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Fabrication of thermoresponsive degradable hydrogel made by radical polymerization of 2-methylene-1,3-dioxepane: Unique thermal coacervation in hydrogel
Polymer 179 (2019) 121633
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Fabrication of thermoresponsive degradable hydrogel made by radical polymerization of 2-methylene-1,3-dioxepane: Unique thermal coacervation in hydrogel
T
Syuuhei Komatsua, Taka-Aki Asohb, Ryo Ishiharaa, Akihiko Kikuchia,* a b
Department of Materials Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo, 125-8585, Japan Department of Applied Chemistry, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-8585, Japan
HIGHLIGHTS
(2-methylene-1,3-dioxepane-co-2-hydroxyethyl acrylate) hydrogel was synthesized. • Poly prepared hydrogel showed thermoresponsive behavior and then formed coacervate droplets in hydrogel. • The • The hydrogel collapsed and degraded to become water-soluble oligomers. ARTICLE INFO
ABSTRACT
Keywords: Thermoresponsive degradable hydrogel Coacervate droplets Cell culture scaffold 2-Methylene-1,3-dioxepane
Thermoresponsive degradable hydrogels have attracted attention because their properties allow for their use as smart drug carriers for drug delivery systems and cell scaffolds in the living body. In this study, we synthesized thermoresponsive degradable hydrogels via radical copolymerization of 2-methylene-1,3-dioxepane and 2-hydroxyethylacrylate in the presence of a crosslinker at various feed ratios. The synthesized hydrogels showed a temperature-dependent swelling/shrinking behavior, which was also observed in the corresponding linear polymers. During shrinking, coacervate droplets were observed within the hydrogel. Under mild alkaline conditions, the hydrogels were degraded and converted into hydrophilic oligomers. Moreover, cell adhesion to the hydrogel surfaces was observed using poly (D-lysine)-modified hydrogels. The number of adhered cells increased on the hydrogels that showed shrinking behavior at 37 °C. The prepared hydrogels are expected to be applicable for use in functional cell scaffolding instead of as non-degradation materials.
1. Introduction Hydrogels have attracted the attention of many researchers in the biomedical fields of drug delivery system carriers [1–3] and tissue engineering [4,5] for their ability to form a 3D network structure, high water content, and similar composition to soft biological tissues [1–5]. Synthetic hydrogels have many advantages since a variety of functions can be introduced by their synthetic route; such representative functions include stimuli responsiveness [6–8], biodegradability [9–11], self-healing [12,13], and biomolecular recognition [14–16]. Therefore, in recent years, many studies using hydrogels have been conducted in various fields. Among these hydrogel properties, stimuli responsiveness has become the focus of much work because the hydrogels change their properties upon stimuli such as hydrophilic/hydrophobic, sol/gel changing, external physical (light [6], temperature [7]), chemical (pH *
[8]), and biological (biomolecules [14–16]) stimuli. Thermoresponsive hydrogels, for example, have widely been studied as biomaterials because drug loading and/or cell adhesion can be achieved through their thermoresponsive swelling/shrinking behaviors [17–19]. For example, poly (N-isopropylacrylamide) (PNIPAAm) is one widely studied thermoresponsive polymer that shows a lower critical solution temperature (LCST)-type liquid-solid phase separation in aqueous media at 32 °C [20–22]. Thermoresponsive polymers can also be synthesized by copolymerization of hydrophobic and hydrophilic monomers by controlling the hydrophobic/hydrophilic balance [23–26]. These hydrogels are sometimes required to have biodegradable properties for their use in the living body. However, to prepare degradable thermoresponsive hydrogel is generally complicated, because it is necessary to multi-step synthesis. Our research group recently reported the thermoresponsive and
Corresponding author. Tel.: +81 3 5876 1415. E-mail address: [email protected] (A. Kikuchi).
https://doi.org/10.1016/j.polymer.2019.121633 Received 23 May 2019; Received in revised form 29 June 2019; Accepted 5 July 2019 Available online 06 July 2019 0032-3861/ © 2019 Published by Elsevier Ltd.
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Scheme 1. Synthesis of poly (MDO-co-HEA) hydrogel by radical polymerization of MDO, HEA, and MBAAm in DMSO.
Japan). DMSO was distilled under reduced pressure (0.5 kPa, 95.0 °C) before use. Chloroacetaldehyde dimethylacetal and poly (D-lysine) (PDL) were purchased from Sigma-Aldrich (MO, USA). Sulfosuccinimidyl 6-(4′azido-2′-nitrophenylamino)hexanoate, Dulbecco's modified Eagle's medium powder, and high glucose pyruvate 10 × 1 L were purchased from Thermo Fisher Scientific Co. (Walthman, MA, USA). MDO was prepared by a two-step reaction of acetal exchange and dehydrochlorination according to previous reports [27–30]. 2.2. Preparation of poly(HEA) hydrogel and poly(MDO-co-HEA) hydrogels The poly (HEA) hydrogel were prepared by free radical polymerization of HEA in the presence of N,N′-methylenebis (acrylamide) as a crosslinker and V-70 (2.0 mol% to monomer) as a radical initiator in DMSO (Scheme 1). And the poly (MDO-co-HEA) hydrogels were prepared by free radical ring opening copolymerization of MDO and HEA in the presence of N,N′-methylenebis (acrylamide) as a crosslinker and V-70 (2.0 mol% to monomer) as a radical initiator in DMSO. Degassed pre-gel solution was injected into two glass plates sandwiching a 0.5mm-thick poly (dimethylsiloxane) spacer and sealed with Teflon taping (Fig. S1). Gelation was carried out for 24 h at 30 °C, after which the gels were washed in methanol and distilled water consecutively to remove unreacted monomers and crosslinker. After purification, the poly (MDO-co-HEA) hydrogels were cut into disk (diameter 0.9 cm) using a cork borer. The swelling ratios (SRs) of the hydrogels were calculated from the weight of the swollen gels (Ws) and of the dry gels (Wd) using the following equation:
Fig. 1. Illustration of the thermoresponsive degradable hydrogel prepared by radical polymerization of MDO and HEA.
biodegradable polymer poly (2-methylene-1,3-dioxepane (MDO)-co-2hydroxyethylacrylate (HEA)) (poly (MDO-co-HEA)) synthesized by radical copolymerization of MDO and HEA [27,28]. Poly (MDO-co-HEA) shows a thermoresponsive LCST-type liquid-liquid phase separation in aqueous media, followed by the formation of coacervate droplets with concentrations of relatively hydrophobic low-molecular-weight molecules. The LCST can also be controlled by varying the copolymer composition. Moreover, the copolymer shows a degradable property induced by a pH change and transforms to a hydrophilic oligomer due to the ester groups in the polymer main chain derived from MDO. It is well known that MDO is a hydrophobic cyclic ketene acetal monomer, and after radical polymerization, the chemical structure of PMDO is similar to poly (ε-caprolactone), a biodegradable polyester [29,30]. Therefore, it was expected that a thermoresponsive and degradable hydrogel could be synthesized using poly (MDO-co-HEA) as a base material in one-pot. Thus, we herein report the synthesis of thermoresponsive and degradable poly (MDO-co-HEA) hydrogels via the radical copolymerization of MDO and HEA in the presence of a crosslinker (Fig. 1) without multi-step synthesis. We also investigated the thermoresponsive behavior and volume phase transition temperatures (VPTTs) of the hydrogels upon altering the monomer feed composition. Above the VPTT, coacervate droplets formed in the hydrogel, which is to the best of our knowledge a novel behavior in thermoresponsive hydrogels thus far. Moreover, the hydrogels showed degradable properties under alkaline conditions (1.0 mmol L−1 NaOH) and became hydrophilic oligomers. Cell adhesion properties via the thermoresponsive behavior were also observed using poly (D-lysine)-modified hydrogels.
SR = (Ws-Wd)/Wd.
(1)
2.3. Thermoresponsive properties of poly(MDO-co-HEA) hydrogels Thermoresponsive swelling changes were examined for the poly (MDO-co-HEA) hydrogels. The hydrogels were incubated in ultra-pure water at 45 °C for 24 h to bring the gels to an equilibrium state. After incubation, the temperature was changed from 45 °C to a predetermined temperature, and the hydrogels were incubated for 48 h to again reach an equilibrium state. The VPTTs were determined from the inflection point in the temperature-dependent swelling ratio changes of the poly (MDO-co-HEA) hydrogels. Microscopic observation of the poly (MDO-co-HEA) hydrogels was performed at 45 °C above the VPTT. The temperature of the sample gel was controlled by means of a thermostat microwarm plate (Kitazato Science, KM-01, Shizuoka, Japan). 2.4. Degradation of poly(MDO-co-HEA) hydrogels Hydrolysis of the poly (MDO-co-HEA) hydrogels was performed under alkaline conditions as an accelerated test [27,28]. For hydrolysis, the poly (MDO-co-HEA) hydrogels were soaked in a 1.0 mmol L−1 NaOH aqueous solution at 37 °C. The degradable properties were estimated by means of the change in swelling ratio and remaining weight of poly (MDO-co-HEA) hydrogels after hydrolysis.
2. Experimental 2.1. Materials Dimethyl sulfoxide (DMSO), 1,4-butanediol, Dowex 50(H+), tetrahydrofuran, 2,2′-azobis (4-methoxy-2,4-dimethylvaleronitrile) (V-70), potassium t-butoxide, N,N′-methylenebis (acrylamide), 2-hydroxyethylacrylate, phosphate buffer saline (PBS), penicillin-streptomycin solution, and 0.5 w/v% trypsin-5,3 mmol L−1 EDTA•4Na solution were purchased from FujiFilm Wako Pure Chemical Industries Co., Ltd. (Osaka,
2.5. Cell adhesion on poly(MDO-co-HEA) hydrogels PDL-modified poly (MDO-co-HEA) hydrogels were prepared 2
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according to previous reports [31]. These poly (MDO-co-HEA) hydrogels were immersed in 0.1 wt/vol % sulfo-SANPAH aqueous solution, and UV irradiation (350 nm) for 5 min. After UV irradiation, sulfoSANPAH modified poly (MDO-co-HEA) hydrogels were washed three times by ultra-pure water. The sulfo-SANPAH modified poly (MDO-coHEA) hydrogels were immersed in 0.1 wt/vol % PDL aqueous solution at 25 °C overnight. PDL-modified poly (MDO-co-HEA) hydrogels were washed three times by ultra-pure water. After PDL modification, the PDL-modified poly (MDO-co-HEA) hydrogels were sterilized sequentially with 70% ethanol and sterilized PBS and finally soaked in DMEM without serum overnight. Cells (bovine endothelial cells from carotid artery) (2 × 104 cells/cm2) were seeded and cultured on the surface of the poly (MDO-co-HEA) hydrogel scaffolds (diameter: 0.9 cm) for 1 d at 37 °C under 5% CO2. DMEM was then removed and the hydrogel surface was washed with sterile PBS at 37 °C. The samples were observed using an inverted microscope (Keyence BZ-8100, Osaka) with a thermostat microwarm plate at 37 °C.
hydrogels decreased with increasing temperature. These hydrogel properties are dependent on the corresponding polymer composition [6–8]. The poly (MDO-co-HEA) hydrogels consist of hydrophilic HEA and hydrophobic MDO, and the balance of these hydrophilic and hydrophobic segments affects the expression of thermoresponsive properties. Therefore, the thermoresponsive property of these hydrogels depends on the balance of hydrophobic and hydrophilic segments. VPTTs were also determined for the hydrogels (determination of VPTT is shown for Fig. S2), which decreased from 34 °C to 20 °C with an increase in MDO content. Hydrogels 2 and 3 in Table 1 did not show thermoresponsive properties within the temperature range of 5 °C–45 °C because of their increased number of hydrophilic segments. Fig. 3a shows the microscopic appearance of a poly (MDO-co-HEA) hydrogel (entry 4 in Table 1) during the temperature-dependent shrinking process at 35 °C. Many small droplets were observed, and the size of these droplets increased until 15 min after raising the temperature to 35 °C. Twenty minutes later, the droplets inside the gels disappeared and the hydrogels became transparent. This behavior was unique phase separation [31] and similar to the coacervation of poly (MDO-co-HEA) [27,28], where the poly (MDO-co-HEA) solutions separated two phase into polymer-rich phase, coacervate droplets and polymer-poor phases above the LCST. The coacervate droplets shows coalescence with other coacervate droplets due to unstable, and increase droplets size. Finally, the coacervate droplets become one large polymer-rich phase [27,28]. Similarly, above the VPTT, the poly (MDOco-HEA) hydrogel became opaque and reverted to transparent with time. The crosslinked and hydrated poly (MDO-co-HEA) gels may have a similar tendency to form a polymer-rich phase at elevated temperatures (Fig. 3b and c). And after 20 min, the all droplets in hydrogel were coalescence and became one large polymer-rich phase. Compared with the droplets observed in a solution of linear poly (MDO-co-HEA) [27], the droplet size in the hydrogel was relatively small. This is likely due to the restrained molecular mobility caused by crosslinking of the polymer. In fact, the highly crosslinked hydrogel prepared using 10 mol % of crosslinking agent to monomer showed less of a swelling behavior than that of entry 4 in Table 1 and did not show coacervate droplet-like structures under microscopic observation (Fig. S3). These results indicated that the droplet formation was dependent on crosslinking density due to restraining of molecular motion within the gels.
3. Results and discussion 3.1. Preparation of thermoresponsive degradable hydrogels Poly (MDO-co-HEA) hydrogels were prepared by radical copolymerization of MDO and HEA in the presence of MBAAm as a crosslinker, the preparation conditions for which are summarized in Table 1. For crosslinker concentrations of 2.5 and 1.0 mol% to the monomer, no gelation was observed and only a viscous liquid was formed at feed MDO:HEA molar ratios of 40:60 and 30:70. This is probably due to the increased feed amount of MDO, since copolymerization of MDO with HEA at a higher MDO feed concentration suppressed polymerization, i.e., lowered the conversion and molecular weight of the formed polymer. At this condition, the lower amount of crosslinker may also contribute to the insufficient formation of 3D polymer networks. 3.2. Characterization of thermoresponsive behavior of hydrogels Poly (MDO-co-HEA) has previously shown a thermoresponsive LCST-type phase transition behavior [27], and thus the corresponding hydrogels should show the same behavior. Therefore, the temperaturedependent swelling changes in the obtained hydrogels were examined. Fig. 2 shows the temperature-dependent swelling ratios for PHEA and poly (MDO-co-HEA) hydrogels with various compositions as indicated in Table 1. The swelling ratios of the PHEA hydrogel were almost identical regardless of temperature. PHEA is a hydrophilic polymer, and hence the PHEA hydrogel did not show a thermoresponsive behavior. On the other hand, the swelling ratios of the poly (MDO-co-HEA)
3.3. Degradation behavior of hydrogels Degradable property is one of the most important properties of a biomaterial for use in living body. We then investigated the degradable properties of the poly (MDO-co-HEA) hydrogels by alkaline hydrolysis (1 mmol L−1 NaOH) as an accelerated test (Fig. 4a). Under alkaline conditions, the weight of the PHEA hydrogel slightly decreased, probably due to hydrolysis of the ester groups in the side chains. On the other hand, the poly (MDO-co-HEA) hydrogels showed a weight decrease that depended on the polymer feed composition. One poly (MDO-co-HEA) hydrogel (30:70 (entry 4 in Table 1)) showed complete degradation after 7 h of incubation in 1 mmol L−1 NaOH. In contrast, another poly (MDO-co-HEA) hydrogel (20:80 (entry 3 in Table 1)) showed only a ten to twenty percent decrease in weight due to the introduction of a smaller amount of MDO to the hydrogel. The swelling ratios of the PHEA and poly (MDO-co-HEA) hydrogels under alkaline conditions were also investigated, and the results are shown in Fig. 4b and c. The swelling ratio of the PHEA hydrogel increased to 50 after 12 h of hydrolysis, which result supports the above consideration that hydrolysis of the side chain ester groups occurred in part under alkaline conditions and generated acrylate anions. Therefore, the PHEA hydrogel became more hydrophilic, which increased the PHEA hydrogel swelling ratio. On the other hand, the poly (MDO-coHEA) hydrogels showed a major change in swelling ratio along with hydrolysis time. Similar to the PHEA hydrogel, the side ester groups were hydrolyzed to become acrylate anions, and more importantly, the
Table 1 Preparation conditions of poly (MDO-co-HEA) hydrogels. Entry
1 2 3 4 5 6 7 8 9 10 11 12 13 a
Feed ratio (molar ratio) MDO/HEA
MDO
HEA
0:100 10:90 20:80 30:70 40:60 10:90 20:80 30:70 40:60 10:90 20:80 30:70 40:60
0.0 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8
2.0 1.8 1.6 1.4 1.2 1.8 1.6 1.4 1.2 1.8 1.6 1.4 1.2
MBAAm (mol%)
DMSO (mL)
Gelationa
5.0 5.0 5.0 5.0 5.0 2.5 2.5 2.5 2.5 1.0 1.0 1.0 1.0
2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0
O O O O O O O O × O O × ×
o: gelation occurred, x: no gelation occurred. 3
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Fig. 2. (a) Temperature-dependent swelling ratios for PHEA and poly (MDO-co-HEA) hydrogels with different compositions. Open circles: entry 1, closed triangles: entry 4, and closed squares: entry 5 in Table 1. Data are expressed as means with standard deviations (n = 3). (b) Squares are 1 cm × 1 cm macroscopic observations of non-thermoresponsive PHEA hydrogels (upper row) and thermoresponsive deswelling behavior of poly (MDO-co-HEA) hydrogel (lower row).
Fig. 3. (a) Micrographical appearance of poly (MDO-co-HEA) hydrogel (entry 4 in Table 1) during deswelling process at 35 °C. All scale bars are 10 μm. (b) Optical change in poly (MDO-co-HEA) hydrogel at 35 °C (entry 4). Left: just after temperature change from 5 °C to 35 °C (time 0 min); and right: 20 min after incubation at 35 °C. (c) Illustration of the proposed thermoresponsive behavior mechanism with coacervate droplet formation in poly (MDO-co-HEA) hydrogel.
ester groups originating from MDO in the polymer backbone were also hydrolyzed simultaneously. As a result, the mesh size of the poly (MDOco-HEA) hydrogel expanded, and then a significant increase in the swelling ratio of the poly (MDO-co-HEA) hydrogels occurred with
hydrolysis time (Fig. S4). One poly (MDO-co-HEA) hydrogel (20:80, entry 3 in Table 1) maintained a 3D network structure after 24 h of hydrolysis. In contrast, another poly (MDO-co-HEA) hydrogel (30:70, entry 4 in Table 1) collapsed and dissolved due to the disruption of 4
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Fig. 5. Cell (bovine endothelial cells from carotid artery) adhesion behavior on poly (MDO-co-HEA) hydrogel surfaces. (a) Optical images of hydrogel surfaces after being cultured at 37 °C for 24 h (upper: entry 3, lower: entry 4). All scale bars are 100 μm. (b) Adhesion and spread cell number of poly (MDO-co-HEA) hydrogel surfaces (entry 3 and entry 4).
living body as biomaterials because the hydrophilic oligomers resulting from hydrolysis would undergo renal excretion. Fig. 4. Degradable properties of poly (MDO-co-HEA) hydrogels by alkaline hydrolysis as an accelerated test (1 mmol L−1 NaOH) at 37 °C. (a) Percentage of weight remaining of poly (MDO-co-HEA) hydrogels after alkaline hydrolysis. Open circles: MDO:HEA = 0:100 (molar ratio) (entry 1 in Table 1); diamonds: MDO:HEA = 20:80 (molar ratio) (entry 3 in Table 1); and triangles: MDO:HEA = 30:70 (molar ratio) (entry 4 in Table 1). (b) Change in swelling ratio of PHEA (circles: MDO:HEA = 0:100 (entry 1 in Table 1)) and poly (MDOco-HEA) hydrogels with hydrolysis (diamonds: MDO:HEA = 20:80 (entry 3 in Table 1), triangles: MDO:HEA = 30:70 (entry 4 in Table 1)). (c) Optical images of PHEA and poly (MDO-co-HEA) hydrogels during alkaline hydrolysis tests.
3.4. Cell adhesion property on poly(MDO-co-HEA) hydrogels PDL-modified poly (MDO-co-HEA) hydrogels were prepared by condensation reaction of activate ester of sulfo-SANPAH modified poly (MDO-co-HEA) hydrogels and amino group, PDL. The cell adhesion properties of the PDL-modified poly (MDO-co-HEA) hydrogel surfaces were then investigated using bovine endothelial cells from the carotid artery incubated for 1 d at 37 °C. The original hydrogels were insufficient for supporting cell adhesion at 37 °C, and thus their surfaces were modified with PDL [32]. The immobilized PDL amount was 20 ng/ mm2, which did not change the bulk hydrogel thermoresponsive characteristics. The tested hydrogels showed cell adhesion and spread on the PDL-modified hydrogel surfaces (Fig. 5a, Fig. S5). At 37 °C, the entry 3 (20:80) hydrogel existed in a swollen state and thus had a relatively hydrophilic surface, whereas the entry 4 (30:70) hydrogel existed in a shrunken state and thus had a relatively hydrophobic surface, which supported cell adhesion and spreading. Thus, the entry 4 (30:70) hydrogel showed more cell adhesion than the entry 3 (20:80) hydrogel (Fig. 5b). This result was in good agreement with previous research on surface-modified thermoresponsive hydrogel scaffolds such as poly (ethyleneimine)-modified PNIPAAm microgels [19]. The results indicated that the thermoresponsive behavior of surface-modified hydrogels promoted the effective adhesion and spreading of cultured cells. Therefore, the poly (MDO-co-HEA) hydrogels are expected to function as degradable materials that can control the adhesion and non-adhesion of cells by their thermoresponsive behavior in the living body.
main chain ester groups, becoming a hydrophilic oligomer after 5 h of hydrolysis. Furthermore, swelling behaviors were compared between poly (MDO-co-HEA) hydrogels with different feed compositions, i.e., entries 3 (20:80) and 4 (30:70, feed molar ratio) in Table 1. The poly (MDO-co-HEA) (20:80) hydrogel existed in a swollen state at 37 °C, and its swelling ratio increased rapidly. The swollen poly (MDO-co-HEA) (20:80) hydrogel was thus considered to be hydrolyzed both at the surface and inside the hydrogel at 37 °C. On the other hand, the poly (MDO-co-HEA) (30:70) hydrogel was in shrunken state at 37 °C, and its swelling ratio thus showed only a minor change for the initial 1 h of incubation. This led to surface degradation occurring initially for the shrunken hydrogel rather than inner degradation. Subsequently, the swelling ratio increased drastically upon further hydrolysis, indicating that hydrolysis occurred within the hydrogel with time. These results suggested that initial hydrolysis could be controlled by the swelling status of the hydrogels. Moreover, we evaluated the molecular weights of soluble polymers in the supernatants after hydrolysis of the poly (MDO-co-HEA) hydrogels (Table 2). After hydrolysis, the number average molecular weights of the polymers in supernatants were between 1.2 × 104 Da and 2.1 × 104 Da. This suggests that the hydrogels could be used in the
4. Conclusion In this study, thermoresponsive degradable hydrogels were synthesized by radical copolymerization of a hydrophobic monomer, MDO, and hydrophilic monomer, HEA, in the presence of a crosslinker, MBAAm. The prepared hydrogels showed VPTT behavior in aqueous media with a change in swelling ratio. These VPTTs could be controlled over a wide temperature range by altering the hydrogel raw material proportions. Above the VPTT, coacervate droplets formed in less crosslinked hydrogels. The results indicated that the hydrogels showed a liquid-liquid phase separation, which is similar to the behavior of the linear poly (MDO-co-HEA). In addition, degradable properties occurred under mild alkaline conditions due to hydrolysis of the ester groups. Upon an increased hydrolysis time, the hydrogels swelled and collapsed. The swelling speed of the shrunken hydrogel (above VPTT) was higher than that of the swelled hydrogel (below VPTT) because alkaline
Table 2 Number average molecular weights of polymers in poly (MDO-co-HEA) hydrogel supernatants after hydrolysis. Entry
3 4 5 a
MDO/HEA
After hydrolysis
(Feed ratio)
Mna ( × 104)
Mw/Mna
20:80 30:70 40:60
1.2 2.1 1.9
12.1 10.5 4.3
Determined by GPC. 5
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hydrolysis occurred on the surface rather than inside the hydrogel. Therefore, the hydrolysis locations in the hydrogels were able to be controlled by thermoresponsive behavior. Moreover, the cell adhesion properties were investigated using PDL-modified hydrogels, which should allow for cell adhesion and spreading. The applicability of the hydrogels for cell cultivation was confirmed by cell assays, and the cells appeared to be well spread on the surface of the shrunken hydrogel at the incubation temperature (37 °C). Accordingly, the thermoresponsive degradable hydrogels synthesized by radical copolymerization of MDO and HEA are expected to function as cell culture scaffolds for regenerative medicine and inhibited cell adhesion materials.
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Polymer journal homepage: www.elsevier.com/locate/polymer
Fabrication of thermoresponsive degradable hydrogel made by radical polymerization of 2-methylene-1,3-dioxepane: Unique thermal coacervation in hydrogel
T
Syuuhei Komatsua, Taka-Aki Asohb, Ryo Ishiharaa, Akihiko Kikuchia,* a b
Department of Materials Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo, 125-8585, Japan Department of Applied Chemistry, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-8585, Japan
HIGHLIGHTS
(2-methylene-1,3-dioxepane-co-2-hydroxyethyl acrylate) hydrogel was synthesized. • Poly prepared hydrogel showed thermoresponsive behavior and then formed coacervate droplets in hydrogel. • The • The hydrogel collapsed and degraded to become water-soluble oligomers. ARTICLE INFO
ABSTRACT
Keywords: Thermoresponsive degradable hydrogel Coacervate droplets Cell culture scaffold 2-Methylene-1,3-dioxepane
Thermoresponsive degradable hydrogels have attracted attention because their properties allow for their use as smart drug carriers for drug delivery systems and cell scaffolds in the living body. In this study, we synthesized thermoresponsive degradable hydrogels via radical copolymerization of 2-methylene-1,3-dioxepane and 2-hydroxyethylacrylate in the presence of a crosslinker at various feed ratios. The synthesized hydrogels showed a temperature-dependent swelling/shrinking behavior, which was also observed in the corresponding linear polymers. During shrinking, coacervate droplets were observed within the hydrogel. Under mild alkaline conditions, the hydrogels were degraded and converted into hydrophilic oligomers. Moreover, cell adhesion to the hydrogel surfaces was observed using poly (D-lysine)-modified hydrogels. The number of adhered cells increased on the hydrogels that showed shrinking behavior at 37 °C. The prepared hydrogels are expected to be applicable for use in functional cell scaffolding instead of as non-degradation materials.
1. Introduction Hydrogels have attracted the attention of many researchers in the biomedical fields of drug delivery system carriers [1–3] and tissue engineering [4,5] for their ability to form a 3D network structure, high water content, and similar composition to soft biological tissues [1–5]. Synthetic hydrogels have many advantages since a variety of functions can be introduced by their synthetic route; such representative functions include stimuli responsiveness [6–8], biodegradability [9–11], self-healing [12,13], and biomolecular recognition [14–16]. Therefore, in recent years, many studies using hydrogels have been conducted in various fields. Among these hydrogel properties, stimuli responsiveness has become the focus of much work because the hydrogels change their properties upon stimuli such as hydrophilic/hydrophobic, sol/gel changing, external physical (light [6], temperature [7]), chemical (pH *
[8]), and biological (biomolecules [14–16]) stimuli. Thermoresponsive hydrogels, for example, have widely been studied as biomaterials because drug loading and/or cell adhesion can be achieved through their thermoresponsive swelling/shrinking behaviors [17–19]. For example, poly (N-isopropylacrylamide) (PNIPAAm) is one widely studied thermoresponsive polymer that shows a lower critical solution temperature (LCST)-type liquid-solid phase separation in aqueous media at 32 °C [20–22]. Thermoresponsive polymers can also be synthesized by copolymerization of hydrophobic and hydrophilic monomers by controlling the hydrophobic/hydrophilic balance [23–26]. These hydrogels are sometimes required to have biodegradable properties for their use in the living body. However, to prepare degradable thermoresponsive hydrogel is generally complicated, because it is necessary to multi-step synthesis. Our research group recently reported the thermoresponsive and
Corresponding author. Tel.: +81 3 5876 1415. E-mail address: [email protected] (A. Kikuchi).
https://doi.org/10.1016/j.polymer.2019.121633 Received 23 May 2019; Received in revised form 29 June 2019; Accepted 5 July 2019 Available online 06 July 2019 0032-3861/ © 2019 Published by Elsevier Ltd.
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Scheme 1. Synthesis of poly (MDO-co-HEA) hydrogel by radical polymerization of MDO, HEA, and MBAAm in DMSO.
Japan). DMSO was distilled under reduced pressure (0.5 kPa, 95.0 °C) before use. Chloroacetaldehyde dimethylacetal and poly (D-lysine) (PDL) were purchased from Sigma-Aldrich (MO, USA). Sulfosuccinimidyl 6-(4′azido-2′-nitrophenylamino)hexanoate, Dulbecco's modified Eagle's medium powder, and high glucose pyruvate 10 × 1 L were purchased from Thermo Fisher Scientific Co. (Walthman, MA, USA). MDO was prepared by a two-step reaction of acetal exchange and dehydrochlorination according to previous reports [27–30]. 2.2. Preparation of poly(HEA) hydrogel and poly(MDO-co-HEA) hydrogels The poly (HEA) hydrogel were prepared by free radical polymerization of HEA in the presence of N,N′-methylenebis (acrylamide) as a crosslinker and V-70 (2.0 mol% to monomer) as a radical initiator in DMSO (Scheme 1). And the poly (MDO-co-HEA) hydrogels were prepared by free radical ring opening copolymerization of MDO and HEA in the presence of N,N′-methylenebis (acrylamide) as a crosslinker and V-70 (2.0 mol% to monomer) as a radical initiator in DMSO. Degassed pre-gel solution was injected into two glass plates sandwiching a 0.5mm-thick poly (dimethylsiloxane) spacer and sealed with Teflon taping (Fig. S1). Gelation was carried out for 24 h at 30 °C, after which the gels were washed in methanol and distilled water consecutively to remove unreacted monomers and crosslinker. After purification, the poly (MDO-co-HEA) hydrogels were cut into disk (diameter 0.9 cm) using a cork borer. The swelling ratios (SRs) of the hydrogels were calculated from the weight of the swollen gels (Ws) and of the dry gels (Wd) using the following equation:
Fig. 1. Illustration of the thermoresponsive degradable hydrogel prepared by radical polymerization of MDO and HEA.
biodegradable polymer poly (2-methylene-1,3-dioxepane (MDO)-co-2hydroxyethylacrylate (HEA)) (poly (MDO-co-HEA)) synthesized by radical copolymerization of MDO and HEA [27,28]. Poly (MDO-co-HEA) shows a thermoresponsive LCST-type liquid-liquid phase separation in aqueous media, followed by the formation of coacervate droplets with concentrations of relatively hydrophobic low-molecular-weight molecules. The LCST can also be controlled by varying the copolymer composition. Moreover, the copolymer shows a degradable property induced by a pH change and transforms to a hydrophilic oligomer due to the ester groups in the polymer main chain derived from MDO. It is well known that MDO is a hydrophobic cyclic ketene acetal monomer, and after radical polymerization, the chemical structure of PMDO is similar to poly (ε-caprolactone), a biodegradable polyester [29,30]. Therefore, it was expected that a thermoresponsive and degradable hydrogel could be synthesized using poly (MDO-co-HEA) as a base material in one-pot. Thus, we herein report the synthesis of thermoresponsive and degradable poly (MDO-co-HEA) hydrogels via the radical copolymerization of MDO and HEA in the presence of a crosslinker (Fig. 1) without multi-step synthesis. We also investigated the thermoresponsive behavior and volume phase transition temperatures (VPTTs) of the hydrogels upon altering the monomer feed composition. Above the VPTT, coacervate droplets formed in the hydrogel, which is to the best of our knowledge a novel behavior in thermoresponsive hydrogels thus far. Moreover, the hydrogels showed degradable properties under alkaline conditions (1.0 mmol L−1 NaOH) and became hydrophilic oligomers. Cell adhesion properties via the thermoresponsive behavior were also observed using poly (D-lysine)-modified hydrogels.
SR = (Ws-Wd)/Wd.
(1)
2.3. Thermoresponsive properties of poly(MDO-co-HEA) hydrogels Thermoresponsive swelling changes were examined for the poly (MDO-co-HEA) hydrogels. The hydrogels were incubated in ultra-pure water at 45 °C for 24 h to bring the gels to an equilibrium state. After incubation, the temperature was changed from 45 °C to a predetermined temperature, and the hydrogels were incubated for 48 h to again reach an equilibrium state. The VPTTs were determined from the inflection point in the temperature-dependent swelling ratio changes of the poly (MDO-co-HEA) hydrogels. Microscopic observation of the poly (MDO-co-HEA) hydrogels was performed at 45 °C above the VPTT. The temperature of the sample gel was controlled by means of a thermostat microwarm plate (Kitazato Science, KM-01, Shizuoka, Japan). 2.4. Degradation of poly(MDO-co-HEA) hydrogels Hydrolysis of the poly (MDO-co-HEA) hydrogels was performed under alkaline conditions as an accelerated test [27,28]. For hydrolysis, the poly (MDO-co-HEA) hydrogels were soaked in a 1.0 mmol L−1 NaOH aqueous solution at 37 °C. The degradable properties were estimated by means of the change in swelling ratio and remaining weight of poly (MDO-co-HEA) hydrogels after hydrolysis.
2. Experimental 2.1. Materials Dimethyl sulfoxide (DMSO), 1,4-butanediol, Dowex 50(H+), tetrahydrofuran, 2,2′-azobis (4-methoxy-2,4-dimethylvaleronitrile) (V-70), potassium t-butoxide, N,N′-methylenebis (acrylamide), 2-hydroxyethylacrylate, phosphate buffer saline (PBS), penicillin-streptomycin solution, and 0.5 w/v% trypsin-5,3 mmol L−1 EDTA•4Na solution were purchased from FujiFilm Wako Pure Chemical Industries Co., Ltd. (Osaka,
2.5. Cell adhesion on poly(MDO-co-HEA) hydrogels PDL-modified poly (MDO-co-HEA) hydrogels were prepared 2
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according to previous reports [31]. These poly (MDO-co-HEA) hydrogels were immersed in 0.1 wt/vol % sulfo-SANPAH aqueous solution, and UV irradiation (350 nm) for 5 min. After UV irradiation, sulfoSANPAH modified poly (MDO-co-HEA) hydrogels were washed three times by ultra-pure water. The sulfo-SANPAH modified poly (MDO-coHEA) hydrogels were immersed in 0.1 wt/vol % PDL aqueous solution at 25 °C overnight. PDL-modified poly (MDO-co-HEA) hydrogels were washed three times by ultra-pure water. After PDL modification, the PDL-modified poly (MDO-co-HEA) hydrogels were sterilized sequentially with 70% ethanol and sterilized PBS and finally soaked in DMEM without serum overnight. Cells (bovine endothelial cells from carotid artery) (2 × 104 cells/cm2) were seeded and cultured on the surface of the poly (MDO-co-HEA) hydrogel scaffolds (diameter: 0.9 cm) for 1 d at 37 °C under 5% CO2. DMEM was then removed and the hydrogel surface was washed with sterile PBS at 37 °C. The samples were observed using an inverted microscope (Keyence BZ-8100, Osaka) with a thermostat microwarm plate at 37 °C.
hydrogels decreased with increasing temperature. These hydrogel properties are dependent on the corresponding polymer composition [6–8]. The poly (MDO-co-HEA) hydrogels consist of hydrophilic HEA and hydrophobic MDO, and the balance of these hydrophilic and hydrophobic segments affects the expression of thermoresponsive properties. Therefore, the thermoresponsive property of these hydrogels depends on the balance of hydrophobic and hydrophilic segments. VPTTs were also determined for the hydrogels (determination of VPTT is shown for Fig. S2), which decreased from 34 °C to 20 °C with an increase in MDO content. Hydrogels 2 and 3 in Table 1 did not show thermoresponsive properties within the temperature range of 5 °C–45 °C because of their increased number of hydrophilic segments. Fig. 3a shows the microscopic appearance of a poly (MDO-co-HEA) hydrogel (entry 4 in Table 1) during the temperature-dependent shrinking process at 35 °C. Many small droplets were observed, and the size of these droplets increased until 15 min after raising the temperature to 35 °C. Twenty minutes later, the droplets inside the gels disappeared and the hydrogels became transparent. This behavior was unique phase separation [31] and similar to the coacervation of poly (MDO-co-HEA) [27,28], where the poly (MDO-co-HEA) solutions separated two phase into polymer-rich phase, coacervate droplets and polymer-poor phases above the LCST. The coacervate droplets shows coalescence with other coacervate droplets due to unstable, and increase droplets size. Finally, the coacervate droplets become one large polymer-rich phase [27,28]. Similarly, above the VPTT, the poly (MDOco-HEA) hydrogel became opaque and reverted to transparent with time. The crosslinked and hydrated poly (MDO-co-HEA) gels may have a similar tendency to form a polymer-rich phase at elevated temperatures (Fig. 3b and c). And after 20 min, the all droplets in hydrogel were coalescence and became one large polymer-rich phase. Compared with the droplets observed in a solution of linear poly (MDO-co-HEA) [27], the droplet size in the hydrogel was relatively small. This is likely due to the restrained molecular mobility caused by crosslinking of the polymer. In fact, the highly crosslinked hydrogel prepared using 10 mol % of crosslinking agent to monomer showed less of a swelling behavior than that of entry 4 in Table 1 and did not show coacervate droplet-like structures under microscopic observation (Fig. S3). These results indicated that the droplet formation was dependent on crosslinking density due to restraining of molecular motion within the gels.
3. Results and discussion 3.1. Preparation of thermoresponsive degradable hydrogels Poly (MDO-co-HEA) hydrogels were prepared by radical copolymerization of MDO and HEA in the presence of MBAAm as a crosslinker, the preparation conditions for which are summarized in Table 1. For crosslinker concentrations of 2.5 and 1.0 mol% to the monomer, no gelation was observed and only a viscous liquid was formed at feed MDO:HEA molar ratios of 40:60 and 30:70. This is probably due to the increased feed amount of MDO, since copolymerization of MDO with HEA at a higher MDO feed concentration suppressed polymerization, i.e., lowered the conversion and molecular weight of the formed polymer. At this condition, the lower amount of crosslinker may also contribute to the insufficient formation of 3D polymer networks. 3.2. Characterization of thermoresponsive behavior of hydrogels Poly (MDO-co-HEA) has previously shown a thermoresponsive LCST-type phase transition behavior [27], and thus the corresponding hydrogels should show the same behavior. Therefore, the temperaturedependent swelling changes in the obtained hydrogels were examined. Fig. 2 shows the temperature-dependent swelling ratios for PHEA and poly (MDO-co-HEA) hydrogels with various compositions as indicated in Table 1. The swelling ratios of the PHEA hydrogel were almost identical regardless of temperature. PHEA is a hydrophilic polymer, and hence the PHEA hydrogel did not show a thermoresponsive behavior. On the other hand, the swelling ratios of the poly (MDO-co-HEA)
3.3. Degradation behavior of hydrogels Degradable property is one of the most important properties of a biomaterial for use in living body. We then investigated the degradable properties of the poly (MDO-co-HEA) hydrogels by alkaline hydrolysis (1 mmol L−1 NaOH) as an accelerated test (Fig. 4a). Under alkaline conditions, the weight of the PHEA hydrogel slightly decreased, probably due to hydrolysis of the ester groups in the side chains. On the other hand, the poly (MDO-co-HEA) hydrogels showed a weight decrease that depended on the polymer feed composition. One poly (MDO-co-HEA) hydrogel (30:70 (entry 4 in Table 1)) showed complete degradation after 7 h of incubation in 1 mmol L−1 NaOH. In contrast, another poly (MDO-co-HEA) hydrogel (20:80 (entry 3 in Table 1)) showed only a ten to twenty percent decrease in weight due to the introduction of a smaller amount of MDO to the hydrogel. The swelling ratios of the PHEA and poly (MDO-co-HEA) hydrogels under alkaline conditions were also investigated, and the results are shown in Fig. 4b and c. The swelling ratio of the PHEA hydrogel increased to 50 after 12 h of hydrolysis, which result supports the above consideration that hydrolysis of the side chain ester groups occurred in part under alkaline conditions and generated acrylate anions. Therefore, the PHEA hydrogel became more hydrophilic, which increased the PHEA hydrogel swelling ratio. On the other hand, the poly (MDO-coHEA) hydrogels showed a major change in swelling ratio along with hydrolysis time. Similar to the PHEA hydrogel, the side ester groups were hydrolyzed to become acrylate anions, and more importantly, the
Table 1 Preparation conditions of poly (MDO-co-HEA) hydrogels. Entry
1 2 3 4 5 6 7 8 9 10 11 12 13 a
Feed ratio (molar ratio) MDO/HEA
MDO
HEA
0:100 10:90 20:80 30:70 40:60 10:90 20:80 30:70 40:60 10:90 20:80 30:70 40:60
0.0 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8 0.2 0.4 0.6 0.8
2.0 1.8 1.6 1.4 1.2 1.8 1.6 1.4 1.2 1.8 1.6 1.4 1.2
MBAAm (mol%)
DMSO (mL)
Gelationa
5.0 5.0 5.0 5.0 5.0 2.5 2.5 2.5 2.5 1.0 1.0 1.0 1.0
2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0
O O O O O O O O × O O × ×
o: gelation occurred, x: no gelation occurred. 3
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Fig. 2. (a) Temperature-dependent swelling ratios for PHEA and poly (MDO-co-HEA) hydrogels with different compositions. Open circles: entry 1, closed triangles: entry 4, and closed squares: entry 5 in Table 1. Data are expressed as means with standard deviations (n = 3). (b) Squares are 1 cm × 1 cm macroscopic observations of non-thermoresponsive PHEA hydrogels (upper row) and thermoresponsive deswelling behavior of poly (MDO-co-HEA) hydrogel (lower row).
Fig. 3. (a) Micrographical appearance of poly (MDO-co-HEA) hydrogel (entry 4 in Table 1) during deswelling process at 35 °C. All scale bars are 10 μm. (b) Optical change in poly (MDO-co-HEA) hydrogel at 35 °C (entry 4). Left: just after temperature change from 5 °C to 35 °C (time 0 min); and right: 20 min after incubation at 35 °C. (c) Illustration of the proposed thermoresponsive behavior mechanism with coacervate droplet formation in poly (MDO-co-HEA) hydrogel.
ester groups originating from MDO in the polymer backbone were also hydrolyzed simultaneously. As a result, the mesh size of the poly (MDOco-HEA) hydrogel expanded, and then a significant increase in the swelling ratio of the poly (MDO-co-HEA) hydrogels occurred with
hydrolysis time (Fig. S4). One poly (MDO-co-HEA) hydrogel (20:80, entry 3 in Table 1) maintained a 3D network structure after 24 h of hydrolysis. In contrast, another poly (MDO-co-HEA) hydrogel (30:70, entry 4 in Table 1) collapsed and dissolved due to the disruption of 4
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Fig. 5. Cell (bovine endothelial cells from carotid artery) adhesion behavior on poly (MDO-co-HEA) hydrogel surfaces. (a) Optical images of hydrogel surfaces after being cultured at 37 °C for 24 h (upper: entry 3, lower: entry 4). All scale bars are 100 μm. (b) Adhesion and spread cell number of poly (MDO-co-HEA) hydrogel surfaces (entry 3 and entry 4).
living body as biomaterials because the hydrophilic oligomers resulting from hydrolysis would undergo renal excretion. Fig. 4. Degradable properties of poly (MDO-co-HEA) hydrogels by alkaline hydrolysis as an accelerated test (1 mmol L−1 NaOH) at 37 °C. (a) Percentage of weight remaining of poly (MDO-co-HEA) hydrogels after alkaline hydrolysis. Open circles: MDO:HEA = 0:100 (molar ratio) (entry 1 in Table 1); diamonds: MDO:HEA = 20:80 (molar ratio) (entry 3 in Table 1); and triangles: MDO:HEA = 30:70 (molar ratio) (entry 4 in Table 1). (b) Change in swelling ratio of PHEA (circles: MDO:HEA = 0:100 (entry 1 in Table 1)) and poly (MDOco-HEA) hydrogels with hydrolysis (diamonds: MDO:HEA = 20:80 (entry 3 in Table 1), triangles: MDO:HEA = 30:70 (entry 4 in Table 1)). (c) Optical images of PHEA and poly (MDO-co-HEA) hydrogels during alkaline hydrolysis tests.
3.4. Cell adhesion property on poly(MDO-co-HEA) hydrogels PDL-modified poly (MDO-co-HEA) hydrogels were prepared by condensation reaction of activate ester of sulfo-SANPAH modified poly (MDO-co-HEA) hydrogels and amino group, PDL. The cell adhesion properties of the PDL-modified poly (MDO-co-HEA) hydrogel surfaces were then investigated using bovine endothelial cells from the carotid artery incubated for 1 d at 37 °C. The original hydrogels were insufficient for supporting cell adhesion at 37 °C, and thus their surfaces were modified with PDL [32]. The immobilized PDL amount was 20 ng/ mm2, which did not change the bulk hydrogel thermoresponsive characteristics. The tested hydrogels showed cell adhesion and spread on the PDL-modified hydrogel surfaces (Fig. 5a, Fig. S5). At 37 °C, the entry 3 (20:80) hydrogel existed in a swollen state and thus had a relatively hydrophilic surface, whereas the entry 4 (30:70) hydrogel existed in a shrunken state and thus had a relatively hydrophobic surface, which supported cell adhesion and spreading. Thus, the entry 4 (30:70) hydrogel showed more cell adhesion than the entry 3 (20:80) hydrogel (Fig. 5b). This result was in good agreement with previous research on surface-modified thermoresponsive hydrogel scaffolds such as poly (ethyleneimine)-modified PNIPAAm microgels [19]. The results indicated that the thermoresponsive behavior of surface-modified hydrogels promoted the effective adhesion and spreading of cultured cells. Therefore, the poly (MDO-co-HEA) hydrogels are expected to function as degradable materials that can control the adhesion and non-adhesion of cells by their thermoresponsive behavior in the living body.
main chain ester groups, becoming a hydrophilic oligomer after 5 h of hydrolysis. Furthermore, swelling behaviors were compared between poly (MDO-co-HEA) hydrogels with different feed compositions, i.e., entries 3 (20:80) and 4 (30:70, feed molar ratio) in Table 1. The poly (MDO-co-HEA) (20:80) hydrogel existed in a swollen state at 37 °C, and its swelling ratio increased rapidly. The swollen poly (MDO-co-HEA) (20:80) hydrogel was thus considered to be hydrolyzed both at the surface and inside the hydrogel at 37 °C. On the other hand, the poly (MDO-co-HEA) (30:70) hydrogel was in shrunken state at 37 °C, and its swelling ratio thus showed only a minor change for the initial 1 h of incubation. This led to surface degradation occurring initially for the shrunken hydrogel rather than inner degradation. Subsequently, the swelling ratio increased drastically upon further hydrolysis, indicating that hydrolysis occurred within the hydrogel with time. These results suggested that initial hydrolysis could be controlled by the swelling status of the hydrogels. Moreover, we evaluated the molecular weights of soluble polymers in the supernatants after hydrolysis of the poly (MDO-co-HEA) hydrogels (Table 2). After hydrolysis, the number average molecular weights of the polymers in supernatants were between 1.2 × 104 Da and 2.1 × 104 Da. This suggests that the hydrogels could be used in the
4. Conclusion In this study, thermoresponsive degradable hydrogels were synthesized by radical copolymerization of a hydrophobic monomer, MDO, and hydrophilic monomer, HEA, in the presence of a crosslinker, MBAAm. The prepared hydrogels showed VPTT behavior in aqueous media with a change in swelling ratio. These VPTTs could be controlled over a wide temperature range by altering the hydrogel raw material proportions. Above the VPTT, coacervate droplets formed in less crosslinked hydrogels. The results indicated that the hydrogels showed a liquid-liquid phase separation, which is similar to the behavior of the linear poly (MDO-co-HEA). In addition, degradable properties occurred under mild alkaline conditions due to hydrolysis of the ester groups. Upon an increased hydrolysis time, the hydrogels swelled and collapsed. The swelling speed of the shrunken hydrogel (above VPTT) was higher than that of the swelled hydrogel (below VPTT) because alkaline
Table 2 Number average molecular weights of polymers in poly (MDO-co-HEA) hydrogel supernatants after hydrolysis. Entry
3 4 5 a
MDO/HEA
After hydrolysis
(Feed ratio)
Mna ( × 104)
Mw/Mna
20:80 30:70 40:60
1.2 2.1 1.9
12.1 10.5 4.3
Determined by GPC. 5
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hydrolysis occurred on the surface rather than inside the hydrogel. Therefore, the hydrolysis locations in the hydrogels were able to be controlled by thermoresponsive behavior. Moreover, the cell adhesion properties were investigated using PDL-modified hydrogels, which should allow for cell adhesion and spreading. The applicability of the hydrogels for cell cultivation was confirmed by cell assays, and the cells appeared to be well spread on the surface of the shrunken hydrogel at the incubation temperature (37 °C). Accordingly, the thermoresponsive degradable hydrogels synthesized by radical copolymerization of MDO and HEA are expected to function as cell culture scaffolds for regenerative medicine and inhibited cell adhesion materials.
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