Polymer Journal (2013) 45, 962–970 & 2013 The Society of Polymer Science, Japan (SPSJ) All rights reserved 0032-3896/13 www.nature.com/pj ORIGINAL ARTICLE Synthesis of diblock functional poly(e-caprolactone) amphiphilic copolymers grafted with bioactive molecules and characterization of their micelles Yi-Ting Huang1, Kang-Yu Peng2, Fang-Chyou Chiu1 and Ren-Shen Lee2 In this study, the grafting of an alkynyl bioactive compound to poly(a-azo-e-caprolactone)-b-poly(e-caprolactone) (PaN3CL-b- PCL) was performed using Huisgen’s 1,3-dipolar cycloaddition, also known as click chemistry. The grafted copolymers were successfully obtained at various ratios, as confirmed by nuclear magnetic resonance, gel permeation chromatography and Fourier transform infrared spectroscopy. The graft-block poly(a-azo-e-caprolactone-graft-bioactive molecule) ((PaN3CL-g-BioM)- b-PCL) copolymers were semicrystalline, with the melting temperature (Tm) depending on the type and the amount of grafting compounds. Grafting of 1-dimethylamino-2-propyne, pent-4-ynyl nicotinate and propargyl N-benzyloxycarbonyl-4-hydroxy prolinate onto the PaN3CL-b-PCL caused these amphiphilic copolymers to self-assemble into micelles in the aqueous phase. The critical micelle concentration (CMC) ranged from 4.6 to 20 mg l À1, and the average micelle size ranged from 105 to 162 nm. The hydrophilicity and the unit of the grafting compounds influenced the stability of the micelle. This study describes the drug-entrapment efficiency and drug-loading content of the micelles, which were dependent on the composition of the graft- block polymers. The results from in vitro cell viability assays showed that (PaN3CL-g-BioM)-b-PCL possessed low cytotoxicity. Polymer Journal (2013) 45, 962–970; doi:10.1038/pj.2012.233; published online 6 March 2013 Keywords: amphiphilic; bioactive molecules; click chemistry; graft-block functional polyester; micelles INTRODUCTION ionic strength, temperature and sample preparation, may also influ- Over the past decade, an increasing amount of attention has been ence aggregation mechanisms.11 Hence, the macromolecular structure paid to environmentally friendly thermoplastics and biomaterials.1,2 of block copolymer aggregates relatively predetermines their nano- Aliphatic polyesters, such as poly(glycolide), poly(lactide) and poly scale morphology.12,13 The hydrophilic–hydrophobic balance of (e-caprolactone) (PCL) combine biodegradability and biocompati- diblock copolymers can be potentially modified by coupling them bility, and are produced on an industrial scale. Nevertheless, a lack of with additional hydrophilic or hydrophobic moieties.14,15 pendent functional groups along these polyester chains is a major Recently, the possibility of grafting functional groups onto the limitation to their potential applications.3 The introduction of chain of PCL through Huisgen’s 1,3-dipolar cycloaddition, known as pendent functional groups along these polyester chains is highly ‘click chemistry,’ was reported. This reaction has received significant desirable to tailor and modulate their physicochemical properties, attention because of its feasibility and mild conditions. Recent such as hydrophilicity, biodegradation rate, bioadhesion, crystallinity research has extensively examined copper-catalyzed azid-alkyne and biological activity.4–6 cycloadditions in biological and material sciences. Riva et al.,16,17 Amphiphilic block copolymers are known for their self-assembly Zednik et al.,18 Suksiriworapong et al.19 and other reports20,21 have into micelles or larger aggregates in solvents that are selective for one shown that small functional groups (for example, benzoate, triethyl block. In aqueous solutions, a core-shell structure commonly ammonium bromide, nicotinic acid, p-aminobenzoic acid and forms consisting of a hydrophobic core surrounded by a hydrated hexyne) and macromolecules (such as poly(ethylene oxide)) could hydrophilic shell. Previous research has extensively studied the be successfully grafted onto a polymer backbone using a click properties of block copolymer micelles in biomedical applications, reaction. Although numerous attempts have been made to attach namely the efficacious delivery of hydrophobic drugs sequestered either functional groups or drug molecules onto polymer backbones, within micellar cores.7–10 few studies have been concerned with attaching drugs onto polyester The parameter that principally governs the self-organization of backbones, in particular PCL.22,23 amphiphilic block copolymers is the hydrophilic–hydrophobic bal- This study investigates using copper-catalyzed azid-alkyne cycload- ance, although other experimental factors, such as concentration, pH, dition as a tool for varying the hydrophilic–hydrophobic balance of 1Department of Chemical and Material Engineering, Chang Gung University, Taoyuan, Taiwan and 2Center of General Education, Chang Gung University, Taoyuan, Taiwan Correspondence: Dr R-S Lee, Center of General Education, Chang Gung University, 259 Wen-Hwa 1st Road, Kwe-Shan, Taoyuan 333, Taiwan. E-mail: [email protected] Received 28 September 2012; revised 15 November 2012; accepted 21 November 2012; published online 6 March 2013 Graft bioactive molecules polyester Y-T Huang et al 963 diblock copolymers in an aqueous medium. This approach ‘clicks’ a nicotinate (PNIC) and propargyl N-benzyloxycarbonyl-4-hydroxy prolinate model AB amphiphilic block copolymer, composed of a PCL (PBHP) were prepared as reported previously.19 Organic solvents such as hydrophobic segment and a poly(a-azo-e-caprolactone-graft-bioactive tetrahydrofuran, methanol, chloroform, toluene, N,N-dimethylformamide and n-hexane were high-pressure liquid chromatography grade and were purchased molecule) (PaN3CL-g-BioM) hydrophilic segment, with additional hydrophilic BioM (Scheme 1). The nitrogen atoms in the grafted from Merck Chemical (Darmstadt, Germany). Ultrapure water was obtained by purification with a Milli-Q Plus system (Waters, Milford, MA, USA). BioM serve as bases and hydrogen bond acceptors. The grafting of BioM onto AB amphiphilic block PCL copolymers has not been reported previously. This work studies the influence of the hydro- Typical click reaction philic/hydrophobic chain lengths of the block copolymers, and the PaN3CL38-b-PCL16 (27.9 mmol, 1.06 mol equiv. of azide) was transferred into grafted BioM on micelle sizes, drug-entrapment efficiency and drug- a glass reactor containing tetrahydrofuran. The alkynyl BioM (1.06 mol), CuI loading content. Fluorescence spectroscopy, dynamic light scattering (2.8 mmol) and triethyl amine (2.8 mmol) were then added to the reactor. The solution was stirred at 60 1C for 24 or 48 h. The cold reaction product was (DLS) and transmission electron microscopy were used to evaluate precipitated in diethyl ether. The purified polymer was dried under vacuum at the micellar characteristics of these graft-block copolymers in an 50 1C for 24 h and then analyzed. Figures 1a–c show the proton nuclear aqueous solution. 1 magnetic resonance ( H-NMR) spectra of (PaN3CL-g-DMAP)-b-PCL, (PaN3CL-g-PNIC)-b-PCL and (PaN3CL-g-PBHP)-b-PCL, respectively. EXPERIMENTAL PROCEDURE Materials Characterization Benzyl alcohol, 2-chlorocyclohexanone, pyrene, 1-dimethylamino-2-propyne 1H-NMR spectra were recorded at 500 MHz (with a WB/DMX-500 spectro- (DMAP), nicotinic acid, 4-pentynl-1-ol, 1,3-dicyclohexyl-carbodiimide, meter; Bruker, Ettlingen, Germany) using chloroform (d 7.24 p.p.m.) as an 4-dimethylamino-pyridine, Z-L-4-hydroxyproline, propargyl bromide, indo- internal standard in chloroform-d (CDCl3). Thermal analysis of the polymer methacin (IMC) and sodium azide were purchased from Aldrich Chemical Co. was performed on a DuPont 9900 system using differential scanning (Milwaukee, WI, USA). m-Chloroperoxybenzoic acid was purchased from calorimetry (DuPont, Newcastle, DE, USA). The heating rate was 20 1CminÀ1. Fluka Chemical Co. (Buchs SG1, Switzerland). Stannous octoate was pur- Glass-transition temperatures (Tgs) were recorded in the middle of the chased from Strem Chemical Co. (Newburyport, MA, USA). e-Caprolactone heat capacity change and were taken from the second heating scan after was dried and vacuum-distilled over calcium hydride. a-Chloro-e-caprolactone quick cooling. Number- and weight-average molecular weights (Mn and Mw, (aClCL) was prepared using a previously reported method.24 Pent-4-ynyl respectively) of the polymers were determined by a gel permeation Cl O O SnOct2 C6H5CH2OH + O C H CH OC O H m toluene 6 5 2 m CI ε-CL O O SnOct 2 C6H5CH2OC O C O H toluene mn CI O O NaN3 C6H5CH2OC O C O H DME mn N3 O O ≡ R-CH2C CH C6H5CH2OC O C O H CuI,Et N 3 mn THF N N N R a. R= N(CH3)2 O C(CH2)2 b. R= N OH O c. R= OC N Z Scheme 1 The synthesis of grafted bioactive molecules diblock (PaN3CL-g-BioM)-b-PCL copolymers. Polymer Journal Graft bioactive molecules polyester Y-T Huang et al 964 a O O ab df ik C6H5CH2OCc O C O H e g m hjln N m i+k N o N N n p h g+l e+j o+p n d+f m a c b 121110987654321p.p.m. b O O ab df ik C6H5CH2OCc O C O H e gmhjln d+f+i+k N m N o O N O n p t q e+j s l N r h g s+a p no t m qr c b 121110987654321p.p.m. c O O ab df ik C6H5CH2OCc O C O H e gmhjln N m p N q i+k o OH N OC r e+j n O N C O O a+t h s l H2C b+s+q t n+g+o c r p+d DMF DMF f m 12 11 10 9 8 7 6 5 4 3 2 1 p.p.m. 1 Figure 1 Representative proton nuclear magnetic resonance ( H-NMR) spectra of (a)(PaN3CL-g-DMAP)-b-PCL, (b)(PaN3CL-g-PNIC)-b-PCL and (c)(PaN3CL-g-PBHP)-b-PCL. DMAP, 1-dimethylamino-2-propyne; PBHP, propargyl N-benzyloxycarbonyl-4-hydroxy prolinate; PCL, poly(e-caprolactone); PNIC, pent-4-ynyl nicotinate.