Supporting Information
Synthesis of Biomimetic Hyperbranched Zwitterionic Polymers
as Targeting Drug Delivery Carriers
Xiaohong Wang†,‡,1, Xinke Sun†,‡,1, Guohua Jiang†,‡∗, Rijing Wang†,‡, Ruanbing Hu†,‡,
†‡
‡
†‡
†‡
Xiaoguang Xi , , Yang Zhou ,Sheng Wang , , Tao Wang ,
†
Key Laboratory of Advanced Textile Materials and Manufacturing Technology (Ministry of
Education), Zhejiang Sci-Tech University, Hangzhou 310018, China
E-mail: polymer_jiang@hotmail.com
Tel: 86-571-86843527
†
Department of Materials Engineering, College of Materials and Textiles, Zhejiang Sci-Tech
University, Hangzhou 310018, China
Experimental section
Materials
HBPO and S-1-Dodecyl-S’-(α,α’-dimethyl-α’’-acetic acid)trithiocarbonate (DMP)
were synthesized according to the previously published procedures.[1, 2]
Azobisisobutyronitrile (AIBN, 98 %) was recrystallized twice from ethanol and dried
in vacuum prior to use. The AIBN was purchased from East China Chemical Co.
(Shanghai,China).
Dicyclohexycarbodiimide
(DCC,
99%),
α-bromoisobutyryl
bromide(98%), folic acid (FOL), Rhodamine B (RB) were purchased from Aladdin
Reagent Co. (Shanghai, China) and used as received. All other reagents and solvents
were of analytical grade and used as received without further purification.
Synthesis of CBB monomer
As shown in Scheme s1, the N-(2-(methacryloyloxy)ethyl-N,N-dimethyl2-isbutyryl bromide (CBB monomer) was synthesized as follows. 11 g DMAEMA
and 17.7 g α-bromoisobutyryl bromide were reacted in 50 ml anhydrous acetonitrile at
50 °C under N2 protection for 24 hours. Upon addition of 250 ml ethyl ether to the
reaction mixture, the formed yellow crystals were isolated and dried. The resulting
CBB monomers were immediately stored in a desiccator at -20 °C (yield 84%). 1H
NMR (DMSO-d6) δ (ppm): 1.89 (s, 3H, CH2=C(CH3)COO-), 2.81(s, 6H,
-CH2N(CH3)2C(CH3)2-), 3.47 (s, 6H, -CH2N(CH3)2C(CH3)2-), 3.74 (t, 2H,
-CH2N(CH3)2C(CH3)2-), 4.51 (t, 2H, -CH2CH2N(CH3)2C(CH3)2-), 5.73 and 6.09 (s,
2H, CH2=C(CH3 )COO-).
Synthesis of HBPO-PCBB and HBPO-PCB
The HBPO-DMP RAFT chain-transfer agent (CTA) was synthesized as reported
previously [3]. The HBPO-PCBB was synthesized through a RAFT path by using
HBPO-DMP as macro-CTAs, AIBN as catalyst. The general procedure was as follows.
The macro-CTAs HBPO-DMP, CBB monomer and AIBN were dissolved in DMF and
the solution was transferred to the flask. After the mixture was added by syringe, the
flask was immersed in liquid nitrogen followed by three cycles of freeze-pump-thaw
procedures. Finally, the flask was flame-sealed under vacuum and placed in a
pre-heated oil-bath at 80 °C for 24 h. The HBPO-PCB copolymers can be generated
by hydrolysis of the HBPO-PCBB in DI water and the final product was obtained by
lyophilization. The feed ratio of CBB to the initiation site of macro-CTAs is
controlled at 80 for HBPO-PCB. 1H NMR (HBPO-PCB, D2O) δ (ppm): 0.8-1.1 (3H,
-CH2C(CH3)(C)COO-),
1.80-2.1
(2H,
-C(CH3)2CH2-),
2.55-2.82
(6H,
-CH2N(CH3)2C(CH3)2-), 3.07-3.28 (6H, -CH2N(CH3)2C(CH3)2-), 3.65-3.74 (2H,
-CH2N(CH3)2C(CH3)2-), 4.15-4.39 (2H, -CH2CH2N(CH3)2C(CH3)2-).
Synthesis of HBPO-PCB-FOL and HBPO-PCB-RB
HBPO-PCB-FOL
(HPC-FOL)
was
synthesized
by
two
steps.
Folic
acid-ethylenediamine (FOL-EDA) was synthesized firstly as follow, 0.66g FOL, 0.82
g DCC, 0.116 ml EDA, 16 ml pyridine, and 40 ml DMSO were added in to a flask
and stirred at room temperature under the protection of N2 for 18h. Insoluble
substances were removed by filtration, and the filtrate was then put into ethyl ether to
make product precipitate. The yellow precipitated solid were washed with ethyl ether
several times to remove residual solvent before dried at 30oC. Then, FOL-EDA was
added into a flask to react with the filtrate for another 24h after the insoluble product
from reaction of 0.28 g HBPO-PCB, 0.109 g NHS, 0.196 g DCC and 10ml DMSO as
solvent under N2 protection removed. After that, insoluble substance was removed
again, and un-reacted monomer FOL-EDA was removed via dialysis against DMSO
for 3 days. The last product was purified and gained by dialysis against DI-water and
freeze drying.
The method used to synthesize HBPO-PCB-RB is the same as that of
HBPO-PCB-FOL. Rhodamine B (RB)- ethylenediamine (EDA) was synthesized
firstly. Then, RB-EDA was grafted to the surface of HBPO-PCB via DCC/NHS
chemistry.
Synthesis of HBPO-PCB/DOX and HBPO-PCB-FOL/DOX
HBPO-PCB/DOX and HBPO-PCB-FOL/DOX were synthesized via dissolution
method. 20 mg HBPO-PCB and HBPO-PCB-FOL were dissolved in 2 ml DMSO
before 2 mg DOX·HCl and 0.6979 mg triethylamine were added and then stirred at
room temperature for 2 h. Resultant complex solution was dropt into 5 ml DI water
slowly and kept stirring for 2 h. Then, the solution was moved into a 3500 Mw
dialysis membrane and dialysis against water to remove un-reacted DOX and
triethylamine. Final complex was gained via freeze drying.
Stability study
To evaluate the stability and protein resistant properties of the resultant
biomimetic hyperbranched colpolymers, the HBPO-PCB was dispersed in 100% fetal
bovine serum (FBS). The particle size was continuously monitored by DLS. Tests in
serum were conducted at 37 °C to mimic physiological conditions.
Characterization
General Characterization and Instrumentation.
The 1H NMR spectra were recorded on an AVANCE AV 400MHz Digital
FT-NMR spectrometer operating at 400 MHz using deuterated DMSO-d6 and D2O as
the solvent. The sizes and morphologies of the resultant samples were characterized
by JSM-2100 transmission electron microscopy (TEM) at an accelerating voltage of
200 kV, whereby a small drop of sample solution was deposited onto a carbon-coated
copper EM grid (200 mesh) and dried at room temperature at atmospheric pressure.
Dynamic light scattering (DLS) measurements were performed in aqueous solution
using a HORIBA Zetasizer apparatus (LB-550 V) equipped with a 5.0 mW laserdiode
operating at 650 nm at room temperature.
Cytotoxicity and Cellular Uptake.
The relatibe cytotoxicity of HBPO-PCB micelles was eatimated by MTT
viability assay and the experiments of intracellular drug release were performed on
confocal laser scanning microscopy (CLSM).
MTT Assay. Hela cells were seeded into 96-well plates at 8000 cells per well in
200 ul of complete DEMEM (Dulbecco’s modified Eagle’s medium), and incubated at
37 oC in 5 % CO2 atmosphere for 24 h. After 24 h of incubation, the culture medium
was removed washed carefully with flesh PBS three times. 200 ul of DMEM
containing appropriate dilutions of HBPO-PCB were added and the cells were grown
for another 24h. After 24 h of incubation, the supernatant fluid was carefully removed
and 90 ul flesh DMEM was added along with 10 ul MTT solution. Then, 20 μL of 5
mg mL-1 MTT assays stock solution in PBS was added to each well. After the cells
were incubated for 4 h, the medium containing unreacted MTT was carefully removed.
The obtained blue formazan crystals were dissolved in 200 μL well-1 DMSO, and the
absorbance wasmeasured in a BioTek Elx800 at a wavelength of 570 nm.
CLSM. Hela cells were seeded into 24-well plates at 40000 cells per well in 500
ul of complete DEMEM (Dulbecco’s modified Eagle’s medium), and incubated at 37
o
C in 5 % CO2 atmosphere for 24 h. After 24 h of incubation, the culture medium was
removed washed carefully with flesh PBS three times. Then, 1 ml of DMEM
containing HBPO-PCB-RB (100 mg/ml) was added and the cells were incubated for
further 6 h. After incubation of 6 h, the supernatant fluid was removed and the cells
was wahed with flesh PBS for three times before investigated by fluorescence
microscope.
Activity Analysis
The cytotoxicity of pure DOX, DOX-loaded HBPO-PCB and DOX-loaded
HBPO-PCB-FOL micelles against HeLa cells was evaluated in vitro by MTT assay.
HeLa cells were seeded into 96-well plates at 1×104 cells per well in 200 μL of
medium and further incubated for 24 h. The cells were then washed with PBS DOX,
DOX-loaded HBPO-PCB and DOX-loaded HBPO-PCB-FOL diluted in complete
DMEM (200 μL) were added to cells, and the cells were further incubated for 48 h.
After the incubation, the culture medium was removed and washed with PBS twice.
Then, 200 μL of DMEM and 20 μL of 5 mg mL-1 MTT assays stock solution in PBS
were added. After the cells were incubated for 4 h, the medium containing unreacted
MTT was carefully removed. The obtained blue formazan crystals were dissolved in
200 μL well-1 DMSO, and the absorbance was measured in a Perkin-Elmer 1420
multilabel counter at a wavelength of 570 nm.
Scheme S1. Synthesis of biomimetic hyperbranched drug carrier with target
delivery property
Figure S1. Release of indomethacin from HBPO-PCB micelles at pH 6.0, 7.0 and 8.0.
References
1. Jiang, G.; Wang, L.; Chen, W. Eur Polym J. 2006, 42, 3333-3340.
2. Lai, J. T.; Filla, D.; Shea, R. Macromolecules. 2002, 35, 6754-6756.
3. Sun, X.; Jiang, G.; Wang, Y.; Xu, Y. Colloid Polym. Sci. 2011, 289, 677-684.