Journal of Biomaterials Science 23 (2012) 1091–1106 brill.nl/jbs
Synthesis, Characteristics and Potential Application of Poly(β-Amino Ester Urethane)-Based Multiblock Co-Polymers as an Injectable, Biodegradable and pH/Temperature-Sensitive Hydrogel System Cong Truc Huynh a , Minh Khanh Nguyen a,∗ , In Ki Jeong a , Sung Wan Kim b,c and Doo Sung Lee a,∗∗ a
Theranostic Macromolecules Research Center, Department of Polymer Science and Engineering, Sungkyunkwan University, Suwon, Gyeonggi-do 440-746, South Korea b Center for Controlled Chemical Delivery, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT 84112, USA c Department of Bioengineering, College of Engineering, Hanyang University, Seoul, South Korea Received 8 March 2011; accepted 15 April 2011
Abstract Physical polymeric hydrogels have significant potential for use as injectable depot drug/protein-delivery systems. In this study, a series of novel injectable, biodegradable and pH/temperature-sensitive multiblock co-polymer physical hydrogels composed of poly(ethylene glycol) (PEG) and poly(β-amino ester urethane) (PEU) was synthesized by the polyaddition between the isocyanate groups of 1,6-diisocyanato hexamethylene and the hydroxyl groups of PEG and a synthesized monomer BTB (or ETE) in chloroform in the presence of dibutyltin dilaurate as a catalyst. The synthesized co-polymers were characterized by nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy and gel-permeation chromatography. Aqueous solutions of the co-polymers showed a sol-to-gel phase transition with increasing pH and a gel-to-sol phase transition with increasing temperature. The gel regions covered the physiological conditions (37°C, pH 7.4) and could be controlled by changing the molecular weight of PEG, PEG/PEU ratio and co-polymer solution concentration. A gel formed rapidly in situ after injecting the co-polymer solution subcutaneously into SD rats and remained for more than 2 weeks in the body. The cytotoxicity tests confirmed the non-cytotoxicity of this co-polymer hydrogel. The controlled in vitro release of the model anticancer drug, doxorubicin, from this hydrogel occurred over a 7-day period. This hydrogel is a potential candidate for biomedical applications and drug/protein-delivery systems. © Koninklijke Brill NV, Leiden, 2012
* Present address: Department of Biomedical Engineering, Case Western Reverse University, Cleveland, OH 44106, USA. ** To whom correspondence should be addressed. Tel.: (82-31) 290-7282; Fax: (82-31) 292-8790; e-mail:
[email protected]
© Koninklijke Brill NV, Leiden, 2012
DOI:10.1163/092050611X575423
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Keywords pH sensitivity, temperature sensitivity, hydrogel, poly(β-amino ester urethane), biodegradability, doxorubicin, controlled release
1. Introduction Stimuli-sensitive physical polymeric hydrogels and their applications, such as injectable depot drug/protein-delivery systems and/or use in tissue engineering, have attracted considerable attention [1–17]. The high water content inside the hydrogels imparts biocompatible properties and biodegradability or a lack of chemical cross-links can enhance the rate of excretion from the human body [1–5]. In contrast to the chemical cross-linked hydrogels, which require chemical reactions (enzyme, photocross-linked and Michael-addition type) for gel formation [18–21], physical polymeric hydrogels can be formed by the self-assembly of amphiphilic block/graft co-polymers in response to external stimuli, such as electric fields, glucose, pH, temperature or their combinations. Temperature-sensitive hydrogels have attracted considerable attention in a variety of polymer systems, such as poly(N-isopropylacrylamide) [22], Pluronic and its derivatives [23–25], triblock co-polymers systems consisting of hydrophilic poly(ethylene glycol) and a hydrophobic block including poly(ε-caprolactone) (PCL–PEG–PCL or PEG– PCL–PEG), poly(caprolactone-co-lactide) (PCLA–PEG–PCLA), poly(lactide-coglycolide) (PLGA–PEG–PLGA) and poly(phosphazene) [26–34] and some natural polymers [4, 5]. These hydrogels exist as an injectable solution at low temperatures but change to a gel at physiological temperature (37°C) or after being injected into the body. However, the neutral property may limit their applications to the delivery of ionic biomolecules. To overcome this problem, the conjugation of ionic groups into hydrogel systems has attracted attention. Oligomer sulfamethazine (OSM) was introduced as an anionic pH-sensitive moiety, and the applications of OSM containing co-polymer hydrogels in the delivery of drugs/proteins have been reported [35–39]. Recently, cationic hydrogels bearing tertiary amine groups, which can form ionic interactions with anionic drugs/proteins, were introduced as excellent materials for drug/protein-delivery systems [40]. Cationic polymeric hydrogels, such as poly(βamino ester) (PAE), poly(amido amine) (PAA), poly(β-amino urethane) (PAU) and their derivatives [41–53], and their potential applications for the delivery of genes, proteins and drug molecules have been studied. Physical polymeric hydrogels containing amino urethane groups have great potential as carriers for the delivery of drugs/proteins owing to their non-cytotoxicity and ability to form hydrogen bonds and ionic interactions with anionic biomolecules. However, the reported gels have high molecular weight and/or are nonbiodegradable hydrogels or showed fast degradation [49–51]. In this study, we focused on the synthesis and examination of an injectable, biodegradable and pH/temperature-sensitive hydrogel system using multiblock co-polymers composed
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of poly(ethylene glycol) and poly(β-amino ester urethane) ([PEG–PEU]x ). The synthesized co-polymers were characterized by nuclear magnetic resonance (NMR) spectroscopy, Fourier transform infrared (FT-IR) spectroscopy and gel-permeation chromatography (GPC). The sol–gel phase transitions and the influencing factors were examined. The in vitro cytotoxicity, in vivo gelation and degradation were examined. The in vitro release of the model anticancer drug, doxorubicin, from this hydrogel was also studied. 2. Materials and Methods 2.1. Materials Poly(ethylene glycol) (PEG), dibutyltin dilaurate (DBTL), 1,6-diisocyanato hexamethylene (HDI), anhydrous dichloromethane (DCM), anhydrous chloroform, N,N trimethylene dipiperidine (TMDP), 2-hydroxylethyl acrylate (HEA), doxorubicin hydrochloride and phosphate-buffered saline (PBS) were obtained from SigmaAldrich and used as received. 4-Hydroxylbutyl acrylate (HBA) was purchased from TCI and used as received. NaOH, HCl, diethyl ether and n-hexane were all products of Samchun. All other reagents were of analytical grade and used without further purification. 2.2. Synthesis of Biodegradable Multiblock Co-Polymers [PEG–PEU]x The multiblock co-polymers [PEG–PEU]x were synthesized by the polyaddition of hydroxyl groups at the ends of PEG and synthesized monomer HBA–TMDP–HBA (BTB) (or HEA–TMDP–HEA, ETE) and the isocyanate groups of HDI in chloroform in the presence of dibutyltin dilaurate as a catalyst (Scheme 1). The monomers (BTB or ETE) were synthesized by the Michael-addition reaction between the vinyl groups of HBA or HEA and the secondary amine groups of TMDP. The detail reaction to synthesize the monomer BTB is as follows: TMDP and HBA were dissolved in anhydrous DCM at ambient temperature at a mole ratio of 1:2 and a reactant concentration of 20 wt%. The flask was then placed in a 45°C oil-bath with constant stirring for 2 h and the reaction solution was concentrated by vacuum evaporation and precipitated in an excess of n-hexane. The precipitated monomer was filtered and dried under vacuum for 48 h prior to use. The structure of the synthesized monomer BTB (ETE) was confirmed by 1 H-NMR. The synthesis processing of [PEG–PEU]x multiblock co-polymer B-20-1 is as follows: PEG (1 mmol) and DBTL (0.002 g) were dried in a 250-ml two-neck round-bottom flask under vacuum at 100°C for 2 h. The temperature was then decreased to 60°C and BTB (9 mmol) was added and dried under vacuum for 30 min. The vacuum was replaced by dried nitrogen followed by the addition of 80 ml anhydrous chloroform. After the reactants had dissolved, HDI (9 mmol) was added and the reaction was continued for a further 3 h. Finally, the reaction solution was concentrated by vacuum evaporation and precipitated in an excess of diethyl ether. The precipitated co-polymer was filtered and dried under vacuum for 48 h. The final
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Scheme 1. Synthesis of multiblock [PEG–PEU]x co-polymers.
yield was approx. 90%. The molecular weight of the co-polymers could be controlled by changing the feed ratio of the reactants and the PEG molecular weight. 2.3. Characterization A 500 MHz spectrometer (Varian Unity Inova 500NB) was used to record the NMR spectra with CDCl3 as the solvent. A FT-IR spectrometer (FT/IR-4100 Type A, TGS, Jasco) was used to record the infrared spectra. The molecular weights of the co-polymers 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-803, KF802.5 and KF-802) columns in series, at a flow rate of 1.0 ml/min (eluent: THF; 40°C). PEG standards (Waters) were used for calibration. 2.4. Sol–Gel Phase Transition Measurement The sol (flow)–gel (non-flow) phase transition of the co-polymer in aqueous solution was determined using the tube inversion method. Briefly, the co-polymer was dissolved in PBS at pH approx. 3 in a 4 ml vial (10 mm diameter) at the given concentration for 4 h. The pH was adjusted with 5 M NaOH and 5 M HCl solutions at 0°C and stabilized at 2°C overnight. The sample vials containing approx. 0.5 ml of the co-polymer solution were placed in a water-bath and heated slowly from 0 to 70°C. The samples were equilibrated for 10 min at 2°C intervals. The sol–gel transition was determined by inverting the vial [53].
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2.5. Rheology A dynamic mechanical analyzer (Bohlin Rotational Rheometer) was used to determine the change in the viscosity of the co-polymer aqueous solutions. Oscillation mode with a controlled stress of 0.4 Pa and a frequency of 1 rad/s was performed. The heating rate was 1°C/min. A co-polymer solution in PBS was placed between a 20 mm diameter plate and a 100 mm diameter plate with a gap of 250 µm and the test was performed [53]. 2.6. In Vitro Cytotoxicity of [PEG–PEU]x Co-Polymer Hydrogel The in vitro cytotoxicity of co-polymer hydrogels was examined according to the ISO/EN 10993 Part 5 Guidelines. These guidelines prescribe the use of the Dulbecco’s modified Eagle’s medium (DMEM) extraction test to assess the possible toxic effects of the components released from the medical polymers during extraction. Different amounts (10–300 mg/ml) of co-polymer were extracted at 37°C for 24 h using the DMEM culture medium as the extraction fluid. After incubation, the extracts were filtered (0.2 µm pore size; Advantec MFS) and 1 ml of each extract was added to L929 fibroblast cells which had been seeded in 24-well plates. Fresh DMEM was used as a negative control. After 48 h incubation, the cell viability and proliferation were determined using an MTT assay. Briefly, 100 µl fresh growth medium containing 50 µg MTT was added to each well and the cells were incubated at 37°C for 4 h. The absorbance at 570 nm (SpectraMax® M5 Microplate Reader, Molecular Devices) was directly proportional to the number of living cells. The percentage survival relative to the mock-treated cells (100% survival) was calculated [15, 35]. 2.7. In Vivo Experiments Male Sprague–Dawley (SD) rats (Hanlim Experimental Animal Laboratory) were used for the in vivo experiments. The rats (5–6 weeks old, average body weight 200 g) were handled in accordance with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals (NIH publication 85-23, revised 1985). To examine the injectability, in vivo gelation and gel stability, an aqueous solution (200 µl, 20 wt%) of B-20-1 co-polymer at pH 6.6 was injected subcutaneously into the back of the male SD rats. After a predetermined time, the rats were killed and the gel morphology was observed [53]. 2.8. In Vitro Release of DOX from the Hydrogel The in vitro release of DOX, an anticancer drug, using this co-polymer hydrogel was examined to assess its potential applications in drugs/proteins delivery. Co-polymer solutions (20 wt% at pH 6.6) were prepared and DOX at a final concentration of 200 or 50 µg/ml was added and stirred at 2°C for 12 h. The pH of the solutions was adjusted to 7.4 with small amounts of 5 M NaOH and 0.5 ml of the DOX-loaded co-polymer solution was placed into 4-ml vials and incubated at 37°C for 30 min
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to allow gel formation. Subsequently, 3 ml fresh release medium (PBS buffer solution, 37°C, pH 7.4) was added to each vial, which was then incubated at 37°C and shaken at 20 rpm. At a predetermined time, 1.5 ml of the release medium was sampled and 1.5 ml of the fresh release medium was added to the vials to maintain a constant volume of release medium. The DOX concentration in the release media and standard samples was analyzed by UV-Vis spectroscopy at 495 nm. The absorbance comparison was used to calculate the DOX concentration and accumulative release. A standard line was determined using different DOX concentrations ranging from 50 to 0.01 µg/ml [54–57]. 3. Results and Discussion 3.1. Synthesis and Characterization of [PEG–PEU]x Multiblock Co-Polymers The novel biodegradable [PEG–PEU]x was synthesized by polyaddition between the isocyanate groups of HDI and the hydroxyl groups at the ends of PEG and the synthesized monomer BTB (or ETE) in chloroform in the presence of dibutyltin dilaurate as a catalyst (Scheme 1). The monomers (BTB or ETE) were synthesized by the Michael-addition reaction between the secondary amine of TMDP and the vinyl groups of HBA or HEA. Figure 1a shows the 1 H-NMR spectrum with the labeled protons of the synthesized monomer BTB. The protons at 2.57–2.94 ppm (peaks e and f) were assigned to new methylene protons, which were born from the Michael-addition reaction. The presence of HBA and TMDP in the monomer was confirmed by peaks a, d and g, respectively. Figure 1b shows the 1 H-NMR spectrum with the labeled protons of the copolymer B-20-1. The protons at 3.56–3.76 ppm (peak a) were assigned to the methylene protons of PEG. The protons at 3.07–3.22 ppm (peak b) and 1.28– 1.56 ppm (peaks c and d) were assigned to the first, second and third methylene protons of HDI in the co-polymer, respectively. Peaks e, h, g and i confirmed the presence of the synthesized monomer BTB in the B-20-1 co-polymer. The relative peak area of peaks a (PEG) and e (BTB) in Fig. 1b was used to calculate the fraction of PEU segments in the synthesized co-polymers. FT-IR spectroscopy was used to confirm the formation of the [PEG–PEU]x copolymer structure. Figure 2 shows the FT-IR spectrum of the B-20-1 co-polymer. As shown in Fig. 2a, the C–O–C ether stretching vibration of PEG showed peaks at 1104 cm−1 (#1). The peak at 3335 cm−1 (#2) was assigned to the N–H stretching band for the formation of urethane groups. The carbonyl stretching vibration of the ester groups in monomer BTB and formation urethane groups were indicated at 1675–1725 cm−1 (#3). The isocyanate groups were reacted completely because of the absence of peaks at 2267 cm−1 (#4). The carbonyl groups with and without hydrogen bonding showed peaks at 1680 and 1720 cm−1 , respectively, as shown in Fig. 2b, further demonstrating the formation of functional urethane groups [50]. The NMR and FT-IR spectra confirmed the formation of a co-polymer structure. In addition, the molecular weight of the [PEG–PEU]x co-polymers and their distribu-
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(a)
(b)
Figure 1. 1 H-NMR spectra of (a) monomer BTB and (b) co-polymer B-20-1.
tion were determined by GPC. Figure 3 shows the GPC traces of co-polymer B-20-1 and PEG (Mn = 2000) for comparison. These characterizations clearly demonstrate the successful synthesis of the [PEG–PEU]x co-polymers. Table 1 lists the characteristics of the synthesized co-polymer. 3.2. Sol–Gel Phase Transition Diagram of [PEG–PEU]x Multiblock Co-Polymers The tube inversion method was used to determine the sol–gel phase transition diagram of the co-polymer aqueous solutions. The sol–gel phase transition of the co-polymer solutions showed the dependence on temperature and pH, as shown in Fig. 4. For example, with co-polymer B-20-1 (Fig. 4a), at room temperature (20°C), a co-polymer solution existed at low pH (i.e., pH 6.6) but it turned into a gel at high pH (i.e., pH 7.4). This is because the tertiary amine groups in the co-polymer were ionized and showed hydrophilic properties at low pH but were deionized and showed hydrophobic properties at high pH. At a basic pH (i.e., 7.4), a gel existed at low temperatures (5°C) due to the completed deionization of the tertiary amine groups as well as the strong hydrophobic interactions and hydrogen bonds between the deionized PEU segments [43, 56]. With increasing temperature, a gel-to-sol
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(a)
(b)
Figure 2. FT-IR spectrum of (a) co-polymer B-20-1 (#1, ether groups of PEG stretching, 1104 cm−1 ; #2, –NH– stretching vibration of urethane groups, 3334 cm−1 ; #3, stretching vibration of the carbonyl group of the ester and the urethane groups, 1720 cm−1 ; #4, disappearance of the –NCO stretching vibration, 2267 cm−1 ) and (b) stretching vibration of the functional carbonyl of the urethane groups.
Figure 3. GPC traces of PEG (Mn = 2000) and co-polymer B-20-1.
transition occurred at high temperature (62°C) due to the network breakage caused by partial dehydration of PEG and breakage of hydrogen bonds between urethane
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Table 1. Characteristics of the synthesized [PEG–PEU]x multiblock co-polymers Sample
PEG
Co-polymer composition ([PEG–PEU]x or [EGn –EUm ]x )
z value
PEU (wt%)
Mn
PDI
E-20-1 B-20-1 B-20-2 B-46-1
2000 2000 2000 4600
[EG45 –E-EU10 ]1.5 [EG45 –B-EU9 ]1.6 [EG45 –B-EU6 ]2.0 [EG104 –B-EU9 ]1.4
1 2 2 2
75.3 75.0 66.6 56.6
12 400 12 800 11 900 14 300
1.77 1.76 1.78 1.69
PEG was provided by Sigma-Aldrich and the wt% PEU was calculated from 1 H-MNR. The copolymer composition, Mn and PDI were measured and calculated from GPC. n, number of repeating units of PEG; m, number of repeating units of the PEU segment.
(a)
(b)
Figure 4. Sol–gel phase transition diagram of the [PEG–PEU]x multiblock co-polymer hydrogels: (a) effect of the PEG/PEU ratio, monomer structure and PEG molecular weight (20 wt%) and (b) different co-polymer B-20-1 concentration. This figure is published in colour in the online edition of this journal, which can be accessed via http://www.brill.nl/jbs
groups in PEU block at high temperatures [43, 44, 56]. Figure 5 shows a schematic diagram for the sol–gel transition mechanism of [PEG–PEU]x hydrogels and the in
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(a)
(b)
Figure 5. (a) Schematic showing the sol–gel phase transition of [PEG–PEU]x hydrogels and (b) photographs of in vitro sol–gel transition upon heating and increasing pH (20 wt% B-20-1): (1) at 20°C, pH 6.6 and (2) at 37°C, pH 7.4. This figure is published in colour in the online edition of this journal, which can be accessed via http://www.brill.nl/jbs
vitro sol–gel phase transition with increase in pH and temperature. A co-polymer solution existed in the sol state (Fig. 5b1) at room temperature and mildly acidic pH (20°C, pH 6.6) due to the hydrophilic character of ionized PEU segments. However, a gel (Fig. 5b2) formed at the physiological conditions (37°C, pH 7.4) due to strongly hydrophobic interaction and hydrogen bonding between PEU segments. Figure 4a shows the effect of the PEU block length (B-20-1 and B-20-2), monomer structure (B-20-1 and E-20-1) and PEG molecular weight (B-20-1 and B-46-1) on the sol–gel phase transition. When the PEU block length was increased, the gel region became broader and shifted to the lower pH due to an increase in the number of hydrophobic interactions and hydrogen bonds between the long PEU segments. The gel region of co-polymer B-20-1 was wider and shifted to a lower pH compared to co-polymer E-20-1 because of the higher hydrophobicity of BTB than that of ETE. At a fixed PEU segment (approx. 6000, 9 repeat units), the gel region became broader and shifted to the lower pH and higher temperature when the PEG molecular weight was increased. This is due to an increase in micelle size of the co-polymer [PEG–PEU]x with the longer PEG segments, triggering the formation of bridged micelles as well as the gels [43, 58].
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Figure 4b shows the influence of the co-polymer concentration on the sol–gel phase transition diagram (co-polymer B-20-1). The gel region shifted to a higher pH and became narrower when the co-polymer concentration decreased. This can be explained by a decrease in the density of hydrogen bond and hydrophobic interactions between the PEU segments. Moreover, at the same pH, the gel region became broader with increasing co-polymer concentration because of the loose order of the gel at low concentrations [57]. 3.3. Rheological Property The change in viscosity of the co-polymer aqueous solution with temperature was examined by dynamic rheological analysis. The sol–gel phase transition of the copolymer can be determined by examining the change in viscosity of the co-polymer solution. Figure 6 shows the change in viscosity of the co-polymer B-20-1 solutions (20 wt%) at different pH as a function of temperature. At pH 6.6, the co-polymer solution showed low viscosity (2000 Pa·s) [43, 50]. With increasing temperature, the viscosity increased slowly due to the increasing hydrophobicity of the PEU segments. However, with further increases in temperature, the viscosity decreased rapidly (at 62°C) due to aggregation of the PEU segments, indicating a gel-to-sol transition [50, 53]. A similar rule was observed with the co-polymer solution at pH 7.0 and 7.2. The gel-to-sol transition temperatures in the viscosity method confirmed the accuracy of the tube inversion method. The red-dashed line in Fig. 6 was used to estimate the change in viscosity in in vivo applications. The co-polymer solution at pH 6.6 and 20°C with low viscosity (from A, approx. 10 Pa·s) was injected into the body and the viscosity
Figure 6. Change in viscosity of a 20 wt% B-20-1 co-polymer aqueous solution as a function of temperature at different pH. This figure is published in colour in the online edition of this journal, which can be accessed via http://www.brill.nl/jbs
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Figure 7. In vitro cytotoxicity of B-20-1 hydrogel (error bars represent SD, n = 3).
increased due to the change in pH and temperature under physiological conditions (to B, approx. 104 Pa · s). 3.4. In Vitro Cytotoxicity of [PEG–PEU]x Hydrogel The in vitro cytotoxicity of this co-polymer hydrogel was examined according to the ISO/EN 10993 Part 5 Guidelines [15, 35]. This test was designed to determine the possible cytotoxic effects of the materials extracted from biomaterials implanted in the body. Various amounts (10–300 mg/ml) of co-polymer were incubated for 24 h at 37°C in DMEM to obtain the extracts. The fibroblast cells L929 were exposed to the extracts for 48 h and the cytotoxic effects were evaluated. Fresh DMEM was used as a negative control. The co-polymer hydrogels did not show serious cytotoxicity up to 300 mg/ml, as shown in Fig. 7. The cell viability was >90% and >80% with co-polymer concentrations of 200 and 300 mg/ml, respectively. This confirms the non-toxicity of this co-polymer hydrogels and their potential applications as a biocompatible material. 3.5. In Vivo Gel Formation and Gel Stability The co-polymer aqueous solution (B-20-1, 200 µl, 20 wt%, pH 6.6) was injected subcutaneously into the back of male SD rats to examine the injectability, in vivo gelation and gel stability in the body. At a designated time, the rats were killed and the morphology of the gel was observed. Figure 8 shows the in situ formed gels at different times after the injection. The gel can be formed in a short time (10 min) due to a change in the pH and temperature under physiological conditions. The formed gels maintained inside the rats for more than 2 weeks with some decrease in size due to the degradation of the co-polymer and the erosion of degraded products [53]. This demonstrates the injectable and biodegradable properties of this hydrogel. 3.6. In Vitro Release of DOX from the [PEG–PEU]x Hydrogel The potential application of this hydrogel as a drug/protein carrier was assessed by examining the in vitro release of DOX, an anticancer drug, from this hydrogel
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Figure 8. Photographs of the in situ gel and in vivo stability of the gels at different times after being injected subcutaneously into SD rats (20 wt%, pH 6.6, B-20-1). This figure is published in colour in the online edition of this journal, which can be accessed via http://www.brill.nl/jbs
Figure 9. In vitro release of DOX from B-20-1 hydrogel (20 wt%) with different concentrations of DOX-loaded (error bars represent SD, n = 3). This figure is published in colour in the online edition of this journal, which can be accessed via http://www.brill.nl/jbs
with different DOX-loaded concentrations. The cumulative release of DOX from this hydrogel was controlled for more than 7 days, as shown in Fig. 9. The release profiles of DOX from the hydrogels with different DOX-loading concentration (50 or 200 µg/ml) were similar, indicating that the concentration of DOX loaded in the hydrogel did not affect the release profiles. Therefore, degradation of the copolymer hydrogel can control the release of DOX. 4. Conclusion A series of novel biodegradable and pH/temperature-sensitive [PEG–PEU]x multiblock co-polymer hydrogels was synthesized. The synthesized co-polymers were
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characterized by NMR, Fourier transform infrared spectroscopy and gel-permeation chromatography. The co-polymers aqueous solutions showed a sol-to-gel phase transition with increasing pH and a gel-to-sol phase transition with increasing temperature, and the gel window covered the physiological conditions (37°C, pH 7.4). The gel region could be controlled by changing the monomer structure, PEG molecular weight, co-polymer solution concentration and PEG/PEU ratio. After a subcutaneous injection of a co-polymer solution into SD rat, in situ a gel formed rapidly that remained in the body for more than 2 weeks. The in vitro cytotoxicity test confirmed the non-cytotoxicity of this co-polymer hydrogel. The in vitro release of DOX from this hydrogel was controlled for more than 7 days. These results confirmed that this novel biodegradable co-polymer hydrogel is a potential candidate for biomedical and drug/protein-delivery applications. Acknowledgement This study was supported by the Basic Science Research Program through a National Research Foundation of Korea (NRF) grant funded by the Korean Government (MEST) (2010-0027955). References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
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