Kim et al.
Synchrotron SAXS Study of Cross-Linked Polymeric Micelles
correlated with the PLA core size.25 Using SANS,
Bhattacharjee et al. showed that the core size of
doxorubicin-loaded poly(ethylene oxide)-poly(propylene
oxide)-poly(ethylene oxide) triblock copolymer did not
change significantly upon drug loading.26 Akiba et al.
carried out a SAXS study of a polymeric micelle solu-
tion made of partially benzyl-esterified poly(ethylene
glycol)-b-poly(aspartic acid) (PEG-P(Asp(Bzl)) and found
that the scattering profiles showed characteristic features
of core–shell spherical micelles. Furthermore, they con-
firmed that PEG and P (Asp(Bzl)) formed a hydrophilic
shell and a hydrophobic core, respectively.27 Although
many microscopy studies on the morphology of polymeric
micelles have been done by using tools such as cryo-TEM,
SANS, and SAXS, to the best of our knowledge, there are
no reports on SAXS study to determine the internal struc-
tures of the cross-linked polymeric micelles in solution.16–27
Herein, we describe the preparation of cross-linked
polymeric micelles (CPMs) using an amphiphilic
methoxypoly(ethylene glycol)-b-poly(DL-lactide)-(L-lysine)
(mPEG-PLA-Lys4) copolymer and an amine-reactive
bifunctional bis(N -hydroxysuccinimide ester) containing
a central disulfide bond. The morphological information
related to size and shape of CPMs was compared to those
of non-cross-linked polymeric micelles (NCPMs) using
DLS and TEM. To obtain more detailed information on
the structure in solution, we investigated the morphology,
USA) at an acceleration voltage of 120 kV. The samples
were stained with uranyl acetate.
2.3. Synthesis of mPEG-PLA-Lys4
mPEG-PLA-Ahx-Boc: mPEG-PLA (2.0 g, 0.25 mmol)
was dissolved in anhydrous dichloromethane (50 mL),
and Boc-6-Ahx-OH (0.12 g, 0.5 mmol) and DCC (0.1 g,
0.5 mmol) were added to the solution. The mixture was
allowed to react for 48 h at room temperature, and
dicyclohexylurea was removed by filtration. Excess ace-
tone was added to the crude product mixture to pre-
cipitate residual dicyclohexylurea. After the precipitates
were filtered out of solution, the solvent was removed
by rotary evaporation. The product was precipitated from
excess of cold diethyl ether. The polymer was col-
lected and dried under vacuum to give mPEG-PDLLA-
Ahx-Boc. Yield = 1.9 g. 1H NMR ꢁ (ppm): 5.18 (m,
–C(O)CH(CH3)O–), 3.63 (m, CH3OCH2CH2O–), 3.40 (s,
CH3OCH2CH2O–), 1.70 (d, –C(O)CH2(CH2ꢂ2CH2NH–),
1.56 (m, –C(O)CH(CH3ꢂO–), 1.49 (d, –C(O)OC(CH3ꢂ3).
mPEG-PLA-NH2: mPEG-PLA-Ahx-Boc (1.5 g) was
dissolved in dichloromethane (10 mL) and the solution was
cooled using an ice-bath. The reaction mixture was treated
with trifluoroacetic acid (TFA, 5 mL) for 1 h. The solution
was concentrated and the crude product was precipitated
from cold diethyl ether. The polymer was collected and
dried under vacuum at room temperature. Yield = 1.2 g.
1H NMR ꢁ (ppm): 5.18 (m, –C(O)CH(CH3ꢂO–), 3.63 (m,
Delivered by Ingenta to: Sibley Library, Eastman School of Music
size, shape, and internal structures of NCPMs and CPMs
IP: 185.14.192.68 On: Sat, 23 Jul 2016 07:25:32
through the fitting of scattering data obtained by SAXS.
CH3OCH2CH2O–), 3.40 (s, CH3OCH2CH2O–), 1.70 (d,
–C(O)CH2(CH2ꢂ2CH2NH–), 1.56 (m, –C(O)CH(CH3ꢂO–).
mPEG-PLA-Lys(Z)4: In a dried flask, mPEG-PLA-NH2
(1.0 g) and Lys(Z)-NCA (0.19 g, 0.62 mmol) were dis-
solved in a mixture of CHCl3 (20 mL) and DMF (5 mL)
under a nitrogen atmosphere. The reaction was allowed
Copyright: American Scientific Publishers
The cross-linked polymeric micelles used in this experi-
ment are underway in vitro and in vivo tests to evaluate
its potential as drug carrier.
2. EXPERIMENTAL DETAILS
2.1. Materials
ꢀ
to proceed for 72 h at 35 C. The mixture was precipi-
tated with excess cold diethyl ether under stirring to give
a white solid. The precipitate was dried under vacuum
at 35 ꢀC. Yield = 0.91 g. 1H NMR ꢁ (ppm): 7.33 (s,
–C(O)OCH2(C6H5)), 5.18 (m, –C(O)CH(CH3)O–), 5.07
(s, –C(O)OCH2(C6H5)) 3.63 (m, CH3OCH2CH2O–), 3.40
(s, CH3OCH2CH2O–), 3.10 (–C(O)CHNH–), 1.70 (d,
–C(O)CH2(CH2ꢂ2CH2NH–), 1.56 (m, –C(O)CH(CH3ꢂO–),
1.25 (–(CH2ꢂ4NH–).
Methoxypoly(ethylene glycol)-b-poly(lactide) (mPEG-
PLA) with a molecular weight of 8000 (mPEG5000,
PLA3000) was purchased from Advanced Polymer Mate-
rial. Boc-aminohexanoic acid (Boc-Ahx-OH), dicyclo-
hexylcarbodiimide (DCC), N 6-carbobenzyloxy-L-lysine,
trichloromethyl chloroformate (TCF), thiohexanoic acid,
and N -hydroxysuccinimide were obtained from Sigma-
Aldrich and were used as received. Conventional chemi-
cals employed in the present study were reagent grade and
used as purchased without further purification.
mPEG-PLA-Lys4: In a dried flask, mPEG-PLA-Lys(Z)4
(0.7 g) was dissolved in TFA (10 mL) and a solution of
HBr (3 mL, 33 wt% in acetic acid) was added. The reac-
tion mixture was stirred under nitrogen for 3 h at 0 ꢀC. The
solvent was removed under vacuum, and the crude prod-
uct was precipitated with excess of diethyl ether to give a
white solid. The resulting polymer was dried under vac-
uum at room temperature for 24 h to give PEG-PLA-Lys4.
2.2. Measurements
Nuclear magnetic resonance (NMR) spectra were obtained
in CDCl3 and DMSO using a Bruker NMR spectrome-
ter (Aspect 3000 FT 400 MHz). The average size and
zeta-potential of the polymeric micelles was measured
via dynamic light scattering (DLS, Zetasizer Nano ZS,
Malvern instrument). To observe the size and distribution
of polymeric micelles, transmission electron microscopy
(TEM) was performed on a Tecnai G2 (FEI company,
1
Yield = 0.54 g. H NMR ꢁ (ppm): 7.71 (–C(O)CH2NH–),
5.18 (m, –C(O)CH(CH3ꢂO–), 3.63 (m, CH3OCH2CH2O–),
3.40 (s, CH3OCH2CH2O–), 3.24 (–C(O)CHNH–), 1.70 (d,
–C(O)CH2(CH2ꢂ2CH2NH–), 1.56 (m, –C(O)CH(CH3ꢂO–),
1.25 (–(CH2ꢂ4NH–).
J. Nanosci. Nanotechnol. 16, 6432–6439, 2016
6433