Thermo-responsive micelles prepared from brush-like block copolymers of proline- and oligo(lactide)-functionalized norbornenes

Polymer 177 (2019) 178–188

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Thermo-responsive micelles prepared from brush-like block copolymers of proline- and oligo(lactide)-functionalized norbornenes

T

Sutthira Sutthasupaa,b,∗, Kajornsak Faungnawakijc, Kenneth B. Wagenerd, Fumio Sandae a

Division of Packaging Technology, Faculty of Agro Industry, Chiang Mai University, Chiang Mai, 50100, Thailand Center of Excellence in Materials Science and Technology, Faculty of Science, Chiang Mai University, Chiang Mai, 50200, Thailand c National Nanotechnology Center, National Science and Technology Development Agency, 111 Thailand Science Park, Paholyothin Rd., Patumthani, 12120, Thailand d Department of Chemistry, University of Florida, Gainesville, FL, 32611-7200, USA e Department of Chemistry and Materials Engineering, Faculty of Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamate-cho, Suita, Osaka, 564-8680, Japan b

H I GH L IG H T S

brush-like block copolymers were synthesized by ROMP. • Thermo-responsive polymers self-assembled in cold water and further aggregated at temperatures above the LCST. • The • The LCST and shape of the aggregates were tunable by amphiphilic balance of the block copolymers.

A R T I C LE I N FO

A B S T R A C T

Keywords: Norbornene Proline Polymer brush ROMP Self-assembly Thermo-responsive micelle

Thermo-responsive micelles were prepared from brush-like block copolymers of proline-derived norbornene and macromonomers bearing oligo(lactide) groups. The brush-like polymers with moderate molecular weights were synthesized by the ring-opening metathesis block copolymerization of a proline-functionalized norbornene (1) with norbornene macromonomers bearing oligo(lactide) groups using Umicore M31 as a catalyst. The prolinefunctionalized polynorbornene [poly(1)] exhibited the lower critical solution temperature (LCST) at 18 °C. Phase separation was reversible on heating and cooling without hysteresis. Poly(1) featured amphiphilic character, it self-assembled to form micelles in water at temperatures below the LCST, and aggregation of micelles was observed above the LCST. The LCST of the block copolymers increased with increasing percentage of the branched oligo(lactide) component, suggesting that the phase transition temperatures are tunable with respect to the monomer composition. The block copolymers self-assembled into micelles below the LCST, and further aggregated into larger particles, presumably due to dehydration at the corona, at temperatures above the LCST. The block copolymers also showed the potential to self-assemble into a variety of shapes determined by the amphiphilic balance of the block components.

1. Introduction Supramolecular architectures in the nanometer size range, such as micelles and vesicles, are commonly obtained by self-assembly of amphiphilic molecules [1]. One strategy for producing such supramolecular architectures is the synthesis of polymers with controlled unit sequences capable of self-assembling in solution [2]. Self-assembling systems respond to external stimuli, such as solvent polarity, pH and temperature [3], with potential applications in drug and gene delivery, and in nanotechnology [1], and they are even considered as nano-smart materials for use as components in nanoelectronics [4]. Thermo∗

responsive polymers are especially useful and important for smart supramolecular design due to their high versatility. Block copolymerization is a procedure to produce macromolecular architectures of vesicles and micelles [5]. Functional groups, block configurations and arrangement play important roles for the control of the properties of block copolymers. It is therefore essential to incorporate temperature-responsive groups into one block to obtain thermo-responsive materials. Amino-acid-based polymers with stimuliresponsive self-organization contribute to the production of intelligent materials for a variety of biological and medical applications, such as controlled release, thermo-responsive vesicles appropriate to drug

Corresponding author. Division of Packaging Technology, Faculty of Agro Industry, Chiang Mai University, Chiang Mai, 50100, Thailand, , E-mail addresses: [email protected], [email protected] (S. Sutthasupa).

https://doi.org/10.1016/j.polymer.2019.05.072 Received 24 February 2019; Received in revised form 3 May 2019; Accepted 28 May 2019 Available online 30 May 2019 0032-3861/ © 2019 Elsevier Ltd. All rights reserved.

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with norbornene monomers bearing leucine and indomethacin moieties. These were used to construct 3D macroporous scaffolds without toxicity toward HEK cells, releasing indomethacin below pH 5.7 [51,52]. Although amino acid-based polynorbornenes show useful features as mentioned above, studies of thermal-response are still limited. In this study, we demonstrate the ROMP of proline-functionalized norbornene and its block copolymerization with a macromonomer based oligo (lactic acid)-derived norbornene to explore amino-acid-functionalized polynorbornenes as novel thermal-responsive materials. The effect of stereo-structures of oligo(lactic acid) on temperature-dependent transmittance, and the effect of temperature on the self-assembly of brushlike block copolymers by dynamic light scattering (DLS) and transmission electron microscopy (TEM) are also investigated.

delivery, biochemical sensing, and biocompatible materials [6–8]. For example, poly(N-isopropylacrylamide) derivatives with ʟ-aspartic acid moieties in the side chains exhibit both temperature- and pH-response [9]. ʟ-Proline-derived well-defined polymers have been successfully synthesized, and the polymers exhibit thermosensitive phase-separation at low critical solution temperatures (LCST) around 15–45 °C in aqueous media [10]. pH- and temperature-responsive block copolymers containing proline have also been obtained [11]. Block copolymers of proline and hydroxyproline segments display upper critical solution temperatures as well as LCSTs [12]. A practical method for constructing polymers with a variety of functional groups is ring-opening metathesis polymerization (ROMP) of norbornene derivatives using ruthenium complex catalysts, including Grubbs 1st, 2nd, 3rd and Grubbs-Hoveyda catalysts, owing to their high tolerance for polar functional groups, such as carboxy, amide and ester moieties [13,14]. Some of these catalysts facilitate not only the livingness of the system, but also the ability to control the molecular weights, polydispersities and tacticities of the polymers [15–17], influencing to the preparation of well-defined block copolymers and micelles suitable for drug carrier and transport [18,19], graft copolymers that self-assemble to amphiphilic nanostructures [20], and polymeric materials with highly controlled structures and advantageous functions [21–23]. Commercially available dichloro[1,3-bis(2,4,6-trimethylphenyl)-2-imidazolidinylidene](3-phenyl-1H-inden-1-ylidene)(pyridyl) ruthenium(II) (Umicore M31) [24,25] efficiently catalyzes the ROMP of various norbornene derivatives to give functionalized polymers in good yields with high cis-stereoregularity [26]. Umicore M31 also catalyzes the block copolymerization of functionalized-norbornenes to give the corresponding block copolymers applicable to thermo-responsive materials [27–29]. However, there are few works investigating aminoacid-functionalized polymers obtained by ROMP catalyzed with Umicore M31. It is useful to investigate the ROMP behavior of amino-acidderived norbornenes using Umicore M31 and the possibility for preparing self-assembling block copolymers. Molecular brushes comprise a unique class of densely grafted polymers. The brush characteristics are tunable by the side chain length, backbone composition and grafting density [30,31]. Many polymer brushes with a variety of properties have been prepared by introducing functional groups using various polymerization techniques. Such molecules exhibit specific properties applicable to therapeutic delivery vehicles [32–34] and super-soft elastomers [35]. Graft-through ROMP of norbornene-functionalized macromonomers is remarkably effective for producing block brush copolymers that self-assemble, due to rapid polymerization with high conversion of macromonomer and simple combination of a diversity of functional polymers into molecular brush architectures, as well as accurate and independent control regarding the lengths and structures of backbones and side chains [36]. Such block brush polymers exhibit amphiphilicity, and core-shell molecular brushes can be prepared with hydrophilic and hydrophobic compartments. They have found useful applications as nanocarriers for drug delivery [30,37], in nano-lithographic pattern transfer [36], and as UV-light responsive nanoparticles [38]. Amino acids and polypeptides have been employed as hydrophilic segments [30,39], while macromonomers with poly(lactide) side chains are common hydrophobic segments [40–44], due to the biocompatibility and biodegradability of poly(lactide). The properties of nanoparticles depend on the stereo structures of poly(lactide) [45–48]. We have synthesized a series of block copolymers of amino-acidbifunctionalized norbornenes by ROMP using Grubbs catalysts. For example, leucine- and phenylalanine-functionalized block copolymers self-assemble to form micelles showing pH-response tunable by the length of each block [49]. Diblock copolymers containing indomethacin and aspartic acid conjugated to norbornene form nanostructures to release indomethacin, with release rates controllable by solvent composition [50]. We have also synthesized polymer brushes by the copolymerization of oligo(lactic acid)-derived norbornene macromonomers

2. Experimental section 2.1. Measurements 1

H (400 MHz) and 13C (100 MHz) NMR were measured on a Bruker DPX 400 NMR, using tetramethylsilane (TMS) as an internal standard in CDCl3. IR spectra were analyzed on a Nicolet FTIR-6700 spectrometer. Elemental analysis was performed on a PerkinElmer 2400 series CHNS/ O analyzer. Number-average molecular weight (Mn) and molecular weight distribution (Đ) of polymers were estimated by size exclusion chromatography (SEC) on a Waters e2695 model 3580 equipped with a Viscotek refractive index (RI) detector and two gel columns (PLgel, bead size 10 μm, MW resolving range 500–10,000,000) using THF as an eluent at a flow rate of 1.0 mL/min, calibrated by polystyrene standards at 35 °C. Alternatively, a Waters 2414 refractive index (RI) detector, equipped with two columns of Styragel HR5E 7.8 × 300 mm column (molecular weight resolving range = 2000–4,000,000), was used with THF as the eluent at a flow rate of 1.0 mL/min at 40 °C, calibrated with polystyrene standards. MALDI-TOF mass measurements were performed with a Bruker microflex MALDI-TOF mass spectrometer using 2,5-dihydroxybenzoic acid as a matrix. The acquisition was operated in linear mode with a scan range of 800–10,000 m/z. The transmittances (%) of polymer solutions were recorded on a PerkinElmer-Lamda 650 UV–vis spectrophotometer. Hydrodynamic diameters of micelles at a set temperature were evaluated on a Malvern Instruments Zetasizer Nano-ZS, wherein each measurement was repeated at least 10 times to obtain the average value. Transmission electron microscopy (TEM) studies were performed on a JEOL JEM-2010 electron microscope. The operation was done at an acceleration voltage of 100 kV. Samples were prepared by drop-casting a given polymer solution onto a carboncoated copper grid, wherein an excess amount of solution was blotted using filter paper. The samples were then air-dried at room temperature before measurement.

2.2. Materials cis-5-Norbornene-exo-2,3-dicarboxylic anhydride, ʟ-proline dimethyl ester hydrochloride, 3,6-dimethyl-1,4-dioxane-2,5-dione (50:50 racemic mixture of ᴅ- and L-lactide), (3S)-cis-3,6-dimethyl-1,4-dioxane2,5-dione (ʟ-lactide), N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC•HCl), triethylamine, 1,8-diazabicyclo[5.4.0] undec-7-ene (DBU) and Umicore M31 were purchased from SigmaAldrich and used as received. 5-Norbornene-2-methanol (mixture of endo-, exo-) was purchased from Tokyo Chemical Industry and used as received. (3R)-cis-3,6-Dimethyl-1,4-dioxane-2,5-dione (ᴅ-lactide) was purchased from Materials Science Research Center, Chiang Mai University. Deionized water was purchased from RCI Labscan., Ltd. Dichloromethane (CH2Cl2) used for polymerization was distilled by the standard procedure before use. 179

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increased and the color changed from orange–yellow to dark yellow. Ethyl vinyl ether was added to the mixture to quench the catalyst. The mixture was poured into hexane (200 mL), and the precipitated polymer was collected by filtration using a membrane filter, and dried under reduced pressure to obtain a block copolymer [poly(1)80-blockpoly(2L12)20] as white solid. Yield 268 mg (91%). Spectroscopic data of the representative polymers. Poly(1) IR: 1735 (ester C=O), 1630 (–N–C=O) cm−1. 1H NMR (400 MHz, CDCl3): δ 1.40–1.42 (m, 1H, norbornene CH2), 1.70–1.73 (m, 1H, norbornene CH2), 1.91–2.25 (m, 8H, proline 4 × –CH2–), 2.78–2.86 (m, 2H, norbornene –CH–), 3.26–3.33 (m, 2H, bridge position), 3.52–3.59 (m, 4H, –CH2N–), 3.69–3.71 (m, 12H, 2 × –COOCH3), 4.37–4.43 (m, 2H, –NCHCOO–), 6.23–6.26 (m, 2H, –CH=CH–). Poly(2L12) 1H NMR (400 Hz, CDCl3): δ 1.46–1.76 (m, 74H, norbornene > CH2CH–, 24 × –CH3], 1.97 (broad, 2H, bridge position), 2.33 [s, broad, 1H, > CH– (norbornene)], 2.79 [broad, 2H, norbornene, –CH2–], 3.40 (m, broad, 1H, –OH), 3.76–4.13 (m, 2H, > CHCH2O–), 4.35–4.40 [m, 1H, > CHOH, (end group)], 5.17–5.32 (m, 23H > CHCH3, 2H, –CH=CH– (mixture of endo, exo)]. IR: 3509 (broad, OH), 1743 (ester C=O) cm−1. Poly(2L20) IR: 3508 (broad, OH), 1741 (ester C=O) cm−1. Poly(1)90-block-poly(2L12)10 IR: 3499 (broad, OH), 1743 (ester C=O), 1647 (–N–C=O) cm−1.

2.3. Monomer synthesis 2.3.1. Synthesis of monomer 1 cis-5-Norbornene-exo-2,3-dicarboxylic anhydride (1.03 g, 6.25 mmol) and ʟ-proline methyl ester hydrochloride (2.07 g, 12.5 mmol) were dissolved in CH2Cl2 (100 mL). Triethylamine (1.75 mL, 25 mmol) and EDC•HCl (2.40 g, 25 mmol) were added to the solution at 0 °C, and the mixture was continuously stirred at room temperature overnight. Then the mixture was subsequently washed with 1 M HCl aq., saturated NaHCO3 aq., and water twice, and then dried over anhydrous MgSO4. It was concentrated on a rotary evaporator to obtain 1 as a white solid. Yield 80%. IR (KBr): 1746 (ester C=O), 1648 (amide C=O) cm−1 (Fig. S1). 1H NMR (400 Hz, CDCl3): δ 1.40–1.42 (m, 1H, norbornene CH2), 1.70–1.73 (m, 1H, norbornene CH2), 1.91–2.25 (m, 8H, proline 4 × –CH2–), 2.78–2.86 (m, 2H, norbornene –CH–) 3.26–3.33 (m, 2H, bridge position), 3.52–3.59 (m, 4H, –CH2N–), 3.69–3.71 (m, 12H, 2 × –COOCH3), 4.37–4.43 (m, 2H, –NCHCOO–), 6.23–6.26 (m, 2H, –CH=CH–). 13C NMR (100 Hz, CDCl3): δ 24.75, 25.06, 45.01, 45.36, 46.27, 46.34, 47.39, 47.67, 58.88, 137.41, 139.40,170.60, 171.93, 173.05, 173.85. Anal. Calcd for C21H28N2O6: C, 62.36; H, 6.98; N, 6.93. Found: C, 62.04; H, 6.92; N; 6.86. 2.3.2. Synthesis of macromonomers Macromonomers 2D12, 2L12, 2D20, 2DL12 and 2DL20 were prepared by the polymerization of ᴅ-, ʟ- and ᴅ,ʟ-lactides according to a method modified from the previously reported procedure [41]. The symbols D, L and DL correspond to ᴅ-, ʟ- and ᴅ,ʟ-, and subscripts 12 and 20 correspond to the degrees of polymerization of lactides. Typical procedure: A solution of ʟ-lactide (3.46 g, 24 mmol) in dry CH2Cl2 (70 mL) was fed into a flask containing norbornene-2-methanol (mixture of endo-, exo-) (0.248 g, 2.0 mmol). The mixture was stirred at room temperature for 30 min, and then DBU (2.4 mmol) was subsequently added to the mixture to initiate the polymerization of lactides. After 1 h, the reaction was quenched by adding acetic acid (several drops), and the resulting mixture was further stirred for another 1 h at room temperature. It was concentrated to one-half volume on a rotary evaporator. Then, the mixture was washed with brine three times, and dried over anhydrous MgSO4. CH2Cl2 was removed on a rotary evaporator and dried under reduced pressure to obtain a colorless sticky solid. It was dissolved in CH2Cl2 (5 mL), and the resulting solution was poured into a large amount of cold methanol to precipitate macromomomer 2L12 as white sticky solid in 76% yield. IR (ATR): 3511 (–OH), 1744 (ester C=O) cm−1 (Fig. S2). 1H NMR (400 Hz, CDCl3): δ 1.24–1.29 (m, 2H, > CH2CH–, norbornene), 1.41–1.66 (m, 72H, 24 × –CH3), 1.80–1.85 (m, 2H, bridge position), 2.39 [s, broad, 1H, > CH– (norbornene)], 2.81–2.84 (m, 2H, norbornene, –CH2–), 3.38 (s, broad, 1H, –OH), 3.71–4.23 (m, 2H, > CHCH2O–), 4.32–4.39 [m, 1H, > CHOH, (end group)], 5.16–5.25, 5.30 (m, s, 23H > CHCH3), 5.78–5.93, 6.07, 6.14–6.16 [m, s, m, 2H, –CH=CH– (mixture of endo, exo)]. 13C NMR (100 Hz, CDCl3): δ 16.61, 16.71, 20.44, 20.72, 28.82, 37.68, 41.54, 42.14, 49.34, 66.68, 68.99, 69.16, 69.42, 131.94, 135.18, 136.99, 169.35, 169.59.

2.5. Determination of the phase separation temperatures of the aqueous solutions of the polymers The turbidities of aqueous solutions of the polymers and di-block copolymers (2 mg/mL) were measured by UV–vis spectroscopy monitoring the % transmittance (%T) changes at 500 nm. The sample solutions were filtered through 0.45 μm membrane filters, transferred to quartz cuvettes, placed in a UV–vis spectrometer and warmed from 0 °C to 50 °C. They were equilibrated at each measurement temperature for 6 min, and the %T values at 500 nm were determined. The LCST temperatures were defined as the temperatures where reductions of 50%T of the polymer solutions were observed. 2.6. Characterization of polymeric micelles The temperature dependence to the particle size distribution, hydrodynamic diameter and aggregation of polymers was characterized by DLS measurement. The formation of micelles and aggregation of the block copolymers were investigated by TEM. 3. Results and discussion 3.1. Monomer and macromonomer synthesis We previously synthesized and copolymerized several norbornene monomers di-substituted with amino acid moieties such as alanine, leucine, phenylalanine and aspartic acid to confirm that micelle formation and stability were tunable by the hydrophilicity and hydrophobicity of the amino acid substituents [21]. We chose proline, the only amino acid containing a secondary amino group, in the present study to prepare a novel norbornene derivative and to study the ROMP activity and thermo-response of the polymers. It is expected that the absence of an N–H group in the present monomer remarkably affects the polymer properties and micelle formation, in a manner different from the previously reported monomers having amide N–H moieties. Monomer 1 was synthesized by the addition-condensation of cis-5norbornene-exo-2,3-dicarboxylic anhydride with ʟ-proline methyl ester hydrochloride in 80% yield as illustrated in Scheme 1. The structure of 1 was confirmed by IR and 1H/13C NMR spectroscopies in addition to elemental analysis. The 1H NMR spectrum of 1 exhibited signals reasonably assignable to the expected structure with proper integration ratios. (Fig. 1a). In this study, macromonomers having oligo(lactide) moieties were

2.4. Polymerization Typical procedure for the synthesis of block copolymers. Monomer 1 (136 mg, 336 μmol), Umicore M31 (3.14 mg, 4.2 μmol), and CH2Cl2 (1.0 mL) were fed into a glass tube equipped with a three-way stopcock under nitrogen, and the mixture was stirred at 35 °C. The polymer solution turned yellow-orange within 5 min, and it was continuously stirred for 4 h to turn into orange. The entire consumption of 1 was confirmed by 1H NMR spectroscopic measurement. A solution of macromonomer 2L12 (160 mg, 84 μmol) in CH2Cl2 (1.0 mL) was fed into the polymerization mixture, and the resulting mixture was continuing stirred at 35 °C overnight. The viscosity of the mixture gradually 180

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Scheme 1. Synthesis of monomer 1, macromonomers 2DX, 2LX and 2DLX (x = 12, 20).

Fig. 2. 1H NMR (400 MHz) spectra of (a) poly(1) and (b) brush-like poly(2L12) measured in CDCl3. * = grease. Table 1 Homopolymerization of monomer 1 and macromonomers.a. monomer

time (h)

yieldb (%)

Mn c

Đc

1 2L12 2D12 2DL12 2L20 2DL20

1 24 24 24 24 24

85 73 91 86 71 61

4000–6000d 52,300 76,500 88,900 126,400 80,000

–e 1.52 1.62 1.35 1.54 1.36

a Conditions: [M]0 = 0.42 M in CHCl2, catalyst: Umicore M31 Ru complex, [M]0/[Ru] = 100 at 35 °C. b Crude yield, hexane-insoluble part. c Determined by SEC eluted with THF calibrated by polystyrene standards. d Determined by MALDI-TOF MS. e Not determined.

Fig. 1. 1H NMR (400 MHz) spectra of (a) monomer 1 and (b) macromonomer 2L12 measured in CDCl3.

employed as hydrophobic segments, wherein enantiomerically isomeric ᴅ- and ʟ-lactides, and racemic ᴅʟ-lactide were used to examine the effect of stereo structures on the polymer properties. Norbornene macromonomers 2L12, 2D12, 2DL12 and 2L20, 2D20, 2DL20 bearing 12- and 20-mers of lactic acid chains were synthesized by ring-opening polymerization of ᴅ-, ʟ- and ᴅʟ-lactides using norbornene-2-methanol (mixture of endo, exo) and DBU as initiator and catalyst, respectively, in 70–80% yields as illustrated in Scheme 1. The polymerization was carried out in dry CH2Cl2 to avoid the effects of water. The

macromonomer solutions in CH2Cl2 were concentrated and poured into large volume of cold methanol, according to the procedure in previous reports, to isolate the macromonomer from the unreacted norbornene and oligo(lactic acid) without norbornene chain end [41,53]. However, the mixture was found miscible in methanol and only small amounts of solid compounds precipitated (less than 10%). In comparison with the previous report, it is likely that the macromonomers have high solubility in methanol, presumably due to the excess acetic acid. Hence, we isolated the macromonomer solution as described in the procedure 181

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Scheme 2. Block copolymerization of monomer 1 and macromonomers 2Dx, 2Lx and 2DLx (x = 12, 20).

oligo(lactide) chains of the macromonomers were determined to be 12 and 20 by the above-mentioned 1H NMR integration ratios, which correlated with the feed molar ratios of norbornene-2-methanol to lactide. Along with signals i and g of methine and methyl protons of oligo(lactide) units, signals i’ and g’ assignable to methine and methyl protons of –CH(CH3)OH at the chain end of oligo(lactide) units were observed around 4.3 and 1.5 ppm in the 1H NMR spectrum of macromonomer 2L12, as shown in Fig. 1b.

Table 2 Block-copolymerization of monomer 1 and macromonomersa. macromonomer

2L12 2L12 2L12 2L12 2D12 2D12 2D12 2DL12 2DL12 2DL12 2L20 2L20 2DL20

feed ratio (mol%) 1

macromonomer

90 80 70 60 90 70 50 90 80 70 80 70 80

10 20 30 40 10 30 50 10 20 30 20 30 20

yieldb (%)

quant 91 74 87 quant 93 82 quant 78 80 85 83 85

Mn c

–e 65,400 93,300 122,400 77,600 104,800 152,400 –e 149,100 190,900 97,900 147,100 104,100

Đc

–e 1.46 1.73 1.63 1.39 1.48 1.63 –e 1.24 1.44 1.40 1.61 1.53

unit ratiod (mol%) 1

macromonomer

94 86 75 59 92 76 42 92 83 72 86 71 85

6 14 25 41 8 24 58 8 17 28 14 29 15

3.2. Polymer synthesis 3.2.1. Homopolymerization Proline-derived monomer 1 was polymerized using Umicore M31 Ru catalyst. The monomer conversion was monitored by 1H NMR spectroscopy, and calculated from the integration ratios between the olefinic protons of the monomer around 6.2 ppm and the protons of the repeat units of the formed polymer [poly(1)] around 5.0–5.4 ppm (Fig. 2). Sixty percent of 1 was converted into poly(1) in less than 4 min, and reached 98% conversion in 20 min as shown in Fig. S4. Monomer 1 was efficiently polymerized with Umicore M31 to give poly (1) in high yield (85%). The molecular weight could not be properly determined by SEC in either THF, CHCl3 or DMF as the eluent, presumably due to the large interaction between the polar groups of poly (1) with the column materials. The molecular weight of poly(1) was estimated to be 4300–6000 by MALDI-TOF (Fig. S5). Macromonomers 2L12, 2L20, 2D12, 2LD20 and 2DL20 were also polymerized, as evidenced by the rapid increase of viscosity soon after initiating the polymerization (ca. 15 min), but the macromonomers were not completely consumed even after 4 h, presumably due to the bulkiness of the brush substituents. The elongation of poly(lactide) units resulted in decreased ROMP activity. In addition, the endo-substituent macromonomers may cause more steric hindrance to polymerization than the exo-forms [54]. Complete consumption was achieved by extending the polymerization time to 24 h and confirmed by the disappearance of the olefin proton signals of the norbornene moiety (endo- and exo-) around 5.9–6.2 ppm in the 1H NMR spectra

a Conditions: [M]total = 0.42 M in CHCl2, catalyst: Umicore M31 Ru complex, [M]0/[Ru] = 100 at 35 °C, polymerization time: first stage 4 h and second stage 24 h. b Hexane-insoluble part. c Determined by SEC eluted with THF calibrated by polystyrene standards. d Determined by 1H NMR. e Not determined.

described above. The macromonomers exhibited monomodal SEC traces as shown in Fig. S3. Nevertheless, due to small differences in the molecular weights between oligo(lactic acid)s with and without norbornene chain end, overlap of the SEC traces probably occurs. This could be clarified by 1H and 13C NMR spectroscopies along with the SEC measurement of the polymers. The structures of the macromonomers were confirmed by IR, 1H and 13C NMR spectroscopies. Compared to the endo- and exo-forms of norbornene-2-methanol, the olefin protons assignable to endo- (5.9 and 6.2 ppm) and exo- (6.1 ppm) isomers were observed in the proper integration ratios with those of the methine and methyl protons of oligo(lactide) units. The polymerization degrees of

Fig. 3. SEC traces of the block copolymers. 182

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3.3. Block copolymerization The block copolymers of proline-derived monomer 1 with macromonomers 2L12, 2L20, 2D12, 2LD12 and 2LD20 were synthesized (Scheme 2). The polymer block from 1 was expected to serve as a hydrophilic/thermo-responsive segment, judging from the report concerning the thermo-response of proline-based polyacrylamide obtained by RAFT polymerization [10–12]. The brush-like oligo(lactide) macromonomer units are the representative hydrophobic segments [44]. The brush-like block copolymers were synthesized by the grafting through manner [36], ring-opening metathesis copolymerization of monomer 1 with macromomomers 2L, 2D and 2DL (Scheme 2). Although the livingness of polymer 1 could not be confirmed by SEC and Đ, the monomer conversion and 1H NMR verified the completion of reaction. In addition, to avoid the possibility of catalyst degradation due to the steric hindrance of macromonomers and the effect of the residual oligo(lactide), the first stage of block copolymerization was performed with 1. Block copolymerizations with different feed ratios of 1 and the macromonomers were carried out to obtain the corresponding block copolymers (Table 2). The complete consumption of all the monomers was confirmed by 1H NMR spectroscopy and SEC. Fig. S6 shows the 1H NMR spectra of poly(1)90-block-poly(2L12)10 and poly (1)80-block-poly(2L12)20. Signal c, assignable to the methyl ester of the poly(1) segment, was observed at 3.7 ppm in the 1H NMR spectra. Signals d′ and e′, assignable to the methine and methyl protons of –CH (CH3)OH at the chain end of oligo(lactide) units, were also observed at 4.3 and 1.4 ppm in the 1H NMR spectra of poly(1)90-block-poly(2L12)10 and poly(1)80-block-poly(2L12)20. All the protons exhibited the proper integration ratios, which were almost comparable to the feed molar ratio, indicating that the small effect of the contamination by oligo (lactide)s without norbornene terminus did not significantly affect the block copolymerization of monomer 1 with the macromonomers. The copolymers with Mn's of 65,400–190,900 (Đ = 1.39–1.73) were successfully obtained as listed in Table 2. The Mn and Đ increased with increasing feed ratios of the macromonomers. Back-biting reaction did not seem to occur remarkably, judging from the relatively small Đ values. The Mn's of the block copolymers with 90% feed ratios of monomer 1 could not be determined by SEC. The high composition of monomer unit 1 possibly contributed to the interaction between the polar substituents with SEC gels as described above. The monomers consumption was analyzed by 1H NMR spectroscopy as shown in Fig. S6. The block copolymerization required 24 h for completion. Since the first poly(1) block could not be properly characterized by SEC as mentioned above, the chain extensions by block copolymerization with the macromonomers were confirmed by comparing the SEC traces of the block copolymers with various compositions (Fig. 3). The SEC peak appeared at a higher molecular weight region when the macromonomer block composition was high. Fig. 4. Temperature dependence of the transmittance at 500 nm of aqueous solutions (2.0 mg/mL) of (a) poly(1) and (b) poly(1)90-block-poly(2L12)10, poly (1)90-block-poly(2D12)10, poly(1)80-block-poly(2L12)20 and poly(1)70-block-poly (2L12)30. Solid line = heating, dashed line = cooling.

3.4. Thermo-responsive properties of polymers in water. (Phase separation temperatures of the polymers in water) Poly(1) exhibited sharp phase separation in water (pH = 7) as shown in Fig. 4 a. The lower critical solution temperature (LCST) was defined as the temperature at which the transmittance of the polymer solution was reduced to 50% upon heating from 0 °C to 40 °C. Poly(1) displayed the LCST at 18 °C as depicted in Fig. 4a. The white opaque solution became clear when it was cooled. The LCST of poly(1) was dependent on the concentration. The LCST decreased from 29 °C to 15–17 °C by increasing the polymer concentration in water from 0.5 to 3.0 mg/mL (Fig. S7). This result indicates that aggregation is of key importance of phase transition [55]. It should be noted that the phase transition was remarkably sensitive and also reversible on heating and cooling without hysteresis, which is commonly caused by the formation of intra- and interchain hydrogen bonds between different chain segments when they are overlapped in the collapsed state [56]. Such

(Fig. 2). Fig. S3 shows the SEC traces of the macromonomers and the polymers obtained by ROMP. Small peaks with area fractions of ca. 10% were observed in the SEC traces of the polymers. The small peaks are assignable to oligo(lactide) without norbornene moiety, likely formed by chain transfer during the ring-opening polymerization of lactides, indicating the partial absence (ca. 10%) of norbornene terminus at the oligo(lactide) macromonomers [41]. The Mn values of the brush-like polymers (52,000–130,000 by SEC) were dependent on the length of the polylactide side chain, as summarized in Table 1. These brush-like polymers were used as hydrophobic segments to obtain amphiphilic block copolymers in the subsequent step.

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Fig. 5. (a) Hydrodynamic diameter distributions of poly(1) micelles formed in water (2 mg/mL) measured at various temperatures; (b) TEM images of the aggregates of poly(1) prepared from an aqueous solution (2 mg/mL) at 20 °C; (c) schematic image of self-assembly of poly(1). Table 3 DLS data for the polymeric micelles a. polymer

temperature (°C) 20 Dh

poly(1) poly(1)90-block-poly(2L12)10 poly(1)90-block-poly(2D12)10 poly(1)90-block-poly(2DL12)10 poly(1)80-block-poly(2L12)20 poly(1)80-block-poly(2DL12)20 poly(1)70-block-poly(2L12)30 poly(1)80-block-poly(2L20)20 poly(1)80-block-poly(2DL20)20 a b

30 b

(nm)

517 ± 13 331 ± 27 18 ± 1 70 ± 8 14 ± 1 49 ± 21 96 ± 4 46 ± 43 42 ± 9

40 b

PDI

Dh

0.12 0.38 0.73 0.92 0.38 0.93 0.75 0.24 0.63

374 820 592 402 773 583 465 981 892

b

(nm)

PDI

Dh

± ± ± ± ± ± ± ± ±

0.10 0.23 0.18 0.16 0.15 0.11 0.03 0.08 0.24

350 685 496 448 536 499 431 745 969

14 47 20 25 52 4 3 20 100

(nm)

PDI

± ± ± ± ± ± ± ± ±

0.03 0.21 0.11 0.12 0.14 0.08 0.14 0.05 0.15

4 5 1 1 10 12 25 2 7

Measured in water (c = 2 mg/mL). Z-average.

30% were insoluble in cold water and exhibited no phase separation. Meanwhile, poly(1)90-block-poly(2L12)10, poly(1)90-block-poly(2D12)10, poly(1)80-block-poly(2L12)20 and poly(1)70-block-poly(2L12)30 exhibited LCST-type phase separation in water without hysteresis. The LCST-type phase separation of the block copolymers was observed at temperatures higher than that of poly(1). The transmittance drastically decreased between 18 and 30 °C. The LCST of poly(1)90-block-poly (2L12)10 was 21.2 °C, which was 1.3 °C lower than that of the ᴅ-counterpart, poly(1)90-block-poly(2D12)10. It has been reported that the LCST of optically active polymers is affected by the presence of enantiomerically isomeric monomer units [59,60], and chain length of oligo(ethylene glycol) units at the side chains [61]. Thus, it is likely that the difference of LCST of the present study is also caused by the difference of stereo-structures of the monomer units in the copolymers. With increased hydrophobic percentage, (2L12)30 block resulted in the slight increase of LCST as shown in Fig. 4b. The LCST of poly(1)80-blockpoly(2L12)20 was 25.1 °C, whereas that of poly(1)70-block-poly(2L12)30 was 26.5 °C. The incorporation of a hydrophilic hydroxy group into the proline unit,(i.e., hydroxyproline) largely increased the LCST [40–44]. It is likely that the interaction between the oligo(lactide) side chains and hydroxy end groups increases the LCST in this study. The present block copolymers exhibited negligibly small hysteresis, and the transition

hydrogen bonds cannot be completely removed in the cooling process, even when water is employed as a good solvent. This phenomenon is primarily observed in polymers containing amide moieties undergoing strong inter- and intramolecular hydrogen bond formation, while the hydrogen bonding interaction between ester moieties is much weaker, and thus the hysteresis is insignificant [57,58]. The present poly(1), which contains an ester but no amide, behaves similarly, and is comparable to copolymers of N-acryloyl-ʟ-proline methyl ester and N,Ndimethylacrylamide via RAFT polymerization. Those acrylamide copolymers show narrower transition curves compared with the broader curves (transition width = 20 °C) of the counterparts obtained via free radical copolymerization. It is suggested that a reversible, sharp thermo-responsive system is constructed by using copolymers with well-designed and controlled structures [10]. We tried synthesizing poly(1) samples with various molecular weights by changing the monomer/catalyst ratios in order to examine the effect of polymer molecular weight on LCST. However, no remarkable difference of molecular weights was observed between the samples obtained by the polymerizations with the monomer/catalyst ratios of 50, 100 and 200 (Fig. S8 and Table S1). The LCST of the poly (1) samples ranged from 18.0 to 19.3 °C (Fig. S9 and Table S1), almost the same irrespective of the monomer/catalyst ratios. The diblock copolymers with hydrophobic percentages larger than 184

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occurred at the same temperature regardless of heating and cooling, even though the inner cores contain polymer brushes, which commonly cause hysteresis. Hydrogen bonding between the polymer side chains of the brushes considerably affects the reversibility [62]. The methyl ester groups of the proline corona possibly suppressed the hysteresis in the present study. 3.5. Formation and characterization of micelles: self-assembly of brush like polymers in water DLS measurements were conducted at temperatures below and above LCST in order to investigate the formation of polymer aggregates upon phase transition, i.e., the influence of temperature on the selfassembly of the polymers. Poly(1) exhibited a trimodal hydrodynamic diameter distribution at 15 °C, below but close to the LCST (18 °C), as shown in Fig. 5a. According to the polydispersity, the size distribution of poly(1) at 15 °C would more realistically represent the hydrodynamic diameter (Dh), as summarized in Table S2. The particles with an average Dh of 11 nm (34%) are attributable to the single chain polymers, while the particles with a Dh around 42 nm (19%) are attributable to micellar aggregates. The particles with Dh around 577 (47%) are attributable to further aggregated micelles. On the contrary, uniform distributions with Dh values of 517, 374 and 350 nm were observed at 20, 30 and 40 °C (temperatures above the LCST), respectively, as summarized in Table 3, indicating the segregation of micelles of poly(1) upon increasing temperature. The number-weighted Dh data of poly(1) (Fig. S10) agreed with the decrease of Z-average Dh values of poly(1) upon increasing the temperature. On the other hand, the Dh values of the block copolymers tended to increase from 20 °C to 30 °C, while slightly decreased from 30 °C to 40 °C (except for poly(1)90-block-poly (2DL12)10 and poly(1)80-block-poly(2DL20)20, probably due to shrinkage of the proline side chains, thus stabilizing the aggregates. The TEM specimens were prepared by depositing the polymer solutions at 20 °C, above the LCST. The TEM results demonstrated the self-assembly of poly(1) into micelles in aqueous solution and further association of the micelles to form larger aggregates with vesicle-like structures as depicted in Fig. 5b. Poly(1) is considered as an amphiphilic homopolymer consisting of a hydrophobic core (polynorbornene main chain) and a thermo-responsive corona (proline methyl ester moieties) according to the results mentioned above. Since water is a poor solvent for polynorbornene, the core contracts to minimize its contact with water [63]. The structures of self-assemblies vary between micelles, vesicles and spheres, depending on the hydrophilic/hydrophobic balance of the polymers [64]. It is expected that the present amphiphilic thermo-responsive norbornenebased polymers will contribute to the development of nanostructured materials featuring a wide range of biological applications. The block copolymers exhibited bimodal hydrodynamic diameter distributions of micelles in water below LCST (20 °C) (Fig. 6), in agreement with the large PDI values (0.38–0.93), as listed in Table 3. The Dh values of DLS peaks with the largest content of poly(1)90-block-poly(2L12)10, poly (1)90-block-poly(2D12)10 and poly(1)90-block-poly(2DL12)10 were 394, 187 and 327 nm, respectively as listed in Table S2. Particles with an average Dh around 10 nm were also observed, presumably attributable to the single chain polymers (see also number-weighted Dh in Fig. S10). The DLS patterns were somewhat different from each other. Poly(1)90block-poly(2L12)10 showed a DLS area ratio higher than that of poly (1)90-block-poly(2D12)10, attributable to aggregated micelles, probably due to the effect of phase transition temperature leading to more rapid aggregation. The results imply that the enantiomeric difference between the oligo(lactide) side chains affected the self-assembling properties. On the other hand, the block copolymers exhibited unimodal distributions above the LCST as shown in Figs. 6 and 7, while the hydrodynamic diameter significantly increased to 400–900 nm compared to the values below the LCST. It seems that the polynorbornene segments with proline moieties in the side chains located in the corona part

Fig. 6. Hydrodynamic diameter distributions of micelles of (a) poly(1)90-blockpoly(2L12)10, (b) poly(1)90-block-poly(2D12)10 and (c) poly(1)90-block-poly (2DL12)10 formed in water (2 mg/mL) measured by DLS at various temperatures. 185

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Fig. 7. Hydrodynamic diameter distributions of micelles of poly(1)70-block-poly(2L12)30 formed in water (2 mg/mL) measured by DLS at various temperatures, and TEM images of the aggregates of poly(1)70-block-poly(2L12)30 prepared from an aqueous solution (2 mg/mL) at 20 °C.

Fig. 8. TEM images of (a) poly(1)70-block-poly(2L12)30, (b) poly(1)80-block-poly(2L12)20, (c), (d) poly(1)80-block-poly(2DL12)20 prepared from aqueous solutions (2 mg/mL) at 20 °C.

to the decomposition of the large agglomerated micelles and subsequent rearrangement into smaller micellar aggregates consisting of shrunken oligo(lactide) side chains in the core and the proline methyl ester side chains that seem to locate in the corona [58]. The other block copolymers also exhibited bimodal hydrodynamic diameter distributions below the LCST (20 °C) (Fig. S11), in agreement with the large PDI values (0.38–0.92). The increase of the hydrophobic segment (2L12 block) affected the Dh (Table 3 and Table S2). The average Dh values of poly(1)70-block-poly(2L12)30 below LCST (20 °C) were 190 nm (82%) and 7 nm (12%) as described in Table S1. The size distribution for poly(1)70-block-poly(2L12)30 was investigated at a temperature near the LCST (25 °C) in order to further examine the thermo-responsive behavior. A unimodal DLS curve was observed at 25 °C with a Dh of 240 nm, as shown in Fig. 7, suggesting the self-assembly to form large micelles. The average Dh increased to 400 nm by elevating the temperature to 30 °C and 40 °C, suggesting an increase in aggregation number. The TEM images of poly(1)70-block-poly(2L12)30 confirmed the aggregation of particles and the large micelles at 20 °C. The bilayer-like structures were randomly observed. Poly(1)80-block-poly(2L20)20 and poly(1)80-block-poly(2DL20)20 with longer lactide repeat units also demonstrated bimodal distributions below the LCST (20 °C), as presented in Fig. S11. The average Dh values of poly(1)80-block-poly(2L20)20 were 12 nm (80%) and poly (1)80-block-poly(2DL20)20 were 7.5 nm (37%) and 220 nm (57%). The DLS results (Table 3) revealed that the elongation of poly(lactide) chains slightly affected the Dh. Larger aggregates of poly(1)80-blockpoly(2L12)20 were observed at temperatures above LCST, probably a result of the increase of core size due to the longer chain length of oligo (lactide) repeat units. Poly(1)80-block-poly(2L20)20 formed aggregates with Dh = 981 and 745 nm whereas poly(1)80-block-poly(2DL20)20 formed aggregates with Dh = 892 and 969 nm at 30 and 40 °C, respectively.

Fig. 9. Proposed self-assembled structures of the block copolymers.

of the micelles dehydrated, and together with the contribution of hydrophobic interaction between thermo-responsive chains in aqueous media, result in inter-micelle hydrophobic interaction to form larger aggregates [42,65]. The temperature rise from 30 to 40 °C resulted in a decrease of the size of aggregates as listed in Table 3. This is attributed 186

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Kathryn R. Williams at the University of Florida for their helpful suggestions and comments.

No remarkable enantiomeric effect of the side chains on self-assembly was observed in the present study. In general, the stereo structures of oligo(lactide) moieties affect the thermal and physical properties of block copolymers, and the micelles are stabilized by the formation of stereo complexes in the micelle cores [40,45]. In the present study, the oligo(lactide) moieties are located in the side chains, and the oligo(lactide) content is quite low, therefore resulting in the small effect on the characteristics of micelles. The overall results indicate that the temperature significantly influences self-assembly. The thermo-responsive corona segment of poly(1) plays an important role in self-assembly in this study, while the lengths of the oligo(lactide) side chains cause an increase of the core sizes, subsequently increasing the size of aggregates. The TEM images of the block copolymers exhibited interesting morphologies. Poly(1)70-block-poly(2L12)30 formed clusters of short rod-like micelles below the LCST as shown in Fig. 8a, while poly(1)80block-poly(2L12)20 and poly(1)80-block-poly(2DL12)20 formed clusters containing brush-like structures and packing of vesicles as shown in Fig. 8 (b–d). It is considered that the shapes of the block copolymers are so-called polymer brushes as illustrated in Fig. 9. The fractions of the block compartments tune the amphiphilic balance, which mainly affects the self-assembly behavior. Hence the block copolymers adopt various architectures, such as vesicles, unimolecular micelles, spherical micelles, cylindrical brush micelles and bilayers, depending on the side chain length and grafting density [66]. Adjustment of the side chain symmetry of the hydrophilic compartment allows the copolymers to assemble into spherical and cylindrical brush micelles in bottlebrush block copolymer systems with poly(lactide)/PEO segments [67]. The bottlebrush block copolymers further aggregated at temperatures higher than LCST in all cases as mentioned above. The DLS results demonstrated a slight decrease in the sizes of vesicles and aggregates owing to the dehydration of the proline moieties in the side chains. Consequently, it is considered that the resulting unstable micelles hydrophobically interact with each other to form larger aggregates [42].

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4. Conclusions In conclusion, we have demonstrated the synthesis of novel temperature-responsive polymeric nanoparticles via ROMP of a prolinefunctionalized norbornene and block copolymerization with oligo(lactide)-functionalized norbornene macromonomers. Polymer-based proline-conjugated norbornene presents as an amphiphilic homopolymer consisting of a thermo-responsive corona and a hydrophobic core of the norbornene main chain. Self-assembly with tunable phase separation was achieved by adjusting the percentages of the temperature-responsive and hydrophobic segments of the oligo(lactide) brush. The polymers self-assembled in cold water and further aggregated at temperatures above the LCST. We believe that the achievement in the present study provides a platform for the preparation of well-defined polymeric nanoparticles from polymer brushes, leading to intelligent materials for biomedical use such as drug delivery systems. Further study concerning the synthesis of multifunctional and stimuli-responsive amino-acid-based polynorbornenes is currently undergoing. Acknowledgements Financial support for this work was provided by a grant from the Thailand Research Fund MRG6080032. This research work was partially supported by Chiang Mai University. The authors are grateful to Prof. Dr. Christian Slugovc at Institute for Chemistry and Technology of Materials, Graz University of Technology (ICTM, TU Graz) for the helpful discussion and suggestion to use Umicore 31, as well as for his assistance in the study of monomer conversion. The authors are grateful to Mr. Wasawat Kraithong at the National Nanotechnology Center for the measurements of temperature dependence of the transmittance, Asst. Prof. Dr. Kayo Terada at Nara Institute of Technology, and Dr. 187

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