Monomethoxy poly(ethylene glycol) (MPEG, Mn 5000) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA), which was purified before use by precipitation against excess diethyl ether and vacuum drying. Triethylamine (TEA), 2-bromopropionyl bromide (BPB), 2-hydroxy nicotinic acid (2-HNA), 1,1′-carbonyldiimidazole (CDI), 3-picolylamine, 4-vinylbenzyl chloride (VBC), 2-methyl-bromopropionate (MBP), copper(I) bromide (Cu(I)Br), potassium carbonate (K2CO3) toluene, and N,N,N’,N”,N”-pentamethyldiethylenetriamine (PMDETA) were also purchased from Sigma-Aldrich. All the liquids were dried and distilled before use. Paclitaxel (PTX) was kindly supplied from Samyang Genex Co. (Daejeon, Korea).
2.2. Synthesis of 2-(4-(vinylbenzyloxy)-N-picolylnicotinamide) (2-VBOPNA)
2-VBOPNA was synthesized by reaction of 2-hydroxy-N
-picolylnicotinamide (2-HPNA) and VBC (). 2-HPNA was prepared by the same method reported previously [19
(30.16 g, 0.220 mol) and 2-HPNA (20 g, 0.088 mol) were suspended in dry acetone (600 mL) at 70°C, and then, VBC (26.64 g, 0.176 mol) was slowly dropped. After reaction for 20 h, the crude solution was filtered and product was isolated by silica gel column chromatography using tetrahydrofuran (THF)/n
-hexane. Pure 2-VBOPNA was obtained from recrystallization in THF/n
-hexane. Yield 75%. Anal. Calcd for C21
: C, 73.03; H, 5.54; N, 12.17. Found: C, 73.19; H, 5.60; N, 12.13.
Synthetic scheme of 2-(4-(vinylbezyloxy)-N-picolylnicotinamide) (2-VBOPNA, a) and PEG-b-P(2-VBOPNA) (b).
2.3. Synthesis of diblock copolymers of PEG and P(2-VBOPNA) (PEG-b-P(2-VBOPNA))
Brominated MPEG (PEG-Br) was prepared by the same method as reported elsewhere [10
]. A series of diblock copolymers of MPEG and P(2-VBOPNA) were synthesized by ATRP of 2-VBOPNA initiated from the PEG-Br (). PEG-Br macroinitiator (0.4 g, 0.08 mmol), 2-VBOPNA (0.4 g, 1.16 mmol), and Cu(I)Br (0.046 g, 0.32 mmol) were added to a flame-dried round-bottom flak. The flask was evacuated and refilled with dry nitrogen twice. Toluene (2 mL) was degassed separately and added into the flask. After the mixture was stirred and purged with N2
for 10 min, PMDETA (0.054 g, 0.32 mmol) was introduced and the flask was placed in a preheated oil bath. During reaction at 80°C for 2 h, the increase in viscosity of the solution was observed. After polymerization, methylene chloride (15 mL) was added and the coppor catalyst was removed by a silica gel column. The block copolymers were repeatedly precipitated against diethyl ether. Further purification was performed by filtration (0.2 μm nylon filter) of block copolymers solution in warm water (50°C), in order to remove P(2-VBOPNA) homopolymer. Final product was obtained by freeze- and vacuum-drying. Diblock copolymers with different P(2-VBOPNA) blocks were synthesized as the same method by changing the monomer/initiator ratio. A homopolymer of P(2-VBOPNA) was polymerized from MBP. PEG-b
-P(2-VBOPNA): Yield 75-80 %; 1
H NMR (CDCl3
): δ 0.83-1.57 (br, 3H), 2.10 (br, 6H), 3.34 (s, 3H), 3.36 (t, J
= 5.0 Hz, 2H), 3.42-3.79 (m, 450H), 3.84 (t, J
= 5.0Hz, 2H), 4. 62 (s, 6H), 5.13 (br s, 6H), 6.40 (br, 9H), 7.09 (br, 9H), 7.52 (br, 3H), 7.69 (br, 3H), 8.51 (br, 9H), 10.19 (br, 3H).
2.4. Characterizations of diblock copolymers of PEG-b-P(2-VBOPNA)
Molecular weight and molecular distribution were obtained from GPC (Agilent 1100 series RI detector, quaternary pump, Santa Clara, CA, USA) using a set of two PLgel 5 μm MIXED-D & E columns (Agilent). THF was used as an eluent and the flow rate was 1mL/min at 35°C. Chromatograms were analyzed based on a PEG standard curve. The chemical compositions of diblock copolymers were confirmed from 1H-NMR (JNM-AL400 FT-NMR spectrometer, 400MHz, JEOL Ltd., Tokyo, Japan).
2.5. Light scattering study
Dynamic light scattering measurement was performed at 25 °C using Brookhaven BI-200SM goniometer and BI-9000AT autocorrelator (Brookhaven Instrument Corp., Holtsville, NY, USA). Sample solutions were prepared by dissolving lyophilized power into deionized water (0.5 mg/mL) followed by brief sonication and filtration through a 0.45 μm syringe filter. The scattered light of a vertically polarized He-Ne laser (633 nm) was measured at the angle of 90° and was collected by the autocorrelator. The hydrodynamic diameters (d) of micelles were calculated by using the Stokes-Einstein equation d = kBT/3πηD, where kB is the Boltzmann constant, T is the absolute temperature, η is the solvent viscosity, and D is the diffusion coefficient.
Fluorescence spectra of PEG-b-P(2-VBOPNA)s were obtained using JASCO FP-6500 fluorospectrometer (Great Dunmow, Essex, UK) at room temperature. Sample solutions were prepared by dissolving polymer powder in double-distilled water (2.5 mg/mL). By serial dilution, concentration of polymer solutions was ranged from 2.5×10-5 to 2.5 mg/mL. All samples were degassed by gentle bubbling of nitrogen for 30 min before measurements. At 330 nm excitation light (slit 2.0 nm), emission spectra were monitored at 380 nm (slit 1.0 nm) and accumulated with an integration of 3 s/nm.
2.7. Solubility test
Excess amount of PTX was added to screw-capped vials containing 1 mL of polymer solutions in phosphate buffered saline (0-10%, w/v). The samples were incubated in a shaking water bath (90 rpm, 37°C) for 24 h. Each solution was quickly filtered through pre-warmed syringe filter (0.2 μm) and analyzed by reverse phase (RP)-HPLC system (Futecs NS-6000A, Daejeon, Korea) using a C18 column (ProntoSIL Co., Chadds Ford, PA, USA). The mobile phase was acetonitrile/water (45:55) mixture and the flow rate was 1.0 mL/min. Solubility values were determined by a calibration curve of various PTX concentrations (0.1-100 μg/mL) versus integrated area (mAU·s), which was monitored at 227 nm.
2.8. Transmission electron microscopy
Sample solution (10 mg/mL) was dropped onto a 400-mesh copper grid coated with carbon. After a minute, the grid was trapped with a filter paper to remove water and dried again. Then, 5% (w/v) of uranyl acetate solution was dropped to the grid. Excess solution was removed and the sample was dried under atmosphere. Transmission electron microscopy (TEM) images were obtained using JEOL JEM-2000EX (Tokyo, Japan), operating at the acceleration voltage of 200 kV.
2.9. PTX release from polymer micelles
release of PTX from PEG-b
-P(2-VBOPNA) micelles was conducted using 0.8 M sodium salicylate solution (pH~6.0) as a sink medium [17
]. The concentration of PTX was 0.25 mg/mL. Polymer micelles loading the same amount of PTX (0.25 mg) were placed into dialysis membrane bag (MWCO 6000-8000 Da). The whole bag was sunk in 40 mL of sodium salicylate solution (37°C). The medium was refreshed at a given time and taken media was stored in refrigerator till HPLC analysis.