- Email: [email protected]
933 Real time lymphnode evaluation during radical prostatectomy with confocal laser endomicroscopy (Cellvizio): First results of a phase II study
European Polymer Journal 68 (2015) 267–277
Contents lists available at ScienceDirect
European Polymer Journal journal homepage: www.elsevier.com/locate/europolj
Macromolecular Nanotechnology
Synthesis of nanogels of poly(e-caprolactone)-b-poly (glycidyl methacrylate) by click chemistry in direct preparation Xuan Thang Cao, Ali Md Showkat, Kwon Taek Lim ⇑ Department of Imaging System Engineering, Pukyong National University, Busan, South Korea
a r t i c l e
i n f o
Article history: Received 28 January 2015 Received in revised form 27 March 2015 Accepted 19 April 2015 Available online 25 April 2015 Keywords: Nanogels Click chemistry Core cross-linked micelles Nonselective solvent
a b s t r a c t The controlled synthesis of nanogels was carried out in a single step using ‘‘click’’ chemistry. Poly(e-caprolactone)-b-poly(glycidyl methacrylate) (PCL-b-PGMA) block copolymers were prepared by the combination of ring-opening polymerization and the reversible addition fragmentation chain transfer polymerization, and subsequently functionalized with azido groups. The direct formation of nanogels of the azide-functionalized PCL-b-PGMA-N3 was controlled using dipropargyl adipate (DPA) as a cross-linking agent from the homogeneous solution in a nonselective solvent. The results revealed that the formation of macrogels or nanogels with core cross-linked block copolymers depends on the concentration of the block copolymer and the cross-linker and the chain length of the PGMA-N3 block, so that the preparation of nanogels was manipulated simply with adjusting the molar ratio of alkyne to azide groups. The nanogels were confirmed by nuclear magnetic resonance, X-ray photoelectron spectroscopy, transmission electron microscopy, dynamic light scattering analyses and gel permeation chromatography. DPA was found to cross-link the block copolymer effectively and afford robust nanostructures, while leaving click-readied azide functionalities throughout the core domain, which are proposed to be readily available for further chemical modification. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction The preparation of well-defined cross-linked polymeric micelles by a novel route has been attracted much interest in recent years. The advantage of polymeric micelles has been intensely studied due to the potential applications such as drug delivery and biomedical devices [1,2]. During the past decades, many studies have focused on the development and the feasibility of polymeric micelles for therapeutic and diagnostics medicine applications [3,4]. However, the micelle stabilization is the biggest obstacle for these applications. The stability of micelles is influenced by various factors such as low concentration, high temperature and different environmental conditions. Under these conditions, the polymer micelles dissociate into unimers, which cause non-targeted drug release and toxicity [1]. Therefore, the cross-linking of micelles is one of the most powerful tools to stabilize the self-assembled structure. Cross-linking of block polymeric micelles can be carried out at the end of core chains, within the core, at the core–shell interface, or at the shell of micelles. In the last decade, there have been numerous articles reported for the crosslinking reaction of block copolymer micelles [5]. Wooley et al. reported shell cross-linked (SCL) polymeric nanoparticles having a glutathione-responsive disulfide cross-linked corona, which is potential for paclitaxel delivery [6]. Core cross-linked (CCL) ⇑ Corresponding author at: Department of Imaging System Engineering, Pukyong National University, 599-1 Daeyeon 3-Dong, Nam-Gu, Busan 608-737, South Korea. E-mail address: [email protected] (K.T. Lim). http://dx.doi.org/10.1016/j.eurpolymj.2015.04.025 0014-3057/Ó 2015 Elsevier Ltd. All rights reserved.
268
X.T. Cao et al. / European Polymer Journal 68 (2015) 267–277
micelles of block polymers were reported using degradable cross-linkers by reversible addition fragmentation transfer (RAFT) polymerization [7,8]. We have reported core-surface cross-linked nanoparticles by interblock RAFT polymerization of poly(ethylene oxide)-b-polystyrene block copolymer using divinyl benzene as a cross-linker [9]. On the other hand, Yoshida et al. have reported cross-linked micelles prepared directly in a nonselective solvent by hydrogen bond cross-linking of nonamphiphilic poly(vinylphenol)-b-polystyrene using 1,4-butanediamine as a cross-linker [10]. The size and aggregation number of the micelles which depended on the copolymer concentration were investigated. In other examples, the protonation of poly[(4-pyridinemethoxymethyl)styrene]-b-polystyrene by perfluoroalkyl dicarboxylic acid led to CCL micelles in a nonselective solvent [11]. In this case, the micellar size and aggregation number were dependent on the chain length of the cross-linker. Amamoto et al. presented the cross-linking of diblock copolymers consists of poly(methyl methacrylate) and poly(methacrylic esters) with alkoxyamine moieties to form CCL star-like nanogels. The structural transformation from star-like nanogels to diblock copolymers was performed by dynamic covalent exchange among alkoxyamine units in the star-like nanogels and excess added alkoxyamine compounds [12,13]. Jackson et al. prepared CCL star polymers which constructed from two types of polymer chains possessing aldehyde and amino functions. These functional groups facilitated cross-linking of polymer chains through the reversible imine bond formation that can be responded to pH and temperature [14,15]. So far, the one-step direct micellization was mostly induced by physical crosslinking or reaction between different types of polymer chains. The click reaction was utilized to the formation of cross-linked micelles by the reaction between alkynyl-functionalized core/shell micelles of block copolymers and azide-terminated cross-linkers [16–18]. Similarly, CCL micelles were prepared from micelles of amphiphilic block copolymers by click chemistry using degradable cross-linkers which responsed to the reducing reagent [19]. The above studies are related to a two-step procedure, where micelles are formed first in a selective solvent and then the cross-linking of micelles occurs subsequently by click chemistry. The prior formation of well-defined micelles, however, need a little complicated procedure of the dialysis method and is also dependent on block copolymer composition, copolymer concentration, type and concentration of added ions and the nature of the common solvent used in micelle preparation [20], which limits its application in a wide range of block copolymers. In this study, a facile one-step preparation method was reported with the click reaction induced micellization of unimers in a nonselective solvent to produce nanogels. The block copolymer of poly(e-caprolactone)-b-poly(glycidyl methacrylate) (PCL-b-PGMA) was prepared by the combination of ring-opening polymerization (ROP) and RAFT polymerization followed by the azidation process. Click reaction, a high yield and sensitive reaction, took place between azide-functionalized PCL-b-PGMA-N3 and a dialkyne cross-linker to form CCL micelles from the homogeneous solution. The size and aggregation phenomena of CCL micelles of the block copolymer were investigated in detail. The formation of nanogels could be controlled simply by adjusting the molar ratio of alkyne to azide groups. To the best of our knowledge, it is the first attempt that nanogels of block copolymers are prepared directly by click reaction in a nonselective solvent. 2. Experimental details 2.1. Materials
e-Caprolactone (97%), 2-mercaptoethanol, carbon disulfide (99%), benzyl bromide (98%), tin(II) 2-ethylhexanoate (95%), sodium azide (NaN3, 99.99%), ammonium chloride (NH4Cl, 99.5%), propargyl alcohol (99%), adipic acid (99.5%), p-toluenesulfonic acid monohydrate (98.5%), ethylenediamine tetraacetic acid (EDTA, 99%), copper (I) bromide (CuBr, 99,99%), and N,N,N0 ,N00 -pentamethyldiethylenetriamine (PMDETA, 99%) were purchased from Sigma–Aldrich (Korea). Tetrahydrofuran (THF) and acetonitrile were dried over CaH2 and distilled prior to use. Glycidyl methacrylate (97%, Sigma–Aldrich) was passed through a neutral alumina column and 2,20 -azobis(isobutyronitrile) (AIBN) (98%, Sigma– Aldrich) was recrystallized in methanol prior to use. 2-Benzylsulfanylthiocarbonylsulfanyl ethanol (RAFT agent) was synthesized as reported previously [21]. 1H NMR (400 MHz, CDCl3, d): 3.62 (t, 2H, CH2AO), 3.88 (t, 2H, CH2AS), 4.60 (s, 2H, CH2-Ph), 7.31 (m, 5H, Ph). Dipropargyl adipate (DPA) was synthesized according to the previous literature [22]. 1H NMR (400 MHz, CDCl3, d): 4.63 (s, 4H, CH2AOCO), 2.43 (s, 2H, CH), 2.32–2.34 (t, 4H, OCOACH2ACH2), 1.64–1.66 (m, 4H, CH2ACH2ACH2AOCO). Other solvents and chemicals of analytical grade were used as received. 2.2. Synthesis of poly(e-caprolactone)-based RAFT agent (PCL-RAFT) The RAFT agent with hydroxyl end-group was used as an initiator for ROP. A solution of e-caprolactone (4.56 g) and RAFT agent (0.24 g) was stirred at 90 °C for 1 h under nitrogen. Afterward, tin(II) 2-ethylhexanoate (0.041 g) was added to the yellow mixture, and the solution was purged with nitrogen and stirred at 140 °C for 12 h. At the end point, the viscosity of the mixture increased dramatically. The product was dissolved in THF, precipitated in methanol, filtered. This procedure was repeated three times. The final product was dried under vacuum for 24 h. 2.3. Synthesis of PCL-b-PGMA block copolymer The block copolymer of PCL-b-PGMA was synthesized by the RAFT method using the PCL-RAFT macroinitiator. In a typical procedure, PCL-RAFT (0.64 g, 0.044 mmol), glycidyl methacrylate (1.137 g, 8 mmol), AIBN (0.008 g, 0.05 mmol), and THF
X.T. Cao et al. / European Polymer Journal 68 (2015) 267–277
269
(5 mL) were added into a round bottom flask, and stirred for 60 min under nitrogen. The flask was placed in an oil bath at 80 °C and stirred for 24 h. The product was dissolved in THF and precipitated in excess methanol. This procedure was repeated two times. The product was dried in a vacuum oven at 40 °C overnight.
2.4. Preparation of PCL-b-PGMA-N3 block copolymer The PCL-b-PGMA-N3 block copolymer was prepared through the epoxide ring-opening reaction of PGMA segments. A mixture of PCL-b-PGMA (0.259 g), NaN3 (0.284 g, 4.37 mmol), NH4Cl (0.234 g, 4.37 mmol) and DMF (5 mL) were placed in a round bottom flask and stirred at 50 °C for 24 h. The product was extracted with a saturated sodium chloride solution, filtered and washed with deionized water several times. The product was dried in a vacuum oven at 40 °C for 24 h.
2.5. CCL micelles of PCL-b-PGMA-N3 block copolymer by click chemistry The PCL-b-PGMA-N3 block copolymer was dissolved in round bottom flask containing acetonitrile (20 mL) and a magnetic spin bar at ambient temperature. DPA and CuBr were added under nitrogen and stirred for 30 min. Afterward, the deoxygenated PMDETA was added to the solution using a syringe, and the color of the solution changed immediately to blue due to the formation of copper complexes. The solution was stirred at 50 °C for 24 h, and the catalysts were removed through the EDTA solution of ethanol/water (v/v: 1/1) [23]. The product was filtered and washed several times with deionized water. The CCL nanoparticles were dried in a vacuum oven at 40 °C for 24 h.
2.6. Characterization 1
H NMR spectra were recorded on a JNM-ECP 400 (JEOL) instrument. Gel permeation chromatography (GPC) was performed using a HP 1100 apparatus, with THF as a solvent at 25 °C and an elution rate of 1 mL/min. The columns were calibrated with commercial polystyrene standards. Fourier transform infrared (FTIR) spectra were measured on a JASCO FT/IR-4100 spectrometer with DLATGS detector in the 4000–400 cm1 spectral region. The samples were finely ground, mix with spectroscopic grade KBr and pressed into pellets. The chemical states were investigated using X-ray Photoelectron Spectroscopy (XPS) (Thermo VG Multilab 2000) in ultra-high vacuum with Al Ka radiation. For transmission electron microscopy (TEM; JEOL JEM-2010), the samples were prepared by depositing a drop of dispersed nanoparticles in acetonitrile (upper portion) on copper grids. No staining was applied to the sample. Dynamic laser light scattering (DLS) measurements for determining the average hydrodynamic diameter of polymeric micelles were performed using an electrophoretic light scattering instrument (ELS-8000, Otsuka Electronics Corporation), equipped with an ELS controller, and a He–Ne laser at wavelength of 632.8 nm. The intensity of scattered light was detected at 90° to an incident beam. All the analyses were performed at 25 °C. The sample solutions (1 mg/mL) was prepared by dispersing the polymers in acetonitrile with ultrasonic oscillation for 30 min and the solution was allowed to settle down a small portion of agglomerate prior to analysis.
Fig. 1. Synthesis of PCL-b-PGMA-N3 block copolymers by ROP and RAFT polymerization, followed by direct preparation of CCL micelles.
270
X.T. Cao et al. / European Polymer Journal 68 (2015) 267–277
3. Results and discussion 3.1. Synthesis and characterization of PCL-b-PGMA-N3 block copolymers by ROP and RAFT polymerization Our primary aim was to develop a novel route for direct preparation of CCL micelles. Block copolymers which consist of PCL as an ‘‘inactive’’ block and azide-functionalized PGMA as an ‘‘active’’ block were crosslinked using click reaction strategy as shown in Fig. 1. The PCL-b-PGMA was synthesized by ROP and RAFT polymerization with 2-benzylsulfanylthiocarbonylsulfanyl ethanol as a co-initiator. Table 1 Characteristics of the PCL-RAFT macroinitiator 1 and PCL-b-PGMA block copolymers 2–4 which were prepared by ROPa and RAFT polymerization.b Sample no.
1 2 3 4 a b c
Polymer
PCL126-RAFT PCL126-b-PGMA23 PCL126-b-PGMA56 PCL126-b-PGMA154
Feed ratio [M]eCL/[M]RAFTagent
[M]GMA/[M]PCL-RAFT
40 – – –
– 32 94 188
Conversionc (%)
Mn (1H NMR)
Mn (GPC)
PDI (GPC)
60.9 35.1 49.6 63.2
15,000 18,300 23,000 37,000
10,700 11,500 9300 13,200
1.52 1.64 1.65 1.59
ROP was carried out at 140 °C for 12 h. Molar ratio [RAFT agent]:[Sn(Oct)2] = 10:1. Polymerization was carried out at 80 °C for 24 h. Molar ratio [PCL-RAFT]:[AIBN] = 5:3. Conversion was determined gravimetrically.
Fig. 2. GPC chromatographs for polymers (see Table 1).
Fig. 3. FT-IR spectra of (a) PCL126-RAFT macroinitiator; (b) PCL126-b-PGMA23, (c) PCL126-b-PGMA56, (d) PCL126-b-PGMA154 (Table 1); and (e) PCL156-bPGMA23-N3, (f) PCL156-b-PGMA56-N3, (g) PCL156-b-PGMA154-N3, (h) CCL micelles of PCL156-b-PGMA56-N3.
X.T. Cao et al. / European Polymer Journal 68 (2015) 267–277
271
Fig. 4. 1H NMR spectra of (a) PCL126-RAFT macroinitiator in CDCl3, (b) PCL126-b-PGMA56, (c) PCL126-b-PGMA56-N3, and (d) CCL micelles of PCL126-b-PGMA56N3 in DMSO-d6.
As shown in Table 1, block copolymers can be prepared with the different chain length of PGMA. Living radical polymerization was evidenced by the linear correlation between monomer conversion and molecular weight of block copolymers. The GPC curves of block copolymers were relatively unimodal and symmetric (Fig. 2). Moreover, the polydispersity (PDI) of the block copolymers 2–4 after the RAFT process was quite close to the precursor of macro-initiator 1 indicating a good level of control in the RAFT-mediate reaction.
272
X.T. Cao et al. / European Polymer Journal 68 (2015) 267–277
Fig. 3 depicts the FT-IR spectra of polymers. In Fig. 3b, the epoxy ring vibration bands of PCL-g-PGMA block copolymers are shown at 844, 905, and 993 cm1. The absorption bands at 2946 and 2999 cm1 could be ascribed to the vibration of methylene and methyl protons of the PGMA segment. These characteristic bands overlap with the methylene vibration of the PCL-RAFT macroinitiator (Fig. 3a), so that they cannot be seen independently in the FT-IR spectrum. Moreover, the intensity of the peaks at 844 and 905 cm1 corresponding to CAO stretching of cis- and trans-epoxy groups increased with the PGMA chain length, indicating more GMA units in the block copolymers (Fig. 3b–d). The azidation of PCL-b-PGMA could be presented by the new absorption band at 2095 cm1 which was assigned to the valence vibration of azide functions in PGMA-N3 moieties (Fig. 3e–g). In addition, the opened epoxy ring generated a stronger and broader band of hydroxyl groups at 3388 cm1, whereas the signal of the ring at 844, 905, and 993 cm1 disappeared completely. After click reaction, the FT-IR spectrum of nanogels showed reduced intensity of the characteristic band of azides, confirming their reaction with alkyne groups of the cross-linker (Fig. 3h). The residual band at 2095 cm1 of the nanogels indicates the unconsumed azide functionalities of the block copolymer. These data demonstrate that the attempted cross-linking reaction of the block copolymer unimers in a nonselective solvent was indeed successful and that this methodology allowed for the incorporation of further click readied azido-functionality within the core of the cross-linked polymer nanoparticle. The structure of polymers was confirmed by 1H NMR spectra. As shown in Fig. 4a, the methylene protons in the main chain of PCL (a–f) at 4.02, 3.61, 2.27, 1.60, and 1.34 ppm were visualized while the peak at 7.27 ppm was assigned to the phenyl ring (g) in the end group of the RAFT agent. After the RAFT polymerization of GMA for PCL-b-PGMA, the additional peaks at 3.21, 2.80, and 2.67 ppm are associated to the methylene proton (s, t) of the epoxy ring as well as signals at 4.31, 3.73, 1.89, 1.82, 1.20, 0.98, and 0.81 ppm contributed to methylene (r, p) and methyl (q) proton of PGMA moieties (Fig. 4b). The epoxide ring-opening process was revealed by change of the proton bearing epoxy ring in spectroscopy, which was depicted in Fig. 4c. The characteristic peaks of the PCL part were maintained even though the signals of the PGMA moiety have already changed. The new peaks of the opened ring appeared at 5.51, and 3.88 ppm, which implied to the proton of CHAO (s0 ), CH2AO (r0 ), and CH2AN3 (t0 ) whereas the epoxy ring protons at 4.31, 3.73, 3.21, 2.80, 2.67 ppm disappeared. The result demonstrated that azido groups were anchored on the backbone of PGMA part. The click reaction between dipropargyl adipate and the azido groups in the PGMA-N3 parts induced CCL micelles owing to the reduced solubility in the crosslinked region. The CCL micelles consist of the PCL shell and the core of cross-linked PGMA-N3. 1H NMR spectrum indicated the depression of the signal pertinent to the PGMA-N3 moiety whose protons were shielded from magnetic
Fig. 5. XPS C 1s core-level spectra of (a) PCL126-b-PGMA56, (b) PCL126-b-PGMA56-N3, and XPS N 1s core-level spectra of (c) PCL126-b-PGMA56, (d) PCL126-bPGMA56-N3, and CCL micelles of (e) PCL126-b-PGMA56-N3.
273
X.T. Cao et al. / European Polymer Journal 68 (2015) 267–277
Fig. 6. Photographic illustration for solution aggregation of (a) core non-cross-linked micelles; (b, d, f) macrogels; and (c, e, g) nanogels of sample 7, 13, 18; and 8, 14, 19 (see Table 2), respectively.
resonance by PCL coronas [11]. Consequently, the proton signal of cross-linked PGMA-N3 for r0 , s0 , t0 , p, and q decreased significantly in comparison with the non-cross-linked PGMA-N3 segment (Fig. 4d). The XPS analyses were performed to demonstrate binding formation of the block copolymer and CCL micelles. The C 1s core-level spectrum of PCL-b-PGMA with three peaks at binding energies (BEs) of 284.8, 286.5, and 288.4 eV (Fig. 5a) implied to CAC/CAH, CAO, and OAC@O bonds. However, the N 1s core-level spectrum was not observed since there is no nitrogen in PCL-b-PGMA (Fig. 5c). After the azidation reaction, the new peak C 1s core-level appeared at BE of 287 eV indicating CAN bond formation (Fig. 5b). The N 1s core-level of the azido groups was observed at BEs of 399.7, 400.7, and 403.8 eV corresponding to AN@N+@N, AN@N+@N, and AN@N+@N bond, respectively (Fig. 5d). Moreover, the relative area of these three peaks with proportion of 1:1:1 was evidence for the chemical structure of three nitrogen atoms of the azido group. The covalent bond between the azide and alkyne groups was demonstrated by the N 1s spectrum in Fig. 5e. Two peaks at 401.5 (NANAN) and 400.2 eV (NANAC) with area ratio of 1:2 approximately were ascribed to the N 1s of the triazole ring, which was obviously different from AN3 in Fig. 5d. The results are consistent with its theoretical values.
3.2. The formation of macrogel or nanogel materials The click chemistry was employed to investigate the formation of gel materials. CCL micelles of PCL-b-PGMA-N3 were prepared by solutions of the block copolymer and DPA in acetonitrile catalyzed by CuBr/PMDETA. The formation of nanogels
Table 2 Characterization of CCL micelles of block copolymers by click reaction. The reaction was catalyzed by CuBr/PMDETA (nCuBr = nPMDETA = 0.3 ncross-linker) in acetonitrile. Block copolymers
a b c
Sample
wt%a
Cross-linker (mmol) (wt%)
Alkyne: azide molar ratiob
Aggregationc
PCL126-b-PGMA23-N3
1 2 3 4 5 6 7 8 9
0.127 0.127 0.127 0.127 0.635 1.270 1.270 1.270 1.270
0.028 0.023 0.020 0.014 0.014 0.140 0.105 0.096 0.070
(0.040) (0.032) (0.029) (0.020) (0.020) (0.200) (0.150) (0.138) (0.100)
1.8:1 1.5:1 1.3:1 0.9:1 0.18:1 0.9:1 0.675:1 0.619:1 0.45:1
Macrogel Macrogel Nanogel Nanogel Nanogel Macrogel Macrogel Nanogel Nanogel
PCL126-b-PGMA56-N3
10 11 12 13 14
0.127 0.127 0.635 1.270 1.270
0.020 0.018 0.018 0.096 0.083
(0.029) (0.025) (0.025) (0.138) (0.119)
0.9:1 0.8:1 0.16:1 0.413:1 0.357:1
Macrogel Nanogel Nanogel Macrogel Nanogel
PCL126-b-PGMA154-N3
15 16 17 18 19
0.127 0.127 0.635 1.270 1.270
0.014 0.011 0.011 0.083 0.070
(0.020) (0.015) (0.015) (0.119) (0.100)
0.4:1 0.3:1 0.06:1 0.226:1 0.200:1
Macrogel Nanogel Nanogel Macrogel Nanogel
Weight concentration of PCL-b-PGMA-N3 block copolymers in acetonitrile. Alkyne: azide molar ratio was calculated from the number of alkyne and azide groups of the block copolymer and DPA, respectively. Aggregation was observed by naked eyes as shown in Fig. 6.
274
X.T. Cao et al. / European Polymer Journal 68 (2015) 267–277
Fig. 7. Schematic illustration for nanogel or macrogel formation through CCL micelle preparation.
and macrogels was investigated with the variation of the PGMA chain length and the concentration of the block polymer and the cross-linker. The concentration of the block copolymer was varied in a range from 0.127 to 1.270 wt% to obtain monodisperse species [14]. The experimentally observed transition of a completely soluble mixture to one having an insoluble fraction was known as nanogels or macrogels [24]. This work demonstrates that the aggregation of the block copolymer solution depends on the concentration of the cross-linker and the block copolymer, and the size of the PGMA-N3 block. In the block copolymer PCL plays a role as ‘‘inactive’’ chain and PGMA-N3 is an ‘‘active’’ segment which undergone the cross-linking reaction. The PCL126-b-PGMA23-N3 was soluble in acetonitrile to form unimer solution without any aggregation as observed by the naked eye (Fig. 6a). Upon click reaction, the unimers were self-assemble into the micellar structure whose PCL chains became corona and the cross-linked PGMA-N3 part was a core. The formation of macrogel or nanogel materials can be distinctly observed as shown in Fig. 6. When the aggregation is macrogel, the resulting solution separates into two parts; the upper part is transparent and lower one is as gel-like substance (Fig. 6b, d, and f). Otherwise, the solution is bluish haze liquid without precipitation due to nanogel particles (Fig. 6c, e, and g). The observation has been explained deeply by experimental data.
Fig. 8. TEM images of CCL micelles of the block copolymer: sample (a) 8, (b) 14, (c) 7, and (d) 13 (see Table 2).
X.T. Cao et al. / European Polymer Journal 68 (2015) 267–277
275
As shown in Table 2, at concentration of 0.127 wt% of PCL126-b-PGMA23-N3, sample 2 and 3 presented transition concentrations of the cross-linker to form macrogels and nanogels corresponding to alkyne:azide (cross-linker to N3 in block copolymer) molar ratio of 1.5:1 and 1.3:1, respectively. The result can be explained by inter-micellar click reaction occurs partially at the higher ratio of the cross-linker relative to the block copolymer. In other words, the cross-linking reaction takes place not only intra- but also inter-micelles when the alkyne:azide molar ratio is above 1.5:1. As a result, the macrogel material was produced in the solution (sample 1 and 2). At a higher concentration of 1.270 wt% of the block copolymer, sample 7 and 8 showed the lower transition molar ratio of alkyne: azide groups for macrogels and nanogels compared with 0.127 wt%, corresponding to the ratio of 0.675:1 and 0.619:1, respectively. The result indicated that the ratio of the cross-linker to the block copolymer should be reduced to foam nanogels at the higher concentration of the block copolymer. This is attributed to the fact that the higher concentration of the PGMA-N3 moiety has more azido groups therefore more reaction probability toward the cross-linker. The experiments were also carried out for the block copolymers of different block ratios, PCL126-b-PGMA56-N3 and PCL126-b-PGMA154-N3 by the same procedure. The data showed that, for the nanogel formation, the less amount of the cross-linker was used when the PGMA-N3 chain length increased at the same concentration of three block copolymers (sample 3, 11, and 16; sample 8, 14, and 19). The more GMA units mean more azido groups, and a less amount of the cross-linker
Fig. 9. Size distribution of (a) PCL126-b-PGMA23-N3 in acetonitrile and CCL micelles of the block copolymer: sample (b) 8 and (c) 14 (see Table 2).
276
X.T. Cao et al. / European Polymer Journal 68 (2015) 267–277
Table 3 Characterization of CCL micelles of the block copolymer analyzed by GPC. Sample (see Table 2)
Maw (g mol1)
Mbn (g mol1)
Mbw (g mol1)
PDIb
Average number of arms per micelle
3 8
18,870 15,350
100,560 126,500
161,900 206,200
1.61 1.63
8 13
Molecular weight of the block copolymer was determined by GPC beforea and afterb CCL micelles, THF was used as an eluent.
is needed to be free from the inter-micellar coupling. The result demonstrated that gel materials could be controlled eventually by an adjustment of the concentration of the cross-linking agent and the block polymer and the size of the PGMA-N3 block in terms of the molar ratio of alkyne to azide groups. Furthermore, it is important to notice that the nanogels contain unreacted, further click-readied azide-functionality within the core of the cross-linked polymer nanoparticles, which can be used to attach a low molecular compound such as drugs, possibly extended to the potential application for a drug carrier. 3.3. The structure of CCL micelles The schematic diagram for the CCL micelles can be represented as Fig. 7. The resulting macrogels or nanogels depend on the reaction condition. The micelle morphology was obtained by TEM as shown in Fig. 8. TEM images display nanoparticles which are dark spheres with an average diameter of 100 and 103 nm corresponding to sample 8 (Fig. 8a) and 14 (Fig. 8b), respectively (Table 2). The discrete nanoparticles were produced with the inter-chain cross-link of block copolymers. Chained nanoparticles with undefined form were obtained by the increased concentration of the cross-linker due to undesirable inter-micellar cross-linking, which inevitably resulted in micelle fusion (Fig. 8c and d). Consequently, they were highly agglomerated and separated from the solution as macrogels. The results are in good agreement with the experiment where the gel materials were observed by naked eyes as shown in Fig. 6. Hydrodynamic diameters of the CCL micelles were analyzed by DLS as shown in Fig. 9. PCL-b-PGMA-N3 produced a homogeneous solution in acetonitrile. The average hydrodynamic diameter of PCL126-b-PGMA23-N3 in acetonitrile was approximately 4 nm, indicating a unimer solution (Fig. 9a). After the formation of nanogels by click reaction, the block copolymer solution showed the particle distributions at 111 and 117 nm of hydrodynamic diameter corresponding to the sample 8 (Fig. 9b) and 14 (Fig. 9c), respectively. The size distribution of nanoparticles becomes broad with the increase of the PGMA block chain length of the precursor. The results are consistent with the TEM analyses. GPC analysis can estimate the average number of arms of CCL micelles by dividing the molecular weight of CCL micelles by the molecular weight of the block copolymer. As shown in Table 3, the number of arms increases with the chain length of PGMA-N3 implying the CCL micelles could be controlled by using click reaction. 4. Conclusion The direct preparation of CCL micelles was achieved by the click reaction between PCL-b-PGMA-N3 and DPA in the homogeneous solution. A series of PCL-b-PGMA polymers varying the PGMA block length were prepared by ROP and RAFT polymerization with a relatively low polydispersity. The structure of polymers was confirmed by FT-IR and 1H NMR spectra. The FT-IR showed the characteristic vibration of azido groups of PCL-b-PGMA-N3. In the 1H NMR spectrum of the block copolymer, the new peaks of the opened epoxy ring appeared at 5.51 and 3.88 ppm while the epoxy ring protons at 4.31, 3.73, 3.21, 2.80, 2.67 ppm disappeared. In the XPS spectra, the N 1s core-level was observed at 399.7, 400.7, and 403.8 eV with relative area of these three peaks with proportion of 1:1:1 indicating the chemical structure of the azido group. Two peaks at 401.5 and 400.2 eV with area ratio of 1:2 were ascribed to the N 1s of the triazole ring in the cross-linked PGMA-N3 segments. The formation of gel materials could be recognized by visual and experimental observations, which was depended on the click reaction condition. The one-step micellization producing the nanogels was controlled by the concentration of the cross-linker and the block copolymer as well as the size of the PGMA-N3 block in terms of the molar ratio of alkyne to azide groups. The nanogels with a diameter of around 100 nm (measured by TEM and DLS) could be produced simply with adjusting the complementary relationship among the three variables. The facile nature of the click chemistry used for cross-linking of the block copolymer also left unreacted azide functionalities located on the core to be detected by IR spectroscopic analysis. This cross-linking strategy and simultaneous incorporation of click-readied functionality provides a facile route for the future development of these nanostructures in biomedical applications. Acknowledgement This work was supported by the BK-21 Plus program. References [1] M.H. Stenzel, RAFT polymerization: an avenue to functional polymeric micelles for drug delivery, Chem. Commun. (2008) 3486–3503. [2] K. Halake, M. Birajdar, B.S. Kim, H. Bae, C.C. Lee, Y.J. Kim, S. Kim, H.J. Kim, S. Ahn, S.Y. An, J. Lee, Recent application developments of water-soluble synthetic polymers, J. Ind. Eng. Chem. 20 (2014) 3913–3918.
X.T. Cao et al. / European Polymer Journal 68 (2015) 267–277
277
[3] R. Duncan, The dawning era of polymer therapeutics, Nat. Rev. Drug Discover. 2 (2003) 347–360. [4] K. Kataoka, A. Harada, Y. Nagasaki, Block copolymer micelles for drug delivery: design, characterization and biological significance, Adv. Drug Deliv. Rev. 47 (2001) 113–131. [5] A. Rösler, G.W.M. Vandermeulen, H.A. Klok, Advanced drug delivery devices via self-assembly of amphiphilic block copolymers, Adv. Drug Deliv. Rev. 64 (2012) 270–279. [6] S. Samarajeewa, R. Shrestha, M. Elsabahy, A. Karwa, A. Li, R.P. Zentay, J.G. Kostelc, R.B. Dorshow, K.L. Wooley, In vitro efficacy of paclitaxel-loaded dualresponsive shell cross-linked polymer nanoparticles having orthogonally degradable disulfide cross-linked corona and polyester core domains, Mol. Pharm. 10 (2013) 1092–1099. [7] Y. Chan, T. Wong, F. Byrne, M. Kavallaris, V. Bulmus, Acid-labile core cross-linked micelles for pH-triggered release of antitumor drugs, Biomacromolecules 9 (2008) 1826–1836. [8] B.M. Blunden, D.S. Thomas, M.H. Stenzel, Analysis of thiol-sensitive core-cross-linked polymeric micelles carrying nucleoside pendant groups using ‘On-line’ methods: effect of hydrophobicity on cross-linking and degradation, Aust. J. Chem. 64 (2011) 766–778. [9] M.D. Hossain, L.T.B. Tran, J.M. Park, K.T. Lim, Facile synthesis of core-surface crosslinked nanoparticles by interblock RAFT polymerization, J. Polym. Sci. Part A: Polym. Chem. 48 (2010) 4958–4964. [10] E. Yoshida, S. Kunugi, Micelle formation of nonamphiphilic diblock copolymers through noncovalent bond cross-linking, Macromolecules 35 (2002) 6665–6669. [11] E. Yoshida, M. Tanaka, T. Takata, Direct preparation of core cross-linked micelles in a nonselective solvent, Colloid Polym. Sci. 284 (2005) 51–57. [12] Y. Amamoto, Y. Higaki, Y. Matsuda, H. Otsuka, A. Takahara, Programmed thermodynamic formation and structure analysis of star-like nanogels with core cross-linked by thermally exchangeable dynamic covalent bonds, J. Am. Chem. Soc. 129 (2007) 13298–13304. [13] Y. Amamoto, M. Kikuchi, H. Masunaga, S. Sasaki, H. Otsuka, A. Takahara, Intelligent build-up of complementarily reactive diblock copolymers via dynamic covalent exchange toward symmetrical and miktoarm star-like nanogels, Macromolecules 43 (2010) 1785–1791. [14] A.W. Jackson, D.A. Fulton, The formation of core cross-linked star polymers containing cores cross-linked by dynamic covalent imine bonds, Chem. Commun. 46 (2010) 6051–6053. [15] A.W. Jackson, D.A. Fulton, PH triggered self-assembly of core cross-linked star polymers possessing thermoresponsive cores, Chem. Commun. 47 (2011) 6807–6809. [16] R.K. O’Reilly, M.J. Joralemon, C.J. Hawker, K.L. Wooley, Preparation of orthogonally-functionalized core click cross-linked nanoparticles, New J. Chem. 31 (2007) 718–724. [17] M.J. Joralemon, R.K. O’Reilly, C.J. Hawker, K.L. Wooley, Shell click-crosslinked (SCC) nanoparticles: a new methodology for synthesis and orthogonal functionalization, J. Am. Chem. Soc. 127 (2005) 16892–16899. [18] S.M. Garg, X.B. Xiong, C. Lu, A. Lavasanifar, Application of click chemistry in the preparation of poly(ethylene oxide)-block-poly(e-caprolactone) with hydrolyzable cross-links in the micellar core, Macromolecules 44 (2011) 2058–2066. [19] Z. Zhang, L. Yin, C. Tu, Z. Song, Y. Zhang, Y. Xu, R. Tong, Q. Zhou, J. Ren, J. Cheng, Redox-responsive, core cross-linked polyester micelles, J. ACS Macro Lett. 2 (2013) 40–44. [20] C. Allen, D. Maysinger, A. Eisenberg, Nano-engineering block copolymer aggregates for drug delivery, Colloid Surf. B 16 (1999) 3–27. [21] A.O. Saeed, S. Dey, S.M. Howdle, K.J. Thurecht, C.J. Alexander, One-pot controlled synthesis of biodegradable and biocompatible co-polymer micelles, Mater. Chem. 19 (2009) 4529–4535. [22] R. Nomura, K. Yamada, J. Tabei, Y. Takakura, T. Takigawa, T. Masuda, Stimuli-responsive organogel based on poly(N-propargylamide), Macromolecules 36 (2003) 6939–6941. [23] I.V. Gürsel, F. Aldiansyah, Q. Wang, T. Noël, V. Hessel, Continuous metal scavenging and coupling to one-pot copper-catalyzed azide-alkyne cycloaddition click reaction in flow, Chem. Eng. J. 270 (2015) 468–475. [24] C.J. Kloxin, T.F. Scott, B.J. Adzima, C.N. Bowman, Covalent adaptable networks (CANs): a unique paradigm in cross-linked polymers, Macromolecules 43 (2010) 2643–2653.
Contents lists available at ScienceDirect
European Polymer Journal journal homepage: www.elsevier.com/locate/europolj
Macromolecular Nanotechnology
Synthesis of nanogels of poly(e-caprolactone)-b-poly (glycidyl methacrylate) by click chemistry in direct preparation Xuan Thang Cao, Ali Md Showkat, Kwon Taek Lim ⇑ Department of Imaging System Engineering, Pukyong National University, Busan, South Korea
a r t i c l e
i n f o
Article history: Received 28 January 2015 Received in revised form 27 March 2015 Accepted 19 April 2015 Available online 25 April 2015 Keywords: Nanogels Click chemistry Core cross-linked micelles Nonselective solvent
a b s t r a c t The controlled synthesis of nanogels was carried out in a single step using ‘‘click’’ chemistry. Poly(e-caprolactone)-b-poly(glycidyl methacrylate) (PCL-b-PGMA) block copolymers were prepared by the combination of ring-opening polymerization and the reversible addition fragmentation chain transfer polymerization, and subsequently functionalized with azido groups. The direct formation of nanogels of the azide-functionalized PCL-b-PGMA-N3 was controlled using dipropargyl adipate (DPA) as a cross-linking agent from the homogeneous solution in a nonselective solvent. The results revealed that the formation of macrogels or nanogels with core cross-linked block copolymers depends on the concentration of the block copolymer and the cross-linker and the chain length of the PGMA-N3 block, so that the preparation of nanogels was manipulated simply with adjusting the molar ratio of alkyne to azide groups. The nanogels were confirmed by nuclear magnetic resonance, X-ray photoelectron spectroscopy, transmission electron microscopy, dynamic light scattering analyses and gel permeation chromatography. DPA was found to cross-link the block copolymer effectively and afford robust nanostructures, while leaving click-readied azide functionalities throughout the core domain, which are proposed to be readily available for further chemical modification. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction The preparation of well-defined cross-linked polymeric micelles by a novel route has been attracted much interest in recent years. The advantage of polymeric micelles has been intensely studied due to the potential applications such as drug delivery and biomedical devices [1,2]. During the past decades, many studies have focused on the development and the feasibility of polymeric micelles for therapeutic and diagnostics medicine applications [3,4]. However, the micelle stabilization is the biggest obstacle for these applications. The stability of micelles is influenced by various factors such as low concentration, high temperature and different environmental conditions. Under these conditions, the polymer micelles dissociate into unimers, which cause non-targeted drug release and toxicity [1]. Therefore, the cross-linking of micelles is one of the most powerful tools to stabilize the self-assembled structure. Cross-linking of block polymeric micelles can be carried out at the end of core chains, within the core, at the core–shell interface, or at the shell of micelles. In the last decade, there have been numerous articles reported for the crosslinking reaction of block copolymer micelles [5]. Wooley et al. reported shell cross-linked (SCL) polymeric nanoparticles having a glutathione-responsive disulfide cross-linked corona, which is potential for paclitaxel delivery [6]. Core cross-linked (CCL) ⇑ Corresponding author at: Department of Imaging System Engineering, Pukyong National University, 599-1 Daeyeon 3-Dong, Nam-Gu, Busan 608-737, South Korea. E-mail address: [email protected] (K.T. Lim). http://dx.doi.org/10.1016/j.eurpolymj.2015.04.025 0014-3057/Ó 2015 Elsevier Ltd. All rights reserved.
268
X.T. Cao et al. / European Polymer Journal 68 (2015) 267–277
micelles of block polymers were reported using degradable cross-linkers by reversible addition fragmentation transfer (RAFT) polymerization [7,8]. We have reported core-surface cross-linked nanoparticles by interblock RAFT polymerization of poly(ethylene oxide)-b-polystyrene block copolymer using divinyl benzene as a cross-linker [9]. On the other hand, Yoshida et al. have reported cross-linked micelles prepared directly in a nonselective solvent by hydrogen bond cross-linking of nonamphiphilic poly(vinylphenol)-b-polystyrene using 1,4-butanediamine as a cross-linker [10]. The size and aggregation number of the micelles which depended on the copolymer concentration were investigated. In other examples, the protonation of poly[(4-pyridinemethoxymethyl)styrene]-b-polystyrene by perfluoroalkyl dicarboxylic acid led to CCL micelles in a nonselective solvent [11]. In this case, the micellar size and aggregation number were dependent on the chain length of the cross-linker. Amamoto et al. presented the cross-linking of diblock copolymers consists of poly(methyl methacrylate) and poly(methacrylic esters) with alkoxyamine moieties to form CCL star-like nanogels. The structural transformation from star-like nanogels to diblock copolymers was performed by dynamic covalent exchange among alkoxyamine units in the star-like nanogels and excess added alkoxyamine compounds [12,13]. Jackson et al. prepared CCL star polymers which constructed from two types of polymer chains possessing aldehyde and amino functions. These functional groups facilitated cross-linking of polymer chains through the reversible imine bond formation that can be responded to pH and temperature [14,15]. So far, the one-step direct micellization was mostly induced by physical crosslinking or reaction between different types of polymer chains. The click reaction was utilized to the formation of cross-linked micelles by the reaction between alkynyl-functionalized core/shell micelles of block copolymers and azide-terminated cross-linkers [16–18]. Similarly, CCL micelles were prepared from micelles of amphiphilic block copolymers by click chemistry using degradable cross-linkers which responsed to the reducing reagent [19]. The above studies are related to a two-step procedure, where micelles are formed first in a selective solvent and then the cross-linking of micelles occurs subsequently by click chemistry. The prior formation of well-defined micelles, however, need a little complicated procedure of the dialysis method and is also dependent on block copolymer composition, copolymer concentration, type and concentration of added ions and the nature of the common solvent used in micelle preparation [20], which limits its application in a wide range of block copolymers. In this study, a facile one-step preparation method was reported with the click reaction induced micellization of unimers in a nonselective solvent to produce nanogels. The block copolymer of poly(e-caprolactone)-b-poly(glycidyl methacrylate) (PCL-b-PGMA) was prepared by the combination of ring-opening polymerization (ROP) and RAFT polymerization followed by the azidation process. Click reaction, a high yield and sensitive reaction, took place between azide-functionalized PCL-b-PGMA-N3 and a dialkyne cross-linker to form CCL micelles from the homogeneous solution. The size and aggregation phenomena of CCL micelles of the block copolymer were investigated in detail. The formation of nanogels could be controlled simply by adjusting the molar ratio of alkyne to azide groups. To the best of our knowledge, it is the first attempt that nanogels of block copolymers are prepared directly by click reaction in a nonselective solvent. 2. Experimental details 2.1. Materials
e-Caprolactone (97%), 2-mercaptoethanol, carbon disulfide (99%), benzyl bromide (98%), tin(II) 2-ethylhexanoate (95%), sodium azide (NaN3, 99.99%), ammonium chloride (NH4Cl, 99.5%), propargyl alcohol (99%), adipic acid (99.5%), p-toluenesulfonic acid monohydrate (98.5%), ethylenediamine tetraacetic acid (EDTA, 99%), copper (I) bromide (CuBr, 99,99%), and N,N,N0 ,N00 -pentamethyldiethylenetriamine (PMDETA, 99%) were purchased from Sigma–Aldrich (Korea). Tetrahydrofuran (THF) and acetonitrile were dried over CaH2 and distilled prior to use. Glycidyl methacrylate (97%, Sigma–Aldrich) was passed through a neutral alumina column and 2,20 -azobis(isobutyronitrile) (AIBN) (98%, Sigma– Aldrich) was recrystallized in methanol prior to use. 2-Benzylsulfanylthiocarbonylsulfanyl ethanol (RAFT agent) was synthesized as reported previously [21]. 1H NMR (400 MHz, CDCl3, d): 3.62 (t, 2H, CH2AO), 3.88 (t, 2H, CH2AS), 4.60 (s, 2H, CH2-Ph), 7.31 (m, 5H, Ph). Dipropargyl adipate (DPA) was synthesized according to the previous literature [22]. 1H NMR (400 MHz, CDCl3, d): 4.63 (s, 4H, CH2AOCO), 2.43 (s, 2H, CH), 2.32–2.34 (t, 4H, OCOACH2ACH2), 1.64–1.66 (m, 4H, CH2ACH2ACH2AOCO). Other solvents and chemicals of analytical grade were used as received. 2.2. Synthesis of poly(e-caprolactone)-based RAFT agent (PCL-RAFT) The RAFT agent with hydroxyl end-group was used as an initiator for ROP. A solution of e-caprolactone (4.56 g) and RAFT agent (0.24 g) was stirred at 90 °C for 1 h under nitrogen. Afterward, tin(II) 2-ethylhexanoate (0.041 g) was added to the yellow mixture, and the solution was purged with nitrogen and stirred at 140 °C for 12 h. At the end point, the viscosity of the mixture increased dramatically. The product was dissolved in THF, precipitated in methanol, filtered. This procedure was repeated three times. The final product was dried under vacuum for 24 h. 2.3. Synthesis of PCL-b-PGMA block copolymer The block copolymer of PCL-b-PGMA was synthesized by the RAFT method using the PCL-RAFT macroinitiator. In a typical procedure, PCL-RAFT (0.64 g, 0.044 mmol), glycidyl methacrylate (1.137 g, 8 mmol), AIBN (0.008 g, 0.05 mmol), and THF
X.T. Cao et al. / European Polymer Journal 68 (2015) 267–277
269
(5 mL) were added into a round bottom flask, and stirred for 60 min under nitrogen. The flask was placed in an oil bath at 80 °C and stirred for 24 h. The product was dissolved in THF and precipitated in excess methanol. This procedure was repeated two times. The product was dried in a vacuum oven at 40 °C overnight.
2.4. Preparation of PCL-b-PGMA-N3 block copolymer The PCL-b-PGMA-N3 block copolymer was prepared through the epoxide ring-opening reaction of PGMA segments. A mixture of PCL-b-PGMA (0.259 g), NaN3 (0.284 g, 4.37 mmol), NH4Cl (0.234 g, 4.37 mmol) and DMF (5 mL) were placed in a round bottom flask and stirred at 50 °C for 24 h. The product was extracted with a saturated sodium chloride solution, filtered and washed with deionized water several times. The product was dried in a vacuum oven at 40 °C for 24 h.
2.5. CCL micelles of PCL-b-PGMA-N3 block copolymer by click chemistry The PCL-b-PGMA-N3 block copolymer was dissolved in round bottom flask containing acetonitrile (20 mL) and a magnetic spin bar at ambient temperature. DPA and CuBr were added under nitrogen and stirred for 30 min. Afterward, the deoxygenated PMDETA was added to the solution using a syringe, and the color of the solution changed immediately to blue due to the formation of copper complexes. The solution was stirred at 50 °C for 24 h, and the catalysts were removed through the EDTA solution of ethanol/water (v/v: 1/1) [23]. The product was filtered and washed several times with deionized water. The CCL nanoparticles were dried in a vacuum oven at 40 °C for 24 h.
2.6. Characterization 1
H NMR spectra were recorded on a JNM-ECP 400 (JEOL) instrument. Gel permeation chromatography (GPC) was performed using a HP 1100 apparatus, with THF as a solvent at 25 °C and an elution rate of 1 mL/min. The columns were calibrated with commercial polystyrene standards. Fourier transform infrared (FTIR) spectra were measured on a JASCO FT/IR-4100 spectrometer with DLATGS detector in the 4000–400 cm1 spectral region. The samples were finely ground, mix with spectroscopic grade KBr and pressed into pellets. The chemical states were investigated using X-ray Photoelectron Spectroscopy (XPS) (Thermo VG Multilab 2000) in ultra-high vacuum with Al Ka radiation. For transmission electron microscopy (TEM; JEOL JEM-2010), the samples were prepared by depositing a drop of dispersed nanoparticles in acetonitrile (upper portion) on copper grids. No staining was applied to the sample. Dynamic laser light scattering (DLS) measurements for determining the average hydrodynamic diameter of polymeric micelles were performed using an electrophoretic light scattering instrument (ELS-8000, Otsuka Electronics Corporation), equipped with an ELS controller, and a He–Ne laser at wavelength of 632.8 nm. The intensity of scattered light was detected at 90° to an incident beam. All the analyses were performed at 25 °C. The sample solutions (1 mg/mL) was prepared by dispersing the polymers in acetonitrile with ultrasonic oscillation for 30 min and the solution was allowed to settle down a small portion of agglomerate prior to analysis.
Fig. 1. Synthesis of PCL-b-PGMA-N3 block copolymers by ROP and RAFT polymerization, followed by direct preparation of CCL micelles.
270
X.T. Cao et al. / European Polymer Journal 68 (2015) 267–277
3. Results and discussion 3.1. Synthesis and characterization of PCL-b-PGMA-N3 block copolymers by ROP and RAFT polymerization Our primary aim was to develop a novel route for direct preparation of CCL micelles. Block copolymers which consist of PCL as an ‘‘inactive’’ block and azide-functionalized PGMA as an ‘‘active’’ block were crosslinked using click reaction strategy as shown in Fig. 1. The PCL-b-PGMA was synthesized by ROP and RAFT polymerization with 2-benzylsulfanylthiocarbonylsulfanyl ethanol as a co-initiator. Table 1 Characteristics of the PCL-RAFT macroinitiator 1 and PCL-b-PGMA block copolymers 2–4 which were prepared by ROPa and RAFT polymerization.b Sample no.
1 2 3 4 a b c
Polymer
PCL126-RAFT PCL126-b-PGMA23 PCL126-b-PGMA56 PCL126-b-PGMA154
Feed ratio [M]eCL/[M]RAFTagent
[M]GMA/[M]PCL-RAFT
40 – – –
– 32 94 188
Conversionc (%)
Mn (1H NMR)
Mn (GPC)
PDI (GPC)
60.9 35.1 49.6 63.2
15,000 18,300 23,000 37,000
10,700 11,500 9300 13,200
1.52 1.64 1.65 1.59
ROP was carried out at 140 °C for 12 h. Molar ratio [RAFT agent]:[Sn(Oct)2] = 10:1. Polymerization was carried out at 80 °C for 24 h. Molar ratio [PCL-RAFT]:[AIBN] = 5:3. Conversion was determined gravimetrically.
Fig. 2. GPC chromatographs for polymers (see Table 1).
Fig. 3. FT-IR spectra of (a) PCL126-RAFT macroinitiator; (b) PCL126-b-PGMA23, (c) PCL126-b-PGMA56, (d) PCL126-b-PGMA154 (Table 1); and (e) PCL156-bPGMA23-N3, (f) PCL156-b-PGMA56-N3, (g) PCL156-b-PGMA154-N3, (h) CCL micelles of PCL156-b-PGMA56-N3.
X.T. Cao et al. / European Polymer Journal 68 (2015) 267–277
271
Fig. 4. 1H NMR spectra of (a) PCL126-RAFT macroinitiator in CDCl3, (b) PCL126-b-PGMA56, (c) PCL126-b-PGMA56-N3, and (d) CCL micelles of PCL126-b-PGMA56N3 in DMSO-d6.
As shown in Table 1, block copolymers can be prepared with the different chain length of PGMA. Living radical polymerization was evidenced by the linear correlation between monomer conversion and molecular weight of block copolymers. The GPC curves of block copolymers were relatively unimodal and symmetric (Fig. 2). Moreover, the polydispersity (PDI) of the block copolymers 2–4 after the RAFT process was quite close to the precursor of macro-initiator 1 indicating a good level of control in the RAFT-mediate reaction.
272
X.T. Cao et al. / European Polymer Journal 68 (2015) 267–277
Fig. 3 depicts the FT-IR spectra of polymers. In Fig. 3b, the epoxy ring vibration bands of PCL-g-PGMA block copolymers are shown at 844, 905, and 993 cm1. The absorption bands at 2946 and 2999 cm1 could be ascribed to the vibration of methylene and methyl protons of the PGMA segment. These characteristic bands overlap with the methylene vibration of the PCL-RAFT macroinitiator (Fig. 3a), so that they cannot be seen independently in the FT-IR spectrum. Moreover, the intensity of the peaks at 844 and 905 cm1 corresponding to CAO stretching of cis- and trans-epoxy groups increased with the PGMA chain length, indicating more GMA units in the block copolymers (Fig. 3b–d). The azidation of PCL-b-PGMA could be presented by the new absorption band at 2095 cm1 which was assigned to the valence vibration of azide functions in PGMA-N3 moieties (Fig. 3e–g). In addition, the opened epoxy ring generated a stronger and broader band of hydroxyl groups at 3388 cm1, whereas the signal of the ring at 844, 905, and 993 cm1 disappeared completely. After click reaction, the FT-IR spectrum of nanogels showed reduced intensity of the characteristic band of azides, confirming their reaction with alkyne groups of the cross-linker (Fig. 3h). The residual band at 2095 cm1 of the nanogels indicates the unconsumed azide functionalities of the block copolymer. These data demonstrate that the attempted cross-linking reaction of the block copolymer unimers in a nonselective solvent was indeed successful and that this methodology allowed for the incorporation of further click readied azido-functionality within the core of the cross-linked polymer nanoparticle. The structure of polymers was confirmed by 1H NMR spectra. As shown in Fig. 4a, the methylene protons in the main chain of PCL (a–f) at 4.02, 3.61, 2.27, 1.60, and 1.34 ppm were visualized while the peak at 7.27 ppm was assigned to the phenyl ring (g) in the end group of the RAFT agent. After the RAFT polymerization of GMA for PCL-b-PGMA, the additional peaks at 3.21, 2.80, and 2.67 ppm are associated to the methylene proton (s, t) of the epoxy ring as well as signals at 4.31, 3.73, 1.89, 1.82, 1.20, 0.98, and 0.81 ppm contributed to methylene (r, p) and methyl (q) proton of PGMA moieties (Fig. 4b). The epoxide ring-opening process was revealed by change of the proton bearing epoxy ring in spectroscopy, which was depicted in Fig. 4c. The characteristic peaks of the PCL part were maintained even though the signals of the PGMA moiety have already changed. The new peaks of the opened ring appeared at 5.51, and 3.88 ppm, which implied to the proton of CHAO (s0 ), CH2AO (r0 ), and CH2AN3 (t0 ) whereas the epoxy ring protons at 4.31, 3.73, 3.21, 2.80, 2.67 ppm disappeared. The result demonstrated that azido groups were anchored on the backbone of PGMA part. The click reaction between dipropargyl adipate and the azido groups in the PGMA-N3 parts induced CCL micelles owing to the reduced solubility in the crosslinked region. The CCL micelles consist of the PCL shell and the core of cross-linked PGMA-N3. 1H NMR spectrum indicated the depression of the signal pertinent to the PGMA-N3 moiety whose protons were shielded from magnetic
Fig. 5. XPS C 1s core-level spectra of (a) PCL126-b-PGMA56, (b) PCL126-b-PGMA56-N3, and XPS N 1s core-level spectra of (c) PCL126-b-PGMA56, (d) PCL126-bPGMA56-N3, and CCL micelles of (e) PCL126-b-PGMA56-N3.
273
X.T. Cao et al. / European Polymer Journal 68 (2015) 267–277
Fig. 6. Photographic illustration for solution aggregation of (a) core non-cross-linked micelles; (b, d, f) macrogels; and (c, e, g) nanogels of sample 7, 13, 18; and 8, 14, 19 (see Table 2), respectively.
resonance by PCL coronas [11]. Consequently, the proton signal of cross-linked PGMA-N3 for r0 , s0 , t0 , p, and q decreased significantly in comparison with the non-cross-linked PGMA-N3 segment (Fig. 4d). The XPS analyses were performed to demonstrate binding formation of the block copolymer and CCL micelles. The C 1s core-level spectrum of PCL-b-PGMA with three peaks at binding energies (BEs) of 284.8, 286.5, and 288.4 eV (Fig. 5a) implied to CAC/CAH, CAO, and OAC@O bonds. However, the N 1s core-level spectrum was not observed since there is no nitrogen in PCL-b-PGMA (Fig. 5c). After the azidation reaction, the new peak C 1s core-level appeared at BE of 287 eV indicating CAN bond formation (Fig. 5b). The N 1s core-level of the azido groups was observed at BEs of 399.7, 400.7, and 403.8 eV corresponding to AN@N+@N, AN@N+@N, and AN@N+@N bond, respectively (Fig. 5d). Moreover, the relative area of these three peaks with proportion of 1:1:1 was evidence for the chemical structure of three nitrogen atoms of the azido group. The covalent bond between the azide and alkyne groups was demonstrated by the N 1s spectrum in Fig. 5e. Two peaks at 401.5 (NANAN) and 400.2 eV (NANAC) with area ratio of 1:2 approximately were ascribed to the N 1s of the triazole ring, which was obviously different from AN3 in Fig. 5d. The results are consistent with its theoretical values.
3.2. The formation of macrogel or nanogel materials The click chemistry was employed to investigate the formation of gel materials. CCL micelles of PCL-b-PGMA-N3 were prepared by solutions of the block copolymer and DPA in acetonitrile catalyzed by CuBr/PMDETA. The formation of nanogels
Table 2 Characterization of CCL micelles of block copolymers by click reaction. The reaction was catalyzed by CuBr/PMDETA (nCuBr = nPMDETA = 0.3 ncross-linker) in acetonitrile. Block copolymers
a b c
Sample
wt%a
Cross-linker (mmol) (wt%)
Alkyne: azide molar ratiob
Aggregationc
PCL126-b-PGMA23-N3
1 2 3 4 5 6 7 8 9
0.127 0.127 0.127 0.127 0.635 1.270 1.270 1.270 1.270
0.028 0.023 0.020 0.014 0.014 0.140 0.105 0.096 0.070
(0.040) (0.032) (0.029) (0.020) (0.020) (0.200) (0.150) (0.138) (0.100)
1.8:1 1.5:1 1.3:1 0.9:1 0.18:1 0.9:1 0.675:1 0.619:1 0.45:1
Macrogel Macrogel Nanogel Nanogel Nanogel Macrogel Macrogel Nanogel Nanogel
PCL126-b-PGMA56-N3
10 11 12 13 14
0.127 0.127 0.635 1.270 1.270
0.020 0.018 0.018 0.096 0.083
(0.029) (0.025) (0.025) (0.138) (0.119)
0.9:1 0.8:1 0.16:1 0.413:1 0.357:1
Macrogel Nanogel Nanogel Macrogel Nanogel
PCL126-b-PGMA154-N3
15 16 17 18 19
0.127 0.127 0.635 1.270 1.270
0.014 0.011 0.011 0.083 0.070
(0.020) (0.015) (0.015) (0.119) (0.100)
0.4:1 0.3:1 0.06:1 0.226:1 0.200:1
Macrogel Nanogel Nanogel Macrogel Nanogel
Weight concentration of PCL-b-PGMA-N3 block copolymers in acetonitrile. Alkyne: azide molar ratio was calculated from the number of alkyne and azide groups of the block copolymer and DPA, respectively. Aggregation was observed by naked eyes as shown in Fig. 6.
274
X.T. Cao et al. / European Polymer Journal 68 (2015) 267–277
Fig. 7. Schematic illustration for nanogel or macrogel formation through CCL micelle preparation.
and macrogels was investigated with the variation of the PGMA chain length and the concentration of the block polymer and the cross-linker. The concentration of the block copolymer was varied in a range from 0.127 to 1.270 wt% to obtain monodisperse species [14]. The experimentally observed transition of a completely soluble mixture to one having an insoluble fraction was known as nanogels or macrogels [24]. This work demonstrates that the aggregation of the block copolymer solution depends on the concentration of the cross-linker and the block copolymer, and the size of the PGMA-N3 block. In the block copolymer PCL plays a role as ‘‘inactive’’ chain and PGMA-N3 is an ‘‘active’’ segment which undergone the cross-linking reaction. The PCL126-b-PGMA23-N3 was soluble in acetonitrile to form unimer solution without any aggregation as observed by the naked eye (Fig. 6a). Upon click reaction, the unimers were self-assemble into the micellar structure whose PCL chains became corona and the cross-linked PGMA-N3 part was a core. The formation of macrogel or nanogel materials can be distinctly observed as shown in Fig. 6. When the aggregation is macrogel, the resulting solution separates into two parts; the upper part is transparent and lower one is as gel-like substance (Fig. 6b, d, and f). Otherwise, the solution is bluish haze liquid without precipitation due to nanogel particles (Fig. 6c, e, and g). The observation has been explained deeply by experimental data.
Fig. 8. TEM images of CCL micelles of the block copolymer: sample (a) 8, (b) 14, (c) 7, and (d) 13 (see Table 2).
X.T. Cao et al. / European Polymer Journal 68 (2015) 267–277
275
As shown in Table 2, at concentration of 0.127 wt% of PCL126-b-PGMA23-N3, sample 2 and 3 presented transition concentrations of the cross-linker to form macrogels and nanogels corresponding to alkyne:azide (cross-linker to N3 in block copolymer) molar ratio of 1.5:1 and 1.3:1, respectively. The result can be explained by inter-micellar click reaction occurs partially at the higher ratio of the cross-linker relative to the block copolymer. In other words, the cross-linking reaction takes place not only intra- but also inter-micelles when the alkyne:azide molar ratio is above 1.5:1. As a result, the macrogel material was produced in the solution (sample 1 and 2). At a higher concentration of 1.270 wt% of the block copolymer, sample 7 and 8 showed the lower transition molar ratio of alkyne: azide groups for macrogels and nanogels compared with 0.127 wt%, corresponding to the ratio of 0.675:1 and 0.619:1, respectively. The result indicated that the ratio of the cross-linker to the block copolymer should be reduced to foam nanogels at the higher concentration of the block copolymer. This is attributed to the fact that the higher concentration of the PGMA-N3 moiety has more azido groups therefore more reaction probability toward the cross-linker. The experiments were also carried out for the block copolymers of different block ratios, PCL126-b-PGMA56-N3 and PCL126-b-PGMA154-N3 by the same procedure. The data showed that, for the nanogel formation, the less amount of the cross-linker was used when the PGMA-N3 chain length increased at the same concentration of three block copolymers (sample 3, 11, and 16; sample 8, 14, and 19). The more GMA units mean more azido groups, and a less amount of the cross-linker
Fig. 9. Size distribution of (a) PCL126-b-PGMA23-N3 in acetonitrile and CCL micelles of the block copolymer: sample (b) 8 and (c) 14 (see Table 2).
276
X.T. Cao et al. / European Polymer Journal 68 (2015) 267–277
Table 3 Characterization of CCL micelles of the block copolymer analyzed by GPC. Sample (see Table 2)
Maw (g mol1)
Mbn (g mol1)
Mbw (g mol1)
PDIb
Average number of arms per micelle
3 8
18,870 15,350
100,560 126,500
161,900 206,200
1.61 1.63
8 13
Molecular weight of the block copolymer was determined by GPC beforea and afterb CCL micelles, THF was used as an eluent.
is needed to be free from the inter-micellar coupling. The result demonstrated that gel materials could be controlled eventually by an adjustment of the concentration of the cross-linking agent and the block polymer and the size of the PGMA-N3 block in terms of the molar ratio of alkyne to azide groups. Furthermore, it is important to notice that the nanogels contain unreacted, further click-readied azide-functionality within the core of the cross-linked polymer nanoparticles, which can be used to attach a low molecular compound such as drugs, possibly extended to the potential application for a drug carrier. 3.3. The structure of CCL micelles The schematic diagram for the CCL micelles can be represented as Fig. 7. The resulting macrogels or nanogels depend on the reaction condition. The micelle morphology was obtained by TEM as shown in Fig. 8. TEM images display nanoparticles which are dark spheres with an average diameter of 100 and 103 nm corresponding to sample 8 (Fig. 8a) and 14 (Fig. 8b), respectively (Table 2). The discrete nanoparticles were produced with the inter-chain cross-link of block copolymers. Chained nanoparticles with undefined form were obtained by the increased concentration of the cross-linker due to undesirable inter-micellar cross-linking, which inevitably resulted in micelle fusion (Fig. 8c and d). Consequently, they were highly agglomerated and separated from the solution as macrogels. The results are in good agreement with the experiment where the gel materials were observed by naked eyes as shown in Fig. 6. Hydrodynamic diameters of the CCL micelles were analyzed by DLS as shown in Fig. 9. PCL-b-PGMA-N3 produced a homogeneous solution in acetonitrile. The average hydrodynamic diameter of PCL126-b-PGMA23-N3 in acetonitrile was approximately 4 nm, indicating a unimer solution (Fig. 9a). After the formation of nanogels by click reaction, the block copolymer solution showed the particle distributions at 111 and 117 nm of hydrodynamic diameter corresponding to the sample 8 (Fig. 9b) and 14 (Fig. 9c), respectively. The size distribution of nanoparticles becomes broad with the increase of the PGMA block chain length of the precursor. The results are consistent with the TEM analyses. GPC analysis can estimate the average number of arms of CCL micelles by dividing the molecular weight of CCL micelles by the molecular weight of the block copolymer. As shown in Table 3, the number of arms increases with the chain length of PGMA-N3 implying the CCL micelles could be controlled by using click reaction. 4. Conclusion The direct preparation of CCL micelles was achieved by the click reaction between PCL-b-PGMA-N3 and DPA in the homogeneous solution. A series of PCL-b-PGMA polymers varying the PGMA block length were prepared by ROP and RAFT polymerization with a relatively low polydispersity. The structure of polymers was confirmed by FT-IR and 1H NMR spectra. The FT-IR showed the characteristic vibration of azido groups of PCL-b-PGMA-N3. In the 1H NMR spectrum of the block copolymer, the new peaks of the opened epoxy ring appeared at 5.51 and 3.88 ppm while the epoxy ring protons at 4.31, 3.73, 3.21, 2.80, 2.67 ppm disappeared. In the XPS spectra, the N 1s core-level was observed at 399.7, 400.7, and 403.8 eV with relative area of these three peaks with proportion of 1:1:1 indicating the chemical structure of the azido group. Two peaks at 401.5 and 400.2 eV with area ratio of 1:2 were ascribed to the N 1s of the triazole ring in the cross-linked PGMA-N3 segments. The formation of gel materials could be recognized by visual and experimental observations, which was depended on the click reaction condition. The one-step micellization producing the nanogels was controlled by the concentration of the cross-linker and the block copolymer as well as the size of the PGMA-N3 block in terms of the molar ratio of alkyne to azide groups. The nanogels with a diameter of around 100 nm (measured by TEM and DLS) could be produced simply with adjusting the complementary relationship among the three variables. The facile nature of the click chemistry used for cross-linking of the block copolymer also left unreacted azide functionalities located on the core to be detected by IR spectroscopic analysis. This cross-linking strategy and simultaneous incorporation of click-readied functionality provides a facile route for the future development of these nanostructures in biomedical applications. Acknowledgement This work was supported by the BK-21 Plus program. References [1] M.H. Stenzel, RAFT polymerization: an avenue to functional polymeric micelles for drug delivery, Chem. Commun. (2008) 3486–3503. [2] K. Halake, M. Birajdar, B.S. Kim, H. Bae, C.C. Lee, Y.J. Kim, S. Kim, H.J. Kim, S. Ahn, S.Y. An, J. Lee, Recent application developments of water-soluble synthetic polymers, J. Ind. Eng. Chem. 20 (2014) 3913–3918.
X.T. Cao et al. / European Polymer Journal 68 (2015) 267–277
277
[3] R. Duncan, The dawning era of polymer therapeutics, Nat. Rev. Drug Discover. 2 (2003) 347–360. [4] K. Kataoka, A. Harada, Y. Nagasaki, Block copolymer micelles for drug delivery: design, characterization and biological significance, Adv. Drug Deliv. Rev. 47 (2001) 113–131. [5] A. Rösler, G.W.M. Vandermeulen, H.A. Klok, Advanced drug delivery devices via self-assembly of amphiphilic block copolymers, Adv. Drug Deliv. Rev. 64 (2012) 270–279. [6] S. Samarajeewa, R. Shrestha, M. Elsabahy, A. Karwa, A. Li, R.P. Zentay, J.G. Kostelc, R.B. Dorshow, K.L. Wooley, In vitro efficacy of paclitaxel-loaded dualresponsive shell cross-linked polymer nanoparticles having orthogonally degradable disulfide cross-linked corona and polyester core domains, Mol. Pharm. 10 (2013) 1092–1099. [7] Y. Chan, T. Wong, F. Byrne, M. Kavallaris, V. Bulmus, Acid-labile core cross-linked micelles for pH-triggered release of antitumor drugs, Biomacromolecules 9 (2008) 1826–1836. [8] B.M. Blunden, D.S. Thomas, M.H. Stenzel, Analysis of thiol-sensitive core-cross-linked polymeric micelles carrying nucleoside pendant groups using ‘On-line’ methods: effect of hydrophobicity on cross-linking and degradation, Aust. J. Chem. 64 (2011) 766–778. [9] M.D. Hossain, L.T.B. Tran, J.M. Park, K.T. Lim, Facile synthesis of core-surface crosslinked nanoparticles by interblock RAFT polymerization, J. Polym. Sci. Part A: Polym. Chem. 48 (2010) 4958–4964. [10] E. Yoshida, S. Kunugi, Micelle formation of nonamphiphilic diblock copolymers through noncovalent bond cross-linking, Macromolecules 35 (2002) 6665–6669. [11] E. Yoshida, M. Tanaka, T. Takata, Direct preparation of core cross-linked micelles in a nonselective solvent, Colloid Polym. Sci. 284 (2005) 51–57. [12] Y. Amamoto, Y. Higaki, Y. Matsuda, H. Otsuka, A. Takahara, Programmed thermodynamic formation and structure analysis of star-like nanogels with core cross-linked by thermally exchangeable dynamic covalent bonds, J. Am. Chem. Soc. 129 (2007) 13298–13304. [13] Y. Amamoto, M. Kikuchi, H. Masunaga, S. Sasaki, H. Otsuka, A. Takahara, Intelligent build-up of complementarily reactive diblock copolymers via dynamic covalent exchange toward symmetrical and miktoarm star-like nanogels, Macromolecules 43 (2010) 1785–1791. [14] A.W. Jackson, D.A. Fulton, The formation of core cross-linked star polymers containing cores cross-linked by dynamic covalent imine bonds, Chem. Commun. 46 (2010) 6051–6053. [15] A.W. Jackson, D.A. Fulton, PH triggered self-assembly of core cross-linked star polymers possessing thermoresponsive cores, Chem. Commun. 47 (2011) 6807–6809. [16] R.K. O’Reilly, M.J. Joralemon, C.J. Hawker, K.L. Wooley, Preparation of orthogonally-functionalized core click cross-linked nanoparticles, New J. Chem. 31 (2007) 718–724. [17] M.J. Joralemon, R.K. O’Reilly, C.J. Hawker, K.L. Wooley, Shell click-crosslinked (SCC) nanoparticles: a new methodology for synthesis and orthogonal functionalization, J. Am. Chem. Soc. 127 (2005) 16892–16899. [18] S.M. Garg, X.B. Xiong, C. Lu, A. Lavasanifar, Application of click chemistry in the preparation of poly(ethylene oxide)-block-poly(e-caprolactone) with hydrolyzable cross-links in the micellar core, Macromolecules 44 (2011) 2058–2066. [19] Z. Zhang, L. Yin, C. Tu, Z. Song, Y. Zhang, Y. Xu, R. Tong, Q. Zhou, J. Ren, J. Cheng, Redox-responsive, core cross-linked polyester micelles, J. ACS Macro Lett. 2 (2013) 40–44. [20] C. Allen, D. Maysinger, A. Eisenberg, Nano-engineering block copolymer aggregates for drug delivery, Colloid Surf. B 16 (1999) 3–27. [21] A.O. Saeed, S. Dey, S.M. Howdle, K.J. Thurecht, C.J. Alexander, One-pot controlled synthesis of biodegradable and biocompatible co-polymer micelles, Mater. Chem. 19 (2009) 4529–4535. [22] R. Nomura, K. Yamada, J. Tabei, Y. Takakura, T. Takigawa, T. Masuda, Stimuli-responsive organogel based on poly(N-propargylamide), Macromolecules 36 (2003) 6939–6941. [23] I.V. Gürsel, F. Aldiansyah, Q. Wang, T. Noël, V. Hessel, Continuous metal scavenging and coupling to one-pot copper-catalyzed azide-alkyne cycloaddition click reaction in flow, Chem. Eng. J. 270 (2015) 468–475. [24] C.J. Kloxin, T.F. Scott, B.J. Adzima, C.N. Bowman, Covalent adaptable networks (CANs): a unique paradigm in cross-linked polymers, Macromolecules 43 (2010) 2643–2653.