Preparation of thermo-responsive graft copolymer by using a novel macro-RAFT agent and its application for drug delivery

Materials Science and Engineering C 62 (2016) 45–52

Contents lists available at ScienceDirect

Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Preparation of thermo-responsive graft copolymer by using a novel macro-RAFT agent and its application for drug delivery Cunfeng Song a, Shirong Yu a, Cheng Liu a,b, Yuanming Deng a,b, Yiting Xu a,b, Xiaoling Chen c,⁎, Lizong Dai a,b,⁎ a b c

Department of Materials Science and Engineering, College of Materials, Xiamen University, Xiamen 361005, China Fujian Provincial Key Laboratory of Fire Retardant Materials, Xiamen University, Xiamen 361005, China Department of Endodontics, Xiamen Stomatology Hospital, Teaching Hospital of Fujian Medical University, Xiamen 361003, China

a r t i c l e

i n f o

Article history: Received 10 September 2015 Received in revised form 19 November 2015 Accepted 11 January 2016 Available online 12 January 2016 Keywords: Graft copolymer Macro-RAFT agent Self-assembly Thermo-response Drug delivery

a b s t r a c t A methodology to prepare thermo-responsive graft copolymer by using a novel macro-RAFT agent was proposed. The macro-RAFT agent with pendant dithioester (ZC(S)SR) was facilely prepared via the combination of RAFT polymerization and esterification reaction. By means of ZC(S)SR-initiated RAFT polymerization, the thermoresponsive graft copolymer consisting of poly(methyl methacrylate-co-hydroxylethyl methacrylate) (P(MMAco-HEMA)) backbone and hydrophilic poly(N-isopropylacrylamide) (PNIPAAm) side chains was constructed through the “grafting from” approach. The chemical compositions and molecular weight distributions of the synthesized polymers were respectively characterized by 1H nuclear magnetic resonance (1H NMR) and gel permeation chromatography (GPC). Self-assembly behavior of the amphiphilic graft copolymers (P(MMA-co-HEMA)g-PNIPAAm) was studied by transmission electron microscopy (TEM), dynamic light scattering (DLS) and spectrofluorimeter. The critical micelle concentration (CMC) value was 0.052 mg mL − 1. These micelles have thermo-responsibility and a low critical solution temperature (LCST) of 33.5 °C. Further investigation indicated that the guest molecule release property of these micelles, which can be well described by a firstorder kinetic model, was significantly affected by temperature. Besides, the micelles exhibited excellent biocompatibility and cellular uptake property. Hence, these micelles are considered to have potential application in controlled drug delivery. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Graft copolymer, containing a linear polymer backbone and densely grafted side chains, has fascinating properties such as wormlike conformation, compact molecular dimension and notable chain end effects [1]. Due to these properties, graft copolymers are attractive in various fields, such as biomedicine [2–5], supersoft elastomers [6], and photonics [7,8]. Typically, graft copolymers can be synthesized by living polymerization, including living anionic polymerization and controlled/living polymerization [9,10]. Controlled/living polymerization techniques such as atom transfer radical polymerization (ATRP), single electron transfer living radical polymerization (SET-LRP), living ring-open polymerization (ROP), ring-open metathesis polymerization (ROMP) and reversible addition–fragmentation chain transfer (RAFT) polymerization have been extensively studied. Generally, by using controlled/living polymerization techniques, there are three major routes to prepare graft copolymer: (1) grafting onto [11–15]; (2) grafting through [16–18]; (3) grafting from [19–22]. The “grafting onto” method is to attach the pre-prepared side chains

⁎ Corresponding authors. Tel.: +86 592 2186178; fax: +86 592 2183937. E-mail addresses: [email protected] (X. Chen), [email protected] (L. Dai).

http://dx.doi.org/10.1016/j.msec.2016.01.026 0928-4931/© 2016 Elsevier B.V. All rights reserved.

onto the backbone by coupling reaction. Unfortunately, the coupling efficiency is usually insufficient [23]. The “grafting through” strategy is to obtain graft copolymer via the polymerization of macromonomers. The resulting graft copolymer always possesses a broad chain-length distribution and low molecular weight [24]. The lately arisen “grafting from” approach utilizes the pendant initiating groups on the backbone to initiate the polymerization of side chains. The gradual growth of side chains can effectively decrease the steric effect and achieve a higher grafting density. Comparing with the former two methods, “grafting from” is a particularly attractive procedure to synthesize well-defined graft copolymer. The initiation-group-containing macromolecule, i.e. macroinitiator and macro-chain transfer agent, which plays a key role in “graft from” approach, can be obtained directly from the initiation-group-containing monomer or by the introduction of initiation functional groups to the backbone. In 1997, Grubbs et al. firstly reported the application of controlled/living polymerization techniques for the preparation of graft and dendrigraft systems [25]. The linear polymer backbone was constituted by styrene and p-(4′-chloromethylbenzyloxymethyl)styrene, which contained the chloromethyl groups as latent ATRP-initiating sites. Without further chemical modification, the side chains consisting of styrene, methyl methacrylate, or n-butyl methacrylate were polymerized directly onto the backbone. Whereafter, a new synthetic strategy for

46

C. Song et al. / Materials Science and Engineering C 62 (2016) 45–52

preparing the graft copolymers by ATRP was proposed, with which 2bromoisobutyryl bromide (BriBuBr) or 2-bromopropionyl bromide coupling onto the backbone was used to initiate the side chains [26–29]. Beers et al. prepared the macroinitiator, poly(2-(2-bromoisobutyryloxy)ethyl methacrylate) via the conjugation of BriBuBr with the precursor (poly(2-trimethylsilyoxyethyl methacrylate)) [30]. Taking advantage of this strategy, a series of graft copolymers with narrow molecular weight distributions were obtained. Similarly, hydroxyl-containing poly(6methyl-1,2-heptadiene-4-ol) (PMHDO) backbone was synthesized by Lu et al. [31]. Through the treatment with 2-chloropropiony chloride to provide the PMHDO-Cl macroinitiator, poly(2(dimethylamino)ethyl acrylate) side chains were constructed by SET-LRP. Additionally, Zhang et al. and Li et al. studied the degradable poly(Ɛ-caprolactone) grafting to hydroxyl-containing polymer backbone by ROP of Ɛ-caprolactone [32,33]. RAFT polymerization possessed the advantages that can be applied to a wide variety of monomers and avoided the contamination of transition metal catalyst [34,35], whereas, preparation of graft copolymer by using the macro-chain transfer agent (so called macro-RAFT agent) for RAFT polymerization has barely been explored. Recently, Tang et al. presented the Menschutkin reaction to introduce tunable macro-RAFT agent which underwent the quaternization between the N,Ndimethylaminoethyl methacrylate unit and 3-bromopropyl 4(benzodithioyl)-4-cyanopentanoate [36]. This graft copolymer has been continuously researched as controlled delivery vehicles. However, the biological toxicity of quaternary ammonium salt in metabolic process cannot be ignored [37]. In this work, we proposed a methodology to prepare graft copolymer using a novel macro-RAFT agent. As shown in Fig. 1, the RAFT polymerization of MMA and HEMA was carried out to obtain the polymer backbone (P(MMA-co-HEMA)). Subsequently, the esterification reaction with 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPAD) facilely converted the copolymer into macro-RAFT agent. Finally, NIPAAm was polymerized through the “grafting from” approach to obtain amphiphilic graft copolymer (P(MMA-co-HEMA)-g-PNIPAAm). As we know, PNIPAAm is a thermo-responsive polymer, which can exhibit a reversible phase transition at a lower critical solution temperature (LCST) of ~ 32 °C in aqueous solutions [38]. So it is necessary to study the thermoresponsive property of P(MMA-co-HEMA)-g-PNIPAAm micelles, and to investigate their biocompatibility and potential application in the field of controlled drug delivery.

2. Materials and methods 2.1. Materials Cumyl dithiobenzoate (CDB), 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPAD), 2-hydroxylethyl methacrylate (HEMA), N,N′dicyclohexylcarbodiimide (DCC) and 4-(dimethylamino)pyridine (DMAP) were purchased from Sigma-Aldrich. Doxorubicin (DOX) in the form of the hydrochloride salt was obtained from Aladdin Reagent Company. Methyl methacrylate (MMA) and triethylamine (TEA) were the products of Sinopharm Chemical Reagent Company. N, N′azobisisobutyronitrile (AIBN) was recrystallized from ethanol. Nisopropylacrylamide (NIPAAm) was purified by recrystallized from nhexane. Dialysis membrane (molecular weight cut-off: 8000– 12,000 g/mol) was obtained from Shanghai Chemical Reagent. 2Butanone, toluene, dimethylformamide (DMF), and dichloromethane (DCM) were dried over molecular sieve and distilled before use. All regents in analytical grade were used as received. 2.2. Polymer synthesis P(MMA-co-HEMA) backbone: the copolymer was prepared by RAFT polymerization of monomers MMA (1.2014 g, 12 mmol) and HEMA (0.5206 g, 4 mmol). The monomers with CDB (27.2 mg, 0.1 mmol) and AIBN (1.6 mg, 0.01 mmol) were dissolved in 3 mL of 2-butanone and then placed in Schlenk flask which was thoroughly deoxygenated by three consecutive freeze–pump–thaw cycles. The sealed flask was immersed in a preheated oil bath at 65 °C. After 18 h, the reaction was stopped by plunging the flask into liquid nitrogen. The copolymer was purified by precipitating in n-hexane and dried under vacuum. Macro-RAFT agent: CPAD (0.4191 g, 1.5 mmol), DCC (0.6190 g, 3 mmol) and DMAP (2.4 mg, 0.02 mmol) were dissolved in 50 mL of dry DCM. After the addition of P(MMA-co-HEMA) (1 mmol hydroxyl groups), the reaction was stirred at ambient temperature for 18 h. The reaction solution was dried on a rotary evaporator to afford the crude product. After further purification by repeated dissolving in ethyl acetate and precipitating into n-hexane, macro-RAFT agent was dried for the next RAFT polymerization. Graft copolymer (P(MMA-co-HEMA)-g-PNIPAAm): in a Schlenk flask, NIPAAm (0.5658 g, 5 mmol), macro-RAFT agent (0.1 mmol ZC(S)SR groups), and AIBN (3.2 mg, 0.02 mmol) were dissolved in 1 mL of DMF. The mixture went through three consecutive freeze–

Fig. 1. Design of macro-RAFT agent for the synthesis of P(MMA-co-HEMA)-g-PNIPAAm amphiphilic graft copolymer.

C. Song et al. / Materials Science and Engineering C 62 (2016) 45–52

47

Fig. 3. GPC traces of P(MMA-co-HEMA), macro-RAFT agent and P(MMA-co-HEMA)-gPNIPAAm.

2.3. Micelle formation P(MMA-co-HEMA)-g-PNIPAAm was dissolved in THF, and then dropwise added into distilled water to make 1 mg mL−1 copolymer solution at ambient temperature. The copolymer solution was dialyzed against distilled water, followed by filtrate through the syringe filter (0.45 μm). Nile red encapsulation: P(MMA-co-HEMA)-g-PNIPAAm with 1 wt.% of Nile red was dissolved in THF, other steps were similar with the micelles formation. Dox-loaded micelles: DOX·HCl (2.5 mg) and 3-fold molar TEA were dissolved in DMF (2 mL) and kept stirring for 2 h to remove hydrochloride. Then, P(MMA-co-HEMA)-g-PNIPAAm (10 mg) was added. Distilled water (10 mL) was added into the solution through a syringe to form micelles. This solution was dialyzed against distilled water for 24 h to remove the residual DMF and unentrapped DOX. The loading amount of DOX in micelles was determined by dissolving 10 mL of freeze-dried micelle solution into DMF before UV–Vis measurements. The drug loading content (DLC) and the entrapment efficiency (EE) were respectively calculated based on the following formulas: DLC ¼ Fig. 2. 1H NMR of (a) P(MMA-co-HEMA), (b) macro-RAFT agent and (c) P(MMA-coHEMA)-g-PNIPAAm in CDCl3.

pump–thaw cycles before heating at 65 °C for 12 h. P(MMA-co-HEMA)g-PNIPAAm was precipitated in cool diethyl ether and dried under vacuum.

EE ¼

weight of loaded drug  100%; total weight of polymer and loaded drug

weight of loaded drug  100%: weight of drug in feed

2.4. In vitro release studies The dialysis bags containing 2 mL of DOX-loaded micelles solution were respectively immersed into 50 mL of phosphate buffer at temperature below or above the LCST (25, 37, and 45 °C). 400 μL of the dialysis solution was withdrawn at predetermined time intervals and analyzed by UV–Vis spectrophotometer (SHIMADZU, UV-1750) at 480 nm.

Table 1 Synthesis of P(MMA-co-HEMA)-g-PNIPAAm amphiphilic graft copolymers.a Copolymer

Time (h)

Cov.b (%)

Mnc (g mol−1)

Mw/Mn

Sample 1 Sample 2 Sample 3 Sample 4

3 6 9 12

9.4 13.8 19.2 23.8

27,100 34,700 41,900 45,300

1.36 1.42 1.40 1.41

a Polymerization condition: [NIPAAm]0:[ZC(S)SR]:[AIBN] = 50:1:0.2, [NIPAAm]0 = 5 mol L−1 in DMF at 65 °C. b Obtained from 1H NMR (400 MHz Bruker). c Measured by GPC (Waters 1515).

2.5. Cellular cytotoxicity The cellular cytotoxicity of the micelles was tested by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HCMEC cells in RPMI 1640 and Hela cells in Dulbecco's modified eagle's medium (DMEM) were respectively seeded into a 96-well culture plate at the concentration of 5 × 103 cells/well. The cells were cultured at 37 °C in 5% CO2 atmosphere for 12 h. Whereafter, the medium was replaced with 200 μL of medium containing micelles suspension. The

48

C. Song et al. / Materials Science and Engineering C 62 (2016) 45–52

Fig 4. (a) TEM and (b) DLS of P(MMA-co-HEMA)-g-PNIPAAm micelles; (c) emission spectra of Nile red in different concentrations of P(MMA-co-HEMA)-g-PNIPAAm solution; (d) CMC of P(MMA-co-HEMA)-g-PNIPAAm.

cells were incubated for another 24 h or 48 h, and then rinsed with PBS. 20 μL of MTT solution (5 mg mL−1, AMRESCO) with 100 μL fresh medium was added to each well. After 4 h of cells incubation, the medium was removed. Then, the purple formazan crystals produced by live cells in each well were dissolved by the later addition of 100 μL of DMSO at 37 °C for 30 min. The absorbance at a wavelength of 570 nm was measured by Microplate Reader (Model 680; Bio-Rad Laboratories Richmond, CA). Cell viability was calculated by the following formula: cell viability ¼

Asample  100% Acontrol

where Asample is the absorbance of the wells in which the cells were incubated with medium containing micelles suspension, and Acontrol is the

absorbance of the wells in which the cells were incubated with medium alone.

2.6. Cellular uptake Cellular uptake was evaluated by tracing the fluorescence of Nile red-loaded micelles. Hela cells (1 × 105 cells/well) were seeded in a 6well plate covered with glasses and cultured in DMEM supplemented with 10% fetal calf serum for 12 h. Subsequently, the cells in each well were incubated with 1 mL of the pre-prepared micelles suspension (concentration was 0.5 mg mL− 1 in serum-free media) at 25 °C for 1 h. In order to avoid the possible interference which was caused by the release of Nile red in the extracellular medium at relatively high

Fig. 5. (a) LCST profile for P(MMA-co-HEMA)-g-PNIPAAm micelles detected by transmittance at 500 nm, [P(MMA-co-HEMA)-g-PNIPAAm] = 1 mg mL−1 (the heating rate was 0.5 °C min−1); (b) Z-average diameter of P(MMA-co-HEMA)-g-PNIPAAm micelles tested by DLS at various temperatures; (c) size variation of P(MMA-co-HEMA)-g-PNIPAAm micelles when alternately warming to 45 °C and cooling to 25 °C.

C. Song et al. / Materials Science and Engineering C 62 (2016) 45–52

49

Fig. 6. Cartoon illustration of guest release from P(MMA-co-HEMA)-g-PNIPAAm micelles below or above the LCST.

temperature, each well was washed with PBS for three times. After incubation for another 1 h at 37 °C, the cells were stained by 100 μL of 2-(4amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) for 10 min. The cells were fixed with 4 % formaldehyde for 30 min and then washed with PBS. The cells were observed by Leica TCS SP5 confocal laser scanning microscopy (CLSM, Leica Microsystems, Mannheim). 3. Results and discussion 3.1. Characterization of the copolymers P(MMA-co-HEMA) as the linear polymer backbone was firstly prepared by RAFT polymerization. The peaks at 3.62 ppm (m) and 4.14 ppm (p), which respectively correspond to COOCH3 of PMMA and COOCH2CH2 of PHEMA, were shown in Fig. 2a. Subsequently, PHEMA provided free hydroxyl groups for the esterification reaction with CPAD. Fig. 2b presented that the signals of the PHEMA segment at 4.14 ppm (p) and 3.86 ppm (q) were weakened, and new signals appeared at 4.34 ppm (g) and 4.20 ppm (h) due to the introduction of ZC(S)SR groups. By comparing these integrated areas, the esterification conversion was 89.3%. The peaks appeared at 7.4–8.0 ppm (PhH of CPAD) also proved that the successful synthesis of the macro-RAFT agent. Finally, hydrophilic PNIPAAm side chains were chosen for the construction of P(MMA-co-HEMA)-based amphiphilic graft

copolymers through the “grafting from” strategy. Fig. 2c showed that the peaks at 4.02 ppm (s) and 1.16 ppm (t) assigned to NHCH(CH3)2 and NHCH(CH3)2 of PNIPAAm segment, respectively. A new flat peak at around 6.55 ppm represented the proton of NHCH(CH3)2. As listed in Table 1, the molecular weights of all the graft copolymers are much higher than that of macro-RAFT agent, which indicated the occurrence of ZC(S)SR-initiated RAFT polymerization. With the extending of polymerization time, the molecular weight increases. GPC measurement using THF as eluent was employed to further characterize P(MMA-co-HEMA), macro-RAFT agent, and P(MMA-coHEMA)-g-PNIPAAm. In Fig. 3, the increase in molecular weight from P(MMA-co-HEMA) (Mn = 12,900 g mol−1, Mw/Mn = 1.07) to macroRAFT agent (Mn = 19,000 g mol−1, Mw/Mn = 1.22) could be explained that quite a lot of pendent groups attached onto the polymer backbone. The increase of the dispersity can be ascribed to the residual hydroxyl groups. Meanwhile, Fig. 3 displayed that a steep increase from macroRAFT agent to P(MMA-co-HEMA)-g-PNIPAAm (Mn = 45,300 g mol−1 Mw/Mn = 1.41). 3.2. Micelle formation In Fig. 4a, TEM (JEOL, JEM-2100) image revealed that the P(MMA-coHEMA)-g-PNIPAAm micelles were dispersed with spherical shape. PNIPAAm chains stretched to the outer layer for stabilizing the micelle, and P(MMA-co-HEMA) backbones formed the core of micelle. DLS (Malvern Nanozetasizer, ZEN-3600) analysis of the micelles showed a narrow size distribution with hydrodynamic diameter of 92 ± 6 nm and a relatively low polydispersity index (PDI) of 1.03 ± 0.02 (Fig. 4b). The micelles were found to be negatively charged with an average value −11.0 ± 0.06 mV, indicating that the micelles were stable in dispersion state [39]. Critical micelle concentration (CMC) was used to describe the selfassembly behavior of amphiphilic polymer [40]. The CMC of P(MMA-

Table 2 The correlation coefficients, kinetic rate constants, and diffusion exponents obtained after fitting in vitro drug release data with various kinetic model equations. Formulation

25 °C 37 °C 45 °C Fig. 7. Cumulative drug release of DOX-loaded P(MMA-co-HEMA)-g-PNIPAAm micelles at 25, 37, and 45 °C respectively.

Zero-order

First-order

Higuchi

R2

K0

R2

K1

R2

Kh

R2

Korsmeyer–Peppas Kp

n

0.586 0.769 0.644

0.099 0.420 0.418

0.963 0.996 0.975

0.158 0.092 0.138

0.721 0.926 0.771

1.520 5.553 6.400

0.816 0.939 0.877

0.218 0.147 0.219

0.337 0.422 0.338

R2 indicates correlation coefficient; K0, K1, Kh, and Kp correspond to kinetic rate constants of zero-order, first-order, Higuchi, and Korsmeyer–Peppas models, respectively.

50

C. Song et al. / Materials Science and Engineering C 62 (2016) 45–52

Fig. 8. First-order model fitting of the P(MMA-co-HEMA)-g-PNIPAAm micelles at 25, 37, and 45 °C respectively.

co-HEMA)-g-PNIPAAm was measured by employing Nile red as a hydrophobic fluorescence probe. Fig. 4c was the fluorescence spectra of Nile red in solutions with different concentrations of P(MMA-coHEMA)-g-PNIPAAm solution. The fluorescence emission intensity at 626 nm (λmax) versus the log of concentration was plotted in Fig. 4d. The tangent of the rapid rising curve was extrapolated to intersect with the baseline, and the x-value at this intersection was defined as the CMC (~0.052 mg mL−1). This means that the micelles were formed and stabilized even at low concentration of P(MMA-co-HEMA)-gPNIPAAm. 3.3. Thermo-response Thermo-responsive property of P(MMA-co-HEMA)-g-PNIPAAm micelles was characterized via the optical transmittance at 500 nm, as shown in Fig. 5a. When the temperature was increased to a certain value, the solution turned from clear to milky and the transmittance was abruptly decreased. The reason of this phenomenon has already been explained in previous study [41]. At temperatures below the LCST, the predominantly intermolecular hydrogen bondings between the PNIPAAm chains and the water molecules make the chains soluble. However, at temperatures above the LCST, the destruction of intramolecular hydrogen bonding between CO and N–H groups in the PNIPAAm chains result in a shrunken conformation of PNIPAAm chains, which make the chains difficult to dissolve in water. LCST was defined as the temperature where half of the optical transmittance between the values below and above transitions was reached [42]. It was found that the LCST of P(MMA-co-HEMA)-g-PNIPAAm micelles was 33.5 °C. In general, PNIPAAm homopolymer chains exhibit a phase transition at around 32 °C. A random copolymer of NIPAAm with a hydrophobic monomer decreases the LCST of PNIPAAm copolymer, while incorporation of a more hydrophilic monomer tends to have the opposite effect [43,44].

However, the LCST of P(MMA-co-HEMA)-g-PNIPAAm micelles was higher than that of PNIPAAm homopolymers. This phenomenon might because of the compact macromolecular architecture. It is also interesting to study the correlation between the size variation of P(MMA-coHEMA)-g-PNIPAAm micelles and the temperature. Fig. 5b displayed the size distribution of P(MMA-co-HEMA)-g-PNIPAAm micelles at temperatures below or above the LCST. Along with the increase of temperature, the micelles start to shrink at about 27 °C. The size of P(MMA-coHEMA)-g-PNIPAAm micelles became smaller and smaller until reaching a minimum value at about 35 °C. Contrary to the other results [45,46], no temperature-induced aggregation was observed. In Fig. 5c, it was noted that the z-average diameter of P(MMA-co-HEMA)-g-PNIPAAm micelles was 98 nm at 25 °C, and then deceased to 53 nm at 45 °C. After cooling again to 25 °C, the diameter almost reverted to the original value. The reversible size variation continuously occurred when the temperature alternately changed below or above the LCST. 3.4. In vitro release The thermo-responsive P(MMA-co-HEMA)-g-PNIPAAm micelles were investigated as carriers for loading hydrophobic drug of DOX. The DLC and EE were 13.2% and 60.8%, respectively. The release process of which was illustrated in Fig. 6. The influence of temperature on the release property was studied in detail. Fig. 7 exhibited the cumulative drug release at 25, 37, and 45 °C, respectively. When the temperature was 25 °C (below the LCST), hydrophilic PNIPAAm stabilized the micelles. DOX was released from the hydrophobic core only with an initial burst of about 10%, while most of DOX still remained in the micelles within 120 h. With the temperature above the LCST, the shrunken PNIPAAm shells accelerated the DOX release from cores. Thereby, the release rate was increased appreciably. 50%–60% of the initial drugs were sustainably released by the DOX-loaded micelles within 120 h. It

Fig. 9. Cell viability of (a) HCMEC and (b) Hela cells after incubation with P(MMA-co-HEMA)-g-PNIPAAm micelles for 24 h and 48 h.

C. Song et al. / Materials Science and Engineering C 62 (2016) 45–52

51

Fig. 10. Cell viability of (a) HCMEC and (b) Hela cells after treatment with free DOX and DOX-loaded micelles for 24 h.

could be explained that the temperature-induced variation of the core– shell structure accelerated the release of DOX. When the temperature increased from 37 °C to 45 °C, the effect became weaker. In order to investigate the drug-release kinetics mechanism of drug release from the micelles, in vitro drug release data were fitted with four different mathematical models: zero-order, first-order, Higuchi, and

Korsmeyer–peppas [47–49]. Correlation coefficients were calculated from the plots using liner regression analyses, which were listed in Table 2. A good linear relationship was observed in Fig. 8 when the data were fitted with a first-order kinetic model (0.963 ≤ R2 ≤ 0.996), which indicated that the drug release highly depends on the amount of drug in the core of the micelle structures.

Fig. 11. CLSM images of Hela cells being treated with (a) free Nile red, 1 h at 25 °C; (b) Nile red-loaded micelles, 1 h at 25 °C; (c) Nile red-loaded micelles, another 1 h at 37 °C. For each panel, the images from left to right show cell nuclei stained with DAPI (blue), Nile red (red) in cells, and overlays of the two images.

52

C. Song et al. / Materials Science and Engineering C 62 (2016) 45–52

3.5. Cellular cytotoxicity It was crucial to evaluate the potential cytotoxicity of P(MMA-coHEMA)-g-PNIPAAm micelles for biomedical application. HCMEC and Hela cells in corresponding culture medium were respectively incubated with P(MMA-co-HEMA)-g-PNIPAAm micelles. Fig. 9 presented that the viabilities of the two cells lines were above 80% even when the final micelle concentration was up to 250 mg L−1. This indicated that the P(MMA-co-HEMA)-g-PNIPAAm micelles have a good biocompatibility. Moreover, the cell viabilities at 48 h were higher than that at 24 h, which suggested that P(MMA-co-HEMA)-g-PNIPAAm micelles cannot hinder the growth of the cells. In vitro cytotoxicities of the free DOX and DOX-loaded micelles were also assessed by MTT assays. As shown in Fig. 10, both the free DOX and DOX-loaded micelles exhibited a dose-dependent cytotoxicity toward the two cells lines (HCMEC and Hela cells). When the DOX concentration was up to 0.1 mg L−1, the DOX-loaded micelles exhibited higher cytotoxicity than the free DOX. One possible reason is the fact that amount of internalized DOX-loaded micelles was larger than that of the free DOX. In addition, the sustained release of DOX-loaded micelles in cells over a long period of time was beneficial to the enhancement of the cytotoxicity. 3.6. Cellular uptake The ability of internalization into the cells is of essential importance to drug carriers. The intracellular distribution of P(MMA-co-HEMA)-gPNIPAAm micelles was showed in Fig. 11. Owing to the free Nile red in water with very low solubility, there was little Nile red in solution interacting with the cells, which resulted in fewer uptakes and almost no fluorescent image in Fig. 11a. In contrast, Fig. 11b revealed that Nile red-loaded P(MMA-co-HEMA)-g-PNIPAAm micelles were taken up by Hela cells after incubation at 25 °C for 1 h. Incubation for another 1 h at 37 °C, a diffuse localization can be clearly seen in the cytoplasm of the cells (Fig. 11c). It was demonstrated that Nile red-loaded micelles were effectively internalized into Hela cells. 4. Conclusion In this study, amphiphilic graft copolymer (P(MMA-co-HEMA)-gPNIPAAm) was prepared through the “grafting from” approach by using a novel macro-RAFT agent. 1H NMR and GPC showed that the amphiphilic graft copolymer was successfully synthesized. The micelles self-assembled from P(MMA-co-HEMA)-g-PNIPAAm show a thermoresponsibility. In addition, their size variation is reversible when the temperature alternately changes below or above the LCST. This inspired us to continuously study the drug (DOX) release behavior of the P(MMA-co-HEMA)-g-PNIPAAm micelles. These micelles exhibit temperature-induced release property, excellent biocompatibility and cellular uptake property, which open up opportunities for controlled drug delivery. Acknowledgment This work was financially supported by the National Natural Science Foundation of China (51373142, 51511130130); the Scientific and Technical Project of Fujian Province of China (2013H6019) and the Scientific and Technological Innovation Platform of Fujian Province (2014H2006); the Medical Innovation in Fujian Province (2012-CXB39) and Xiamen Benefiting the People of Science and Technology Plan Project (3502Z20154075).

References [1] C. Feng, Y. Li, D. Yang, J. Hu, X. Zhang, X. Huang, Chem. Soc. Rev. 40 (2011) 1282–1295. [2] B. Li, G. Chen, F. Meng, T. Li, J. Yue, X. Jing, Y. Huang, Polym. Chem. 3 (2012) 2421–2429. [3] A. Nese, N.V. Lebedeva, G. Sherwood, S. Averick, Y. Li, H. Gao, L. Peteanu, S.S. Sheiko, K. Matyjaszewski, Macromolecules 44 (2011) 5905–5910. [4] K. Yao, Y. Chen, J. Zhang, C. Bunyard, C. Tang, Macromol. Rapid Commun. 34 (2013) 645–651. [5] J.B. Matson, R.H. Grubbs, J. Am. Chem. Soc. 130 (2008) 6731–6733. [6] K. Mukumoto, S.E. Averick, S. Park, A. Nese, A. Mpoukouvalas, Y. Zeng, K. Koynov, P.R. Leduc, K. Matyjaszewski, Macromolecules 47 (2014) 7852–7857. [7] L. Zhao, M. Byun, J. Rzayev, Z. Lin, Macromolecules 42 (2009) 9027–9033. [8] J. Rzayev, Macromolecules 42 (2009) 2135–2141. [9] S. Ito, R. Goseki, T. Ishizone, A. Hirao, Polym. Chem. 5 (2014) 5523–5534. [10] N. Hadjichristidis, H. Iatrou, M. Pitsikalis, J. Mays, Prog. Polym. Sci. 31 (2006) 1068–1132. [11] H. Gao, K. Matyjaszewski, J. Am. Chem. Soc. 129 (2007) 6633–6639. [12] A.C. Engler, H.I. Lee, P.T. Hammond, Angew. Chem. Int. Ed. 48 (2009) 9334–9338. [13] Y. Li, Y. Zhang, D. Yang, Y. Li, J. Hu, C. Feng, S. Zhai, G. Lu, X. Huang, Macromolecules 43 (2010) 262–270. [14] Q. Fu, W. Lin, J. Huang, Macromolecules 41 (2008) 2381–2387. [15] Y.Y. Durmaz, V. Kumbaraci, A.L. Demirel, N. Talinli, Y. Yagci, Macromolecules 42 (2009) 3743–3749. [16] L. Petton, E.P.C. Mes, H. Van Der Wal, S. Claessens, F. Van Damme, S. Verbrugghe, F.E.D. Prez, Polym. Chem. 4 (2013) 4697–4709. [17] C. Weber, C. Remzi Becer, W. Guenther, R. Hoogenboom, U.S. Schubert, Macromolecules 43 (2010) 160–167. [18] K. Yamada, M. Miyazaki, K. Ohno, T. Fukuda, M. Minoda, Macromolecules 32 (1999) 290–293. [19] Z. Zheng, J. Ling, A.H. Muller, Macromol. Rapid Commun. 35 (2014) 234–241. [20] X. Song, W. Yao, G. Lu, Y. Li, X. Huang, Polym. Chem. 4 (2013) 2864–2875. [21] A. Touris, K. Kostakis, S. Mourmouris, V. Kotzabasakis, M. Pitsikalis, N. Hadjichristidis, Macromolecules 41 (2008) 2426–2438. [22] G.L. Morandi, S. Pascual, V.R. Montembault, S.P. Legoupy, N. Delorme, L. Fontaine, Macromolecules 42 (2009) 6927–6931. [23] M. Zhang, A.H.E. Müller, J. Polym. Sci. A Polym. Chem. 43 (2005) 3461–3481. [24] S.H. Lahasky, L. Lu, W.A. Huberty, J. Cao, L. Guo, J.C. Garno, D. Zhang, Polym. Chem. 5 (2014) 1418–1426. [25] R.B. Grubbs, C.J. Hawker, J. Dao, J.M.J. Frechet, Angew. Chem. Int. Ed. 36 (1997) 270–272. [26] M. Wang, S. Zou, G. Guerin, L. Shen, K. Deng, M. Jones, G.C. Walker, G.D. Scholes, M.A. Winnik, Macromolecules 41 (2008) 6993–7002. [27] I. Javakhishvili, M. Tanaka, K. Ogura, K. Jankova, S. Hvilsted, Macromol. Rapid Commun. 33 (2012) 319–325. [28] G. Cheng, A. Böker, M. Zhang, G. Krausch, A.H.E. Müller, Macromolecules 34 (2001) 6883–6888. [29] H.G. Börner, K. Beers, K. Matyjaszewski, S.S. Sheiko, M. Möller, Macromolecules 34 (2001) 4375–4383. [30] K.L. Beers, S.G. Gaynor, K. Matyjaszewski, S.S. Sheiko, M. Möller, Macromolecules 31 (1998) 9413–9415. [31] G. Lu, Y. Li, H. Guo, W. Du, X. Huang, Polym. Chem. 4 (2013) 3132–3139. [32] J.-F. Zhang, W.-M. Ren, X.-K. Sun, Y. Meng, B.-Y. Du, X.-H. Zhang, Macromolecules 44 (2011) 9882–9886. [33] S. Li, C. Ye, G. Zhao, M. Zhang, Y. Zhao, J. Polym. Sci. A Polym. Chem. 50 (2012) 3135–3148. [34] M. Benaglia, E. Rizzardo, A. Alberti, M. Guerra, Macromolecules 38 (2005) 3129–3140. [35] S. Wang, Z. Cheng, J. Zhu, Z. Zhang, X. Zhu, J. Polym. Sci. A Polym. Chem. 45 (2007) 5318–5328. [36] D. Tang, X. Jiang, H. Liu, C. Li, Y. Zhao, Polym. Chem. 5 (2014) 4679–4692. [37] B. Dizman, M.O. Elasri, L.J. Mathias, Macromolecules 39 (2006) 5738–5746. [38] D. Schmaljohann, Adv. Drug Deliv. Rev. 58 (2006) 1655–1670. [39] K.Y. Win, S.-S. Feng, Biomaterials 26 (2005) 2713–2722. [40] C.J. Chen, G.Y. Liu, Y.T. Shi, C.S. Zhu, S.P. Pang, X.S. Liu, J. Ji, Macromol. Rapid Commun. 32 (2011) 1077–1081. [41] C.S. Chaw, K.W. Chooi, X.M. Liu, C.W. Tan, L. Wang, Y.Y. Yang, Biomaterials 25 (2004) 4297–4308. [42] P. Sun, Y. Zhang, L. Shi, Z. Gan, Macromol. Biosci. 10 (2010) 621–631. [43] K.S. Soppimath, D.W. Tan, Y.Y. Yang, Adv. Mater. 17 (2005) 318–323. [44] S. Kim, K.E. Healy, Biomacromolecules 4 (2003) 1214–1223. [45] C. Chang, H. Wei, C. Quan, Y. Li, J. Liu, Z. Wang, S. Cheng, X. Zhang, R. Zhou, J. Polym. Sci. A Polym. Chem. 46 (2008) 5318–5328. [46] H. Wei, X. Zhang, W. Chen, S. Cheng, R. Zhou, J. Biomed. Mater. Res. A 83A (2007) 980–989. [47] S.A. Bravo, M.C. Lamas, C.J. Salomon, J. Pharm. Pharm. Sci. 5 (2002) 213–219. [48] W.I. Higuchi, J. Pharm. Sci. 56 (1967) 315–324. [49] R.W. Korsmeyer, R. Gurny, E. Doelker, P. Buri, N.A. Peppas, Int. J. Pharm. 15 (1983) 25–35.