1 Supplementary Information for ‘Ultrasound and pH Dually Responsive Polymer Vesicles for Anticancer Drug Delivery’ Wenqin Chen and Jianzhong Du* School of Materials Science and Engineering, 4800 Caoan Road, Shanghai, 201804, China. E-mail: [email protected]Supplementary Figures Figure S1. Synthetic route to (A) TMA monomer and (B) block copolymers: PEO-b-PDEA (polymer 1), PEO-b-PTMA (polymer 2) and PEO-b-P(DEA-stat-TMA) (polymers 3 and 4).
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Supplementary Information for ‘Ultrasound and pH Dually Responsive Polymer Vesicles
for Anticancer Drug Delivery’
Wenqin Chen and Jianzhong Du*
School of Materials Science and Engineering, 4800 Caoan Road, Shanghai, 201804, China. E-mail:
N,N,N',N",N"-pentamethyldiethylenetri-amine (PMDETA, 98%) and triethylamine were purchased from Aladdin
Chemistry, Co. and used as received. Polyethylene glycol monomethylether (MeO-PEO-OH; Mn = 1900) was
purchased from Alfa Aesar and dried azeotropically by using anhydrous toluene to remove traces of water before
use. 2-(Diethylamino)ethyl methacrylate (DEA) was purchased from Aladdin Chemistry, Co. and was passed
through a basic Al2O3 column before use. Anisole, methanol, dichloromethane (DCM), tetrahydrofuran (THF) and
dialysis tubing with molecular weight cutoff from 8000 to 14000 were purchased from Sinopharm Chemical
Reagent Co., Ltd (SCRC, Shanghai, China).
General Experimental
Proton nuclear magnetic resonance (1H NMR) spectra were recorded using a Bruker AV 400 MHz spectrometer at
ambient temperature using CDCl3 as solvent. Chemical shifts are in ppm with respect to TMS (tetramethylsilane)
using the manufacture indirect referencing method. All chemical shifts are quoted on the scale in ppm using
residual solvent as the internal standard (1H NMR: δ = 7.26 for CDCl3). Coupling constants (J) are reported in Hz
with the following splitting abbreviations: s = singlet, d = doublet, t = triplet and m = multiplet.
Gel permeation chromatography (GPC) analysis were carried out with a Waters Breeze 1525 GPC analysis system
with two PL mix-D columns, using THF as the eluent at a flow rate of 1.0 mL/min at 35 oC. The copolymers were
dissolved in THF and filtered prior to analysis.
Dynamic light scattering (DLS) measurements were carried out with Zetasizer Nano series instrument (Malvern
Instruments ZS 90) equipped with a multipurpose autotitrator (MPT-2). DLS studies of aqueous polymer vesicles
were carried out at 25 oC and a fixed scattering angle of 90o. Each reported measurement was conducted three
runs.
Transmission electron microscopy (TEM) images were obtained using a H7560 (Hitachi Limited Corporation)
electron microscope operating at an acceleration voltage of 80 kV. To prepare TEM samples, a drop of aqueous
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vesicle solution (0.4 mg/mL) was placed on a copper grid coated with thin carbon film. The pH of phosphotungstic
acid (PTA, 1.0 wt%) was adjusted by NaOH aqueous solution to ca. 7.4 and then used as the stain. The solution
droplet was dried by evaporation under ambient conditions overnight.
Differential scanning calorimetry (DSC) data were recorded by DSC Q100 (TA Instruments). In the dynamic DSC
measurements, freshly extruded samples were kept for 3 min at –80 oC and heated at the rate of 10 oC/min to 80
oC.
Determination of critical vesicle formation concentration of PEO43-b-P(DEA33-stat-TMA47) diblock copolymer An initial solution of pyrene was made by dissolving pyrene (3.0 mg, 15 μmol) in acetone (25 mL) to form a 6 ×
10-5 M solution. The pyrene solution (10 μL) was dropped into 11 centrifuge tubes. The acetone was evaporated
overnight in a vacuum oven. The PEO43-b-P(DEA33-stat-TMA47) polymer vesicle stock solution was serially
diluted with deionized water starting with a concentration of 0.5 mg/mL down to 4.9 × 10-4 mg/mL by
half-and-half dilution. Each polymer solution (4.0 mL) was transferred to a centrifuge tube containing pyrene and
stirred overnight.1 Fluorescence determinations were made by exciting samples at 334 nm, using a 5 nm slit width
for excitation and a 5 nm slit width for emission. Emission wavelengths were scanned from 350 to 500 nm. The
intensities of the I1 (372.1 nm) vibronic bands were evaluated for each sample. The intensity values were plotted
against the log of the concentration of each polymer vesicle sample. The critical vesicle formation concentration
(CVC) was taken as the intersection of two regression lines calculated from the linear portions of the graphs.
Synthesis of poly(4-vinylpyridine) (P4VP)
Distilled 4-vinylpyridine (2.00 g, 19.0 mmol) and recrystallized AIBN (0.0300 g, 0.200 mmol) were dissolved in
ethanol, and then the solution was heated at 60 oC reacting for 24 h under argon atmosphere while stirring. The
obtained viscous solution was poured into deionized water to obtain poly(4-vinylpyridine) (P4VP). The polymer
was purified by reprecipitation with water from ethanol solution to remove unreacted 4VP monomers. At last it
was dried in a vacuum oven to constant weight at 40 oC.
Preparation of poly(4-vinylpyridine) hydrochloride (P4VP•HCl)
P4VP (0.62 g) was dissolved in ethanol, and then hydrochloric acid (1.0 M, 2.5 mL) was added into the solution
while stirring at room temperature. The reaction was carried out for 4.0 h when a fit amount of white precipitates
appeared. A white powder product (0.56 g) was obtained after vacuum filtration and vacuum dry for 24 h.
Synthesis of (2-tetrahydrofuranyloxy)ethyl methacrylate (TMA)
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TMA was synthesized by the addition reaction between 2-hydroxyethyl methacrylate and 2,3-dihydrofuran in
methanol with P4VP•HCl as out-phase catalyst. A literature method2 was modified as following. 2-Hydroxyethyl
1.1 Hz, 3H, CH3O), 1.96 (s, 6H, (CH3)2C). 1H NMR spectrum is shown in Fig. S4 A.
Supplementary References
1. Greene, A. C., Zhu, J., Pochan, D. J., Jia, X. & Kiick, K. L. Poly(acrylic acid-b-styrene) Amphiphilic Multiblock Copolymers as Building Blocks for the Assembly of Discrete Nanoparticles. Macromolecules 44, 1942-1951, (2011).
2. Xuan, J. A., Pelletier, M., Xia, H. S. & Zhao, Y. Ultrasound-Induced Disruption of Amphiphilic Block Copolymer Micelles. Macromol. Chem. Phys. 212, 498-506, (2011).
3. Lu, H. et al. Preparation of water-dispersible silver-decorated polymer vesicles and micelles with excellent antibacterial efficacy. Polym. Chem. 3, 2217-2227, (2012).