aspartic acid conjugated norbornenes and characterization of their self-assembled nanostructures as drug carriers

European Polymer Journal 85 (2016) 211–224

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European Polymer Journal journal homepage: www.elsevier.com/locate/europolj

Synthesis of diblock copolymers of indomethacin/aspartic acid conjugated norbornenes and characterization of their self-assembled nanostructures as drug carriers Sutthira Sutthasupa a,⇑, Fumio Sanda b a

Division of Packaging Technology, Faculty of Agro Industry, Chiang Mai University, Chiang Mai 50100, Thailand 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

a r t i c l e

i n f o

Article history: Received 31 August 2016 Received in revised form 6 October 2016 Accepted 12 October 2016 Available online 20 October 2016 Keywords: Norbornene Indomethacin Micelle ROMP Polymer-drug conjugate Drug release

a b s t r a c t Aspartic acid derived norbornene monomer 1 and indomethacin-conjugated norbornene monomer 2 were block copolymerized via ring-opening metathesis polymerization (ROMP) using the Grubbs 2nd generation ruthenium complexes. The block copolymers with number-average molecular weights ranging from 11,700 to 15,300 were obtained in good yields. Well-controlled structures of the block copolymers were verified by 1H NMR and size exclusion chromatography (SEC) characterizations. The block composition significantly affected the solubility of the block copolymers in organic media. 1H NMR spectroscopic, turbidity, and dynamic light scattering measurements revealed that poly(1)block-poly(2) in the ratio of 1:2 = 50:50, 62:38 and 75:25 formed compound micelles with a diameter around 100 nm in a selective solvent, DMSO. The micelle size increased as the length of the poly(2) block increased. It was observed that the addition of water into the micelle solution resulted in the increase of the micelle size (around 1000 nm). TEM observation indicated that the sizes of the block copolymers were around 80–100 nm, and the formation of compound micelle clusters by the addition of water. Indomethacin was gradually released from the compound micelles after incubation in an acidic environment (pH = 3) at room temperature. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Synthesis of copolymers with controlled unit sequences is a powerful approach for producing materials with ordered structures and molecular self-assembly [1,2]. Block copolymerization is a practical method that leads to various macromolecular architectures such as fibers, nanotubes, emulsifiers, vesicles and micelles, some of which are applicable to nanosized drug containers [3–6]. The use of molecular assemblies as delivery vehicle is a promising way to develop the drug delivering system under physical or chemical stimuli-sensitivity that is controllable by temperature and pH [7,8]. The properties of block copolymers depend on the functional groups, block compositions and sequences. It is necessary to incorporate stimuli-responsive groups into one block in order to construct stimuli-triggered drug release materials based on block copolymers [8–11]. Block copolymers are commonly synthesized by living polymerization, including anionic, cationic,

⇑ Corresponding author. E-mail addresses: [email protected], [email protected] (S. Sutthasupa). http://dx.doi.org/10.1016/j.eurpolymj.2016.10.029 0014-3057/Ó 2016 Elsevier Ltd. All rights reserved.

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Scheme 1.

atom-transfer radical polymerization, and ring-opening metathesis polymerization (ROMP) [4], leading to control over the degrees of polymerization of the polymer blocks in a reaction process. ROMP of norbornene derivatives achieves a high level of control over polydispersity, tacticity and backbone configuration [12–14]. ROMP with Grubbs ruthenium (Ru) complexes with high tolerance toward polar functional groups [15] allows the polymerization of highly functionalized olefin monomers producing polymers with low polydispersity. Consequently, Ru-based ROMP makes it possible to synthesize block copolymers with wide varieties of functionalities [16–20]. Besides, ROMP produces an unsaturated polymer backbone that limits the conformational degrees of freedom, which restrains the hydrophobic chain stretching parameter for self-assembly. This feature emphasizes the importance of ROMP producing amphiphile nanoparticle assemblies as unique entities [21]. Conjugation of amino acids and peptides into synthetic polymers is of particular interest, because synthetic polymers are not only biologically relevant but also benefit synthetic materials in terms of providing highly pure chiral sources that provide biomimetic materials for chiral recognition [22,23]. Introduction of amino acids into synthetic polymers offers other advantageous characteristics such as viability of chemical modifications with bioactive molecules [24,25], and interactions with proteins and genes. Amino acid based polymers form amphiphilic, pH- and thermo responsive vesicles applicable to a drug delivery system [8,11,26–29]. Block copolymers containing L-phenylalanine and L-valine in the side chain are used for drug delivery and gene transfer materials, which are responsive to pH due to the primary amino groups in the side chain [11]. Poly(N-isopropylacrlyamide) derivatives bearing L-aspartic acid moieties in the side chains exhibit both thermo and pH responsiveness [8]. Indomethacin-incorporated poly(ethylene oxide)-poly(b-benzyl L-aspartate) micelles release indomethacin according to the pH [30]. Various drug conjugated polynorbornene nanoparticles and micelles have been synthesized by ROMP [31–33]. An amphiphilic block copolymer in which one block is conjugated with hydrophobic indomethacin and the other block is hydrophilic hexa(ethylene oxide)-containing monomers forms a core-shell structure. After incubation in an acidic environment, 20% of the indomethacin is released from the nanoparticles [34]. Folate-derived polynorbornene nanocarriers carrying multiple chemotherapeutic agents (doxorubicin, indomethacin) mildly release drug molecules in response to pH [35]. We have previously reported that amino acid bifunctionalized norbornene derivatives efficiently undergo ROMP to give the corresponding polymers in a living fashion. We synthesized amphiphile block copolymers forming micelles by utilizing the living ROMP of amino acid derived norbornene monomers, and prepared pH responsive micelles upon modification of the amino acid end groups [36,37]. Much attention has been paid to the preparation of side-chain amino acid based polymers in a controlled fashion in recent years [38,39], since introducing amino acids into synthetic polymers results in many advantages characteristics and applications. In this paper, we demonstrate the synthesis of diblock copolymers composed of indomethacinconjugated polynorbornene and aspartic acid derived polynorbornene segment (Scheme 1). We report the characterization of the block copolymers, and the formation of micelles in selective solvents, together with physicochemical properties of the micelles using DLS and TEM. We further report the release of indomethacin from the micelles in response to acidic condition.

2. Experimental section 2.1. Measurements 1 H (400 MHz) and 13C (100 MHz) NMR spectra were recorded using tetramethylsilane (TMS) as an internal standard in CDCl3 on a Bruker DPX 400 NMR spectrometer. The IR spectra were measured on a Nicolet FTIR-6700 spectrometer. The number- and weight-average molecular weights (Mn and Mw) of the polymers were determined by size exclusion chromatography (SEC) on a Waters e2695 separations modules, model 3580 equipped with a Viscotek refractive index (RI) detector and two polystyrene gel columns (PL gel, bead size 10 lm, MW resolving range 500–107) using THF as an eluent at a flow rate of 1.0 mL/min, calibrated by polystyrene standards at 35 °C. The UV–vis spectra were recorded on a Thermo Scientific Genesys 10S UV–vis spectrophotometer. Elemental analyses were performed by Perkin-Elmer 2400 series CHNS/O analyzer. Mass spectra were acquired on a Bruker Daltonics-micrOTOF ESI-TOF mass spectrometer. The hydrodynamic diameters of the micelles were measured using a Malvern Instrument Zetasizer Nano-S, wherein each measurement was repeated at least

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10 times to obtain the average values. Transmission electron microscopy (TEM) studies were performed on a JEOL JEM-2010 electron microscope operating at an acceleration voltage of 100 kV. Samples were prepared by drop-casting a given micelle solution onto a carbon-coated copper grid. An excess solution was carefully blotted off using filter paper. The samples were then air-dried at room temperature before measurement. 2.2. Materials 5-Norbornene-2-exo,3-exo-dimethanol, cis-5-norbornene-exo-2,3-dicarboxylic anhydride, L-aspartic acid dimethyl ester hydrochloride, N-(3-(dimethylamino)propyl)-N0 -ethylcarbodiimide hydrochloride (EDCHCl), triethylamine, N,N-dimethyl4-aminopyridine (DMAP) and the Grubbs second generation catalyst were purchased from Sigma-Aldrich and used as received. 1-(4-Chlorobenzoyl)-5-meyhoxy-2-methyl-3-indoleacetic acid (indomethacin) was purchased from Tokyo Chemical Industry and used as received. Dichloromethane (CH2Cl2) used for polymerization was distilled by the standard procedure before use. 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 L-aspartic acid dimethyl ester hydrochloride (2.47 g, 12.5 mmol) were dissolved in CH2Cl2 (100 mL). Triethylamine (1.75 mL, 25 mmol) and EDCHCl (1.20 g, 12.5 mmol) were added to the solution at 0 °C, and the resulting mixture was stirred at room temperature overnight. The mixture was subsequently washed with 1 M HCl aq., saturated NaHCO3 aq., and water twice, and then dried over anhydrous MgSO4. It was filtered and concentrated on a rotary evaporator to obtain 1 as white solid. Yield 70%. IR (KBr): 3273 (N–H), 3059, 2958, 1747, 1728 (ester C@O), 1651 (amide C@O), 1537, 1435, 1415, 1398, 1344, 1262, 1242, 1207, 1157, 1127, 1088, 991, 746 cm1. 1H NMR (400 Hz, CDCl3): d 1.51–1.53 (m, 1H, norbornene CH2), 2.28–2.30 (m, 1H, norbornene CH2) 2.47 (s, 2H, bridge position), 2.81–3.02 (m, 6H, 2  >CHCOA, 2  ACH2COOA), 3.60–3.72 (m, 12H, 4  ACOOCH3), 4.76–4.83 (m, 2H, ANHCHA), 6.22 (s, 2H, ACH@CHA), 6.64–6.73 (m, 2H, ACONHA). 13C NMR (100 Hz, CDCl3): d 35.79, 35.83, 44.61, 45.29, 46.12, 48.34, 48.44, 51.19, 52.64, 138.22, 138.18, 171.37, 172.34, 172.50. Anal. Calcd for C21H28N2O10: C, 53.84; H, 5.98; N, 6.02. Found: C, 53.02; H, 5.96; N; 5.77. ESI-TOF [M + Na]+. Calcd for C21H28N2O10: 491.1598 Found: 491.1693. 2.3.2. Synthesis of monomer 2 5-Norbornene-2-exo,3-exo-dimethanol (0.775 g, 5.0 mmol) and indomethacin (3.575 g, 10.0 mmol) were dissolved in CH2Cl2 (100 mL). N,N-Dimethylpyridin-4-amine, DMAP (0.31 g, 2.5 mmol) and N-(3-(dimethylamino)propyl)-N0 -ethylcarbo diimide hydrochloride (EDCHCl, 1.36 g, 11.0 mmol) were added to the solution at 0 °C, and the resulting mixture was stirred at room temperature overnight. The mixture was subsequently washed with 1 M HCl aq., saturated NaHCO3 aq., and saturated brine twice, then dried over anhydrous MgSO4. It was concentrated on a rotary evaporator to obtain 2 as yellow solid. Yield 78%. IR (KBr): 3298 (NAH), 3064, 2930, 1732 (ester C@O), 1660 (amide NAC@O), 1591, 1477, 1457, 1356, 1317, 1260, 1222, 1209, 1166, 1143, 1088, 1067, 996, 754 cm1. 1H NMR (400 Hz, CDCl3): d 1.30–1.39 (m, 2H, norbornene CH2), 2.38 (s, 6H, 2  >CCH3), 2.41 (S, 2H, >CHCH2O), 2.58–2.59 (m, 2H, bridge position), 3.67 (4H, 2  ACOCH2), 3.85 (S, 6H, ACOCH3), 3.96–4.23 (m, 4H, 2  >CCH2A), 6.07–6.09 (m, 2H, 2  ACH = CHA), 6.66–6.69 (m, 2H, aromatic AH), 6.87–6.91 (m, 2H, aromatic AH), 6.93–6.91 (m, 2H, aromatic AH), 7.44–7.49 (m, 2H, aromatic AH), 7.64–7.67 (m, 2H, aromatic –H). 13C NMR (100 Hz, CDCl3): d 29.69, 39.78, 42.58, 44.72, 55.70, 65.85, 101.66, 111.66, 114.86, 129.09, 131.14, 137.63, 139.22, 156.06, 168.32, 170.56. Anal. Calcd for C47H42Cl2N2O8: C, 67.71; H, 5.08; N, 3.36. Found: C, 67.65; H, 556; N; 3.45. ESI-TOF [M + Na]+. Calcd for C47H42Cl2N2O8: 855.2198 Found: 855.2275. 2.4. Polymerization 2.4.1. General procedure for the synthesis of block copolymers Monomer 1 (92.5 mg, 0.21 mmol), Grubbs 2nd generation Ru catalyst (3.6 mg, 4.2  103 mmol), 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 38 °C for 4 h. The complete consumption of 1 was confirmed by 1H NMR measurement. A solution of 2 (168 mg, 0.21 mmol) in CH2Cl2 (1.0 mL) was fed into the polymerization mixture, and the resulting mixture was further stirred for 24 h, during which the mixture viscosity increased and the color of the polymerization mixture gradually changed from pink to dark brown. Then, ethyl vinyl ether was added to the mixture to quench the reaction. The mixture was poured into hexane (200 mL), and the precipitated polymer was separated by filtration using a membrane filter and dried under reduced pressure. Block copolymers could be obtained as white solid. Yield: 160 mg (80%). 2.4.2. Spectroscopic data of the polymers. Poly(1) IR 3369, 3009, 2955, 1729 (ester C@O), 1664, 1514, 1437, 1368, 1285, 1206, 1170, 1050, 995, 862, 756 cm1. 1H NMR (400 Hz, CDCl3): (400 Hz, CDCl3): d 1.45 (m, 1H, norbornene CH2), 1.76 (broad, 1H, norbornene CH2), 2.64 (broad, 8H, bridge position, 2  >CHCOA, 2  ACH2COOA), 3.59 (s, 12H, 4  ACOOCH3), 4.48–4.62 (broad, 2H, ANHCHA), 5.11 (s, 1H, ACH = CHA), 5.30 (s, 1H, ACH = CHA) 7.77–7.88 (broad, 2H, ACONHA). Poly(2). IR 3084, 2930, 1732 (ester C@O), 1680,

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1591, 1477, 1457, 1400, 1356, 1317, 1260, 1222, 1166, 1143, 1088, 1067, 1036, 1015, 996, 925, 832, 754 cm1. 1H NMR (400 Hz, CDCl3): d 1.30–1.44 (m, 2H, norbornene CH2), 2.38 (s, 6H, 2  >CCH3), 2.41 (S, 2H, >CHCH2O), 2.56 (m, 2H, bridge position), 3.63–3.68 (m, 4H, 2  ACOCH2), 3.81 (S, 6H, ACOCH3), 3.85–4.23 (m, 4H, 2  >CCH2A), 5.15 (broad, 2H, 2  ACH@CHA), 6.65–6.69 (m, 2H, aromatic AH), 6.85–6.88 (m, 2H, aromatic AH), 6.94–6.97 (m, 2H, aromatic AH), 7.35– 7.72 (m, 4H, aromatic AH). Poly(1)38-block-poly(2)62. IR 3369 (ANH), 2954, 2927, 1734 (ester C@O), 1677 (amide C@O), 1523, 1478, 1438, 1321, 1261, 1171, 1127, 1088, 1068, 835, 754 cm1. 2.5. Determination of the critical micelle concentration (CMC) The block copolymers solutions in DMSO (selective solvent) and CH2Cl2 (good solvent) were prepared at various concentrations. The turbidity of the polymer solutions were evaluated as the difference in transmittance at 435 nm of the copolymer in good solvent compared to a selective solvent (Tgood  Tselective). CMC can be determined by a turning point of the plot between the transmittance and the concentrations. 2.6. In vitro indomethacin release experiments 2.6.1. Typical procedure A drug conjugated micelle solution (0.5 wt% in DMSO 3 mL) was transferred to a prewashed dialysis bag (Spectrum, molecular weight cut-off: 3500 g/mol), and incubated in 60 mL of DMSO/THF = 80/20 (v/v, pH = 3, HCl-adjusted) with rapid stirring at room temperature. Meanwhile the drug conjugated micelle solution (0.15 wt% in DMSO/water = 85/15 (v/v) and (0.15 wt%) in DMSO/water = 80/20 (v/v) were incubated in 60 mL of DMSO/water/THF = 68/12/20 (v/v/v), and DMSO/water/ THF = 64/16/20 (v/v/v, pH = 3, HCl-adjusted), respectively. The amount of released indomethacin was determined by measuring the absorbance at 320 nm of the solution in the outer chamber at 5 h, 24 h and 48 h. 3. Results and discussion 3.1. Monomer synthesis Monomer 1 was synthesized by the reaction of cis-5-norbornene-exo-2,3-dicarboxylic anhydride and L-aspartic acid dimethyl ester hydrochloride in 60% yield (Scheme 2). Indomethacin-conjugated monomer 2 was synthesized by the reaction of 5-norbornene-2-exo,3-exo-dimethanol and indomethacin in 78% yield as illustrated in Scheme 3. The structures of the monomers were confirmed by IR and 1H/13C NMR spectroscopies. The 1H NMR spectra of 1 and 2 exhibited signals reasonably assignable to the expected structures with proper integration ratios [40] (Fig. S1). Two kinds of signals corresponding to a- and b-methyl esters of 1 could be clearly observed with 1:1 integration ratio around 3.60 ppm, indicating no contamination of compounds formed by the reaction of the amino and a-/b-methyl esters. The ester groups of monomer 1 were hydrolyzed to obtain a monomer having free carboxy groups derived from aspartic acid moieties. As our previous reported on the monomer with leucine having free carboxy groups were successfully polymerized using the Grubbs 2nd catalyst [36]. Unfortunately, the expected monomer could not be successfully isolated due to the high solubility in the water phase, which made the separation difficult upon extraction. As a result, we decided to move on the polymerization using monomer 1 bearing ester groups to study the polymerization behavior and the ability of block copolymerization with the drug conjugated monomer 2 together with the self assembly, properties of the formed polymers and potential for drug carriers. 3.2. Homopolymerization Monomers 1 and 2 were polymerized with the Grubbs second generation Ru catalysts. The monomers underwent homopolymerization to produce the polymers [poly(1) and poly(2)] in good yields (98% and 73%) with Mn’s of 2 100 and 18,000, respectively as listed in run 1 and run 7 in Table 1. The fast propagation rate compared to initiation may be responsible for the large PDI of poly(2) [41,42]. Besides, the bulky indomethacin moiety seems to retard the polymerizability of 2. As a result, the residue of monomer 2 originating from the incomplete polymerization was observed as shown in Fig. 1.

Scheme 2. Synthesis of monomer 1.

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Scheme 3. Synthesis of monomer 2.

Table 1 Homo and block copolymerizations of 1 and 2.a Run

1 1 2 3 4 5 6 7 a b c d e f

Yieldc (%)

Feed ratio

100 75 62 50 38 25 0

Mnd

Mw/Mn

2 b

0 25 38 50 62 75 100b

98 91 92 80f 72f 85f 73

2100 11,700 15,000 14,000 13,000 15,300 18,000

1.30 2.60 2.20 2.60 2.30 2.20 1.90

Unit ratioe 1

2

100 75 62 52 45 38 0

0 25 38 48 55 62 100

Conditions: [M]total = 0.42 M in CH2Cl2, catalyst Grubbs 2nd generation, [M]total/[Ru] = 100, 38 °C, first stage 5 h and second stage 24 h. Polymerization time = 5 h. Hexane-insoluble part. Determined by SEC (THF, polystyrene calibration). Determined by 1H NMR measured in CDCl3. Calculated from the 1H NMR integration ratio of the residual monomer and the weight of hexane-insoluble polymer.

Five-hours may not be enough to polymerize monomer 2 quantitatively. On the contrary, the small PDI (1.30) of poly(1) suggested the livingness of ROMP of monomer 1. Thus, it is expected that the block copolymerization of monomers 1 and 2 is achieved by using 1 and 2 as the first and second feed monomers, although the second stage polymerization does not take place in a completely living manner. 3.3. Block copolymerization Block copolymerization of 1 and 2 was carried out (Scheme 1). After the first stage polymerization of 1 for 5 h (the complete consumption was confirmed by 1H NMR spectroscopy), 2 was added to the polymerization mixture, and the resulting mixture was stirred for another 24 h. The SEC peak top of the polymer, which was obtained by the feeding of 2 to poly(1), appeared at a higher molecular weight region than that of poly(1) before the addition of 2 as shown in Fig. 2. This confirmed that block copolymerization took place to yield poly(1)-block-poly(2). The block copolymerizations of 1 and 2 with various feed ratios were carried out to obtain block copolymers with Mn’s of 11,700–15,300 (Mw/Mn = 2.20–2.60) in good yields as listed in runs 2–6 in Table 1. The unit ratios of 1 and 2 in the polymer chain were determined from the 1H NMR integration ratios. The unit ratios of 2 in the block copolymers of runs 4–6 in Table 1 were larger than the feed ratios, especially in run 5 and run 6. This result is reasonable according to the incomplete consumption of 2 confirmed by the 1H NMR spectra as shown in Fig. S2, presumably responsible for the bulkiness of 2 as mentioned above. Table 2 summarizes the solubility of the monomers and polymers. The formation of block copolymers is supported by the different solubility between, poly(1), poly(2) and the copolymers. Poly(1) was soluble in CH2Cl2, CHCl3, THF, acetone, MeOH, DMSO and DMSO/H2O = 85/15, while insoluble in H2O, 0.1 M HCl and 0.5 M NaOH. Poly(2) was soluble in CH2Cl2, CHCl3, THF but insoluble in acetone, DMSO and aqueous solvents. The copolymers with the poly(1) block content of 62% and more were soluble in acetone, and those with 50% and more were soluble in DMSO. On the other hand, the copolymers with the poly(1) block content less than the above-mentioned percentages were insoluble in acetone and DMSO. The solubility in the polar solvents decreased with the decrease in the hydrophobic poly(2) block. The copolymer solutions in DMSO were not transparent but opaque when the polymers looked completely dissolved [poly(1) content = 50%], suggesting the self-assembly of the polymers in DMSO. Meanwhile, the copolymers with less than 50% of poly(1) block were insoluble in DMSO. It is suggested that the hydrophilic block length influenced the balance of self-assembly in a non-aqueous polar solvent (DMSO). The addition of water into the block

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Fig. 1. 1H NMR spectra (400 MHz) of poly(1) measured in DMSO-d6 and poly(2) measured in CDCl3. *Residue of monomer 2. **See note [40] and Fig. S1.

Fig. 2. SEC traces of the first stage [poly(1)] and second stage [poly(1)-block-poly(2)] in the block copolymerization of 1 and 2. The feed molar ratio of 1:2 = 62:38.

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S. Sutthasupa, F. Sanda / European Polymer Journal 85 (2016) 211–224 Table 2 Solubility of the polymers.a

a

Polymer

Hexane

CH2Cl2

CHCl3

THF

Acetone

MeOH

DMSO

DMSO/H2O = 85/15

H2O

HCl (0.1 M)

NaOH aq. (0.5 M)

Poly(1) Poly(1)-block-poly(2) 75:25 62:38 50:50 38:62 25:75 Poly(2)



+

+

+

+

+

+

+







     

+ + + + + +

+ + + + + +

+ + + + + +

+ +    

± ± ±   

+ + +   

+ + +   

     

     

     

Symbols: (+) soluble; (±) partly soluble; () insoluble.

copolymer solution in DMSO resulted in an increase in turbidity. The maximum amount of water that could be mixed with the polymer solution of poly(1)-block-poly(2) in the ratios of 75:25, 62:38, and 50:50 was 15% (v/v). Further addition of water, that is, more than 15%, induced disaggregation and precipitation of the copolymers. DMSO is an aprotic polar solvent. DMSO/H2O mixtures are often used as solvent for organic chemistry as well as chemical technology and biology, since DMSO/H2O mixtures display interesting features in density, viscosity, adiabatic, and isothermal compressibility [43]. DMSO is suitable for screening organic and organic compounds during preclinical and drug development stages. It also enhances the topical penetration of drugs for transdermal delivery [44–46]. DMSO is safely usable as a co-solvent at low concentrations (65%) with H2O for the controlled delivery of hydrophobic drugs, while higher concentrations of DMSO are possibly used as formulations for external applications such as burn and transdermal patches [47]. Despite the fact that poly(1)-block-poly(2) showed poor solubility in aqueous media, its solubility in DMSO and the mixture of DMSO/H2O [15% (v/v) H2O] expresses the possibility of using the polymer as material for the purpose of external applications. Anyway, the water-soluble micelles are the best choice of drug carriers for oral delivery, thus the further modification of polymer structures to achieve water-soluble polymers is still necessary for us in the next project. The polymers with higher block contents of poly(1) were partly soluble in MeOH.

3.4. Formation of self-assemblies As mentioned above, the copolymers with at least 50% poly(1) block content were soluble in a polar aprotic solvent (DMSO). In order to clarify the self-assembly of the copolymers, the solutions were analyzed by 1H NMR spectroscopy and DLS. Fig. 3 shows the 1H NMR spectra of poly(1)62-block-poly(2)38 measured in CDCl3, DMSO-d6 and DMSO-d6/ D2O = 90/10 (v/v) mixture. Poly(1) exhibited two olefinic proton signals at 5.11 ppm and 5.30 ppm, while poly(2) exhibited one olefinic proton signal at 5.15 ppm and aromatic proton signals around 6.66 ppm to 7.70 ppm as shown in Fig. 1. In CDCl3 [good solvent for both poly(1) and poly(2)], poly(1)-block-poly(2) exhibited olefinic proton signals i and l [Fig. 3(i)], together with the poly(1)-based methoxy proton (a, c), ANHC⁄H < (e) and aromatic proton signals (s, u, v, w, x, y, z), methyl proton (t) of the poly(2) block. On the other hand in DMSO-d6 [Fig. 3(ii)] the block copolymer clearly exhibited signals based on the poly(1) block especially olefinic protons (i, j), NHC⁄H < (e) and methoxy protons (a, c), whereas aromatic protons, bridge position (m) and (n) proton signals based on the poly(2) block were attenuated. This could be due to the suppression of the molecular motion of aggregated poly(2) segments surrounded by the poly(1) block. Therefore, it is indicated that the smaller and unclear proton signals of poly(2) block in DMSO-d6 are attributable to the formation of micelles consisting of poly(2) core and poly(1) corona. When block copolymers form micelles, the 1H NMR signals of the corona part are clearly observed compared to those of the core part due to the larger mobility [48]. The above mentioned results clarify that DMSO-d6 is a selective solvent for poly(1)-block-poly(2) to form micelles. The 1H NMR spectra of poly(1)-block-poly(2) were measured in DMSO-d6 as increasing the turbidity upon addition of water into the solution. The block copolymer clearly exhibited signals based on the poly(1) block especially methyl protons (a, c), whereas signals based on the poly(2) block such as aromatic protons, bridge position (m) and (n) are attenuated and some proton signals disappeared. This fact suggests that poly(1)-block-poly(2) also formed micelles in the mixture of DMSO/water. The increase in the turbidity was presumably due to the aggregation of micelles. This phenomenon is further determined by critical micelle concentration (cmc), DLS measurement and TEM images as mentioned in the following part. 3.5. Characterization of micelles Critical micelle concentration (cmc) is designated from sharp changes in measurable quantities. Various properties such as solubilization, surface tension, and turbidity express abrupt changes at the cmc [49]. Turbidity is a simple index to detect aggregates because it reflects changes in the sizes of particles dispersed in solution, and it is commonly used to

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Fig. 3. 1H NMR (400 MHz) spectra of poly(1)62-block-poly(2)38 measured in (i) CDCl3 and (ii) DMSO-d6. (iii) DMSO-d6/D2O = 90/10 (v/v).

determine cmc, micelle formation, and stability [37,50,51]. In this study, the turbidity of polymer solutions was evaluated as the difference in transmittance at 435 nm of copolymer solutions in a good solvent compared to a selective solvent (Tgood  Tselective). Fig. 4(a) is a plot of (Tgood  Tselective) vs concentration of poly(1)62-block-poly(2)38 solutions in CHCl2 and DMSO as good and selective solvents, respectively. The plot exhibits a turning point which can be regarded as the cmc at c = 3.5  102 wt%. The cmc of poly(1)75-block-poly(2)25 was observed at c = 2.5  102 wt% [Fig. 4(c)]. The polymer

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Fig. 4. Plots of transmittance difference at 435 nm (Tgood  Tselective) vs concentration of copolymer solutions, wherein CH2Cl2 was used as a good solvent in every case. (a) CH2Cl2 and DMSO solutions of poly(1)62-block-poly(2)38. (b) CH2Cl2 and DMSO/water = 85/15 (v/v) solutions of poly(1)62-block-poly(2)38. (c) CH2Cl2 and DMSO solutions of poly(1)75-block-poly(2)25. (d) CH2Cl2 and DMSO/water = 85/15 (v/v) solutions of poly(1)75-block-poly(2)25.

particles became precipitated when the water content was increased higher than 15% in DMSO/water mixtures, suggesting the aggregation of micelles. Thus, the polymer solutions were prepared in DMSO/water = 85/15 (v/v) at various concentrations, and the transmittance was compared with that of the polymer solution in CH2Cl2. Fig. 4 (b) and (d) show the turning points at lower concentrations compared with those in DMSO. In the case of poly(1)62-block-poly(2)38, the turning point was observed at c = 2.0  103 wt%, while poly(1)75-block-poly(2)25 presented the turning point at c = 6.0  103 wt%. It seems that block copolymers form larger micelles in DMSO/water mixtures than in DMSO, judging from the lower of the cmc, presumably due to the self-aggregation of micelles. Dynamic light scattering (DLS) measurements were taken to confirm the micelle formation and self-aggregation of the block copolymers. Particles with an average hydrodynamic diameter (Dh) around 119 nm (PDI = 0.086) were detected in a 0.5% (w/v) poly(1)75-block-poly(2)25 solution in DMSO, while particles with Dh around 136 nm (PDI = 0.094) and 154 nm (PDI = 0.198) were found in poly(1)62-block-poly(2)38 and poly(1)50-block-poly(2)50 solutions, respectively as shown in Fig. 5. The longer the indomethacin block was, the larger the particle size was. The PDI data suggested the monodispersities of the particle sizes in DMSO. The particle size of poly(1)62-block-poly(2)38 and poly(1)75-block-poly(2)25 increased upon raising water content in the DMSO/water = 85/15 (v/v), as shown in Fig. 6. The PDI’s of poly(1)62-block-poly(2)38 under the conditions of (i), (ii), and (iii) were 0.094, 0.125, and 0.157, while those of poly(1)75-block-poly(2)25 were 0.086, 0.125, and 1.0, respectively. Therefore, the DLS results confirmed the formation of compound micelles in DMSO, and the increase in the turbidity of compound micelles solutions upon raising the water content is a result of the self-aggregation of the compound micelles. TEM measurements were taken to demonstrate the formation of micelles beside the aggregation of block copolymers in the mixture of water/DMSO. Although the TEM observation could not clarify the outer shells and inner cores, it was clear that poly(1)62-block-poly(2)38 and poly(1)75-block-poly(2)25 formed spherical compound micelles in shape with the average size of 50–100 nm as shown in Fig. 7(a) and (d). The sizes measured by TEM were smaller than those observed by DLS, because DLS measures swollen compound micelles in solution whereas TEM measures dried ones. The self-aggregation of the compound micelles in the mixing of water/DMSO could be clearly observed as shown in Fig. 7(b), (c), (e) and (f). The TEM images

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Fig. 5. The particle size distribution of 0.5% (w/v) poly(1)-block-poly(2) solutions in the ratios of 1:2 = 75:25, 62:38, and 50:50 in DMSO (refractive index = 1.479, viscosity = 1.99 mPs, at 25 °C). Dh = hydrodynamic diameter.

apparently demonstrate that the aggregate size becomes larger when the polymer concentration rises, as shown in Fig. 7(c) and (f). DMSO is miscible with water in all proportions and forms strong hydrogen bonds with water [52]. It seems that the aspartate-containing outer shells of the poly(1) segment prefer interacting with each other to form large compound micelles rather than staying as micelles when the water content increases.

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Fig. 6. Comparison of the particle size distribution of solutions of a) poly(1)62-block-poly(2)38 and b) poly(1)75-block-poly(2)25, at water concentrations of (i) 0.5% (w/v) in DMSO, (ii) 0.15% (w/v) in DMSO/water = 85/15 (v/v, refractive index = 1.4589, viscosity = 3.11 mPs, at 25 °C), and (iii) 0.5% in DMSO/ water = 85/15 (v/v).

Fig. 7. TEM images of poly(1)62-block-poly(2)38 in the solution of (a) 0.5 wt% in DMSO, (b) 0.05wt% in DMSO/water = 85/15 (v/v), (c) 0.5 wt% in DMSO/ water = 85/15 (v/v); poly(1)75-block-poly(2)25 (d) 0.5 wt% in DMSO, (e) 0.05 wt% in DMSO/water = 85/15 (v/v). (f) 0.5 wt% in DMSO/water = 85/15 (v/v).

3.6. In vitro indomethacin release The release of drugs from nanoparticles depends on physical and chemical parameters such as drug diffusion rate, partition coefficient between the drug and hydrophobic segment, and copolymer degradation. These parameters change as a function of environmental surrounding in stimuli-responsive systems, leading to triggered release processes. The solubilities of indomethacin in buffered aqueous media with pH = 1.2 and 7.2 are 0.011 and 2.1 mmol/L, respectively [7,53]. The release of indomethacin is apparently triggered by the lowering of the pH of the media. In this study, a calibration curve (Fig. S5) was

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plotted as a function of absorbance value and concentration of indomethacin, in order to calculate the amount of indomethacin released. This curve also revealed that indomethacin is completely connected to the polymer chain. Fig. 8 demonstrates the percentage of indomethacin released over time at pH 3.0. In the case of the micelle solution of 0.5% (w/v) poly(1)62-block-poly(2)38 DMSO solution as shown in Fig. 8(a), the amount of indomethacin released from the micelles slightly increased as a function of time. In the first 5 h, only 5% of indomethacin was released. After 24 h, 14% of indomethacin was released from the core of micelles and the amount increased to 24% after 48 h. Ester linkages are commonly responsive toward hydrolysis in acidic media, yielding the corresponding carboxylic acids and alcohols [54]. The degradation of the nanoparticles occurred by the hydrolysis of the methyl ester groups of the polymer side chains after one-week incubation at 60 °C. Meanwhile cytotoxicity due to methanol was negligibly small for exposure over 12 h, presumably because residence periods of nanoparticles in the human body are only a few hours [55,56]. They are cleared from blood through the renal system. We consider that methanol is slowly released from the present micelles according to the previous research. In the present study, we did not analyze the amount of methanol released during the incubation in the acidic media. It is the next project for us to study the cytotoxicity effect of methanol released by ester hydrolysis. In this work, indomethacin is released by cleavage from the polymeric backbone [7]. Various polynorbornene-based drug carriers connect drugs via cleavable chemical linkages such as ester, hydrazine and carbamate. The release of the drug corresponds to the degradation of micelles besides the cleavage of chemical linkage under acidic or basic conditions [32,33,35,57]. It is likely that the release of indomethacin in this work is cooperatively caused by the disaggregation of micelle and ester hydrolysis under acidic condition. Indomethacin was released at a much higher rate in the case of 0.15% (w/v) of poly(1)62-block-poly(2)38 in DMSO/water = 85/15 as shown in Fig. 8(b), wherein the indomethacin release reached an equilibrium state at 28% after 24 h. As mentioned above, the copolymer precipitated when the water content became higher than 15% in DMSO/water. Then the unstable micelles disaggregate and the indomethacin segments seems to be easily hydrolyzed at the ester linkages and released faster than the stable micelles. For the reason, we studied further regarding in the release of indomethacin under this condition. A polymer solution was prepared in DMSO/water = 80/20 (v/v), resulting in the formation of unstable micelles with certain amount of precipitates regarding to the free polymers. After dialysis under pH 3, indomethacin was quantitatively released after 24 h as shown in Fig. 8(c). Judging from the results, it is suggested that the micelles are stable in DMSO with the indomethacin segments at the core of micelles, and indomethacin is released by cleavage from the polymeric backbone via the cleavable ester linkages. We previously prepared pH responsive micelles from block copolymers consisting of the hydrophilic polynorbornene block having unprotected phenylalanine and the hydrophobic poly7-oxanorbornene block having leucine [37]. Indomethacin was successfully loaded into the micelles and in vitro release was subsequently performed under conditions that were the same as those in the present study. After a rapid release of ca. 30% of indomethacin during 5 h, a quite stable plateau was observed. The percentage release at 24 and 48 h became stable at ca. 40% (Fig. S6 in the Supporting Information). Indomethacin is probably located at the interface between the core and the corona of the micelles. The micelles disaggregated upon lowering the pH, and this was followed by the release of the drug at a faster rate compared with the conjugated indomethacin in the present study.

Fig. 8. In vitro release of indomethacin from the micelles of poly(1)62-block-poly(2)38 determined by the UV absorbance at 320 nm at room temperature. (a) 0.5% (w/v) in DMSO, incubated in DMSO/THF = 80/20 (v/v, pH = 3, HCl-adjusted), (b) 0.15% (w/v) in DMSO/water (v/v) = 85/15, incubated in DMSO/water/ THF = 68/12/20 (v/v/v, pH = 3, HCl-adjusted), and (c) 0.15% (w/v) in DMSO/water = 80/20 (v/v), incubated in DMSO/water/THF = 64/16/20 (v/v/v, pH = 3, HCl-adjusted).

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4. Conclusions In the present paper, we successfully demonstrated the block copolymerization of norbornene monomer 1 derived from aspartic acid and monomer 2 conjugated with indomethacin via ROMP using the Grubbs 2nd generation catalyst. The corresponding block copolymers could be obtained in good yields. The block copolymers with poly(1) segment with 50% and more formed micelles in a selective solvent; DMSO. The average diameters were in the range of 100–150 nm (DLS) and 70–100 nm (TEM). The micelles further aggregated to form clusters upon addition of water into the micelle solution in DMSO. The nanoparticles prepared from poly(1)62-block-poly(2)38, released 14% and 24% of indomethacin conjugated after 24 and 48 h, respectively in an acidic environment at room temperature. The achievement in this study would contribute to the progress of amino acid based polynorbornene synthesis by ROMP that self-assembles into nanoparticles, and extends the application of ROMP-based polymers as drug delivery materials for the purpose of external applications. Acknowledgements This research was financially supported by a grant from the National Research Council of Thailand (NRCT). The authors are grateful to Dr. Kajornsak Faungnawakij at the National Nanotechnology Center (NANOTEC) for the measurements of the elemental analysis. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.eurpolymj.2016.10.029. References and note [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40]

[41] [42] [43] [44]

I.W. Hamley, Nanotechnology 14 (2003) 39–54. S. Mann, Nat. Mater. 8 (2009) 781–792. R. Duncan, Nat. Rev. Drugs Discov. 2 (2003) 347–360. T.P. Lodge, Macromol. Chem. Phys. 204 (2003) 265–273. M. Antonietti, S. Forster, Adv. Mater. 15 (2003) 1323–1333. S.F.M. van Dongen, H.P.M. de Hoog, R.J.R.W. Peters, M. Nallani, R.J.M. Nolte, J.C.M. van Hest, Chem. Rev. 109 (2009) 6212–6274. C. Giacomelli, V. Schmidt, R. Borsali, Macromolecules 40 (2007) 2148–2157. C. Luo, Y. Liu, Z. Li, Macromolecules 43 (2010) 8101–8108. C. Li, Z. Ge, J. Fang, S. Liu, Macromolecules 42 (2009) 2916–2924. A. Klaikherd, C. Nagamani, S. Thayumanavan, J. Am. Chem. Soc. 131 (2009) 4830–4838. S. Kumar, R. Archarya, U. Chatterji, P. De, Langmuir 29 (2013) 15375–15385. R.H. Grubbs, W. Tunas, Science 243 (1989) 907–915. R.R. Schrock, Acc. Chem. Res. 23 (1990) 158–165. G.C. Bazan, R.R. Schrick, E. Khosravi, W.J. Feast, V.C. Gibson, M.B. O’regan, J.K. Tomas, W.M. Davis, J. Am. Chem. Soc. 112 (1990) 8378–8387. T.M. Trnka, R.H. Grubbs, Acc. Chem. Res. 34 (2001) 18–29. J.A. Love, J.P. Morgan, T.M. Trnka, R.H. Grubbs, Angew. Chem. Int. Ed. 41 (2002) 4035–4037. C. Slugovc, Macromol. Rapid Commun. 25 (2004) 1283–1297. A. Leigeb, J. Wappel, C. Slugovc, Polymer 51 (2010) 2927–2946. R. Singh, C. Czekelins, R.R. Schrock, Macromolecules 39 (2006) 1316–1317. C.W. Bielawski, R.H. Grubbs, Prog. Polym. Sci. 32 (2001) 1–29. S.A. Barnhill, N.C. Bell, J.P. Patterson, D.P. Olds, N.C. Gianneschi, Macromolecules 48 (2015) 1152–1161. F. Sanda, T. Endo, Macromol. Chem. Phys. 200 (1999) 2651–2661. K. Bauri, S. Pant, S.G. Roy, P. De, Polym. Chem. 4 (2013) 4052–4060. H.D. Maynard, S.Y. Okada, R.H. Grubbs, Macromolecules 33 (2000) 6239–6248. H.D. Maynard, S.Y. Okada, R.H. Grubbs, J. Am. Soc. 123 (2001) 1275–1279. H. Mori, I. Kato, S. Saito, T. Endo, Macromolecules 43 (2010) 1289–1298. H. Xu, Q. Yao, C. Cai, J. Gou, Y. Zhang, H. Zhong, X. Tang, J. Control. Release 199 (2015) 84–97. Y. Bae, K. Kataoka, Adv. Drug Deliv. Rev. 61 (2009) 768–784. A. Lalatsa, A.G. Schatzlein, M. Mazza, T.B. Hang-Le, I.F. Uchegbu, J. Control. Release 161 (2012) 523–536. S.B. La, T. Okano, K. Kataoka, J. Pharm. Sci. 85 (1996) 85–90. P.A. Bertin, J.M. Gibbs, C.K-F. Shen, C.S. Thaxton, W.A. Russin, C.A. Mirkin, S.T. Nguyen, J. Am. Chem. Soc. 128 (2006) 4168–4169. D. Smith, S.H. Clark, P.A. Bertin, B.L. Mirkin, S.T. Nguyen, J. Mater. Chem. 19 (2009) 2159–2165. P.A. Bertin, D. Smith, S.T. Nguyen, Chem. Commun. (2005) 3793–3795. P.A. Bertin, K.J. Watson, S.T. Nguyen, Macromolecules 37 (2004) 8364–8372. N.V. Rao, H. Dinda, M.N. Ganivada, J.D. Sarma, R. Shunmugam, Chem. Commun. 50 (2014) 13540–13543. S. Sutthasupa, F. Sanda, T. Masuda, Macromolecules 41 (2008) 305–311. S. Sutthasupa, M. Shiotsuki, H. Matsuoka, T. Masuda, F. Sanda, Macromolecules 43 (2010) 1815–1822. H. Mori, T. Endo, Macromol. Rapid Commun. 33 (2012) 1090–1107. R.K. O’Reilly, Polym. Int. 59 (2010) 568–573. The monomer 2 was contaminated with compound 2 (17%, calculated based on the integration ratios of the 1H NMR spectrum) (Fig. S1). The proton signal around 8.2 ppm referred to the OH moiety and other protons of monomer 2 as marked with A⁄, B⁄, and C⁄. [M + Na]+ of compound 2 at 516.1571 was observed by mass spectrometry. T.-L. Choi, R.H. Grubbs, Angew. Chem. Int. Ed. 42 (2003) 1743–1746. S. Sutthasupa, K. Terada, F. Sanda, T. Masuda, Polymer 48 (2007) 3026–3032. C.G. Elles, N.E. Levinger, Chem. Phys. Lett. 317 (2000) 624–630. A.C. Williams, B.W. Barry, Adv. Drug Deliv. Rev. 56 (2004) 603–618.

224 [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57]

S. Sutthasupa, F. Sanda / European Polymer Journal 85 (2016) 211–224 M. Moniruzzaman, Y. Tahara, N. Tamura, N. Kamiya, M. Goto, Chem. Commun. 46 (2010) 1452–1454. B. Kumar, K. Jain, S.K. Prajapati, Int. J. Drug Deliv. 3 (2010) 83–94. T. Ur-Rehman, S. Tavelin, G. Grobner, G. Int, J. Pharm. 394 (2010) 92–98. K. Stubenrauch, C. Moitzi, G. Fritz, O. Glatter, G. Trimmel, F. Stelzer, Macromolecules 39 (2006) 5865–5874. I.W. Hamley, Introduction to Soft Matter: Polymers, Colloids, Amphiphiles and Liquid Crystal, Wiley-VCH, Weinheim, Germany, 2000. Chapter 4.6, pp. 217. S. Halacheva, S. Rangelov, C. Tsvetanov, Macromolecules 39 (2006) 6845–6852. J.X. Zhang, L.Y. Qiu, Y. Jin, K. Zhu, J. Colloids Surf., B 43 (2005) 123–130. A.K. Soper, A. Luzar, J. Phys. Chem. 100 (1996) 1357–1367. A. Nokhodchi, Y. Javadzadeh, M.R. Siahi-Shadbad, M. Barzegar-Jalali, J. Pharm. Pharm. Sci. 8 (2005) 18–25. H. Soyez, E. Schacht, S. Vanderkerken, Adv. Drug Deliv. Rev. 21 (1996) 81–106. J.A. Portilla-Arias, M. Gracia-Alvarez, A.M. de Ilarduya, E. Holler, J.A. Galbis, S. Munoz-Guerra, Macomol. Biosci. 8 (2008) 540–550. A. Lanz-Landazuri, M. Gracia-Alvarez, J. Portilla-Arias, A.M. de Ilarduya, R. Patil, E. Holler, J.Y. Ljubimova, S. Munoz-Guerra, Macomol. Biosci. 11 (2011) 1370–1377. N.V. Rao, S.R. Mane, A. Kishore, J.D. Sarma, R. Shunmugam, Biomacromolecules 13 (2012) 221–230.