Preparation and swelling properties of “click” hydrogel from polyaspartamide derivatives using tri-arm PEG and PEG-co-poly(amino urethane) azides as crosslinking agents

Polymer 54 (2013) 1341e1349

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Preparation and swelling properties of “click” hydrogel from polyaspartamide derivatives using tri-arm PEG and PEG-copoly(amino urethane) azides as crosslinking agents Ngoc-Thach Huynh, Young-Sil Jeon, Dukjoon Kim, Ji-Heung Kim* School of Chemical Engineering, Polymer Technology Institute, Sungkyunkwan University, Suwon, Kyunggi 440-746, Republic of Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 June 2012 Received in revised form 24 December 2012 Accepted 2 January 2013 Available online 10 January 2013

Copper-assisted azide-alkyne cycloaddition (CuAAC) became a very interesting tool lately for synthesizing biocompatible polymer-based materials such as hydrogels or microgels under mild conditions. As our previous works, modified polyaspartamides with pendent alkyne or azide groups are synthesized by successful ring-opening aminolysis reaction of the polysuccinimide (PSI) using “click” functional amine compounds and ethanolamine. In this work, 3-arm poly(ethylene glycol) (PEG) and 3-arm PEG-co-poly(amino urethane), PEG-(PAU)3, were modified with 3-azido-1-propionic acid (APrA) to obtain the corresponding azido-terminal polymeric crosslinkers, and used to prepare hydrogels with alkynated polyaspartamide via click chemistry. The obtained click hydrogels were transparent and physically strong probably due to highly efficient and homogeneous click reaction. The hydrogel from PEG-(PAU)3 was also pH-sensitive caused by its amino urethane groups in the structure. The hydrogels were characterized by the in-situ gelation and swelling behavior, in vitro cytotoxicity, and morphology. These novel hydrogels have potentials for bioapplications in the fields of drug delivery and tissue engineering. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: “Click” gel Polyaspartamide CuAAC

1. Introduction Hydrogels are three-dimensional, hydrophilic polymer networks capable of retaining large amount of water. Hydrogels have received great attention over the past decades since they are suitable for a variety of biomedical applications ranging from drug delivery systems to tissue engineering scaffolds [1e4]. Hydrogels provide three-dimensional frameworks with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Moreover, their high water content also contributes to their excellent biocompatibility, as already indicated by Ovsianikov et al. [5]. Also hydrogels show minimal tendency to adsorb proteins from body fluids because of their low interfacial tension [6]. Many polymers, synthetic or natural, have been utilized to create hydrogels for biomedical applications. For example, derivatives of poly(ethylene glycol) (PEG) macromers have been widely used due to their tissue-like elasticity, well-defined chemistry, and tunable biochemical, biophysical, and biomechanical mechanism. Coupling with photo-polymerizations as a gelation mechanism, PEG hydrogels can be synthesized with spatio* Corresponding author. E-mail address: [email protected] (J.-H. Kim). 0032-3861/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.polymer.2013.01.001

temporally defined features and properties to control cellular activities, such as spreading, migration, and differentiation [7e10]. On the other hand, copper(I)-catalyzed azide-alkyne 1,3-dipolar Huisgen cycloaddition (CuAAC), so called “click” chemistry [11e16], has received much attention in polymer and material science due to its high efficiency, high tolerance of functional groups and solvents, as well as moderate reaction temperature [17]. Furthermore, the utility of “click” chemistry inside living cells has also recently been demonstrated [18,19], therefore one may envision incorporation of azide and alkyne functional groups into synthetic or natural biodegradable polymer for formation of in-situ hydrogels, they also called ‘click-gels’. Hilborn and coworker were the first to apply click chemistry to prepare hydrogels [20]. They used poly(vinyl alcohol) (PVA) functionalized with either acetylene or azide groups. Hawker and coworkers used PEG as the main structural component to prepare well-defined hydrogel with improved mechanical properties [21]. Lamanna and coworkers developed hydrogels based on hyaluronan derivatives and examined the suitability of them as scaffolds for tissue engineering [22]. Several other groups have reported click hydrogels composed of natural polymer along with synthetic polymers [23,24]. Polypeptides and their related synthetic poly(amino acid)s have become important in biomedical applications due to their

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biocompatibility and biodegradability [25]. Poly(N-2-hydroxyethyl-D,L-aspartamide) (PHEA) is one of these synthetic polymers: it is water-soluble, biodegradable, biocompatible and non-toxic [26e 30]. As we have shown in our previous works, a variety of PHEA derivatives and the crosslinked gels were successfully synthesized by ring-opening reaction of the polysuccinimide (PSI) using nucleophilic amine compounds with hydrophilic/hydrophobic, pH/temperature sensitive, or other reactive moieties. The star-shaped structure is known to affect the gelation behavior of block copolymer hydrogels. Choi et al. first reported star-shaped copolymers with 8-arm PEG as the inner block and thermosensitive poly(L-lactide) (PLLA) (PEG(-PLLA)8) and PCL (PEG(-PCL)8) as the outer blocks [31]. Subsequently, PLLA(-PEG)3 with 3-arm PLLA was reported [32]. The gelation of these copolymers is due to the hydrophobic interactions of PLLA or PCL blocks. The gelation in the case of mixture of star-shaped PEG(PLLA)8 and PEG-poly(D-lactide) (PEG(-PDLA)8) was attributed to the formation of a stereocomplex between the enantiomeric PLLA and PDLA segments [33]. Recently, PEG(-PLLA)8 end-capped by cholesterol exhibited a sol-to-gel transition, while PEG(-PLLA)8 itself did not [34]. The gelation was triggered by the hydrophobic interaction of the cholesterol groups. Recently, a series of novel pH/temperature-sensitive hydrogels based on poly(amino urethane)-based copolymers and their potential application as a drug carrier were reported. The PAU copolymers were obtained by the addition polymerization of the isocyanate groups of 1,6-diisocyanato hexamethylene (HDI) and hydroxyl groups at the end of PEG and 1,4-bis(hydroxyethyl) piperazine (HEP) in toluene in the presence of dibutyltin dilaurate (DBTDL) as a catalyst. PAU exhibits hydrophilic properties at relatively low pH, but turns into hydrophobic properties at neutral pH. Poly(amino urethane)-based (PAU) copolymers are usually biocompatible and have been reported as candidates for drug carrier [35]. Hydrogels normally shows weak mechanical properties, and this limits their applications especially in hard tissue repair. As our previous report, the copper mediated 1,3-cycloaddition reaction of an azide with an ethynyl has been employed to fabricate PHEA hydrogels. The click gels were obtained by the polymer reaction between two PHEA components multiply functionalized with azide and alkyne groups [36]. Those hydrogels exhibited high swelling capacities and significantly improved mechanical properties compared to hydrogels previously synthesized by others chemical crosslinking process. The difference might be due to the formation of rigid 1,2,3triazole ring structure as the network forming reaction with its high efficiency and high tolerance of functional groups and solvents. In this work, a series of azido-terminal crosslinkers was synthesized by modification of 3-arms poly(ethylene glycol) (PEG) and 3-arms poly(ethylene glycol)-poly(amino urethane) (PEG-(PAU)3) with 3-azido-1-propionic acid (APrA). Then the “click” hydrogels can be obtained by reactions between PHEA components multiply functionalized alkyne groups and crosslinkers. The effects of various reaction parameters such as temperature and the concentration of catalysis as well as PEG-based crosslinker structure on the gel formation and physical properties were investigated. HCT 116 cells were used as model cells to explore the suitability of using these hydrogels for testing the cytotoxicity. The effect of PAU component on the pH-sensitive swelling behavior and degradability of the PHEA-based hydrogels was also investigated.

ethanolamine (EA; 99%), N,N-dimethylformamide (DMF; anhydrous 99.8%), sodium azide (NaN3; >99.5%) and phosphate buffered saline (PBS; pH ¼ 7.4), (þ)-sodium L-ascorbate, Copper (II) sulfate were purchased from Aldrich Chemical Co. and used as received. Diethyl ether (99%) was purchased from DaeJung Chemical Co. (Korea). Tri-arm PEG (Mn ¼ 1000) (glycerol ethoxylate) was purchased from ID Biochem, Inc. (Seoul, Korea) and used as received. 1,4-Bis-(2-hydroxyethyl)piperazine (HEP), hexamethylene diisocyanate (HDI), dibutyltin dilaurate, phosphate buffer saline (PBS) and anhydrous toluene were obtained from SigmaeAldrich. All other chemicals were purchased of quality sufficient to use without purification. PSI was prepared and purified using a previously reported procedure [37]. The weight-average molecular weight of PSI was determined to be approximately 190,000 g mol1 (PDI: 1.17 by GPC), as calculated from empirical equation that relate the solution viscosity to the molecular weight. PHEA, poly(2-hydroxyethyl aspartamide), was prepared from PSI via reaction with EA using a previously reported method. 1H NMR (500 MHz, D2O; ppm): 2.68e2.78 (m, 2H, eCH2CHCONH), 4.5e4.7 (m, 1H, eCHCH2CONH), 3.4-3.65 (br, 2H, eCH2OH), 3.2e3.4 (br, 2H, eCH2CH2OH). Polyaspartamide derivatives with alkyne pendent groups, PHEA-PPGA, were synthesized from PSI via successive nucleophilic ring-opening reaction using propargyl amine and ethanolamine. We could confirm the presence of propargyl moiety by additional peaks from the 1 H NMR (500 MHz, D2O; ppm): 2.5 (1H, terminalhCH), 3.6e3.75 (2H, eNHCH2e), and approximately 20 mol% of propargyl groups was found to be introduced. 2.2. Synthesis of 3-azido-1-propionic acid (APrA) 4 g (26.1 mmol) of 3-bromopropionic acid was dissolved in 20 mL of DMF and 20 mL of water was added. To this solution, 1.7 g (26.1 mmol) of sodiumazide was added and the mixture was stirred   at 55 C for 18 h and at 67 C for 6 h. After cooling to room temperature the crude reaction mixture was extracted with ethylether (300 mL) and the combined organic phase was removed the solvent in vacuum vaporatory leaving 2.7 g of 3-azidopropionic acid (23.5 mmol, yield 90%). Fig. 1: 1H NMR (500 MHz, CDCl3; d, ppm): 2.57e2.59 (2H, e CH2COOH), 3.51e3.53 (2H, eCH2N3). IR (film): 2933, 2866 cm1 (CeH stretching), 2100 cm1 (eN3 asymmetric stretching). 2.3. Synthesis of 3-arms poly(ethylene glycol)-(azide)3 (PEG(azide)3) Tri-azido glycerolethoxylate, termed as 3-arm PEG-(azide)3, was synthesized by esterification of glycerolethoxylate with azidopropionic acid in the presence of DMAP and DCC. Typically, 2 g of glycerolethoxylate (2 mmol) and 0.49 g of DMAP (4 mmol, 2 equiv) were dried in a vacuum oven overnight. Then 20 mL of anhydrous DCM was added to the flask, and any trace of water in the system was removed by rotary evaporator. Then the flask was cooled down to room temperature, 50 mL of DCM, 2.76 g of azidopropionic acid (12 equiv) and 7.42 g of DCC (18 equiv) were added sequentially and the reaction mixture was stirred at room temperature under a nitrogen atmosphere for 24 h. DCU, the byproduct, was removed by filtration and 1.85 g of the product (yellow oil) was collected after removing the solvent by rotary evaporator (yield w 92.5%).

2. Experimental 2.1. Materials

2.4. Synthesis of 3-arms poly(ethylene glycol)-(PAU)3 (PEG-(PAU)3) and its azido derivatives

L-Aspartic acid (>98%), o-phosphoric acid (98%), 2-bromo-1aminoethane hydrobromide (99%), propargylamine (PPGA; 98%),

The PEG(-PAU)3 block copolymers were synthesized by addition polymerization of the isocyanate groups of HDI and hydroxyl

N.-T. Huynh et al. / Polymer 54 (2013) 1341e1349

1343

Fig. 1. 1H NMR spectra of the APrA and PEG-(azide)3 cross-linker.

groups at the end of the 3-arm PEG and HEP in toluene in the presence of dibutyltin dilaurate as a catalyst (Scheme 1) [38]. The feed ratio of the components was calculated so as to obtain hydroxyl groups at the ends of the resulting copolymer. The synthetic process of PEG(-PAU)3 was as follows: 2.0 g (2.0 mmol) of 3-arm PEG (Mn ¼ 1000) and 0.008 g of dibutyltin dilaurate were added into a 250 mL two-neck round-bottom flask equipped with a magnetic stir-bar. The flask was placed into an oil-bath and dried for 1 h under vacuum at 100  C and then cooled to 70  C. After that, 1.4 g (8.0 mmol) of HEP was added into the flask and the mixture was dried under vacuum for 30 min. Then, 60 mL of anhydrous toluene was added. After the reactants were completely dissolved, 1.3 mL (8.0 mmol) of HDI was added to the flask and the reaction was continued for 1 h at 70  C. Finally, the reaction mixture was evaporated under vacuum, dissolved in chloroform and then precipitated in excess diethyl ether. The precipitated polymer was filtrated and dried under vacuum at room temperature for 48 h. The product yield was over 85%.

Following the procedure of esterification of hydroxyl group in PEG-(PAU)3 by propionic acid in the present of DMAP and DCC catalyst system, we can obtain the PEG-(PAU-azide)3 with high reaction yield w90%. 2.5. Preparation of crosslinked gels by click chemistry Clickable functional alkyne-groups can be introduced onto polysuccinimide (PSI) through aminolysis to obtain PHEA-PPGA which was reported in our previous work [36]. Then 0.1 g of PHEA-PPGA was dissolved in 1 mL of degassed water with 10 wt% of concentration. 0.37 g of PEG-(azide)3 or 0.40 g of PEG-(PAU-azide)3 (33.3 mol% of PHEA-PPGA) with 0.005 g (2.5 wt%) of CuSO4 and 0.005 g of sodium ascorbate (2.5 wt%) was dissolved in the polymeric solution. The gelation occurred after finishing the catalyst addition. The crosslinked gel was then washed with a large amount of water for a day, followed by freeze-drying for 3 days to provide the dried gels. 2.6. Measurements 1

Scheme 1. Synthesis route of 3-arms poly(ethylene glycol)-(azide)3 (PEG-(azide)3).

H NMR spectra were recorded on a Unity Inova-500 (Varian,  USA) spectrometer using D2O or DMSO-d6 as the solvent at 5 C. Fourier-transform infrared (FTIR) spectra were obtained using a PerkinElmer FTIR spectrometer (SPECTRUM 2000). The molecular weights of the copolymers and their distributions were measured by gel permeation chromatography (GPC) using a Waters Model 410 instrument with a refractive index detector (Shodex, RI-101) and three Styragel (KF-803L, KF-802.5 and KF802) columns in series, at a flow rate of 1.0 mL/min (eluent: DMF; 40  C). Poly(ethylene glycol) standards (Waters) were used to determine the molecular weights. The modulus variation of the copolymer aqueous solutions was determined by dynamic mechanical analysis (Bohlin Rotational Rheometer). A polymer solution (30 wt%) in water was placed

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between a 20 mm diameter plate and a 100 mm diameter plate with a gap of 250 mm. Oscillation mode with a stress value of 0.4 Pa and frequency of 1 rad/s was used. The heating rate was 1  C/min. The dried click gels were quickly quenched and then crosssectioned in liquid nitrogen. The morphology of the freeze-dried click gels was observed using field emission scanning electron microscopy (FE-SEM, JEOL 6320, Japan). Porous gel samples were mounted onto a metal stub with double-sided carbon tape and coated with platinum for 30 s under vacuum (103 Torr) using a plasma sputtering method (HC-21 ion sputter coater). 2.7. Equilibrium swelling of gel The swelling capacity of the prepared click gels, SR, is defined as the ratio between the weight of the swollen gels (Ws) after extensive dialysis against distilled water minor the weight of the dry gels and the weight of the dry gels (Wd):

SR ¼

ðW s  W d Þ Wd

The dried samples were placed in different aqueous solutions and then removed from the solutions at regular intervals. The weights of the click-gels were recorded after wiping off the solution on the surface of the gels with moistened filter paper. 2.8. In vitro cytotoxicity of hydrogel The HCT 116 cells were obtained from the Korean Cell Line Bank (Seoul, Korea). The cell culture medium (DMEM, cellgro) and heatinactivated fetal bovine serum (FBS) were obtained from Invitrogen Canada (Burlington, Ont., Canada). The HCT 116 cells were maintained in a DMEM medium containing 10% FBS, 1% penicillin G/ streptomycin. For the in vitro cytotoxicity studies, the cells were harvested by trypsinization and resuspended to a concentration of 1  105 cells/mL in fresh culture medium. The cells were seeded into 96-well flat-bottomed tissue-culture plates at 1  105 cells/ well and incubated for 24 h in a humidified atmosphere containing

5% CO2 at 37  C. The solutions which were extracted from click hydrogel, were diluted with the culture medium. 10 mL of each solution was added to the individual wells of a 96-well culture plate. After incubation for 24 h, the cytotoxicity of the amphiphilic copolymer was analyzed quantitatively using MTT assays to measure the metabolic reduction of 3-(4,5-dimethylthiazol-2yl)-2,5diphenyl tetrazolium bromide to formazan by the viable cells. 3. Results and discussion 3.1. Synthesis and characterizations of 3-arms PEG and PEG-PAU compounds with azide terminal groups The PEG-(azide)3 were synthesized by the esterification of the carboxylic groups of 3-azido propionic acid (APrA) and hydroxyl groups at the end of the PEG. The synthesis route is shown in Scheme 1. Fig. 1 shows the 1H NMR of the APrA and PEG(azide)3. The methylene protons of original PEG (or glycerol ethoxylate) are shown at 3.5e3.7 ppm (c), and peaks a and b are assigned to the methylene protons from APrA. Also peak at 4.24 ppm (d) is assignable to the methylene protons adjacent to oxygen of ester group. The conversion from hydroxyl groups of PEG to the terminal azido groups was determined from the relative integration between peak b (d ¼ 2.64) and c. These results demonstrate that PEG derivatives have been well synthesized by the esterification reaction. FTIR (film): 2933, 2866 cm1 (CeH stretching), 1100 cm1 (Ce OeC stretching), 2100 cm1 (eN3 asymmetric stretching). The PEG(-PAU)3 copolymers were synthesized by the addition polymerization of the isocyanate groups of HDI and hydroxyl groups at the end of the 3-arm PEG and HEP. The synthesis route is shown in Scheme 2. The block length of PAU was controlled by varying the feed ratio of the monomers. The feed ratio of the reactants were calculated to obtain the hydroxyl-terminated copolymers (the number of hydroxyl groups is four equivalents larger than the number of isocyanate groups), as shown in Table 1. The chemical structure of the synthesized copolymers was characterized by 1H NMR and FTIR spectroscopy. Fig. 2 shows the 1 H NMR spectrum of the copolymer PEG(-PAU)3 (P-02). The signals

Scheme 2. Synthesis route of 3-arms poly(ethylene glycol)-(PAU)3 (PEG-(PAU)3) and its modification of azido functioned terminal groups.

N.-T. Huynh et al. / Polymer 54 (2013) 1341e1349 Table 1 Characteristics of PEG(-PAU)3 copolymers. Feed ratio (mol)

P-01 P-02 a b c d

PEG

HEP

HDI

1.0 1.0

6.0 12.0

6.0 12.0

B

PEGa

Mnb of PEG-(PAU)3

PDIb

Mnc of PAU/arm

pKad

1000 1000

2200 2400

1.09 1.11

400 450

6.44 6.52

provided by ID Biochem, Inc. measured by GPC. calculated from the molecular weight of PEG and copolymers. Huynh CT et al., Polymer 51:3843 (2010).

at 3.51e3.78 ppm were assigned to the methylene protons of PEG (peak a). The signals at 3.12e3.22 ppm were assigned to the first methylene protons of HDI (eOCOeNHeCH2eCH2e, b). The signals at 1.43e1.58 ppm and 1.28e1.37 ppm were assigned to the second (eNHeCH2eCH2eCH2e, c) and third methylene protons (eNHe CH2eCH2eCH2eCH2eCH2e, d) of HDI, respectively. The signals at 4.16e4.23 ppm and 2.49e2.71 ppm were assigned to the methylene protons of HEP (peaks e, f, g). The signals at 3.39e3.42 ppm were assigned to the methylene protons near the eOH groups at the end of the copolymer (peak h). The isocyanate peak at 122.9 ppm was not observed in Fig. 2, indicating that the HDI monomer was completely consumed during the polymerization. The FTIR measurements further confirmed the formation of the copolymers. Fig. 3 shows the IR spectrum of the P-02 copolymer. The peak at 1104 cm1 was attributed to the CeOeC stretching of 3arm PEG. The peak at 3334 cm1 corresponded to the eNHe stretching band of the urethane, and the absence of the peaks at 2267 cm1 indicates that the isocyanate groups completely reacted. The peak at 1720 cm1e1530 cm1 were attributed to the carbonyl stretching and hydrogen bond carbonyl groups, respectively, further indicating the formation of the functional urethane groups. The NMR and FTIR results confirmed the formation of PEG(-PAU)3 copolymer. Furthermore, the molecular weight of copolymers and their distributions were determined by GPC. Fig. 4 shows the GPC results of 3-arm PEG (Mn ¼ 1000) and PEG(-PAU)3 (P-02) copolymer. The above characterizations clearly indicate the successful synthesis of the PEG(-PAU)3 copolymers. 3-arm PEG with the molecular weight of 1000 was used for all experiments and copolymers with different of PAU block length were achieved by changes in the feed ratios of the reactants. The repeat unit in PAU (x value in Scheme 2) of copolymer P-01 and P-02 are 2.0 and 4.0, respectively. The characteristics of the synthesized copolymers are summarized in Table 1.

a O

g n O

N H

6N

H

N

O

A

A: PEG(PAU)3 B: PEG(PAU-Azide)3 4000

3500

3000

2500

2000

1500

Fig. 3. FT-IR spectra of the PEG-(PAU)3 and PEG-(PAU-azide)3 cross-linker.

Fig. 5 shows the 1H NMR of PEG(PAU)3 and PEG(PAU-azide)3 which was synthesized by the esterification reaction between the hydroxyl groups at the end of PEG(PAU)3 and the carboxyl groups from the APrA in the presence of DCC and DMAP as the catalyst system. In Fig. 5, excepting the specific peaks for the PEG(PAU)3 which was discussed above, there are 2 peaks (j & k) which are from the APrA. Furthermore, Fig. 3 also shows that the PEG(PAU-azide)3 chemical structure can be confirmed with some specific peaks for PEG(PAU)3 and the peak at 2100 cm1 which is assigned for azide stretching band. These results demonstrate that the PEG(PAUazide)3 copolymers were synthesized successfully. 3.2. Gel formation by ‘click’ chemistry On the basis of 1,3-cycloaddition reactivity of alkyne- and azidemodified polymers, we reasoned that it, therefore, should be possible to prepare hydrogels by simply mixing of prepared PHEAPPGA and azido-terminal PEG crosslinkers in the presence of a Cuþ catalyst system (Schemes 1 and 2). As expected, gelation was observed in the aqueous solutions within a few seconds to minutes, depending on the solution concentration (weight of total polymer/ volume of water), depending on the kinds of crosslinkers

h

N

e

e

OH

x

f c g

b

d e h

PEG-(PAU) -02 3

PEG-(PAU) -01 3

8

7

6

5

4

3

Chemical Shift (δ)

2

Fig. 2. 1H NMR spectra of the PEG-(PAU)3.

1

1000

-1

Wavenumber (cm )

a

f

b,c,d O

O

Transmittance (%)

No.

1345

0 ppm

Fig. 4. GPC traces of PEG and PEG(-PAU)3 copolymer (P-02).

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N.-T. Huynh et al. / Polymer 54 (2013) 1341e1349

a g

O N H

i

6N

O

H

N

h

N

e

e

i

j

16000

Gel4-0.15

14000

N3

OCO

k

x

12000

G', G"

n O

(a)

f

b,c,d O

O

a

8000

f PEG(PAU-Azide)3 i

g h

10000

k e dj

b

c

6000

Gel point 4000

G' G"

2000

PEG(PAU)3

0 0

8

7

6

5

4

3

2

1

50

100

0 ppm

Chemical Shift (δ)

150

200

Time (s)

(b)

Fig. 5. 1H NMR spectra of the PEG-(PAU)3 and PEG-(PAU-azide)3 cross-linker.

Gel5-0.15 14000 12000 10000

G', G"

(PEG(azide)3 & PEG(PAU-azide)3) we can obtain the Gel4 & Gel5. Click gels are formed by chemoselective 1,3-cycloaddition between alkyne functional groups of PHEA-PPGA and the azido-terminal groups of crosslinkers with the formation of multiple triazole crosslinkages. The prepared gels were transparent. In the other words, the aim of the rheological measurements was to characterize the course of the Cu(I)-catalyzed crosslinking process of the PHEA-based click gels in terms of “gel point”, the time at which G0 ¼ G00 , and of the plateau G0 value (Fig. 6a and b). The latter are the values of the elasticity modulus for each given gel when the crosslinking reaction has been completed. In all of the testing processes, we needed approximately 30 s to prepare gelation solutions separately. Then two solutions were mixed and stirred rapidly before placing into the machine. So, the results indicate that for the two gels considered the gelation process is characterized by a quite short time period (70 and 100 s without preparation time), and the increasing G0 values give evidence for an increase in the product’s elasticity. Results of frequency-sweep and stress-sweep experiments from Table 2 show also that the G0 values can be considered reliable ones. Our study is an approach which should provide several advantages with respect to the PHEA-based hydrogel formation including high crosslinking rates and controllable crosslinking density which can affect the macroscopic properties of the obtained hydrogels.

8000 6000

Gel point 4000

G' G"

2000 0 0

50

100

150

200

250

Time (s) Fig. 6. Solegel transition for Gel4-0.15 (a) and Gel5-0.15 (b).

behavior is expected due to the presence of tertiary amine groups on the structure, which are ionized by protonation at low pH, but they remain mostly unprotonated at alkaline pH (pKa of piperazine moiety at room temperature is 5.35  0.04) [39]. From these results, we hypothesized that a broad range of hydrogel materials based on the polyaspartamide backbone polymers may be obtained by facile click reactions both in water and in organic solvents using

3.3. Swelling ratio of click hydrogel The prepared click gels were tested to determine their swelling ratios in aqueous solution using the tea-bag method, and their water absorption was measured as a function of time. Fig. 7a and b show the swelling curves of the click hydrogels (4 & 5) prepared at different polymer concentrations. Fig. 8 shows the swelling curves of click gels 5 which were formed from PHEA-PPGA and using PEG(PAU-azide)3 as the crosslinker with the polymer concentration (15%). As shown in Figs. 7 and 8, the initially fast swelling appeared to level off within 3e4 h in distilled water. The degree of swelling decreased with respect to increasing concentration due to the higher crosslinking density (estimated by the MW between crosslinks, Mc). Fig. 8 demonstrates that the click gel 5 can be sensitive with pH; the degree of swelling decreased as the pH was increased. At higher pH 10, the swelling ratio was about 12 g/g, but at lower pH 4 the swelling increased dramatically up to 42 g/g. This

Table 2 Rheometry results of click hydrogels. Click hydrogel

Gel4

Gel5

a

Concentration (%) Solution

Crosslinkera

5 10 15 15

5

5 10 15 15

based on monomer weight.

3 5 7 5

3 5 7

Gel point (s)

G0 (Pa)

185 135 74 121 74 70 250 186 124 176 124 116

8500 11,235 13,070 10,167 13,070 14,051 8617 10,561 13,458 9804 13,458 13,796

N.-T. Huynh et al. / Polymer 54 (2013) 1341e1349

a different crosslinking system. Additionally, the resulting gel can possess a rather broad range of mechanical strength and degree of swelling, which are controllable by changing the concentration of the polymer solution or length chain of the crosslinkers. Fig. 9 shows the equilibrium swelling ratio of the gel5-0.15 triggered by pH changes. The gel sample was reversibly swollen and shrunken by cycling pH of aqueous medium between pH 10 and pH 4 at room temperature.

(a) 40

Swelling ratio

30

20

3.4. In vitro cytotoxicity test

10

Gel4-0.15 Gel4-0.1 Gel4-0.05

0 0

5

10

15

20

25

Time (h)

(b) 50

Swelling ratio

An in vitro cytotoxicity test (MTT assay) was performed using the HCT 116 cell line, and Fig. 10 shows the microscopic images of cell growth in the medium containing hydrogel after 1 day and 2 days culture (c & d, respectively), as compared with the control cells that had been incubated in a culture dish without the hydrogel (a & b). The cell growth of the hydrogel-containing medium after 2 days was found to be approximately 113% as compared to those obtained from the control. These results suggest that the click hydrogel has low cytotoxicity, and has potential use in biomedical applications, such as tissue engineering or drug delivery systems. 3.5. Morphology and degradation behavior of click hydrogel

40

30

20

10

Gel5-0.15 Gel5-0.1 Gel5-0.05

0 0

5

10

15

20

25

Time (h) Fig. 7. Swelling value for Gel4 (a) and Gel5 (b) with the function of gelation concentrations.

Fig. 11 shows differences in the cross-sectional morphology of freeze-dried click hydrogel (Gel5-0.15) induced by changing pH of the medium. Dramatic changes in porous structure are observed for the various environmental conditions. As shown in the left image of Figure, a well regulated micrometer-sized porous structure with 3e 5 mm pores is observed at room temperature in a neutral condition. The click hydrogel expands to a higher swollen state on decreasing the pH to an acidic state (pH 7 & 4), resulting in a macroporous structure. This change in gel porosity induced by changing pH of the medium clearly demonstrates the pH-responsible behavior of this crosslinked polymeric system. These hydrogel characteristics are important in various biomaterial applications because a microporous scaffold with controlled pore size is necessary for efficient biological function. Biodegradability of materials is required and can provide advantages in many biomedical applications. As shown in Fig. 12, the

50 50

1347

Gel5-0.15

Gel5-0.15 40

Swelling Ratio

30

20

30

20

10

0

10

pH10 pH7 pH4

4

pH

Swelling ratio

40

10 0

0 0

5

10

15

20

25

4

8

12

16

20

24

Time (h)

Time (h) Fig. 8. Swelling value for Gel5-0.15 with the function of buffer pH.

Fig. 9. Reversible swelling curves of click hydrogel (Gel5-0.15) as a function of pH of buffer solution.

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Fig. 10. In vitro cytotoxicity (a) the control cell after 1day, (b) the control cell after 2 days, (c) the HCT 116 cell on click hydrogel after 1 day, (d) the HCT 116 cell on click hydrogel after 2 days.

Fig. 11. Morphology of click hydrogel (Gel5-0.15) at different pH (SEM).

in vitro hydrolytic degradation of click hydrogels was evaluated by recording weight loss as a function of immersion time in phosphate buffer solution of pH ¼ 7.4 (Na2HPO4, 0.01 mol L1; NaCl,  0.138 mol L1; KCl, 0.0027 mol L1) at 37 C. The click hydrogels exhibit progressive weight loss over the 7 days period ranging from 10 to 60%, with different rates depending on the kind of crosslinker. The hydrolytic degradation decreases due to higher crosslinking density in the gel matrix. Attack of water molecules to the urethane groups should result in weight loss of the click hydrogel (Gel5-0.15) by breaking bonds through the formation of CO2 and organic fragments. Here, ion-catalyzed hydrolysis may contribute to the overall hydrolytic degradation process [40].

Novel azide-modified PEG and PEG-PAU copolymers were synthesized successfully and were used as the crosslinkers for preparing hydrogels with alkyne-modified polyaspartamide derivative. Their chemical gels were prepared using the CuAAc click reaction based on chemoselective crosslinking between the two complementary click functional groups. The degrees of swelling and morphology of the porous structure of the prepared gels were varied by changing the reaction parameters and the medium pH. These novel hydrogels possessed well-developed microporous structure and good mechanical strength. Also these hydrogels were found to be non-cytotoxic to suggest potential use in biomedical applications including drug delivery system and tissue engineering. Acknowledgments

100 90

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (#20110011464).

80 70

Weight (%)

4. Conclusion

60

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Gel5-0.15 Gel4-0.15

40 30 20 10 0 0

1

2

3

4

5

6

7

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Time (day) Fig. 12. In vitro degradation behavior of click hydrogels (Gel4-0.15 & Gel5-0.15) contents in PBS (pH ¼ 7.4).

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