Preparation of intermediary layer crosslinked micelles from a photocrosslinkable amphiphilic ABC triblock copolymer

European Polymer Journal 45 (2009) 1918–1923

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Macromolecular Nanotechnology

Preparation of intermediary layer crosslinked micelles from a photocrosslinkable amphiphilic ABC triblock copolymer Jin Sook Kim, Hyun Jeong Jeon, Mi Seon Park, Young Chang You, Ji Ho Youk * Department of Advanced Fiber Engineering, Division of Nano-Systems, Inha University, Incheon 402-751, Republic of Korea

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a r t i c l e

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Article history: Received 14 October 2008 Received in revised form 5 March 2009 Accepted 29 March 2009 Available online 5 April 2009

Keywords: Amphiphilic ABC triblock copolymer Atom transfer radical polymerization (ATRP) Photocrosslinkable micelle Intermediary layer Cinnamate group

a b s t r a c t To prepare intermediary layer crosslinked micelles, a photocrosslinkable amphiphilic ABC triblock copolymer, poly(ethylene glycol)-b-poly(2-cinnamoyloxyethyl methacrylate)-bpoly(methyl methacrylate) (PEG–PCEMA–PMMA), was synthesized and its micellar characteristics were investigated. The triblock copolymer of PEG-b-poly(2-hydroxyethyl methacrylate)-b-PMMA (PEG–PHEMA–PMMA) (Mn = 9800 g/mol, Mw/Mn = 1.33) was first polymerized by activators generated by electron transfer (AGET) atom transfer radical polymerization (ATRP) using a PEG macroinitiator in a mixed solvent of anisole/2-isopropanol (3/1 v/v). The middle block of the copolymer was then functionalized with cinnamoyl chloride. The degrees of polymerization of the PEG, PHEMA, and PMMA blocks were 113, 18 and 21, respectively. The critical micelle concentration (CMC) of the PEG–PCEMA–PMMA was 0.011 mg/mL. The PEG–PCEMA–PMMA micelles were spherically shaped with an average diameter of 43 nm. The intermediary layer of the PEG–PCEMA–PMMA micelles was crosslinked by UV irradiation. Not all of the cinnamate groups underwent photocrosslinking probably due to a lack of other cinnamate groups in their immediate vicinity. However, the degree of photocrosslinking of the intermediary layer of the PEG–PCEMA–PMMA micelles was sufficient to give excellent colloidal stability, even in different external environments. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Amphiphilic block copolymers can self-assemble to form polymeric micelles in aqueous solution. The shell of such a micelle consists of a protective corona that stretches outside to stabilize the micelle, and the hydrophobic micelle core can provide a carrier compartment for hydrophobic agents. However, amphiphilic block copolymer micelles often dissociate into unimers when diluted or subjected to elevated temperatures. In order to overcome this drawback, amphiphilic block copolymer micelles have been transformed into robust nanoparticles with well-defined core-shell morphologies via covalent crosslinking of their cores or shell domains [1–17]. Shell crosslinked (SCL) micelles have cores with improved stability and exhibit unique physical properties that are not

* Corresponding author. Tel.: +82 32 860 7498; fax: +82 32 873 0181. E-mail address: [email protected] (J.H. Youk). 0014-3057/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.eurpolymj.2009.03.022

obtainable from a single nanoparticle [1–8]. Their properties are intermediate between those of micelles, microgels, nanoparticles, and dendrimers. SCL micelles have been suggested for various applications [2,17]. However, one major drawback in the synthesis of SCL micelles is that the shell crosslinking must be carried out in a highly dilute solution (typically 0.1–0.5% solids) in order to avoid extensive intermicellar crosslinking. Liu and coworkers [9–13] synthesized various poly(2-cinnamoylethyl methacrylate) (PCEMA) block copolymers and used them to prepare stable polymer micelles with photocrosslinked cores. Core crosslinking is beneficial because it leaves the micelle shell available for tailoring to a variety of uses. On the other hand, the crosslinked core domain may limit core mobility and guest loading. ABC triblock copolymers have been reported to offer great advantages over conventional AB diblock copolymers for the preparation of crosslinked micelles, since they can allow crosslinking of the central B block at relatively high solid

J.S. Kim et al. / European Polymer Journal 45 (2009) 1918–1923

2. Experimental part 2.1. Materials Methyl methacrylate (MMA, Duksan, 99.8%, Korea) and 2-hydroxyethyl methacrylate (HEMA, Acros, 96%) were passed through a column filled with neutral alumina, stirred over calcium hydride (CaH2), and then distilled under reduced pressure. Anisole (Acros, 99%) and isopropyl alcohol (Duksan, 99.5%) were distilled under vacuum. Tetrahydrofuran (THF, Duksan, 99.5%) was dried over Na and benzophenone. Poly(ethylene glycol) methyl ether (MPEG, Mn = 5000 g/mol, Fluka), ethyl 2-bromoisobutyrate (Aldrich, 98%), copper(II) bromide (CuBr2, Acros, 98%), (PMDETA, N,N,N0 ,N00 ,N00 -pentamethyldiethylenetriamine Acros, 99+%), cinnamoyl chloride (Aldrich, 98%), tin(II) 2ethylhexanoate (Sn(EH)2, Aldrich, 95%), triethylamine (Acros, 99%), pyrene (Aldrich, 98%), N,N-dimethylformamide (DMF, Duksan, 99.5%), acetone (Duksan, 99.5%) and diethyl ether (Duksan, 99%) were all used as received. 2.2. Synthesis of poly(ethylene glycol) (PEG) macroinitiator

bled with N2 for 10 min, and then PMDETA (0.075 mL, 0.36 mmol), HEMA (1.16 mL, 9.6 mmol), and Sn(EH)2 (0.116 mL, 0.36 mmol) were added. The mixture was then placed in an oil bath preheated at 80 °C. When the HEMA conversion had reached over 90% (determined by 1H NMR), degassed MMA (2.6 mL, 24 mmol) was added to the reaction flask. After 24 h, the mixture was diluted with THF and passed though a silica gel column to remove the copper catalyst. After evaporation of all of the solvent, drying in a vacuum oven at room temperature yielded a colorless copolymer. 2.4. Synthesis of PEG–PCEMA–PMMA triblock copolymer PEG–PHEMA–PMMA (3.5 g, 0.37 mmol), cinnamoyl chloride (2.197 g, 13.19 mmol), and TEA (2.29 mL, 16.43 mmol) were dissolved in dry THF (100 mL) contained in a reaction flask (250 mL). The solution was stirred overnight and then the salt formed was filtered off. The solvent was removed by a rotary evaporator. The polymer was subsequently precipitated in a large excess of ether. The yield was 2.9 g (64%). 2.5. Critical micelle concentration (CMC) of PEG–PCEMA– PMMA triblock copolymer To determine the CMC of the triblock copolymer, pyrene was used as a hydrophobic probe. A stock solution of pyrene (0.12 mg/mL) in acetone was added to deionized water. The acetone was then evaporated off. Known amounts of triblock copolymer stock solution (1.0 mg/ mL) in acetone were added to the pyrene solution, and deionized water was added dropwise very slowly. In order to remove the acetone, the solutions were stirred at 65 °C for 6 h. While the final pyrene concentration in each solution was 1.2  106 M, the polymer concentration varied from 1.0  105 to 1.0 mg/mL. The final solutions were allowed to stand for one day for equilibration at room temperature. Steady-state fluorescence spectra were recorded on a Shimadzu RF-5301 fluorescence spectrometer with a bandwidth of 5 nm for excitation and emission. The excitation spectra were obtained using an emission wavelength of 390 nm.

MPEG (6.0 g) was dissolved in THF (100 mL). TEA (0.60 g, 5.9 mmol) and 2-bromoisobutyryl bromide (0.44 g, 1.9 mmol) were added dropwise to this solution, which was being kept in an ice bath. After stirring for 48 h, the amine salt that had formed was removed by filtration. After concentrating the solution, the resulting polymer was precipitated in a large excess of diethyl ether. For further purification, the polymer solution in THF was reprecipitated in diethyl ether to give a yield of 5.2 g (84%).

2.6. Photocrosslinking of the intermediary layer of the PEG– PCEMA–PMMA micelles

2.3. Synthesis of PEG-b-poly(2-hydroxyethyl methacrylate)b-PMMA (PEG–PHEMA–PMMA) triblock copolymer

The molecular weight (MW) and polydispersity of the resulting polymers were determined by a gel permeation chromatography (GPC) system (Young Lin SP930D solvent delivery pump) coupled with an RI detector (RI 750F) and two columns (GPC KD-G and KF-806, Shodex). DMF at 40 °C was used as the eluent at a flow rate of 1.0 mL/min. Poly(ethylene oxide) standards were used for calibration.

A reaction flask (100 mL) with a magnetic stirrer and a rubber septum was charged with the PEG macroinitiator (2.4 g, 0.48 mmol), CuBr2 (0.05 g, 0.024 mmol), anisole (15 mL), and 2-isopropanol (5 mL). The mixture was bub-

The intermediary layer of the PEG–PCEMA–PMMA micelles was crosslinked at room temperature by UV irradiation using a 500 W high-pressure mercury lamp (Ushio UI-501-C) located 15 cm from the sample surface. 2.7. Characterization

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contents (10 wt%) with negligible intermicellar crosslinking [2,6]. The outer block chains can stabilize the micelles during the crosslinking, thereby minimizing micelle aggregation. These intermediary layer crosslinked micelles have potential applications in well-defined nanoreactors, agents for encapsulation, transduction, and drug delivery, etc [7]. In this study, to prepare intermediary layer crosslinked micelles, a photo-crosslinkable amphiphilic ABC triblock copolymer, poly(ethylene glycol)-b-poly(2-cinnamoyloxyethyl methacrylate)-b-poly(methyl methacrylate) (PEG– PCEMA–PMMA), was synthesized via activators generated by electron transfer (AGET) atom transfer radical polymerization (ATRP) and its micellar characteristics were investigated. The intermediary layer of its micelles was crosslinked by UV irradiation in aqueous solution, while the outer PEG blocks stabilized the micelles. This amphiphilic triblock copolymer has the potential to be used to prepare encapsulated functional nanomaterials having high colloidal stability in water.

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J.S. Kim et al. / European Polymer Journal 45 (2009) 1918–1923

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1

H NMR spectroscopy was performed using a Varian VXRUnity NMR spectrometer (400 MHz) with CDCl3 or DMSOd6 as the solvent. The excitation and luminescence spectra were measured using a Shimadzu RF-5301PC spectrofluorophotometer at room temperature. The micellar size and size distribution were determined by dynamic light scattering (DLS) using a BI-200SM particle size analyzer. Each analysis interval lasted 360 s and was performed at room temperature with an angle detection of 90°. The concentration of the polymer solution was 0.1 mg/mL. The morphologies of the copolymer micelles were investigated using a JEM-2100F transmission electron microscope operating at an accelerating voltage of 120 kV. A drop of aqueous PEG–PCEMA–PMMA triblock copolymer solution (20 mg/mL) was deposited onto a 200 mesh copper grid that had been coated with carbon. The size and shape of the micelles were directly determined using transmission electron microscopy (TEM). FTIR spectra were obtained from 64 scans at 2 cm1 resolution using a Vertex 80 V FTIR spectrometer (Bruker). The samples to be studied by infrared spectroscopy were prepared as thin films by dropping the aqueous PEG–PCEMA–PMMA solutions (20 mg/ mL) directly onto ZnSe discs. The thin films were UV irradiated for different times. 3. Results and discussion Scheme 1 shows a general reaction route for the synthesis of the PEG–PCEMA–PMMA triblock copolymer. The PEG–PHEMA–PMMA triblock copolymer was first synthesized via AGET ATRP using a PEG macroinitiator in a mixed solvent of anisole/2-isopropanol (3/1 v/v) at 80 °C. ATRP is one of the most successful controlled radical polymerization techniques for the preparation of polymers having controlled MWs and MW distributions with well-defined architectures [18]. A recent development in ATRP is the use of AGET to initiate the reaction. In AGET ATRP, essentially all of the Cu(II) species are quickly reduced to Cu(I). Normal ATRP starts in the presence of >1000 ppm of catalyst [19,20]. In this study, the Cu(I) active catalyst was

CuBr2/PMDETA Sn(EH)2

O H3C

O

O

O H3C

Br

113

formed by the reaction between the Sn(EH)2 reducing agent and the deactivator. When the HEMA conversion had reached over 90% (after 2 h), degassed MMA was added to the reaction solution for a consecutive ATRP. The PEG–PHEMA–PMMA triblock copolymer obtained was subsequently functionalized with cinnamoyl chloride in order to introduce photocrosslinkable groups into the PHEMA middle block. The chemical structures of PEG– PHEMA–PMMA and PEG–PCEMA–PMMA were verified by analyzing their 1H NMR spectra, which are shown in Fig. 1. The degree of PHEMA block functionalization by cinnamate groups was determined to be above 95%. The cinnamate group is hydrophobic and hence the hydrophilic PHEMA block was changed rather than hydrophobic block. Therefore, the block lengths of the hydrophobic units were increased after the functionalization step. The molecular characteristics of the triblock copolymer were determined by a combination of GPC and 1H NMR spectroscopy. Fig. 2 shows the GPC traces of the PEG macroinitiator, PEG–PHEMA diblock precursor, and PEG–PHEMA–PMMA triblock copolymer. All of the GPC traces were monomodal without any tailing caused by the residual PEG macroinitiator, indicating the high initiation efficiency. The clean and clear shift toward the higher molar mass region with successive AGET ATRP revealed the successful formation of PEG–PHEMA–PMMA (Mn = 9,800 g/ mol, Mw/Mn = 1.33). The degrees of polymerization of the PHEMA and PMMA blocks were determined by 1H NMR spectroscopy to be 18 and 21, respectively, on the basis of the PEG block. The Mn of PEG–PHEMA–PMMA was calculated to be 9400 g/mol, which is similar to that determined by GPC. The micellar characteristics of PEG–PCEMA–PMMA in aqueous solution were investigated by using a fluorescence technique, DLS, and TEM (Figs. 3–5). The CMC of PEG–PCEMA–PMMA was determined by a fluorescence technique using pyrene as a fluorescence probe. Fig. 3 shows the intensity ratios (I337/I334) of the pyrene excitation spectra according to the PEG–PCEMA–PMMA concentration. A negligible change in the intensity ratio was detected in

O

Br O

18

113

O O

O

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OH

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OH O H3C

O

O

Br

O

O

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H3C

21

18

113

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O

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Br

O

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113

O

Scheme 1. Reaction pathway for the synthesis of PEG–PCEMA–PMMA.

O

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Cl OH

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J.S. Kim et al. / European Polymer Journal 45 (2009) 1918–1923

O

H3C

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c

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H 3C

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d Fig. 3. Plot of the intensity ratio (I337/I334) of the pyrene excitation spectra vs. log C for PEG–PCEMA–PMMA.

a

h

g'

-5

Log C (g/L)

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21

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hi

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8

f

I337 /I334

g

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c

1

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δ (ppm) Fig. 1. 1H NMR spectra of (a) PEG–PHEMA–PMMA and (b) PEG–PCEMA– PMMA.

Mn

Mw/Mn

(a) 5100 (b) 7800 (c) 9800

1.06 1.15 1.33

(c) (b) (a)

Fig. 4. TEM image of the PEG–PCEMA–PMMA micelles.

16

17

18

19

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21

Elution time (min) Fig. 2. GPC curves of (a) the PEG macroinitiator, (b) PEG–PHEMA, and (c) PEG–PHEMA–PMMA.

the low concentration range. However, at a certain concentration, the intensity ratio started to increase substantially, which indicated the incorporation of pyrene into the hydrophobic core of the micelles. The CMC was determined from the crossover point in the low concentration range. The CMC of PEG–PCEMA–PMMA was 0.011 mg/mL, which is comparable with values reported for other polymeric amphiphiles [21]. The CMC value of a block

Scattering intensity

100

Dn= 43nm

75

50

25

0 0

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Diameter (nm) Fig. 5. Particle size distribution of the PEG–PCEMA–PMMA micelles.

copolymer is dependent on the block composition. The CMC of a given system is a measure of the ease of

J.S. Kim et al. / European Polymer Journal 45 (2009) 1918–1923

1.0

0.8

min.

Absorbance

formation of micelles and is therefore an indirect measurement of the stability of the micelles. A lower CMC value indicates higher stability. TEM was used to confirm the formation of the micelle and their sizes. Fig. 4 shows a TEM image of the PEG–PCEMA–PMMA micelles. The micelle solution of PEG–PCEMA–PMMA was prepared by adding deionized water dropwise to a PEG–PCEMA–PMMA solution in acetone (80 mg/mL). The acetone was removed by stirring the solution at 65 °C for 6 h. The final triblock copolymer concentration was 20 mg/mL. The micelles were spherically shaped. DLS investigation of the particle size and size distribution of the micelles revealed diameters in the range of 20–100 nm with an average diameter of 43 nm (Fig. 5). These results were consistent with the TEM results, considering that the DLS data reflected the micelle sizes in solution. The intermediary layer of the PEG–PCEMA–PMMA micelles was crosslinked by UV irradiation. Photocrosslinking of the PCEMA block has previously been used to prepare various block copolymer nanostructures [9–13]. The photocrosslinking chemistry of cinnamate groups is nontoxic, cost-effective, and does not produce any byproducts requiring removal after the reaction. In addition, it is known that the micellar characteristic features are not affected by the photocrosslinking process [22]. When cinnamate groups are exposed to UV, they undergo either photoisomerization or photodimerization [23–28]. The photodimerization of cinnamate groups essentially arises from the head-to-head and head-to-tail [2p + 2p] cycloaddition between the double bonds with favorable relative geometries. Due to the short lifetimes of the excited states of benzene derivatives, the two cinnamate groups must be very close to each other in order to facilitate the dimerization reaction [25]. Fig. 6 shows the FTIR spectra of the PEG– PCEMA–PMMA micelles with increasing UV irradiation time. The two major bands at 1637 and 1715 cm1, which were attributed to the AC@CA and unsaturated AC@O stretching vibrations of the cinnamate groups, respectively, are distinct and obvious. It is clear that the intensity of the band at 1637 cm1 decreased with increasing UV irradiation time as the photodimerization of the cinnamate groups proceeded. Concurrently, the saturated AC@O stretching band at 1730 cm1 increased in intensity and shifted to higher wavenumbers with increasing UV irradiation time. The extent of conversion of the cinnamate groups was evaluated by comparing the ratios of the areas of the AC@O and AC@CA peaks (area of AC@O/area of AC@CA) during the photocrosslinking process (Fig. 7). The ratio gradually increased with increasing UV irradiation time and then leveled off after 1 h. However, the AC@CA stretching vibration band was still observable after 90 min, indicating that not all of the cinnamate groups underwent photocrosslinking, probably due to a lack of other cinnamate groups in their immediate vicinity. The effectiveness of the crosslinking reaction was judged from the stability of the crosslinked PEG–PCEMA–PMMA micelles in THF, which can solubilize PEG–PCEMA–PMMA. Fig. 8 shows a TEM image of the PEG–PCEMA–PMMA micelles prepared by UV irradiation for 1 h, followed by dissolution in THF. The spherical structures of the crosslinked

0 15

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1750

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Wavenumbers cm-1 Fig. 6. Changes in the FTIR spectra of the PEG–PCEMA–PMMA micelles with increasing UV irradiation time.

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Area ratio (-C=O/-C=C-)

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30

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UV irradiation time (min) Fig. 7. Increase in the ratio of the AC@O and AC@CA peak areas with increasing UV irradiation time for the PEO–PCEMA–PMMA micelles.

Fig. 8. TEM image of the PEG–PCEMA–PMMA micelles prepared by UV irradiation for 1 hr, followed by dissolution in THF.

J.S. Kim et al. / European Polymer Journal 45 (2009) 1918–1923

4. Conclusion A PEG–PHEMA–PMMA triblock copolymer (Mn = 9,800 g/mol, Mw/Mn = 1.33) was synthesized via AGET ATRP using a PEG macroinitiator in a mixed solvent of anisole/ 2-isopropanol (3/1 v/v) at 80 °C. The degrees of polymerization of the PHEMA and PMMA blocks were determined by 1H NMR spectroscopy to be 18 and 21, respectively. The middle block of PEG–PHEMA–PMMA was functionalized with cinnamoyl chloride and the degree of functionalization was over 95%. The cinnamate group is hydrophobic and hence the hydrophilic PHEMA block was changed rather than hydrophobic block. The CMC of PEG–PCEMA–PMMA was 0.011 mg/mL. The PEG–PCEMA–PMMA micelles were spherically shaped with an average diameter of 43 nm. The photocrosslinking of the intermediary layer of the PEG–PCEMA–PMMA micelles was monitored by FTIR spectroscopy. The intensity of the AC@CA stretching vibration band of the cinnamate group at 1637 cm1 decreased with increasing UV irradiation time. The photocrosslinked PEG–PCEMA–PMMA micelles exhibited excellent colloidal stability, even when exposed to different external environments. Acknowledgements This work was supported by a Korea Research Foundation grant, which was funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2006003-D00140).

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micelles remained intact, confirming the successful photocrosslinking of the intermediary layer of the PEG–PCEMA–PMMA micelles and that indicating the degree of photocrosslinking was sufficient to produce excellent colloidal stability, even in different external environments.

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