however overlapped with other peaks corresponding to trace of residual urea form of EDC (Mohan et al., 2015; Nakajima & Ikada, 1995). For SPG-SH, we also found a newly formed peak at 173 ppm, which indicates that cysteamine was covalently immobilized to SPG-COOH via amide bond formation. To quantify the immobilized thiol groups of SPG-SH, Ellman’s assay was conducted. As a result, thiol groups per a unit mass of SPG was about 0.05 mmol/g, indicating 13
that 3.24 thiol groups exsited every a hundred of repeating units (three glucoses in backbone and a branched glucose) (Fig. S3). FT-IR results also confirmed such structural changes of SPG derivatives by comparing their carbonyl group peaks at around 1605-1650 cm-1 (Fig. S4) (Synytsya & Novak, 2014; Lee, Park, & Ki, 2016). Next, we examined the average molecular weights of SPG and its derivatives. The molecular weights varied with types of SPG while their molecular weight distributions did not alter significantly (Table S1 and Fig. S5). Mn of SPG-COOH was about 183 kDa, which was much lower than that of ultrasonicated SPG. Such a severe molecular weight reduction might be due to alkali treatment in carboxymethylation process. Moreover, its actual molecular weight should be lower than the measured value when SPG-COOH hydrodynamic volume is taken account since it could increase by ionic repulsion of carboxylic groups in mobile phase. After the immobilization of either norbornene or thiol group, the amount of free carboxylic groups should decrease. Consequently, both SPG-NB and SPG-SH showed much lower molecular weights (i.e., SPG-NB, 23 kDa; SPG-SH, 95 kDa) compared with SPG-COOH. Especially, the lowest Mn value of SPG-NB might be explained by chain aggregation due to hydrophobic interaction of immobilized norbornene groups.
3.2. Formation and property of SPG hydrogels To analyze the gelation kinetics of SPG hydrogels, shear elastic and loss moduli (G′ and G′′) were monitored by in situ photo-rheometry as shown in Fig. 2B (3 wt% SPG-NB with 5 mM DTT). After shining UV light, G′ of the precursor solution rapidly increased and reached the plateau (~1200 Pa). The gel point was about 3 s and it did not significantly change even at 14
different concentrations of SPG-NB (Fig. S6A), indicating the gelation occurred very quickly and was completed within 1 min. Next, we measured SPG hydrogel modulus with varying amount of dithiol cross-linker (DTT) at fixed SPG-NB concentrations (i.e., 3, 5, and 8 wt%). Fig. 4 shows shear elastic moduli of SPG hydrogels formed with different SPG-NB and DTT concentrations. G′ increased with an increase of SPG-NB concentration ranging from 0.5 to 10 kPa. A higher shear modulus at a higher polymer concentration can be explained by an increase of cross-linking density. Interestingly, G′ of SPG hydrogel formed with 3 wt% SPG-NB increased with an increase of DTT concentration up to 5 mM and decreased after marking the highest G′ at 5 mM DTT. For other SPG-NB concentrations, similar trends were observed. For 5 and 8 wt% SPG-NB, the peak G′ values were recorded at 8 and 13 mM of DTT, respectively. Such a phenomenon could be caused by the hydrogel network formation mechanism. Since thiolene click-reaction utilized in the hydrogel fabrication was orthogonal reaction, the homogeneity of the network could be maximized when the stoichiometric ratio of thiol and ene groups was balanced (Mũnoz et al., 2014). Therefore, the hydrogels exhibited the highest shear elastic moduli at their own optimum DTT contents. Accordingly, we found that an optimum ratio of DTT (mM) to SPG-NB (wt%) was about 1.6, regardless of SPG-NB concentrations and assumed that the numbers of thiol and norbornene groups were balanced at that ratio. Additionally, based on G′ measurement result, we could determine the amount of immobilized norbornene groups of SPG-NB as 0.32 mmol/g, which indicated that about 2.1 norbornene groups attached per 10 repeating units of SPG. Fig. 5 presents gel fractions and swelling ratios of SPG hydrogels. Hydrogels formed with 5 and 8 wt% SPG-NB solutions showed similar gel fractions at varying DTT content while the gel fractions of 3 wt% SPG-NB hydrogels were relatively lower (< 0.8), indicating the cross15
linking did not completely occur at 3 wt% of SPG-NB. However, for each SPG-NB concentration, the highest gel fractions were obtained at the specific ratio of DTT (mM)/SPG-NB (wt%) (~1.6) and the gel fraction decreased over the ratio as shown in Fig. 5A. Swelling ratio measurement also presented consistent results. Although the swelling ratio tended to decrease with an increase of SPG-NB concentration, it showed the lowest value at the specific DTT content for each SPG-NB concentration (Fig. 5B). Such a result obviously indicates that the exact stoichiometric balance of SPG-NB and dithiol cross-linker is necessary to achieve not only a higher cross-linking density but also efficient network formation of SPG hydrogel.
3.3. Formation and property of PEG/SPG hybrid hydrogels The property of natural polymer-based material can be manipulated by blending with synthetic polymer as compensating its limited performance. (Sionkowska, 2011). Herein, PEG was blended with SPG-SH for hydrogel formation due to its well-known characteristics such as non-toxicity, non-immunogenicity, and bio-inert property (Peppas et al., 2006). The PEG/SPG hybrid hydrogels were fabricated with 4 wt% PEG4NB and varying concentration of SPG-SH (0-3 wt%) in the presence of 1 mM LAP (Fig. 2C). The gelation occurred rapidly within 4 s after shining UV light (Fig. 2D). The gel point was not significantly affected by not only SPG concentration (Fig. S6B) but the presence of co-cross-linker DTT (Fig. S6C). Fig. 6 presents properties of PEG/SPG hybrid hydrogels (i.e., gel fraction, mass swelling ratio, and shear elastic modulus). As shown in Fig. 6A, the hybrid hydrogels showed high gel fraction values (> 0.95) regardless of SPG-SH content, which indicates that almost all PEG4NB and SPG-SH molecules in the precursor solution participated in network formation. Therefore, multi-thiol cross-linker 16
SPG-SH did not interfere with thiol-ene reaction in spite of its relatively huge size compared with DTT. In swelling ratio measurement, the pure PEG hydrogel marked 32 and the swelling ratio of PEG/SPG hybrid hydrogel gradually decreased with an increase of SPG-SH content (Fig. 6B). On the other hand, G′ slightly increased as an increase of content of SPG-SH in the range of 2900-3700 Pa (Fig. 6C). In general, the longer distance between cross-linking points in the network results in the larger mesh size at a fixed number of cross-links (Lee et al., 2016; Rubinstein & Colby, 2003). Therefore, one could expect the use of longer cross-linker causes the hydrogel formation with a lower network density at equilibrium swelling state. Nevertheless, PEG/SPG hybrid hydrogel has shown converse relationship between SPG-SH content and gel property as considering that thiol groups of SPG-SH are relatively far apart each other compared with those of DTT co-cross-linker, which results in a larger mesh size. Such a phenomenon implies other factors might affect the increase of network density of PEG/SPG hybrid hydrogel.
We hypothesized that the triple-helix formation of SPG played an important role in the decrease of swelling ratio as well as the improvement of mechanical property of PEG/SPG hybrid hydrogel. To evidence the presence of triple-helix in the hybrid hydrogel, Congo red assay which is used for determination of conformational change of β-glucan chains in aqueous solution was conducted. The bathochromic shift can be seen in visible light absorption spectrum of Congo red solution when single β-glucan chains form triple-helices (Semedo et al., 2015). We observed a significant bathochromic shift for both neutral SPG-SH solution and PEG/SPG hybrid hydrogel whereas such an evident shift did not appear for PEG hydrogel and alkali treated SPGSH solution (Fig. S7). Such a result indicates the presence of SPG triple-helix in SPG-SH 17
solution as well as PEG/SPG hybrid hydrogel and the triple-helical formation might contribute to an increase of network density as physical cross-linking.
3.4. Degradation of SPG and PEG/SPG hybrid hydrogels The hydrolytic degradations of SPG and PEG/SPG hybrid hydrogels were evaluated by measuring their shear storage modulus, as shown in Fig. 7. For all SPG hydrogels, the significant change both in shear modulus and in bulk volume was not observed at all for 30 days in PBS (Fig. 7A). On the other hand, the shear moduli of PEG/SPG hybrid hydrogels gradually decreased as time progress showing a typical bulk degradation profile (Fig. 7C). To quantitatively compare degradation behaviors, logarithmic plotting of G′/G′0 was implemented (Fig. 7B and 7D) and degradation rate constants (k′) was determined by linear fitting (Table S2). As expected, k′ of all the SPG hydrogels were close to zero indicating that the hydrolytic degradation did not occur. Because norbornene groups were linked to SPG by amide bonds which are stable in physiological condition, the hydrolysis of hydrogel network hardly occurred. For PEG/SPG hybrid hydrogels, larger k′ values were obtained with varying SPG content indicating that hydrogel network degraded. It was due to the hybridization of PEG4NB which contains hydrolytically cleavable ester bonds. Especially, for PEG/SPG hybrid hydrogels, we found that k′ decreased as SPG-SH content increased. It means that the hydrogel with a higher content of SPG more slowly degraded compared with either the pure PEG hydrogel or less SPG contained hydrogel. Such a phenomenon is possibly explained by two reasons. In PEG/SPG hydrogel, SPG-SH should play a role as a multi-thiol cross-linker, forming lots of cross-linking point with PEG4NB while DTT had only two thiol groups. Therefore, the hybrid hydrogel was 18
able to well maintain the network structure even though some of cross-links were cleaved compared with the pure PEG hydrogel (Lee et al., 2016). In addition, physical interaction of SPG chains might contribute to a slower degradation of PEG/SPG hybrid hydrogel by forming triple helix conformation.
3.5. BSA release and three-dimensional culture of A549 cells For application study of thiol-ene SPG hydrogel, we conducted BSA release assay and three-dimensional (3D) cell culture with SPG and PEG/SPG hybrid hydrogels, respectively. As shown in Fig. 8A, BSA was very rapidly released out within 6 h regardless of SPG content. After 24 h, the amount of released BSA from 3 wt% was about 70% compared with the initially loaded BSA amount and did not further increase. For 5 wt% SPG hydrogel, only 20% BSA was released out during the first 6 h and the cumulative BSA release very slowly increased for 78-h observation period, which indicates that BSA release could be controlled by SPG-NB content in hydrogels. For 3D cell culture, A549 cells were encapsulated in pure PEG and PEG/SPG hybrid hydrogels (4 wt% PEG4NB and 1 wt% SPG-SH). Live/Dead assay result has shown that the presence of SPG-SH in hydrogel did not influence the cell survival rate (> 90%) during the encapsulation process (Fig. 8B and Table S3). It implies thiol-ene cross-linking reaction between SPG-SH and PEG4NB was not cytotoxic for tumor cell encapsulation. During 7-day culture, single A549 cells (day 1, Fig. 8C) formed spherical clusters (day 7, Fig. 8D) and their metabolic activities increased (Fig. 8E), indicating cell proliferation in hydrogel matrix. However, there was no significant difference between pure PEG and PEG/SPG hybrid hydrogel groups. 19
Although the distinct effect of SPG on A549 cells was not observed, such results suggest that PEG/SPG hybrid hydrogel would be a candidate as a 3D cell culture matrix. 4. Conclusion To form SPG hydrogel via thiol-ene photopolymerization, we synthesized various SPGderivatives (i.e., SPG-COOH, SPG-NB, and SPG-SH) from ultrasonicated SPG. The depolymerized SPG solution showed a proper viscosity for subsequent synthesis processes. With SPG-NB and SPG-SH, we fabricated SPG and PEG/SPG hybrid hydrogels by orthogonal thiolene click-reaction, respectively. Physical properties (i.e., swelling ratio, shear modulus, degradability) of such SPG-based hydrogels could be manipulated by modular formulation of polymers and thiol cross-linkers. Especially, the triple-helix structure of SPG in PEG/SPG hybrid hydrogel might induce an increase of shear modulus and slower degradation in PBS as physical cross-linking. We could also verify potential applications (i.e., drug carrier, 3D cell culture matrix) of SPG-based hydrogels as biomaterials via BSA release assay and A549 cell 3D culture.
Acknowledgement This study was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2015R1A2A2A03002680) and “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ012268012017)” Rural Development Administration, Republic of Korea.
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Rubinstein, M., & Colby, R. H. (2003). Polymer Physics. (1st ed.). Oxford: Oxford University Press. Ryu, S., Kim, H. H., Park, Y. H., Lin, C. C., Um, I. C., & Ki, C. S. (2016). Dual mode gelation behavior of silk fibroin microgel embedded poly(ethylene glycol) hydrogel. Journal of Materials Chemistry B, 4, 4574-4584. Semedo, M. C., Karmali, A., & Fonseca, L. (2015). A high throughput colorimetric assay of β-1, 3-D-glucans by Congo red dye. Journal of Microbiological Methods, 109, 140-148. Sionkowska, A. (2011). Current research on the blends of natural and synthetic polymers as new biomaterials: Review. Progress in Polymer Science, 36(9), 1254-1276. Synytsya, A., & Novak, M. (2014). Structural analysis of glucans. Annals of Translational Medicine, 2(2), 17-17. Van Vlierberghe, S., Dubruel, P., & Schacht, E. (2011). Biopolymer-based hydrogels as scaffolds for tissue engineering applications: A review. Biomacromolecules, 12(5), 1387-1408. Xiao, Y., Xu, W., Zhu, Q., Yan, B., Yang, D., Yang, J., et al. (2009). Preparation and characterization of a novel pachyman-based pharmaceutical aid. II: A pH-sensitive, biodegradable and biocompatible hydrogel for controlled release of protein drugs. Carbohydrate Polymers, 77(3), 612-620. Zhang, H., Nishinari, K., Williams, M. A. K., Foster, T. J., & Norton, I. T. (2002). A molecular description of the gelation mechanism of curdlan. International Journal of Biological Macromolecules, 30(1), 7-16. Zhang, Y., Kong, H., Fang, Y., Nishinari, K., & Phillips, G. O. (2013). Schizophyllan: a review on its structure, properties, bioactivities and recent developments. Bioactive Carbohydrates and Dietary Fibre, 1(1), 53-71. 25
Figure captions
Fig. 1. Schematic of schizophyllan (SPG) derivative synthesis. Both schizophyllan-norbornene (SPG-NB) and schizophyllan-thiol (SPG-SH) were synthesized via EDC chemistry with carboxymethyl schizophyllan (SPG-COOH). Fig. 2. SPG and PEG/SPG hybrid hydrogel formations by orthogonal thiol-ene photo-click reaction. (A) Schematic of SPG hydrogel formation with SPG-NB and DTT dithiol cross-linker. (B) In situ photo-rheometry result of SPG hydrogel formed with 3 wt% SPG-NB and 5 mM DTT. (C) Schematic of PEG/SPG hybrid hydrogel formation with PEG4NB, SPG-SH, and DTT. (D) In situ photo-rheometry result of SPG hybrid hydrogel formed with 4 wt% PEG4NB, 3 wt% SPGSH, and DTT as a co-cross-linker. Fig. 3. 13C NMR spectra of intact SPG and derivatives. Intact and ultrasonicated SPG were measured after dissolving in deuterated dimethyl sulfoxide; SPG-NB and SPG-SH were measured after dissolving in deuterated water. Assignment of SPG peaks: δ = 40 (DMSO); 61 (C6, A/C/D, free); 68 (C4); 70 (C6, B, bound); 72 (C2, A/B/C); 73 (C2, D); 74 (C3, D, free); 76 (C5); 86 (C3, A/B/C, bound); 102 (C1) (Münzberg et al., 1995). Fig. 4. Shear elastic moduli of SPG hydrogels formed with varying amount of DTT in prepolymer solution at different SPG-NB concentrations (3, 5, and 8 wt%) (n = 3, mean ± SD). Fig. 5. (A) Gel fractions and (B) equilibrium swelling ratios of SPG hydrogels formed with varying content of DTT in prepolymer solution at different SPG-NB concentrations (3, 5, and 8 wt%) (n = 3, mean ± SD).
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Fig. 6. (A) Gel fractions, (B) equilibrium swelling ratios, and (C) shear elastic moduli of PEG/SPG hybrid hydrogels formed with varying SPG-SH content (0-3 wt%) at a fixed concentration (4 wt%) of PEG4NB in prepolymer solution (n = 3, mean ± SD). Fig. 7. Hydrolytic degradation of SPG and PEG/SPG hybrid hydrogels in pH 7.4 PBS for 30 days. (A) Shear elastic modulus (G′) change and (B) logarithmic plots of G′/G0′ of SPG hydrogels formed with varying SPG-NB concentrations. (C) Shear elastic modulus (G′) change and (D) logarithmic plots of G′/G0′ of PEG/SPG hybrid hydrogels formed with varying SPG-SH concentrations (n = 3, mean ± SD). Fig. 8. (A) Cumulative BSA release profiles from SPG hydrogels (3 wt% and 5 wt% SPG-NB) in pH 7.4 PBS at 37 °C (n = 3, mean ± SD). (B) Live/Dead images of encapsulated A549 cells in pure PEG hydrogel (-SPG, 4 wt% PEG4NB) and PEG/SPG hybrid hydrogel (+SPG, 4 wt% PEG4NB/1 wt% SPG-SH) 1-h post-encapsulation. (C, D) Phase-contrast images of encapsulated A549 cells in PEG/SPG hybrid hydrogel on day 1 and 7, respectively. (E) Metabolic activities of encapsulated A549 cells in PEG and PEG/SPG hybrid hydrogels for 7 days (n = 3, mean ± SD).
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S0144-8617(17)30298-9 http://dx.doi.org/doi:10.1016/j.carbpol.2017.03.042 CARP 12131
To appear in: Received date: Revised date: Accepted date:
13-1-2017 28-2-2017 12-3-2017
Please cite this article as: Lee, Sora., Tae, Hyunhyuk., & Ki, Chang Seok., Fabrication of schizophyllan hydrogel via orthogonal thiol-ene photopolymerization.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2017.03.042 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Fabrication of schizophyllan hydrogel via orthogonal thiol-ene photopolymerization
Sora Lee, Hyunhyuk Tae, Chang Seok Ki*
Department of Biosystems and Biomaterials Science and Engineering, Seoul National University, Seoul 08826, Republic of Korea
Sora Lee and Hyunhyuk Tae equally contributed to this study as co-first authors.
*To whom correspondence should be sent: Chang Seok Ki, Ph D Assistant Professor Department of Biosystems and Biomaterials Science and Engineering, Seoul National University, Seoul 08826, Republic of Korea Email: [email protected]
Highlights
SPG and PEG/SPG hydrogel fabrication via orthogonal thiol-ene reaction.
The properties of SPG-based hydrogel could be modulated by controlling components.
Mechanical enhancement of hydrogel has been achieved by dual mode crosslinking.
1
Abstract Schizophyllan (SPG) is a water soluble β-glucan, which is obtained from Schizophyllum commune. SPG has been widely explored with their unique properties such as triple helical structure, immune-modulation, and anti-tumoral activity. In this study, we tried to fabricate SPG hydrogel via thiol-ene photopolymerization. SPG-norbornene and SPG-thiol were synthesized from ultrasonicated SPG. Two types of SPG hydrogels (SPG and PEG/SPG hybrid) were formed via thiol-ene photo-click reaction. By controlling the SPG content and stoichiometric balance of norbornene and thiol groups, swelling ratio and shear elastic modulus of SPG hydrogel could be controlled in the range of 20-60 and 0.5-10 kPa, respectively. For PEG/SPG hybrid hydrogel, we found that the triple-helical structure of SPG played a role in physical network formation with thiol-ene cross-linking. The degradability of SPG hydrogel could be also controlled by varying formulation. Therefore, such highly tunable SPG hydrogels would be utilized for various applications.
Key words: schizophyllan, hydrogel, thiol-ene reaction, photopolymerization
1. Introduction Hydrogel is composed of three-dimensional polymer network retaining a large amount of water, resulting in unique physical and mechanical properties. For centuries, hydrogels have 2
been utilized in a wide variety of industries such as food, pharmaceutics, and cosmetics. Recently, soft tissue-like property of hydrogels elicits great potential in biomedical applications (e.g., tissue culture matrix, wound-care material) (Hoffman, 2012; Peppas, Hilt, Khademhosseini, & Langer, 2006). In general, hydrogel can be formed with various hydrophilic polymers by crosslinking (Hennink & van Nostrum, 2012). Polymer type for hydrogel fabrication is one of key factors which determine the hydrogel property and roughly categorized into synthetic and natural polymers (Drury & Mooney, 2003). Among abundant natural polymers, polysaccharides are especially advantageous in hydrogel fabrication for their abundance, low toxicity, structural diversity, and chemical functionality. In addition, they allow various chemical modifications, resulting in versatile manipulation of hydrogel property (Van Vlierberghe, Dubruel, & Schacht, 2011). Hence, for decades, hydrogels formed with various polysaccharides (e.g., hyaluronic acid (Gramlich, Kim, & Burdick, 2013), carrageenan (Popa, Gomes, & Reis, 2011), xanthan (Bueno, Bentini, Catalani, & Petri, 2013), chitosan (Mirahmadi, Tafazzoli-Shadpour, Shokrgozar, & Bonakdar, 2013), dextran (Corrente, Amara, Pacelli, Paolicelli, & Casadei, 2013)) have been extensively explored in biomedical field. β-glucan commonly refers to a family of glucopyranose polysaccharides containing 1,3-β-glycosidic linkages with 1,6-branch structure of varying degrees. β-glucans can be obtained from a wide variety of sources such as fungi, bacteria, algae, lichen, and some plants (Lehtovaara & Gu, 2011). Compared with other polysaccharides, the most distinctive feature of β-glucan is the triple-helix of which three β-glucan chains are self-assembled by hydrogen bonds. The triple-helical structure of β-glucan can be dissociated by changing solvent types or pH condition, resulting in a single-helix (Miyoshi, Uezu, Sakurai, & Shinkai, 2004). Such a structural uniqueness of β-glucan is very important in processing as well as its utilizations. 3
Meanwhile, tremendous studies have dedicated to evaluation of bioactivity of β-glucan. For centuries, herbal medicines which contain β-glucan have been clinically used and recent efforts have unveiled the multi-bioactivity of β-glucan (e.g., immune-stimulation, anti-inflammatory, anti-microbial, and anti-tumoral activities) (Chen & Seviour, 2007). One of key mechanisms that β-glucan exhibits bioactivity is triggered by phagocytic action of macrophage (Chan, Chan, & Sze, 2009). Such an activation mechanism is a product of long-term evolution of immune system against bacterial or fungal pathogens. For decades, β-glucan has been widely utilized as food additive, nutrition factor, and direct therapeutic material for enhancing immune response (Divyasri, Gunasekar, Benny, & Ponnusami, 2014). For β-glucan-based hydrogel fabrication, a considerable number of both physical and chemical cross-linking techniques have been proposed. Physically cross-linked β-glucan hydrogels can be easily fabricated by controlling temperature or pH of β-glucan solution. For example, linear β-glucan solution forms a reversible ‘low-set’ gel between 55-80 °C and it becomes sol by cooling. At a higher temperature range of 85-145 °C, an irreversible ‘high-set’ gel can be also formed (Maeda, Saito, Masada, Misaki, & Harada, 1967; Zhang, Nishinari, Williams, Foster, & Norton, 2002). Alternatively, β-glucan hydrogel can be prepared by neutralizing the alkali β-glucan solution (Kanzawa, Harada, Koreeda, & Harada, 1987). Such a hydrogel is easily changed to sol-state by increasing pH, as disrupting hydrogen bond. However, the conventional physical cross-linking methods are not physiologically compatible. Such βglucan hydrogels are therefore not suitable in biological uses (e.g., delivery vehicles for biomolecules or cells) (Kim et al., 2000). In recent years, carboxymethylated β-glucan has been cross-linked via ionic cross-linking and semi-interpenetrating hydrogel of β-glucan and other synthetic polymers has been reported (Aalaie, Rahmatpour, & Vasheghani‐ Farahani, 2009; 4
Corrente et al., 2009). However, such cross-linking techniques were somehow sensitive to external conditions (e.g., pH, ionic strength, temperature). Meanwhile, chemical cross-linking methods have been explored in β-glucan hydrogel fabrication. It can form more stable network compared with physically constructed β-glucan network. Xiao et al. (2009) and Itagati et al. (2016) formed β-glucan hydrogels with epichlorohydrin and hexamethylenediisocyanate, respectively. Without chemical cross-linkers, β-glucan hydrogel could be also fabricated by irradiation of gamma-ray. Gamma-ray of high energy might produce chemically functional groups on β-glucan, resulting in cross-links (Lim et al., 2015). Alternatively, β-glucan was oxidized and cross-linked with gelatin via imine linkage formation. This gelation could be quickly completed at a narrow pH range (Fang, Takahashi, & Nishinari, 2005). Recently, Qi et al. (2017) developed a salecan-based hydrogel by grafting 2-acrylamido-2-methyl-1propanesulfonic acid (AMPS) onto salecan chains using N,N’-methylenebis(acrylamide) as a cross-linker. Although such chemical cross-linking techniques are relatively handy to control cross-linking density and gelation kinetics, potential toxicity of cross-linking agent or crosslinking condition should be considered as drawback (Nussinovitch, 2010). In this study, we report β-glucan hydrogel fabrication with a kind of branched βglucans, schizophyllan (SPG) via thiol-norbornene photopolymerization. SPG is one of water soluble β-glucans obtained from Schizophyllum commune and contains a 1,3-β-linked backbone with single 1,6-β-linked glucose side chains at every third residue (Zhang, Kong, Fang, Nishinari, & Phillips, 2013). The thiol-ene photopolymerization is a radical mediated step-growth reaction via orthogonal cross-linking between thiol and norbornene groups, resulting in rapid gelation as well as ideal network formation. Besides, the reaction causes low cytotoxicity in cell encapsulation and less damage to bioactive molecules during gelation process due to less radical 5
generation compared with other radical polymerizations (Lin, Ki, & Shih, 2015; Mũnoz, Shih, & Lin, 2014). For the thiol-ene photopolymerization, SPG-derivatives (i.e., SPG-norbornene, SPGthiol) were synthesized. Next, SPG and PEG/SPG hybrid hydrogels were fabricated and their physical properties, degradability, and applicability as a protein drug carrier as well as a cell culture matrix were evaluated.
2. Materials and methods 2.1. Materials Schizophyllum commune lysate was purchased from CoSeedBioPharm (Korea). The crude SPG was obtained by precipitating with isopropanol after filtering the lysate, followed by vacuum drying. The SPG flake was re-dissolved in 0.1 M sodium hydroxide solution and vigorously stirred at 80 °C for 2 h. The alkali treated SPG was filtered and neutralized with diluted hydrochloric acid, followed by precipitation and thorough washing with isopropanol. Finally, the precipitate was washed again with chloroform to remove residual lipids and proteins. To reduce molecular weight of SPG, the refined SPG was re-dissolved in de-ionized water at 10 mg/mL. 30 mL SPG solution was transferred in to a 50 mL conical tube and ultrasonicated by an ultra-sonic processor at 130 W (VCX-130, SONICS). The ultrasonication time was controlled by measuring the shear viscosity change of SPG solution using a rheometer (Hakke MARSIII, ThermoScientific). 4-arm poly(ethylene oxide) (PEG4OH, 20 kDa), isopropanol, and 5norbornene-2-methylamine were obtained from JenKem Technology, Samchun Chemical, and Tokyo Chemical Industry, respectively. All the other chemicals were purchased from SigmaAldrich and used without further purification. 6
2.2. Synthesis For synthesis of carboxymethyl-schizophyllan (SPG-COOH), ultrasonicated SPG was dissolved in de-ionized water at 20 mg/mL and sodium hydroxide (24 eq. of pyranose of SPG) was added to activate hydroxyl groups of SPG, followed by addition of isopropanol of equal volume. Then, chloroacetic acid (14 eq. of pyranose of SPG) was drop-wisely added and the mixture was subsequently stirred at 60 °C for 3 h. To quench the reaction, de-ionized water of 2fold volume was added and dialyzed using a cellulose acetate tube (MWCO = 12-14 kDa) for 3 days, followed by lyophilization. The degree of substitution (DS) was determined by ASTM D1439-94 standard method (ASTM, 1994). Schizophyllan-norbornene (SPG-NB) was synthesized via EDC chemistry with 5-norbornene-2-methylamine (NB-amine). SPG-COOH was dissolved in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (0.1 M, pH 6) at 16 mg/mL and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) (1 eq. of pyranose of SPG) was added. Then, 5-norbornene-2-methylamine (2 eq.) was added and pH of the mixture solution was adjusted around 6 with hydrochloric acid. The solution was stirred for 18 h, followed by dialysis against de-ionized water at pH 6 for 3 days and lyophilization. Schizophyllan-thiol (SPG-SH) was synthesized according to the previous SPG-NB synthesis protocol with slight modification (Fig. 1). After dialysis, mercaptoethanol was added for reducing di-sulfide bond and pH of the solution was adjusted to 8 with sodium hydroxide, followed by stirring for 30 min. Then, the solution was re-dialyzed against de-ionized water at pH 5 for 3 days and subsequently lyophilized. The amount of SPG-SH thiol groups was determined by Ellman’s assay. PEG-ester linked-tetra-norbornene (PEG4NB) was synthesized by reacting 4-arm PEG-OH with 57
norbornene-2-carboxylic acid (Fairbanks et al., 2009; Ryu et al., 2016) and lithium arylphosphinate (LAP) were synthesized according to published protocols (Fairbanks, Schwartz, Bowman, & Anseth, 2009). 2.3. SPG hydrogel formation Fig. 2 presents schematics for SPG hydrogel formations via thiol-ene photo-click reaction. For SPG hydrogels, SPG-NB was dissolved in phosphate-buffered saline (PBS) at a desired concentration (i.e., 3, 5, 8 wt%) with 1 mM LAP and dithiothreitol (DTT). The concentration of DTT was varied in the range of 2-20 mM according to the concentration of SPG-NB in prepolymer solutions. For PEG/SPG hydrogel, the precursor solutions were prepared with SPG-SH of varying concentration (0-3 wt%), 4 wt% PEG4NB, and DTT in the presence of 1 mM LAP. DTT was used as a co-crosslinker to balance the stoichiometric ratio between norbornene and thiol moieties ([NB] = [SH]) (Fig. 2C). The precursor solution was injected into the gap between glass slides separated by 1 mm-thick spacers and subsequently exposed to ultraviolet (UV) light (5 mW/cm2, 365 nm) for 2 min to initiate thiol-ene photopolymerization. Then, formed hydrogel slabs were collected and immediately immersed in pH 7.4 PBS. 2.4. Nuclear magnetic resonance (NMR) and Fourier transform infrared (FT-IR) Spectroscopies For NMR analysis, intact and ultrasonicated SPGs were dissolved in deuterated dimethyl sulfoxide at 40 mg/mL for analysis. For SPG derivatives, each sample was dissolved in D2O at 40 mg/mL. 13C NMR spectra were obtained by using a 600 MHz NMR spectrometer (AVANCE 600, Bruker). To obtain FT-IR spectra, lyophilized intact SPG and SPG derivative samples were powdered and analyzed by an attenuated total reflectance Fourier transforminfrared (ATR FT-IR) spectrometer (Nicolet 6700, ThermoScientific). FT-IR spectra were 8
obtained with 32 scans at 4 cm-1 resolution.
2.5. Gel filtration chromatography (GFC) Molecular weights of SPG samples were measured by a high performance liquid chromatography (HPLC) (Ultimate 3000, Dionex) with sodium azide 0.1 M solution as an eluent through a serialized column of Waters Ultrahydrogel 120, 500, and 1000. Each SPG sample was dissolved in de-ionized water at 10 mg/mL and filtered with a 0.2 μm pore syringe filter. The flow rate was 1 mL/min and eluted SPG concentration was measured by a refractive index detector. The number and weight average molecular weights were determined based on a standard curve obtained with pullulan standards via software for GFC (Chromeleon 6.8 Extention-pak).
2.6. Rheometry To determine ultrasonication time, shear viscosity change of SPG solution (10 mg/mL) was measured using a MARSIII rheometer (ThermoScientific, rotational ramp CR (controlled rate) mode with continuous type, gap size: 0.1 mm) with a parallel-plate geometry at room temperature. Shear viscosity of SPG solution at a specific shear rate (416 s-1) was measured at different ultrasonication time. To observe gelation behavior, in situ photorheometry was conducted using a MARSIII rheometer (ThermoScientific, oscillation time-sweep mode with 5% strain, 1 Hz frequency, gap size: 80 μm) with a UV cure cell at room temperature. The prepolymer solution (50 μL) was placed on a quartz plate in the UV cure cell and irradiated with 9
UV light (Omnicure S1000, 5 mW/cm2, 365 nm) via a liquid light guide. UV light was shined 60 s after the onset of rheometrical measurements. Gel point (i.e., crossover time) was defined as the time when the elastic modulus (G′) surpassed the loss modulus (G″). For shear modulus measurement, 1 mm-thick SPG and PEG/SPG hydrogel slabs were fabricated and swollen in pH 7.4 PBS at 37 °C for 24 h. Then, circular gel discs (8 mm in diameter) were punched out from the gel slabs using a biopsy punch. Gel moduli were measured by performing oscillatory rheometry in strain-sweep mode (0.1-5%) with 0.8 mm gap-size using a parallel plate geometry (diameter = 8 mm). The average G′ value of each sample was obtained from the linear viscoelastic region (LVR) (Fig. S1) (Ki, Shih, & Lin, 2013).
2.7. Gel fraction and swelling ratio measurements To measure gel fraction and mass swelling ratio, 1 mm-thick circular hydrogels (50 μL) were formed and immersed in pH 7.4 PBS at 37 °C for 24 h until reaching equilibrium swelling. The swollen weight (WSwollen) of hydrogel was measured. Next, hydrogels were immersed in deionized water for 24 h to remove uncross-linked components (i.e., sol fraction) as well as salts. The hydrogels were then vacuum dried and weighed (WDry). The gel fraction and the mass swelling ratio were obtained by the following equations. Gel fraction = WDry / Theoretical weight of solid components Swelling ratio = WSwollen / WDry
(1) (2)
2.8. Degradability test 10
SPG hydrogels were formed with variable contents (i.e., 3, 5, 8 wt%) of SPG in prepolymer solutions. The DTT concentrations were 5, 8, and 13 mM for 3, 5, and 8 wt% SPG, respectively. For PEG/SPG hydrogels, the concentration of PEG was fixed at 4 wt% and SPGSH concentration was varied (i.e., 0, 1, 3 wt%). The formed hydrogels were incubated in pH 7.4 PBS at 37 °C for 30 days. PBS was replaced every 2 days. The shear elastic modulus (G′) change of the incubated hydrogel was tracked. The degradation rate constant (k′) was calculated from exponential curve fitting to G′ plots by the following equation (Metters, Bowman, & Anseth, 2000). ln G′/G′0 = - k′ t
(3)
where G′0 is the shear elastic modulus on day 1 and t is the incubation time.
2.9. Bovine serum albumin release assay Bovine serum albumin (BSA, Sigma Aldrich) was added in the precursor solutions of different concentrations (i.e., 3% and 5% SPG-NB) at 0.5 wt%. Then, the precursor solutions containing BSA were shined by UV light. Then, BSA-contained hydrogels were subsequently immersed in 2 mL of pH 7.4 PBS, followed by incubation at 37 °C for 78 h. The amount of released BSA from SPG hydrogel was quantified by Bradford assay (BioRad) using a microplate reader (Synergy HT, BioTek Instruments).
2.10. Cell encapsulation 11
Adenocarcinoma human alveolar basal epithelial cells (A549) were kept in high glucose Dulbecco's modified Eagle's medium (DMEM) (Corning) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco) and 1% (v/v) antibiotic–antimycotic solution (Gibco) at 5% CO2 and 37 °C. Before cell encapsulation, precursor solution was filtered using a 0.2 μm pore syringe filter for sterilization. A549 cells were trypsinized with 0.25% trypsin–EDTA solution and suspended in the precursor solution at 2 × 106 cell/mL. The cell suspended precursor solution of 20 μL was transferred into a 1 mL syringe mold with a cut-open tip and exposed to UV light (5 mW/cm2, 365 nm) for 2 min. Cell-laden hydrogels were incubated in culture media at 5% CO2 and 37 °C. Metabolic activity was measured by CellTiter-Blue assay (Promega). A cell-laden hydrogel was incubated in 500 μL of 1× reagent medium for 4 h. Then, 200 μL medium was transferred into a black 96-well plate and fluorescence was measured using the microplate reader (Ex/Em: 540/590 nm). Cell survival was evaluated by Live/Dead fluorescence staining. Cellladen hydrogels were washed with PBS 1-h post-encapsulation and stained with 1 μM calcein AM (AnaSpec Inc.) and 4 μM ethidium homodimer-1 (AnaSpec Inc.) for 1 h. The stained cell images were acquired by a fluorescence microscope (BX51, Olympus) and encapsulated cell morphology was observed using a phase-contrast microscope (CKX41, Olympus).
3. Results and discussion 3.1. Characterization of ultrasonicated SPG and SPG derivatives The intact SPG solution showed extremely high viscosity even at a low concentration due to its high molecular weight (891 kDa, Table S1), resulting in difficulty in subsequent processes. In order to decrease the solution viscosity, depolymerization process was therefore 12
conducted by ultrasonication prior to SPG derivative synthesis. The shear viscosity of SPG solution dramatically decreased with treatment time until 40 min. However, the viscosity did not change over 40 min (Fig. S2). After ultrasonication, the depolymerized SPG was characterized by GFC and NMR spectroscopy to confirm its structural change. As a result, it turned out that the molecular weight of SPG decreased to 338 kDa (Table S1) without distinct structural change (an identical 13C NMR spectrum of intact and ultrasonicated SPG (Fig. 3)) (Münzberg, Rau, & Wagner, 1995). Hence, the ultrasonic treatment time was fixed at 40 min and the resulted SPG was utilized in all the later synthesis processes. To synthesize both SPG-NB and SPG-SH, we first conducted carboxymethylation on the ultrasonicated SPG, resulting in SPG-COOH as an intermediate product (Fig. 1). Then, NBamine and cysteamine were immobilized to SPG-COOH via EDC chemistry for SPG-NB and SPG-SH, respectively. Fig. 3 shows 13C NMR spectra of SPG and SPG derivatives. In SPGCOOH spectrum, a newly formed peak at 178 ppm is attributed to carbonyl group (Mohan et al., 2015), indicating that carboxylic group was successfully immobilized to SPG. Its DS value determined by titration method was about 0.28. For SPG-NB, characteristic peaks of norbornene group appeared at 132 and 137.9 ppm with a peak at 173 ppm attributed to carbonyl group of newly formed amide bond. In addition, multiple peaks at 48.9, 43.7, 41.4, 40.4, and 29.2 ppm also indicate norbornene group (Dinda, Venu, Sarma, & Shunmugam, 2014). These peaks were however overlapped with other peaks corresponding to trace of residual urea form of EDC (Mohan et al., 2015; Nakajima & Ikada, 1995). For SPG-SH, we also found a newly formed peak at 173 ppm, which indicates that cysteamine was covalently immobilized to SPG-COOH via amide bond formation. To quantify the immobilized thiol groups of SPG-SH, Ellman’s assay was conducted. As a result, thiol groups per a unit mass of SPG was about 0.05 mmol/g, indicating 13
that 3.24 thiol groups exsited every a hundred of repeating units (three glucoses in backbone and a branched glucose) (Fig. S3). FT-IR results also confirmed such structural changes of SPG derivatives by comparing their carbonyl group peaks at around 1605-1650 cm-1 (Fig. S4) (Synytsya & Novak, 2014; Lee, Park, & Ki, 2016). Next, we examined the average molecular weights of SPG and its derivatives. The molecular weights varied with types of SPG while their molecular weight distributions did not alter significantly (Table S1 and Fig. S5). Mn of SPG-COOH was about 183 kDa, which was much lower than that of ultrasonicated SPG. Such a severe molecular weight reduction might be due to alkali treatment in carboxymethylation process. Moreover, its actual molecular weight should be lower than the measured value when SPG-COOH hydrodynamic volume is taken account since it could increase by ionic repulsion of carboxylic groups in mobile phase. After the immobilization of either norbornene or thiol group, the amount of free carboxylic groups should decrease. Consequently, both SPG-NB and SPG-SH showed much lower molecular weights (i.e., SPG-NB, 23 kDa; SPG-SH, 95 kDa) compared with SPG-COOH. Especially, the lowest Mn value of SPG-NB might be explained by chain aggregation due to hydrophobic interaction of immobilized norbornene groups.
3.2. Formation and property of SPG hydrogels To analyze the gelation kinetics of SPG hydrogels, shear elastic and loss moduli (G′ and G′′) were monitored by in situ photo-rheometry as shown in Fig. 2B (3 wt% SPG-NB with 5 mM DTT). After shining UV light, G′ of the precursor solution rapidly increased and reached the plateau (~1200 Pa). The gel point was about 3 s and it did not significantly change even at 14
different concentrations of SPG-NB (Fig. S6A), indicating the gelation occurred very quickly and was completed within 1 min. Next, we measured SPG hydrogel modulus with varying amount of dithiol cross-linker (DTT) at fixed SPG-NB concentrations (i.e., 3, 5, and 8 wt%). Fig. 4 shows shear elastic moduli of SPG hydrogels formed with different SPG-NB and DTT concentrations. G′ increased with an increase of SPG-NB concentration ranging from 0.5 to 10 kPa. A higher shear modulus at a higher polymer concentration can be explained by an increase of cross-linking density. Interestingly, G′ of SPG hydrogel formed with 3 wt% SPG-NB increased with an increase of DTT concentration up to 5 mM and decreased after marking the highest G′ at 5 mM DTT. For other SPG-NB concentrations, similar trends were observed. For 5 and 8 wt% SPG-NB, the peak G′ values were recorded at 8 and 13 mM of DTT, respectively. Such a phenomenon could be caused by the hydrogel network formation mechanism. Since thiolene click-reaction utilized in the hydrogel fabrication was orthogonal reaction, the homogeneity of the network could be maximized when the stoichiometric ratio of thiol and ene groups was balanced (Mũnoz et al., 2014). Therefore, the hydrogels exhibited the highest shear elastic moduli at their own optimum DTT contents. Accordingly, we found that an optimum ratio of DTT (mM) to SPG-NB (wt%) was about 1.6, regardless of SPG-NB concentrations and assumed that the numbers of thiol and norbornene groups were balanced at that ratio. Additionally, based on G′ measurement result, we could determine the amount of immobilized norbornene groups of SPG-NB as 0.32 mmol/g, which indicated that about 2.1 norbornene groups attached per 10 repeating units of SPG. Fig. 5 presents gel fractions and swelling ratios of SPG hydrogels. Hydrogels formed with 5 and 8 wt% SPG-NB solutions showed similar gel fractions at varying DTT content while the gel fractions of 3 wt% SPG-NB hydrogels were relatively lower (< 0.8), indicating the cross15
linking did not completely occur at 3 wt% of SPG-NB. However, for each SPG-NB concentration, the highest gel fractions were obtained at the specific ratio of DTT (mM)/SPG-NB (wt%) (~1.6) and the gel fraction decreased over the ratio as shown in Fig. 5A. Swelling ratio measurement also presented consistent results. Although the swelling ratio tended to decrease with an increase of SPG-NB concentration, it showed the lowest value at the specific DTT content for each SPG-NB concentration (Fig. 5B). Such a result obviously indicates that the exact stoichiometric balance of SPG-NB and dithiol cross-linker is necessary to achieve not only a higher cross-linking density but also efficient network formation of SPG hydrogel.
3.3. Formation and property of PEG/SPG hybrid hydrogels The property of natural polymer-based material can be manipulated by blending with synthetic polymer as compensating its limited performance. (Sionkowska, 2011). Herein, PEG was blended with SPG-SH for hydrogel formation due to its well-known characteristics such as non-toxicity, non-immunogenicity, and bio-inert property (Peppas et al., 2006). The PEG/SPG hybrid hydrogels were fabricated with 4 wt% PEG4NB and varying concentration of SPG-SH (0-3 wt%) in the presence of 1 mM LAP (Fig. 2C). The gelation occurred rapidly within 4 s after shining UV light (Fig. 2D). The gel point was not significantly affected by not only SPG concentration (Fig. S6B) but the presence of co-cross-linker DTT (Fig. S6C). Fig. 6 presents properties of PEG/SPG hybrid hydrogels (i.e., gel fraction, mass swelling ratio, and shear elastic modulus). As shown in Fig. 6A, the hybrid hydrogels showed high gel fraction values (> 0.95) regardless of SPG-SH content, which indicates that almost all PEG4NB and SPG-SH molecules in the precursor solution participated in network formation. Therefore, multi-thiol cross-linker 16
SPG-SH did not interfere with thiol-ene reaction in spite of its relatively huge size compared with DTT. In swelling ratio measurement, the pure PEG hydrogel marked 32 and the swelling ratio of PEG/SPG hybrid hydrogel gradually decreased with an increase of SPG-SH content (Fig. 6B). On the other hand, G′ slightly increased as an increase of content of SPG-SH in the range of 2900-3700 Pa (Fig. 6C). In general, the longer distance between cross-linking points in the network results in the larger mesh size at a fixed number of cross-links (Lee et al., 2016; Rubinstein & Colby, 2003). Therefore, one could expect the use of longer cross-linker causes the hydrogel formation with a lower network density at equilibrium swelling state. Nevertheless, PEG/SPG hybrid hydrogel has shown converse relationship between SPG-SH content and gel property as considering that thiol groups of SPG-SH are relatively far apart each other compared with those of DTT co-cross-linker, which results in a larger mesh size. Such a phenomenon implies other factors might affect the increase of network density of PEG/SPG hybrid hydrogel.
We hypothesized that the triple-helix formation of SPG played an important role in the decrease of swelling ratio as well as the improvement of mechanical property of PEG/SPG hybrid hydrogel. To evidence the presence of triple-helix in the hybrid hydrogel, Congo red assay which is used for determination of conformational change of β-glucan chains in aqueous solution was conducted. The bathochromic shift can be seen in visible light absorption spectrum of Congo red solution when single β-glucan chains form triple-helices (Semedo et al., 2015). We observed a significant bathochromic shift for both neutral SPG-SH solution and PEG/SPG hybrid hydrogel whereas such an evident shift did not appear for PEG hydrogel and alkali treated SPGSH solution (Fig. S7). Such a result indicates the presence of SPG triple-helix in SPG-SH 17
solution as well as PEG/SPG hybrid hydrogel and the triple-helical formation might contribute to an increase of network density as physical cross-linking.
3.4. Degradation of SPG and PEG/SPG hybrid hydrogels The hydrolytic degradations of SPG and PEG/SPG hybrid hydrogels were evaluated by measuring their shear storage modulus, as shown in Fig. 7. For all SPG hydrogels, the significant change both in shear modulus and in bulk volume was not observed at all for 30 days in PBS (Fig. 7A). On the other hand, the shear moduli of PEG/SPG hybrid hydrogels gradually decreased as time progress showing a typical bulk degradation profile (Fig. 7C). To quantitatively compare degradation behaviors, logarithmic plotting of G′/G′0 was implemented (Fig. 7B and 7D) and degradation rate constants (k′) was determined by linear fitting (Table S2). As expected, k′ of all the SPG hydrogels were close to zero indicating that the hydrolytic degradation did not occur. Because norbornene groups were linked to SPG by amide bonds which are stable in physiological condition, the hydrolysis of hydrogel network hardly occurred. For PEG/SPG hybrid hydrogels, larger k′ values were obtained with varying SPG content indicating that hydrogel network degraded. It was due to the hybridization of PEG4NB which contains hydrolytically cleavable ester bonds. Especially, for PEG/SPG hybrid hydrogels, we found that k′ decreased as SPG-SH content increased. It means that the hydrogel with a higher content of SPG more slowly degraded compared with either the pure PEG hydrogel or less SPG contained hydrogel. Such a phenomenon is possibly explained by two reasons. In PEG/SPG hydrogel, SPG-SH should play a role as a multi-thiol cross-linker, forming lots of cross-linking point with PEG4NB while DTT had only two thiol groups. Therefore, the hybrid hydrogel was 18
able to well maintain the network structure even though some of cross-links were cleaved compared with the pure PEG hydrogel (Lee et al., 2016). In addition, physical interaction of SPG chains might contribute to a slower degradation of PEG/SPG hybrid hydrogel by forming triple helix conformation.
3.5. BSA release and three-dimensional culture of A549 cells For application study of thiol-ene SPG hydrogel, we conducted BSA release assay and three-dimensional (3D) cell culture with SPG and PEG/SPG hybrid hydrogels, respectively. As shown in Fig. 8A, BSA was very rapidly released out within 6 h regardless of SPG content. After 24 h, the amount of released BSA from 3 wt% was about 70% compared with the initially loaded BSA amount and did not further increase. For 5 wt% SPG hydrogel, only 20% BSA was released out during the first 6 h and the cumulative BSA release very slowly increased for 78-h observation period, which indicates that BSA release could be controlled by SPG-NB content in hydrogels. For 3D cell culture, A549 cells were encapsulated in pure PEG and PEG/SPG hybrid hydrogels (4 wt% PEG4NB and 1 wt% SPG-SH). Live/Dead assay result has shown that the presence of SPG-SH in hydrogel did not influence the cell survival rate (> 90%) during the encapsulation process (Fig. 8B and Table S3). It implies thiol-ene cross-linking reaction between SPG-SH and PEG4NB was not cytotoxic for tumor cell encapsulation. During 7-day culture, single A549 cells (day 1, Fig. 8C) formed spherical clusters (day 7, Fig. 8D) and their metabolic activities increased (Fig. 8E), indicating cell proliferation in hydrogel matrix. However, there was no significant difference between pure PEG and PEG/SPG hybrid hydrogel groups. 19
Although the distinct effect of SPG on A549 cells was not observed, such results suggest that PEG/SPG hybrid hydrogel would be a candidate as a 3D cell culture matrix. 4. Conclusion To form SPG hydrogel via thiol-ene photopolymerization, we synthesized various SPGderivatives (i.e., SPG-COOH, SPG-NB, and SPG-SH) from ultrasonicated SPG. The depolymerized SPG solution showed a proper viscosity for subsequent synthesis processes. With SPG-NB and SPG-SH, we fabricated SPG and PEG/SPG hybrid hydrogels by orthogonal thiolene click-reaction, respectively. Physical properties (i.e., swelling ratio, shear modulus, degradability) of such SPG-based hydrogels could be manipulated by modular formulation of polymers and thiol cross-linkers. Especially, the triple-helix structure of SPG in PEG/SPG hybrid hydrogel might induce an increase of shear modulus and slower degradation in PBS as physical cross-linking. We could also verify potential applications (i.e., drug carrier, 3D cell culture matrix) of SPG-based hydrogels as biomaterials via BSA release assay and A549 cell 3D culture.
Acknowledgement This study was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2015R1A2A2A03002680) and “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ012268012017)” Rural Development Administration, Republic of Korea.
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Figure captions
Fig. 1. Schematic of schizophyllan (SPG) derivative synthesis. Both schizophyllan-norbornene (SPG-NB) and schizophyllan-thiol (SPG-SH) were synthesized via EDC chemistry with carboxymethyl schizophyllan (SPG-COOH). Fig. 2. SPG and PEG/SPG hybrid hydrogel formations by orthogonal thiol-ene photo-click reaction. (A) Schematic of SPG hydrogel formation with SPG-NB and DTT dithiol cross-linker. (B) In situ photo-rheometry result of SPG hydrogel formed with 3 wt% SPG-NB and 5 mM DTT. (C) Schematic of PEG/SPG hybrid hydrogel formation with PEG4NB, SPG-SH, and DTT. (D) In situ photo-rheometry result of SPG hybrid hydrogel formed with 4 wt% PEG4NB, 3 wt% SPGSH, and DTT as a co-cross-linker. Fig. 3. 13C NMR spectra of intact SPG and derivatives. Intact and ultrasonicated SPG were measured after dissolving in deuterated dimethyl sulfoxide; SPG-NB and SPG-SH were measured after dissolving in deuterated water. Assignment of SPG peaks: δ = 40 (DMSO); 61 (C6, A/C/D, free); 68 (C4); 70 (C6, B, bound); 72 (C2, A/B/C); 73 (C2, D); 74 (C3, D, free); 76 (C5); 86 (C3, A/B/C, bound); 102 (C1) (Münzberg et al., 1995). Fig. 4. Shear elastic moduli of SPG hydrogels formed with varying amount of DTT in prepolymer solution at different SPG-NB concentrations (3, 5, and 8 wt%) (n = 3, mean ± SD). Fig. 5. (A) Gel fractions and (B) equilibrium swelling ratios of SPG hydrogels formed with varying content of DTT in prepolymer solution at different SPG-NB concentrations (3, 5, and 8 wt%) (n = 3, mean ± SD).
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Fig. 6. (A) Gel fractions, (B) equilibrium swelling ratios, and (C) shear elastic moduli of PEG/SPG hybrid hydrogels formed with varying SPG-SH content (0-3 wt%) at a fixed concentration (4 wt%) of PEG4NB in prepolymer solution (n = 3, mean ± SD). Fig. 7. Hydrolytic degradation of SPG and PEG/SPG hybrid hydrogels in pH 7.4 PBS for 30 days. (A) Shear elastic modulus (G′) change and (B) logarithmic plots of G′/G0′ of SPG hydrogels formed with varying SPG-NB concentrations. (C) Shear elastic modulus (G′) change and (D) logarithmic plots of G′/G0′ of PEG/SPG hybrid hydrogels formed with varying SPG-SH concentrations (n = 3, mean ± SD). Fig. 8. (A) Cumulative BSA release profiles from SPG hydrogels (3 wt% and 5 wt% SPG-NB) in pH 7.4 PBS at 37 °C (n = 3, mean ± SD). (B) Live/Dead images of encapsulated A549 cells in pure PEG hydrogel (-SPG, 4 wt% PEG4NB) and PEG/SPG hybrid hydrogel (+SPG, 4 wt% PEG4NB/1 wt% SPG-SH) 1-h post-encapsulation. (C, D) Phase-contrast images of encapsulated A549 cells in PEG/SPG hybrid hydrogel on day 1 and 7, respectively. (E) Metabolic activities of encapsulated A549 cells in PEG and PEG/SPG hybrid hydrogels for 7 days (n = 3, mean ± SD).
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