Biomacromolecules
Article
on the surface of a plastic Petri dish to form a small bead. A TEM grid
was then floated on top of the bead with the hydrophilic face
contacting the solution. The TEM grid was carefully removed with a
pair of tweezers, wicked with a filter paper to remove excess liquid, and
then dried in air for 1 min.
4.40 (m, 2H, OCH ), 6.21 (br, NH) ppm. 13C NMR (100 MHz,
2
CDCl δ): 14.08, 20.9, 21.6, 23.7, 23.8, 23.1, 28.9, 30.1, 30.3, 31.1,
3
32.5, 34.3, 39.4, 40.3, 41.3, 45.6, 174.1, 174.5, 208.7 ppm.
2.8. Synthesis of p(NIPAM)-b-p(His). Bn-His-NCA was
previously synthesized for the ROP initiated by p(NIPAM) -NH .
5
5
2
2
.3. Synthesis of Tosyl-Protected 2-Amino Ethanol. 2-Amino
Boc-L-His-(Bn)-OH (2.5 g) was suspended in anhydrous 1,4-dioxane
10 mL) to which a solution of PCl (1.8 g) in 1,4-dioxane (20 mL)
ethanol (1.50 mL, 25 mmol) and triethylamine (TEA, 7 mL, 50
mmol) dissolved in anhydrous THF (20 mL) were fed to a 100 mL
(
5
was added to form the Bn-His-NCA at 25 °C under stirring. Within a
few minutes, a clear solution was obtained, which was then filtered
through a glass filter. Crystals of Bn-His-NCA were obtained after the
addition of the filtrate to an excess of diethyl ether. The product was
subsequently washed and dried under vacuum. For the ROP of Bn-
His-NCA by using p(NIPAM)-NH2 as a macroinitiator, the
Schlenk flask fitted with a N inlet and a rubber septum and were
2
cooled in an ice bath. A THF solution of para-toluene sulfonyl
chloride (4.76 g, 25 mmol) was added to the mixture dropwise. White
precipitate of triethylammonium salt was observed and the reaction
was allowed to stir at room temperature overnight. After the
precipitate was filtered off, THF was removed by rotary evaporation.
The residue was purified with flash column (silica gel, hexane/ethyl
p(NIPAM)-NH (0.61 g, 0.10 mmol) and predetermined amount of
2
1
Bn-His-NCA were dissolved in DMF in two separate Schlenk flasks
and subsequently combined using a transfer needle under nitrogen.
The reaction mixture was stirred for 72 h at room temperature under a
nitrogen atmosphere. After polymerization, the solvent was concen-
trated under high vacuum. The concentrated DMF solution was
precipitated in anhydrous diethyl ether and dried under vacuum to
yield p(NIPAM) -b-p(Bn−His) (n = 50, 75, 100, 125).
acetate, v/v 4/2), affording the product as a white solid. Yield: 61%. H
NMR (400 MHz, CDCl δ): 2.10 (t, 1H, OH), 4.30 (t, 1H, NH), 2.35
3
(
(
s, 3H, CH ), 3.36 (m, 2H, NHCH ), 3.82 (m, 2H, HOCH ), 7.34
3
2
2
m, 2H, Hmeta), 7.82 (m, 2H, Hortho) ppm. 1 C NMR (100 MHz,
3
CDCl δ): 20.8, 43.2, 63.4, 125.8, 129.6, 136.3, 141.4 ppm.
3
2
.4. Synthesis of Tosyl-Protected RAFT Chain Transfer
Agent (CTA-2). A dry dichloromethane (10 mL) solution of CTA
(0.364 g, 1 mmol) and tosyl-protected 2-amino ethanol (0.215 g, 1
mmol) was cooled in an ice bath. 4-(N,N′-Dimethylamino)pyridine
DMAP, 0.025 g, 0.2 mmol) and 1,3-dicyclohexylcarbodiimide (DCC,
.26 g, 1.25 mmol) were added in 30 min, and the mixture was stirred
55
n
1
For the deprotection of the benzyl groups, a round-bottomed flask
was charged with a solution of the p(NIPAM) -b-p(Bn-His) in
55
n
(
0
trifluoroacetic acid (100 mg, 3 mL). Then, a 4-fold molar excess of a
3 wt % solution of HBr in acetic acid was added, and the reaction
3
at room temperature for 24 h. The resultant reaction mixture was
filtered, concentrated, and the residue was purified by flash column
mixture was stirred for 2 h at 0 °C. Finally, the reaction mixture was
precipitated in anhydrous diethyl ether. Polymer is isolated and
subjected to aminolysis in presence of hexylamine and few drops of
Na S O in DMF, and then the reaction mixture was precipitated in
(
silica gel, petroleum ether/ethyl acetate, v/v 5/1), affording the
1
product as a pale yellow solid. Yield: 41%. H NMR (400 MHz,
2
2
4
CDCl δ): 0.98 (t, 3H, CH C H CH -), 1.64 (m, 6H, SC(CH ) -,
3
3
10 20
2
3 2
ether. Finally, the product was furthur purified by dialysis and
subsequently freeze-dried to yield p(NIPAM) -b-p(His) (n = 50, 75,
1
.30−1.96 (m, 20H, CH C H CH -), 2.35 (s, 3H, CH ), 2.90 (t, 2H
3
10 20
2
3
5
5
n
SCH ), 3.50 (m, 2H, NHCH ), 4.30 (t, 1H, NH), 4.40 (m, 2H,
2
2
1
OCH ), 7.34 (m, 2H, Hmeta), 7.82 (m, 2H, Hortho) ppm. 1 C NMR
3
100, 125). H NMR (400 MHz, CDCl
3
δ): 1.25−1.43 (br, CH(CH
.51 (br, CH ), 3.01 (d, nH, CH ), 3.47 (br, CH(CH ), 3.95 (t, nH,
) ,
3 2
2
2
2
2
2
(
100 MHz, CDCl δ): 14.8, 20.9, 22.8, 23.1, 28.9, 30.2, 30.4, 31.1, 32.5
3
CH), 6.87 (br, Ar-H), 6.97 (br, Ar-H), 8.10 (br, NH), 9.35 (br, Ar-
3
4.3, 40.8, 52.8, 67.5, 125.4, 129.3, 136.4, 140. 2, 174.5, 208.7 ppm.
.5. Polymerization of NIPAM from CTA-2. A Schlenk tube
NH) ppm. 13C NMR (100 MHz, CDCl δ): 21.6, 23.7, 34.5, 36.2,
2
3
3
9.5, 41.6 45.3, 60.7, 122.3, 148.5, 175.4 ppm.
.9. Fabrication of Micelles. Micelles were prepared by
combining a self-assembly derived precipitation with a membrane
dialysis method. p(NIPAM) -b-p(His) (10 mg) was dissolved in
containing reaction solution (4 mL) of NIPAM (1.1760 g, 10.4
mmol), CTA-2 (66.1 mg, 0.1 mmol), and AIBN (3.344 mg, 0.02
mmol) in DMF was degassed by freeze−pump−thaw cycles for three
2
times and the solution was stirred at 70 °C under N for 24 h.
55
n
2
Precipitation in ethyl ether twice afforded ω-tosylated p(NIPAM)
DMAc and then deionized water (3 mL) was added into the polymer
solution. The turbid mixture was then dialyzed aginist deionized water
for 2 d using a dialysis membrane (regenerated cellulose) with a
molecular weight cut-off 2000 (MWCO = 2000) at 25 °C. The outer
phase was replaced at 3 h intervals with fresh water. The solution was
subsequently lyophilized after filtering through a 0.2 μm syringe filter
in order to remove any impurities and non−micellar aggregates (yield
1
[
p(NIPAM)-Ts] as a white powder (Mn,GPC = 13800). H NMR (400
MHz, CDCl δ): 0.98 (t, 3H, CH C H CH -), 1.28 (br, CH(CH ) ,
3
3
10 20
2
3 2
1
3
7
.30−1.96 (br, 20H, CH C H CH -), 2.52 (br, CH ), 3.47 (br, CH),
3
10 20
2
2
.94 (br, CH), 4.30 (t, 1H, NH), 4.40 (m, 2H, OCH ), 6.21 (br, NH)
2
.34 (m, 2H, Hmeta), 7.82 (m, 2H, Hortho) ppm. 13C NMR (100 MHz,
CDCl δ): 14.08, 20.9, 21.6 23.1, 23.81, 23.1, 28.9, 30.3, 30.9, 31.7,
3
3
1
2.5, 34.1, 39.4, 40.3,41.3, 45.6, 52.8, 67.5, 125.4, 129. 3, 136.4, 140.2,
74.1, 174.5, 208.7 ppm.
=
63%).
For Dox-loaded micelles, Dox (20 mg) dissolved in DMAc (7 mL)
2
.6. Synthesis of p(NIPAM)-NH ·HBr. p(NIPAM)-Ts (0.91 g,
2
and TEA (1.5 equivalent to Dox) was added to the solution of block
copolymer (20 mg) dissolved in DMAc (7 mL) at room temperature
with mixing. After adding 6 mL of water into the solution, the resulting
drug-loaded micelle solution was then dialyzed against deionized water
using a dialysis membrane (MWCO = 2000) at 25 °C for 2 d (yield =
0
.130 mmol) was dissolved in methanol (4 mL) at 0 °C. HBr in acetic
acid (2 mL) was then added dropwise. After stirring for 2 h, an excess
amount of methanol was added. The polymer was precipitated in
diethyl ether twice and dried under vacuum at 40 °C for 24 h, yielding
1
the targeting polymer as a pale yellow powder (Mn,GPC = 14700). H
5
8%).
NMR (400 MHz, CDCl δ): 0.98 (t, 3H, CH C H CH -), 1.25−
3
3
10 20
2
2
.10. Determination of Drug-Loading Content (DLC),
1
.96 (br, 20H, CH(CH ) , 2.52 (br,CH ) 2.90 (t, 2H SCH ), 3.47 (br,
3 2 2 2
Efficiency (DLE), and In Vitro Drug Release. For the quantification
of the amount of drug encapsulated, aliquots of the drug-loaded
micelle solution were lyophilized and broken up by adding 2 mL of
DMSO. The obtained solution was analyzed using the UV−vis
spectroscopy. The characteristic absorbance of Dox (485 nm) was
recorded and compared with a standard curve generated in DMF of
drug concentrations varying from 0−100 mg/mL. The percentages of
DLC and DLE were calculated according to the following equations.
CH), 3.94 (br, CH), 4.30 (t, 1H, NH), 4.40 (m, 2H, OCH ), 6.21 (br,
2
NH) ppm. 13C NMR (100 MHz, CDCl δ): 14.08, 20.9, 21.6, 23.7,
3
2
3.8, 23.1, 28.9, 30.1, 30.3, 31.1, 32.5, 34.3, 39.4, 40.3, 41.3, 45.6,
1
74.1, 174.5, 208.7 ppm.
2
.7. Synthesis of p(NIPAM)-NH . KOH in MeOH solution (1
2
mol/L) was dropped at room temperature to the solution of
p(NIPAM)-NH ·HBr (0.80 g, 0.112 mmol) in MeOH (4 mL) to
2
adjust the pH to 11. After stirring for 1 h, the polymer was precipitated
in diethyl ether twice, yielding the polymer as a white powder (0.71 g,
1
9
0%, Mn,GPC = 13300). H NMR (400 MHz, CDCl δ): 0.94 (t, 3H,
DLC(%) = (weight of drug in the micelle
3
CH C H CH -), 1.25−1.96 (br, 20H, CH(CH ) , 2.52 (br,CH ),
.90 (t, 2H SCH ), 3.47 (br, CH), 3.94 (br, CH), 4.30 (t, 1H, NH),
2
3
10 20
2
3
2
2
/
weight of drug‐loaded micelle) × 100
2
C
dx.doi.org/10.1021/bm400089m | Biomacromolecules XXXX, XXX, XXX−XXX