Supporting Information
Preparation of Multi-responsive Micelles for Controlled Release of Insulin
Tengteng Jiang 1,2, Guohua Jiang 1,2,3,*, Hua Chen 1,2, Lei Li 1,2, Yongkun Liu 1,2, Huijie Zhou 4, Yu
Feng 4, Jinhua Zhou 4
1
Department of Materials Engineering, Zhejiang Sci-Tech University, Hangzhou 310018, P. R.
China
2
National Engineering Laboratory for Textile Fiber Materials and Processing Technology
(Zhejiang), Hangzhou 310018, P. R. China
3
Key Laboratory of Advanced Textile Materials and Manufacturing Technology (ATMT),
Ministry of Education, Hangzhou 310018, P. R. China
4
*
Qixin Honours School, Zhejiang Sci-Tech University, Hangzhou 310018, P. R. China
To whom correspondence should be addressed. Tel: +86 571 86843527; E-mail address:
ghjiang_cn@zstu.edu.cn (G. Jiang)
Experiment
Materials
Azobisisobutyronitrile (AIBN, 98%) was supplied by East China Chemical Co.,
Ltd. (Shanghai, China) and purified by recrystallization twice from ethanol and dried
in vacuum prior to use. Methoxypolyethylene glycol amine (MePEG111-NH2)
(Mw=5000), acryloyl chloride, 3-amino boronic acid, other regular reagents and
solvents were supplied by Aladdin Reagent Co., Ltd. and used without further
purification.
MilliQ
water
was
used
in
all
experiments.
S-1-Dodecyl-S’-(α,α’-dimethyl-α’’-acetic acid) trithiocarbonate (DMP) was prepared
according to preciously published procedures.1 N,N’-bis(acryloyl)cystamine (BAC)
was obtained as the previous report described.2 2-nitrobenzyl acrylate (NBA) was
synthesized according to the previous report.3
Synthesis of 3-acrylamide phemylboronic acid (APBA)
APBA was prepared as the previous report.4 Briefly, 3-amino boronic acid (5.0 g,
32.2 mol) was dissolved in 10 wt% NaOH solution (50 ml) in a bottom flask. The
mixture was ice-cooled to 0 °C. Acryloyl chloride (5.2 ml, 64.4 mol) was introduced
dropwise into the flask over a period. The mixture reacted for 3 h when returned to
room temperature. Beige precipitated was filtered through Buchner funnel when the
solution acidified to pH of 8 with dilute HCl solution (0.1 M). The precipitated was
washed with cooled deionized water and dried in vacuo at room temperature for 12 h.
The resultant powder was dissolved in aqueous ethanol and filtered. Large and beige
crystals could be obtained by crystallized from the filtrate for 1 or 2 days. APBA is
prepared by filtered and stored in desiccator with a yield of over 40%. 1H-NMR
spectrum (DMSO-d6): δ=10.04 (s, 1H), 7.99 (s, 2H), 7.85, 7.80-7.78, 7.47-7.46,
7.27-7.24 (s, d, d, t, 1H each), 6.44-6.39, 6.23-6.19 (2d, dd, 1H each), 5.71-5.69 (dd,
1H).
Synthesis of the MePEGA-macroinitiators via RAFT polymerization
Methoxypolyethylene glycol amine (0.05 g, 0.01 mmol) and triethylamine (10 μL,
0.136 mmol) were dissolved in 6 mL of dichloromethane, cooling for 30 min with
ice-bath. Acryloyl chloride (1.37 μL, 0.02 mmol), dissolved in 3 mL of
dichloromethane, was added dropwise into the solution. The mixture was kept stirring
at 0 °C for 12 h. The chloride product was obtained through filtering, rotary
evaporating, and drying in vacuum with a yield of over 90%. MePEGA
macroinitiators were fabricated in a one-pot synthesis via RAFT using DMP as the
initiator. DMP (0.0030g, 0.08mmol), AIBN (0.0005g, 0.017mmol) and chloride
product (0.02528g, 0.005mmol), dissolved in THF were added to a dry flask, which
was then immersed in liquid nitrogen for a while. Then, the reaction mixture was
degassed three times using the freeze-pump-thaw procedure. Finally, the flask was
flame-sealed under vacuum and placed in a pre-heated oil-bath at 70℃ for 12 h. The
MePEGA-macroinitiators was obtained through dialysis and freeze-drying with a
yield of over 80%. 1H-NMR spectrum (CDCl3): δ=11.09 (s, H), 8.09 (s, H), 3.75 (d,
2H), 3.41 (d, 2H), 3.20 (d, 2H), 3.01 (m, H), 1.93 (t, 2H), 1.86 (t, 2H), 1.73 (t, 2H),
1.42 (t, 2H), 1.24 (m, 3H), and 0.86 (d, 3H).
Synthesis of MePEGA-b-(PNBMA-co-PAAPBA) polymer micelles
MePEGA-macroinitiators (0.02 g, 0.0046 mmol), NBA (0.02 g, 0.0965 mmol),
APBA (0.02 g, 0.1047 mmol), BAC (0.052 g, 0.2 mmol), AIBN (0.0010 g, 0.0340
mmol) were added into a mixed solution of cyclohexane/water (v/v=1:10). The
mixture was stirred for 10 min before added into a flask. Followed with two minutes
of ultrasonic, the mixture was kept stirring at 60℃ for 24 h after three times of
vacuumed and pumped with N2. The final polymer micelles lyophilization after 3
days of dialysis against water with a yield of over 60%. 1H-NMR spectrum (CDCl3):
δ=11.04 (s, 1H), 8.00 (s, H), 7.05, 7.29, 7.40, 7.56, 7.66, 8.18 (s, d, d, t, 1H each),
5.38 (t, 3H), 3.63 (t, 2H), 3.37 (t, 2H), 3.24 (d, 3H), 2.73 (m, H), 2.70 (m, H), 2.31 (m,
H), 1.98 (m, 2H), 1.96 (t, 2H), 1.88 (m, 2H), 1.63 (t, 2H), 1.33 (t, 2H), 1.29 (t, 2H),
1.24 (m, 3H) and 0.96 (d, 3H).
Synthesis of MePEGA-b-(PNBA-co-PAPBA) polymer micelles with encapsulation of
insulin
MePEGA-macroinitiators (0.02 g, 0.0046 mmol), NBA (0.02 g, 0.0965 mmol),
APBA (0.02 g, 0.1047 mmol), BAC (0.052 g, 0.2 mmol), AIBN (0.0010 g, 0.0340
mmol), insulin from porcine pancreas (0.01 g, 0.0017 mmol) were added into a mixed
solution of cyclohexane/water (v/v=1:10). The mixture was stirred for 10 min before
added into a flask. Followed with two minutes of ultrasonic, the mixture was kept
stirring at 60℃ for 24 h after three times of vacuumed and pumped with N2. The final
polymer micelles lyophilization after 3 days of dialysis against water with.
Characterization
1
HNMR spectroscopy was performed on an AVANCE AV 400 MHz Digital
FT-NMR operating at 400 MHz using deuterated methyl sulfoxide (DMSO-d6),
deuterated chloroform (CDCl3) as solvent and tetramethylsilane as internal standard.
Dynamic light scattering (DLS) measurements were performed in aqueous solution
using a HORIBA Zetasizer LB-550V apparatus at 25°C. Transmission election
microscopy (TEM) was performed using a JEM-2100 TEM operated at an
accelerating voltage of 200 kV, whereby a small drop of solution was deposited onto a
copper EM grid and dried at 40°C under atmospheric pressure. UV-visible spectrum
were obtained using a Hitachi U-330 spectrophotometer. Circular dichroism spectra
were collected on a Jasco J-710 spectropolarimeter.
MTT assay
The relative cytotoxicity of micelles was estimated by MTT assay. The MTT
assay is a colorimetric assay for measuring the activity of cellular enzymes that
reduce the tetrazolium dye, MTT, to its insoluble formazan, giving a purple color. It
measures cellular metabolic activity via cellular oxidoreductase enzymes succinate de
hydrogenase and under defined conditions, reflect the number of viable cells. Hela
cells were seeded into 96-well plates at 8000 cells per well in 200 μL of complete
DEMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% fetal
bovine serum, 2% sodium bicarbonate, 1% amino acid, and 1% sodium pripate at
37℃ in 5% CO2 atmosphere for 24 h. After the incubation, 200 μL of DMEM
containing appropriate dilutions of micelles were added to the cells. 24 h laters, the
supernatant fluid was carefully removed, and 90 μL of fresh DMEM was added along
with 10 μL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT,
5mg/mL) solution to the cells. Then, 20 μL of MTT (5 mg/mL) assays stock solution
in PBS was added to each well. After 4 h incubation, the medium containing
unreacted MTT was carefully removed. The obtained blue formazon crystals were
dissolved in 200 μL well-1 DMSO, and the absorbance was measured in a Bio Tek Elx
800 at a wavelength of 570 nm.
Stability Study
To evaluate the stability and protein resistant properties of the resultant, the
micelles were mixed in 100% metal bovine serum (FBS). The particle size was
continuously monitored by DLS. Tests in serum were conducted at 37 ℃ to mimic
physiological conditions.
Insulin Release Study
Insulin-loaded micelle solution was injected in a dialysis bag with 6000 D
MWCO, and then the dialysis bag was placed in PBS (pH=7.4, 0.01 M) with different
glucose concentrations and under UV irradiation (365 nm, 75 mW/cm2). At certain time
intervals, 1 mL of the buffer solution was taken to measure the insulin concentration,
and then replaced by the same volume of release medium. The concentration was
detected by measuring UV-vis absorbance at 235 nm.
Figure S1. 1HNMR spectrum of MePEGA-macroinitiator.
Figure S2. Photograph of micelles dispersed in water before (left) and after (right) UV
irradiation.
Reference
[1] John T. Lai, Debby Filla, Ronald Shea. Functional polymers from novel
carboxyl-terminated trithiocarbonates as highly efficient RAFT agents.
Macromolecules, 2002, 35, 6754-6756.
[2] Hua Yu, David W. Grainger. Amphiphilic thermosensitive N-isopropylacrylamide
terpolymer hydrogels prepared by micellar polymerization in aqueous media.
Macromoleculs, 1994, 27, 4554-4560.
[3] X.G.Jiang; C.A.Lavender; J.W.Woodcock; Bin Zhao, Macromolecules 2008, 41,
2632-2643.
[4] Jianfeng zhang; Ning Ma; Fu Tang; Qianling Cui; Fang He and Lidong Li, ACS
Appl. Mater. Interfaces 2012, 4, 1747-1751.