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of the cells when the cells were cultured in the MSM-DOX for
h.
However, the fluorescence remained nearly constant when
tages of traditional covalent-linked copolymer micelles. There-
fore, this type of shell-responsive micelles are very promising
candidates for improvements in drug delivery systems.
2
the cells were cultured in the MSM-DOX for 4 h. After 12 h in-
cubation, much stronger fluorescence of DOX was observed
and partly localized in the cell nucleus. As observed in Fig-
ure 7A and C, MSM-DOX was internalized through an endocy-
tosis pathway and then the DOX molecules were released and
diffused through endocytic compartments to the nucleus
Experimental Section
Materials
Uracil (ꢁ98.0%), e-caprolactone (e-CL), mono-methoxy poly(ethy-
À1
lene glycol) (MPEG) with M =5000 gmol , 2-mercaptoethanol,
[
15]
n
eventually.
mercapto acetic acid, N,N-dicyclohexylcarbodiimide (DCC), and 4-
dimethylaminopyridine (DMAP) were purchased from Alfa Aesar
Co., Ltd. Stannous octoate (Sn(Oct) ), ethylene carbonate, N-vinyl-
2
In Vitro Cytotoxicity of DOX-Loaded Micelles
caprolactam, azobisisobutyronitrile (AIBN), and 2,2-dimethoxy-2-
phenylacetophenone (DMPA) were purchased from Sigma–Aldrich
Chemical Reagent Co., Ltd. Doxorubicin hydrochloride (DOX·HCl)
was purchased from Adamas Chemical Reagent Co., Ltd. N,N-Dime-
thylformamide (DMF), 1,4-dioxane, and dichloromethane (CH Cl )
Figure 8 shows the viability of BGC-823 cells after incubation
with SM-DOX, MSM-DOX (1:1), and free DOX at different doses
2
2
were purchased from Sinopharm Chemical Reagent Co., Ltd.
Shanghai, China), and dried over calcium hydride for 48 h and
(
then distilled before use. p-Toluenesulfonyl chloride (TsCl) was pur-
chased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai,
China). A clear polystyrene tissue culture treated 12-well and 96-
wellplates were obtained from Beijing Dingguo Changsheng Bio-
technology Co., Ltd. Dialysis bag (molecular weight cut off: 1 KD)
was obtained from Shanghai Baoman Biological Technology Co.,
Ltd. All other reagents and solvents were of analytical grade and
used as received unless otherwise mentioned.
Figure 8. Cytotoxicity of BGC-823 cells against SM-DOX and MSM-DOX (1:1)
after cultured for 48 h with different micelle concentrations.
Synthesis of MPEG-U
[15]
MPEG-U was prepared according to the literature. First, MPEG
and TsCl reacted to obtain MPEG-sulfanilic acid ester (MPEG-OTs).
À1
Then, MPEG-OTs was treated with uracil to give MPEG-U in 82%
of DOX from 0–5.0 mgmL . The IC value (a concentration at
5
0
1
yield. H NMR (300 MHz, CDCl ): d=8.27 (s, 1H, -CONHCO-), 7.37 (d,
À1
3
which 50% of cells were killed) for free DOX was 1.3 mgmL ,
1
H, -NCHCH-), 5.65 (d, 1H, -CHCHCO-), 3.92 (t, 2H, -CH CH N-),
2 2
while that for SM-DOX and MSM-DOX were 3.8 and
3
.65–3.74 (m, 4H, -OCH CH -,), 3.54 ppm (s, 3H, CH OCH -).
2 2 3 2
À1
4
.3 mgmL , respectively. The results demonstrate that DOX-
13
C NMR (300 MHz, [D ]DMSO): d=47.14 (-CH CH N-), 58.02
6
2
2
loaded micelles are able to enter the cells and produce the de-
sired pharmacological action. DOX-loaded micelles show
a slightly lower cytotoxicity than that of free DOX, which can
be attributed to the slow release of DOX from micelles and
delay nuclear uptake in BGC-823 cells, as evidenced by the in
vivo DOX release.
(CH O-), 67.84 (-CH CH N-), 69.72 (-CH CH O-), 71.31 (CH OCH -),
3 2 2 2 2 3 2
102.12 (-CHCHCO-), 146.31 (-CHCHCO-), 150.11 (-NCONH-),
167.32 ppm (-CHCONH-).
Synthesis of Diaminotriazine (DAT)
A
0
(
mixture containing 2,4-diamino-6-vinyl-s-triazine (4.11 g,
.03 mol), DMPA (0.380 g, 0.0015 mol), and 2-mercaptoethanol
4.4 mL, 0.06 mol) in DMF (100 mL) was irradiated by UV at 365 nm
Conclusions
for 1 h. The DMF was removed under reduced pressure and the
product was washed with a mixture of acetone and ethanol
Stimuli-responsive drug delivery systems that can release
drugs in a controllable manner are highly desirable, especially
for the delivery of anticancer drugs. We have successfully de-
veloped stimuli-responsive mixed shell micelles as a drug
nanocarrier. In the first step, MPEG-U, DAT-PCL, and PNVCL-U
were synthesized and their self-assembly in solution was stud-
ied. In addition, we have demonstrated that the complex mi-
celles have strong response to mild acid pH and are capable of
rapidly releasing DOX inside the cells to yield significantly en-
hanced drug efficacy. The resulting micelles are nontoxic. Im-
portantly, the DOX-loaded micelles could be successfully inter-
nalized into cancer cells. These mixed shell micelles based on
complementary multiple hydrogen bonds are very appealing
drug carriers, because they can potentially combine the advan-
(
80 mL, v/v=1:1) three times to give a white powder in 55% yield.
1
1
The H NMR spectrum of the DAT is shown in Figure S12. H NMR
(
2
MS (Bruker Reflex III time-of-flight mass spectrometer) m/z: calcd.
for C H N OS 215.27; found 216.00. The C NMR and FTIR spectra
(300 MHz, [D ]DMSO) of DAT are shown in Figure S13 and Fig-
ure S14 in the Supporting Information, respectively.
300 MHz, [D ]DMSO): d=6.63 (s, 4H, -NH ), 2.82 (t, 2H, -CH CH S-),
6 2 2 2
.57 (t, 4H, -CH SCH -), 3.51 (q, 2H, -CH OH), 4.80 ppm (s, 1H, -OH),
2 2 2
13
7
13
5
6
Synthesis of DAT-PCL
DAT-PCL was synthesized by ring-opening polymerization of e-CL
initiated by DAT with Sn(Oct) as a catalyst. The necessary amounts
of DAT, e-CL, and Sn(Oct) were added into a 50 mL round flask fol-
lowed by six cycles of evacuation-purging with purified nitrogen.
2
2
Chem. Asian J. 2016, 11, 112 – 119
116
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