Supporting Information

Transcription

1 Supporting Information Wiley-VCH Weinheim, Germany

2 Direct Synthesis of Polymer Nanocapsules with a Noncovalently Tailorable Surface Using a Rigid, Disk-shaped Host Molecule with a Cavity and Multiple Polymerizable Groups at the Periphery** Dongwoo Kim, Eunju Kim, Jeeyeon Kim, Kyeng Min Park, Kangkyun Baek, Minseon Jung, Young Ho Ko, Wokyung Sung, Hyung Seok Kim, Ju Hyung Suh, Chan Gyung Park, Oh Sung Na, Dong-ki Lee, Kyung Eun Lee, Sung Sik Han, Kimoon Kim* General Procedures. All the reagents and solvents employed were commercially available and used as supplied without further purification. HS(CH 2 CH 2 O) 4 CH 2 CH 2 SH [S1] and FITC-spermine conjugate 6 [S2] were synthesized according to literature. Photoreaction was performed in a quartz tube by irradiating UV light at 29 o C using a Rayonet photochemical reactor (Model: RMR-600) equipped with four 254 nm lamps and four 300 nm lamps. All the NMR data were recorded on a Bruker DPX-300 or DRX500 spectrometer. UV-visible absorption spectra were recorded on a Hewlett-Packard 8453 diode array spectrophotometer. All fluorescence measurements were performed with 10-mm quartz cells on a Shimadzu RF-5301PC spectrofluorometer. Dialysis was performed using a Spectra/Por RC membrane (MWCO: 8,000). FT-IR spectra were recorded on a Perkin-Elmer Spectrum GX FT-IR spectrophotometer. Dynamic and static light scattering experiments were performed on a DLS-7000 instrument (Otsuka Electronics) using an argon ion laser operating with vertically polarized light at λ = 488 nm. SEM images were taken using a Phillips XL30S FEG scanning electron microscope operating at 5 kv. High-resolution TEM images were recorded on a JEOL-2010F electron microscope operating at 200 kv. Cryo-TEM images were recorded on a Tecnai 12 electron microscope (Philips, Eindhoven, Netherlands) at approximately 170 C and with 120 kv acceleration voltage equipped with a Multiscan 600W CCD camera (Gatan, Inc., Warrendale, PA). Fluorescence images were observed on a Carl Zeiss LSM510 confocal microscope. Human squamous cell carcinoma cell line of the oral cavity (KB cell) was obtained from the Korean Cell Line Bank (KCLB). High-resolution mass (FAB) was carried out at the Korea Basic Science Institute (Daegu). Synthesis of polymer nanocapsule made of (allyloxy) 12 cucurbit[6]uril. Synthesis of polymer nanocapsule 3a. α,ω-dithiol 2a (43.7 mg, 240 µmol) was added to a solution of 1 (10.4 mg, 5.0 µmol) in methanol (10 ml). After purged with N 2, the mixture was irradiated with UV light (254 nm and 300 nm) for 20 h. The product was purified by dialysis using methanol for 2 days to give a colloidal solution of polymer nanocapsule 3a in methanol, which was usually used for further experiments. Removal of the solvent under a reduced pressure followed by drying under vacuo yielded polymer nanocapsule 3a (20.5 mg; 87% based on 1). Elemental analysis calcd for 3a [(C 72 H 96 N 24 O 24 ) 2 (C 6 H 12 O 2 S 2 ) 31 (CH 4 O) 6 ] n : C 44.13, H 6.48, N 7.35, S 21.74; found: C 44.25, H 6.10, N 7.03, S The elemental analysis data indicate that polymer nanocapsule 3a is composed of 1 and 2a in a ratio of 1:15.5 and contains 87 % of the starting material 1. FT-IR spectrum and 13 C CP-MAS NMR spectrum of 3a are given in Figure S1 and S2, respectively. Treatment of polymer nanocapsule 3a with excess ethyl vinyl ether under UV light: S1

3 To a dispersion of polymer nanocapsule 3a in methanol prepared as described above was added excess ethyl vinyl ether (505 mg, 7.0 mmol). After purged with N 2, the mixture was irradiated with UV light (254 nm and 300 nm) for 20 h. The product was purified by dialysis against methanol for 2 days. After removal of most of the solvent, addition of diethyl ether yielded polymer nanocapsule 3a (13.8 mg, 81 %). Elemental analysis calcd for 3a [(C 72 H 96 N 24 O 24 )(C 6 H 12 O 2 S 2 ) 7.4 (C 4 H 9 O 1 ) 2.7 (CH 4 O) 7 (C 4 H 10 O) 4 ] n : C 48.53, H 7.39, N 9.18, S 12.88; found: C 48.15, H 6.62, N 8.79, S The elemental analysis data indicate that polymer nanocapsule 3a is composed of 1 and 2a in a ratio of ~1:7.4. The FT-IR spectrum of 3a is given in Figure S1. Synthesis of polymer nanocapsule 3a in different solvents: Polymer nanocapsule 3a was synthesized in chloroform or acetonitrile using the same procedure as described above. The average diameters of the nanocapsules were measured by SEM studies. Synthesis of polymer nanocapsule 3b and 3c. Dispersion of polymer nanocapsule 3b and 3c in methanol was synthesized using α,ω-dithiol 2b and 2c (1:dithiol = 1:48), respectively, following the same procedure for 3a. The average diameters of the nanocapsules were determined by DLS studies. Synthesis of polymer nanocapsule composed of triphenylene. Synthesis of 2,3,6,7,10,11-hexakis(allyloxy)triphenylene 4. Sodium hydride (740 mg, 18.5 mmol) was added to a solution of 2,3,6,7,10,11- hexahydroxytriphenylene (500 mg, 1.54 mmol) in anhydrous DMSO and the mixture was stirred for 1 h at room temperature. Allyl bromide (2.23 g, 18.5 mmol) was then added to the reaction mixture slowly and stirred for 12 h. After addition of water and dichloromethane to the reaction mixture, the product was extracted with dichloromethane three times. The combined organic layer was washed twice with water and dried over Na 2 SO 4. After removal of solvent in vacuo, the crude product was recrystallized from methanol-dichloromethane at 4 o C to give 4 (420 mg, 48 %). 1 H NMR (500 MHz, CDCl 3 ) d 7.80 (s, 6H), 6.22 (m, 6H), 5.55 (d, J = 17.2 Hz, 6H), 5.37 (d, J = 10.4 Hz, 6H), 4.83 (d, J = 5.2 Hz, 12H); 13 C NMR (125 MHz, CDCl 3 ) d 148.5, 133.9, 123.8, 118.0, 107.6, 70.6; FAB-MS m/z [M] + ; Elemental analysis calcd for C 36 H 36 O 6 : C 76.57, H 6.43; found: C 76.39, H Synthesis of polymer nanocapsule 5a: α,ω-dithiol 2a (43.7 mg, 240 µmol) was added to a solution of 4 (5.6 mg, 10 µmol) in acetonitrile (10 ml). After purged with N 2, the mixture was irradiated with UV light (254 nm and 300 nm) for 20 h. The product was purified by dialysis against acetonitrile for 2 days. After removal of most of the solvent, addition of diethyl ether yielded polymer nanocapsule 5a (10.2 mg, 52 %). Elemental S2

4 analysis calcd for 5a [(C 36 H 42 O 6 )(C 6 H 12 O 2 S 2 ) 6.4 (CH 3 CN) 2 (C 4 H 10 O) 2 ] n : C 53.09, H 7.47, N 1.43, S 21.00; found: C 51.8, H 6.33, N 0.51, S Encapsulation of dye molecules into polymer nanocapsule. Synthesis of polymer nanocapsule encapsulating carboxyfluorescein, CF@3a. α,ω- Dithiol 2a (43.7 mg, 240 µmol) and carboxyfluorescien (3.80 mg, 10 µmol) was added to a solution of 1 (10.4 mg, 5.0 µmol) in methanol (10 ml). After purged with N 2, the mixture was irradiated with UV light (254 nm and 300 nm) for 20 h. The resulting dispersion was purified by dialysis for 2 days to give a colloidal solution of dyeencapsulated polymer nanocapsule CF@3a in methanol. The resulting dispersion was used for fluorescence experiments. Fluorescence experiments. The emission of the encapsulated carboxyfluorescien at 536 nm was monitored with two different excitation wavelengths (503 nm and 458 nm) simultaneously. In a typical experiment, a dispersion of polymer nanocapsule, CF@3a in methanol (1 ml) prepared as described above was diluted with methanol (1 ml) in a cuvette and fluorescence intensities, I 503 and I 458 as well as the intensity ratio, I 503 /I 458, was recorded as a function of time. One hundred seconds later, aqueous HCl solution (30 µl, 0.5 M in water) or methyl viologen (MV 2+ ) solution (400 µl, 5.0 M in methanol) was added to the cuvette and the intensity ratio, I 503 /I 458, continued to be monitored (Figure S8). Surface decoration of polymer nanocapsule using host-guest interactions. Estimation of accessible host molecules constituting polymer nanocapsule 3a using fluorescent probe 6. To a dispersion of polymer nanocapsule 3a in methanol (1 ml) was added FITC-spermine conjugate 6 (0.30 mg, 0.44 µmol; 1.0 equiv with respect to the amount of the CB[6] host present in the nanocapsule) was added and the resulting dispersion was gently shaken for 12 h. Unbound 6 was then removed and collected by dialysis for 12 h. The amount of unbound 6 was measured by fluometry. This experiment was repeated several times with but was usually µmol ( equiv). It indicates that approximately 85 % of the host molecules 1 constituting the polymer nanocapsules 3a are accessible by the fluorescent probe 6. When 0.75, 0.50 or 0.25 equiv of 6 was used to decorate the surface of 3a by following the same procedure a negligible amount (< 5%) of unbound 6 was recovered (measured by fluometry) upon dialysis. This result indicates that near quantitative binding of 6 to the surface of the polymer nanocapsule is achieved when 0.8 or less equivalents (with respect to all the constituting host molecules) of 6 are added to a dispersion of 3a. Monitoring the release of free 6 from polymer nanocapsule 3a decorated with 6. A dispersion of polymer nanocapsule 3a decorated with 6 in methanol (5 ml) was prepared as described above. After the removal of unbound 6 by dialysis for 2 days, the dispersion of the surface decorated nanocapsule was further subjected to dialysis against methanol (2 L) for 70 h and the release of free 6 was monitored by fluometry. A negligible amount (< 3%) of 6 was found to be released during the dialysis indicating the exceptional stability of the noncovalently modified polymer nanocapsule. Preparation of polymer nanocapsule 3a decorated with 6 for physical property measurements. To a dispersion of polymer nanocapsule 3a in methanol (5 ml) was added FITC-spermine conjugate 6 (1.5 mg, 2.2 µmol; 1.0 equiv with respect to the amount of the CB[6] host present in the nanocapsule) was added and the resulting S3

5 dispersion was gently shaken for 12 h. The unbound 6 was removed by dialysis for 2 days. The resulting dispersion was used for further experiments including dynamic light scattering, UV-visible absorption, emission measurements, SEM and TEM experiments. Intracellular delivery of surface decorated polymer nanocapsules. Synthesis of folate-spermidine conjugate 7. To a solution of folic acid (205.0 mg, 460 µmol) in DMSO (10 ml) was added dicyclohexylcabodiimide (28.0 mg, 150 µmol), N-hydroxysuccinimide (15.6 mg, 150 µmol) and triethylamine (20?µL). The reaction mixture was stirred at room temperature until white precipitate appeared. After the white precipitate was filtered off, N 2,N 3 -ditert-butoxycarbonylspermidine [S3] (52.4 mg, 170 µmol) was added to the filtrate. The mixture was stirred at room temperature for 8 h before diethyl ether was added to the mixture to produce yellow precipitate. The precipitate was washed with 20 % aqueous NaOH solution to remove unreacted folic acid and redissolved in trifluoroacetic acid (1 ml). The solution was stirred at room temperature for 30 min before evaporated to dryness under a reduced pressure. After the residue was dissolved in acetonitrile (5 ml), tetrabutylammonium chloride (50.0 mg, 170 µmol) in acetonitrile (3 ml) was added to produce precipitate which was washed with acetonitrile and dried under vacuo to yield 7 (46.0 mg, 38%). The product is a mixture of α- and γ-substituted isomers in a ratio of 1: H NMR (500 MHz, D 2 O) d 1.75 (m, 4H), 1.84 (t, J = 14.7 Hz, 0.55 H, γ- substituted), 1.93 (t, J = 14.7 Hz, 0.45 H, α-substituted), (m, 2H), 2.47 (t, J = 12.7 Hz, 0.55 H, γ-substituted), 2.58 (t, J = 14.2 Hz, 0.45H, α-substituted), (m, 6H), 3.25 (m, 1.1 H, γ-substituted), 3.37 (m, 0.9 H, α-substituted), 4.42 (m, 0.45H, α-substituted), 4.52 (m, 0.55 H, γ-substituted), 4.70 (s, 2H), 7.49 (d, J = 7.8 Hz, 2H), 8.32 (d, J = 7.6 Hz, 2H), 8.60 (s, 1H); HRMS (FAB) m/z calcd for C 26 H 37 N 10 O 5 [M+H- 2HCl] ; found, Preparation of polymer nanocapsule 3a decorated with 6 for cell experiments. To a dispersion of polymer nanocapsule 3a in methanol (5 ml), FITC-spermine conjugate 6 (0.152 mg, µmol, 0.1 equiv. with respect to the CB[6] host present in the nanocapsule) were added, and the resulting dispersion was gently shaken for 12 h. The dispersion of polymer nanocapsule 3a decorated with 6 in methanol was used for cell S4

6 experiments without further purification. Preparation of polymer nanocapsule 3a decorated with 6 and 7 for cell experiments. To a dispersion of polymer nanocapsule 3a in methanol (5 ml), FITCspermine conjugate 6 (0.152 mg, µmol, 0.1 equiv. with respect to the CB[6] host present in the nanocapsule) and folate-spermidine conjugate 7 (0.140 mg, µmol, 0.1 equiv.) were added, and the resulting dispersion was gently shaken for 12 h. The dispersion of polymer nanocapsule 3a decorated with 6 and 7 in methanol was used for cell experiments without further purification. Confocal laser microscopy. KB cells were seeded on a poly-lysine coated cover glass in a 24-well plate at a density of cells per well in RPMI 1640 medium containing 10 % FBS and 1 % penicillin/streptomycin (PS) and incubated in a humidified 5 % CO 2 atmosphere at 37 o C for 24 h. The culture medium was replaced with 1 ml of RPMI 1640 medium including 10 µl of polymer nanocapsule 3a decorated with 6 and 7 ( M in methanol), polymer nanocapsule 3a decorated with 6 ( M in methanol) or 10 µl of methanol, respectively. After 4 h incubation at 37 C, the cells were washed two twice with RPMI 1640 medium and PBS and examined by confocal laser scanning microscope using a 488 nm excitation wavelength. S5

7 Figure S1. FT-IR spectra of a) 1, b) 2a, c) 3a and d) 3a. The FT-IR spectrum of 3a reveals two characteristic peaks of the CB[6] unit of 1 at 1765 cm 1 and 1457 cm 1 corresponding to the C=O and C N stretching vibrations, and complete disappearance of the S H stretching peak at 2557 cm -1 of 2a, the C=C stretching peak at 1647 cm 1, βc H stretching peak at 3017 cm -1 and =CH 2 antisymmetric stretching peak at 3084cm 1 of 1. Figure S2. 13 C CP-MAS NMR spectra of a) 1 and b) 3a. The NMR spectrum of 3a reveals complete disappearance of the olefin peaks of 1 at 120 and 136 ppm as well as appearance of a thioether peak at 35 ppm and an ether peak at 68 ppm, which indicates that all the allyl groups of 1 have been successfully converted to thioether linkages upon formation of 3a. S6

8 Excess dithiol (HS L SH) = Loosely cross-linked polymer nanocapsules = = O N N CH 2 RO OR N N CH 2 O 6 S L S R = CH 2 CH 2 CH 2 n = S L S S L S S L S 3a, n ~ 6 n Highly cross-linked polymer nanocapsules containing disulfide loops Excess ethyl vinyl ether hn = S L S (CH 2 ) 3 OCH 2 CH 3 Figure S3. Schematic representation of the formation of a highly cross-linked polymer nanocapsule with disulfide loops protruding on the surface. The loosely cross-linked polymer nanocapsule initially formed from curved oligomeric patches has unreacted allyl groups. Upon further reaction with excess dithiol, some of the remaining allyl groups form additional thioether bridges between neighboring CB[6] units in the shell to produce a highly cross-linked polymer nanocapsule while the rest of the allyl groups form disulfide loops protruding on the nanocapsule surface. The disulfide loops on the surface can be removed by treating the polymer nanocapsule with excess ethyl vinyl ether under UV light. For example, elemental analysis (based on the N:S ratio) shows that the ratio of 1 and 2a incorporated into 3a is 1:15.5, but decreases to ~1:7.4 after treatment of 3a with excess ethyl vinyl ether. This result suggests that upon reaction with 2a, ~9 of 12 allyl groups of 1 form thioether bridges with a composition of O(CH 2 ) 3 S L S(CH 2 ) 3 O (L = CH 2 CH 2 (OCH 2 CH 2 ) 2 ) linking neighboring CB[6] units to produce a two-dimensional (2D) polymer network that constitutes the shell of the nanocapsule, and the remaining ~3 allyl groups form disulfide loops with an average composition of O(CH 2 ) 3 S L S (S L S) n S L S(CH 2 ) 3 O (n ~ 6), protruding on the nanocapsule surface as illustrated here. S7

9 Figure S4. TEM images of polymer nanocapsules 3a: a) HR-TEM image and b) Cryo-TEM image of 3a. The samples for HR-TEM studies were stained with uranyl acetate. The TEM studies confirmed that the polymer nanocapsules have a hollow structure with a thin wall (average thickness 2.1 ± 0.3 nm). Figure S5. Change in the average size of the product (observed by DLS) during the formation of polymer nanocapsule 3a from 1 and 2a (1:48) (inset: size change during the first 60 min). S8

10 Figure S6. Change in IR spectrum during the formation of polymer nanocapsule 3a from 1 and 2a (1:48). Note that the intensity of C=C stretching peak of 1 at 1647 cm -1 slowly decreases as the reaction proceeds. Figure S7. (A) Size distribution of polymer nanocapsule CF@3a (solid line) and 3a (dotted line). (B) SEM image of CF@3a. (C) Absorption spectrum of CF@3a in methanol. (D) Emission spectra of polymer nanocapsule CF@3a (solid line, λ ex = 458 nm, λ em = 542 nm) and carboxyfluorescein (dotted line, λ ex = 458 nm, λ em = 514 nm) in methanol. Note that the encapsulation of the dye is accompanied by a significant redshift of its emission maximum. S9

11 Figure S8. Changes in emission intensity (at 542 nm) of dye-encapsulated polymer nanocapsule in methanol after addition of 100 µl of 0.5 M HCl aqueous solution (a), and addition of an excess amount of methyl viologen (b). Note that the fluorescence of the encapsulated dye is quenched instantly upon addition of HCl whereas is quenched slowly upon addition of methylviologen, which illustrates sizeselective permeability of the nanocapsule shell. References [S1] S.-T. Huang, H.-S. Kuo, C.-L Hsiao, Y.-L. Lin, Bioinorg. Med. Chem. 2002, 10, [S2] S. Y. Jon, N. Selvapalam, D. H. Oh, J.- K. Kang, S.-Y. Kim, Y. J. Jeon, J. W. Lee, K. Kim, J. Am. Chem. Soc. 2003, 125, [S3] M. Humora, J. Quick, J. Org. Chem. 1979, 44, S10