Thermo-responsive shell cross-linked PMMA-b-P(NIPAAm-co-NAS) micelles for drug delivery

Article abstract of DOI:10.1016/j.ijpharm.2011.08.038

Thermo-responsive amphiphilic poly(methyl methacrylate)-b-poly(N- isopropylacrylamide-co-N-acryloxysuccinimide) (PMMA-b-P(NIPAAm-co-NAS)) block copolymer was synthesized by successive RAFT polymerizations. The uncross-linked micelles were facilely prepared by directly dissolving the block copolymer in an aqueous medium, and the shell cross-linked (SCL) micelles were further fabricated by the addition of ethylenediamine as a di-functional cross-linker into the micellar solution. Optical absorption measurements showed that the LCST of uncross-linked and cross-linked micelles was 31.0 °C and 40.8 °C, respectively. Transmission electron microscopy (TEM) showed that both uncross-linked and cross-linked micelles exhibited well-defined spherical shape in aqueous phase at room temperature, while the SCL micelles were able to retain the spherical shape with relatively smaller dimension even at 40 °C due to the cross-linked structure. In vitro drug release study demonstrated a slower and more sustained drug release behavior from the SCL micelles at high temperature as compared with the release profile of uncross-linked micelles, indicating the great potential of SCL micelles developed herein as novel smart carriers for controlled drug release.

Full text of DOI:10.1016/j.ijpharm.2011.08.038

International Journal of Pharmaceutics 420 (2011) 333–340
Contents lists available at SciVerse ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical Nanotechnology
Thermo-responsive shell cross-linked PMMA-b-P(NIPAAm-co-NAS) micelles for
drug delivery
Cong Changa,b, Hua Weia, De-Qun Wua, Bin Yanga, Ni Chena, Si-Xue Chenga, Xian-Zheng Zhanga,∗
,
Ren-Xi Zhuoa
a Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072, PR China
b Key Laboratory of Traditional Chinese Medicine Resource and Compound Prescription, Ministry of Education, Hubei University of Chinese Medicine, Wuhan 430061, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 April 2011
Received in revised form 12 August 2011
Accepted 23 August 2011
Available online 27 August 2011
Thermo-responsive amphiphilic poly(methyl methacrylate)-b-poly(N-isopropylacrylamide-co-N-
acryloxysuccinimide) (PMMA-b-P(NIPAAm-co-NAS)) block copolymer was synthesized by successive
RAFT polymerizations. The uncross-linked micelles were facilely prepared by directly dissolving the
block copolymer in an aqueous medium, and the shell cross-linked (SCL) micelles were further fabricated
by the addition of ethylenediamine as a di-functional cross-linker into the micellar solution. Optical
absorption measurements showed that the LCST of uncross-linked and cross-linked micelles was 31.0 ◦C
and 40.8 ◦C, respectively. Transmission electron microscopy (TEM) showed that both uncross-linked and
cross-linked micelles exhibited well-defined spherical shape in aqueous phase at room temperature,
while the SCL micelles were able to retain the spherical shape with relatively smaller dimension even
at 40 ◦C due to the cross-linked structure. In vitro drug release study demonstrated a slower and more
sustained drug release behavior from the SCL micelles at high temperature as compared with the release
profile of uncross-linked micelles, indicating the great potential of SCL micelles developed herein as
novel smart carriers for controlled drug release.
Keywords:
Thermo-responsive
Shell cross-linked (SCL) micelle
RAFT
Controlled drug release
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
most extensively investigated temperature-sensitive polymer,
poly(N-isopropylacrylamide) (PNIPAAm), exhibits
a reversible
The self-assembly of amphiphilic block copolymers into core-
shell micelle is a subject of much concern for their potential
applications in biomedicine as nano-sized carriers for drug and
gene delivery (Wang et al., 2005b, 2007; Wu et al., 2009;
Ten Cate and Borner, 2007; Tian et al., 2006). Reversible
addition–fragmentation chain transfer radical (RAFT) polymer-
ization, as one of the controlled living radical polymerizations
(CLRP), is a powerful synthetic technique for the preparation
of amphiphilic block copolymers with well-defined structure,
pre-determined composition, and narrow-distributed molecular
weight. It gives access to a wide range of various monomers, such
as acrylic acid (AA), hydroxyethyl methacrylate (HEMA), and N-
isopropylacrylamide (NIPAAm), which can insert into the chain
transfer agent (CTA) or macro-CTA (de Lambert et al., 2005, 2007).
During the past decade, the fabrication of intelligent particles
capable of changing its structure according to the alteration of
environmental signals (e.g., temperature and pH) has attracted
increasing attention (Rapoport et al., 2002; Ramesh Babu et
al., 2006; Mundargi et al., 2010; Reddy et al., 2008). The
thermo-responsive phase transition at around 33 ◦C in aqueous
medium, termed as the lower critical solution temperature (LCST).
It undergoes a transition from hydrophilic to hydrophobic state in
aqueous solution when the temperature is increased from below
to above its LCST (Zhang and Zhuo, 2001; Shelke et al., 2008). Our
previous study demonstrated that the drug release from PNIPAAm-
b-poly(methyl methacrylate) (PNIPAAm-b-PMMA) micelles was
accelerated dramatically at the high temperature above the LCST
due to the deformation of micellar structure (Wei et al., 2006b).
Although polymeric micelles exhibit kinetic stability (Yokoyama
et al., 1996), the inevitable dissociation of micellar structure into
individual polymer chains under dilution by gastric, blood, or other
body fluids in the biological environment after administration ham-
pers their practical applications (Jiang et al., 2007; Huo et al., 2006).
To address such crucial issue, the strategies of covalent stabilization
via cross-linking of the micellar core or shell were explored in order
to enhance the structural stability and integrity of the supramolec-
ular architecture (Murthy et al., 2001; Ding and Liu, 1998). For
example, Bontha and co-workers reported the construction of
core cross-linked (CCL) micelles based on poly(ethylene oxide)-b-
poly(methacrylic acid) (PEO-b-PMA) using divalent metal cations
(Ca2+) as the cross-linker, and further investigated the release
behavior of cisplatin from the CCL micelles (Bontha et al., 2006).
∗
Corresponding author. Tel.: +86 27 68754509; fax: +86 27 68754509.
E-mail address: xz-zhang@whu.edu.cn (X.-Z. Zhang).
0378-5173/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijpharm.2011.08.038

334
C. Chang et al. / International Journal of Pharmaceutics 420 (2011) 333–340
Hu et al. (2006) used glutaraldehyde (GA) to cross-link stearic
acid grafted chitosan oligosaccharide (CSO-g-SA), and studied the
release of paclitaxel from the resultant shell cross-linked (SCL)
micelles. Sairam et al. (2006) investigated the encapsulation effi-
ciency and drug release behaviors of cross-linked poly(acrylamide)
particles prepared by dispersion polymerization.
bar. The mixture was stirred for 30 min prior to the addition of
carbon disulfide (6.0 mL, 0.1 mol) dropwise. The resulting yellow
solution was stirred overnight at room temperature. Thereafter 2-
bromopropionic acid (15.3 g, 0.1 mol) was added dropwise to the
yellow solution, and the mixture was further stirred overnight at
room temperature. Then the reaction mixture was acidified by the
addition of concentrated hydrochloric acid, and the resulting pre-
cipitate was extracted with acetic ether. Finally, the concentrated
yellow acetic ether solution was poured into petroleum to pre-
cipitate the product. The product was separated by filtration and
further purified twice by redissolving/reprecipitating with acetic
ether/petroleum, and dried under vacuum overnight. 1H NMR (d6-
DMSO) ı (ppm): 1.53 (d, –CH3–CH–), 2.70 (t, –CH2–COOH), 3.56 (t,
–S–CH2–), 4.68 (quar, –S–CH–), 12.8 (s, –COOH).
In this study, we synthesized amphiphilic diblock copolymer
PMMA-b-poly(NIPAAm-co-N-acryloxysuccinimide)
(PMMA-b-P
(NIPAAm-co-NAS)) by successive RAFT polymerization techniques
and further constructed SCL micelles using ethylenediamine as a
di-functional cross-linker. Reactive NAS units were introduced into
the polymer structure as the cross-linking sites by RAFT copoly-
merization given that NAS could offer an optimized accessibility
for compound containing amino group (Li et al., 2006; Hakwere
and Perrier, 2009; Zhang et al., 2007). PMMA was chosen as the
hydrophobic building block for its good biocompatibility (Cheng
et al., 2009; Wei et al., 2008a; Hayashi et al., 2005; de Brabander
et al., 2003). In comparison to the uncross-linked micelle, the
resultant SCL micelle exhibited enhanced stability at the high
temperature as well as in the organic solvent. Moreover, in vitro
drug release study demonstrated that the resultant SCL micelle
could achieve slower and steadier drug release at 45 ◦C, indicating
its potential as intelligent carrier for sustained drug release.
2.4. Synthesis of PMMA (macro-CTA) by RAFT polymerization
MMA (776 mg, 7.76 mmol), CPA(25.4 mg, 0.1 mmol), and AIBN
(1.64 mg, 0.01 mmol) were dissolved in 15 mL of freshly distilled
THF in a 50 mL round-bottom flask with a magnetic stir bar. The
flask was purged with dry N2 for 30 min prior to being immersed
in a preheated oil bath at 70 ◦C. After reaction for 22 h, the reaction
mixtures were poured into a large excess of n-hexane to precipitate
the resulting PMMA. The product was collected by filtration and
further purified twice by redissolution/reprecipitation with THF/n-
hexane, and finally dried under vacuum overnight.
2. Materials and methods
2.1. Materials
2.5. Synthesis of PMMA-b-P(NIPAAm-co-NAS) by RAFT
polymerization
N-isopropylacrylamide (NIPAAm) was purchased from Acros
and used as received. Methyl methacrylate (MMA), carbon
disulfide (CS2), 1,4-dioxane, tetrahydrofuran (THF), chloro-
form and triethylamine (TEA) were purchased from Shang-
hai Chemical Reagent Company and used after distillation.
N-hydroxysuccinimide (NHS) was purchased from Shanghai
Chemical Reagent Company. N-acryloxysuccinimide (NAS) and 2-
(2-Carboxyethylsulfanylthiocarbonylsulfanyl)propionic acid (CPA)
were prepared according to the reported methods (Li et al., 2006;
Wang et al., 2005a; Chang et al., 2009). N, Nꢀ-azobisisobutyronitrile
(AIBN) was purchased from Shanghai Chemical Reagent Company
and used after recrystallization with 95% ethanol. Dialysis tube
(molecular weight cut-off: 8000–12,000 g/mol) and pore-sized
syringe filter (0.45 ␮m) were purchased from Shanghai Chemical
Reagent Company. All other reagents and solvents were used as
received.
In a 50 mL round-bottom flask charged with a magnetic stir
bar, NIPAAm (1.14 g, 10 mmol), NAS (84.5 mg, 0.5 mmol), macro-
CTA PMMA (150 mg, 0.025 mmol), and AIBN (0.8 mg, 0.005 mmol)
were dissolved in 8 mL of freshly distilled 1,4-dioxane. The flask
was purged with dry N2 for 30 min prior to being immersed
in a preheated oil bath at 70 ◦C. After reaction for 22 h, the
reaction mixtures were poured into a large excess of diethyl
ether to precipitate the resulting PMMA-b-P(NIPAAm-co-NAS). The
product was collected by filtration and purified twice by redissolu-
tion/reprecipitation with THF/diethyl ether, and finally dried under
vacuum overnight.
2.6. 1H NMR characterization
1H NMR spectra were recorded on a Varian Unity 300 MHz spec-
trometer using CDCl3 as a solvent. The testing temperature was
fixed at 20 ◦C without specific clarification.
2.2. Synthesis of N-acryloxysuccinimide (NAS)
NAS monomer was synthesized according to reference (Li et al.,
2006). Briefly, NHS (4.25 g) and TEA (4.10 g) were dissolved in 80 mL
of distilled chloroform in a flask preserved in an ice–salt bath. Acry-
loyl chloride (3.34 g) in 20 mL of distilled chloroform was added
dropwise under stirring. After 3 h, the reaction mixture was rinsed
with distilled water and saturated salt solution three times respec-
tively, then dried over anhydrous magnesium sulfate overnight.
After removal of chloroform by rotary evaporation, NAS was har-
vested by recrystallization in ethyl acetate/n-hexane (volume ratio,
1:1) as colorless crystalline. 1H NMR (CDCl3, 300 MHz): ı (ppm from
TMS) 2.9 (s, –CH2–CH2–), 6.2 (d, –CH CH2), 6.4 (t, –CH CH2), 6.7
(d, –CH CH2).
2.7. SEC–MALLS measurements
The size-exclusion chromatography and multi-angle laser light
scattering (SEC–MALLS) analysis were used to determine the
molecular weight of the as-prepared polymers. A dual-detector sys-
tem, consisting of a MALLS device (DAWN EOS, Wyatt Technology)
and an interferometric refractometer (Optilab DSP, Wyatt Technol-
ogy) were used. The columns used were styragel HR1 and HR4. The
concentration of the polymer was kept constant at 10 mg/mL and
THF(chromatographicgrade)was used as the eluentat a flow rate of
0.3 mL/min. The MALLS detector was operated at a laser wavelength
of 690 nm. The dn/dc of PMMA and PMMA-b-P(NIPAAm-co-NAS)
were 0.185 and 0.077, respectively.
2.3. Synthesis of
2-(2-carboxyethylsulfanylthiocarbonylsulfanyl)propionic acid
(CPA)
2.8. Micelle formation
3-mercaptopropionic acid (10.6 g, 0.1 mol), distilled water
(100 mL), and 50 wt.% NaOH solution (16.0 g, 0.2 mol) were added
to a 250 mL round-bottomed flask equipped with a magnetic stir
The uncross-linked micelle was prepared by directly dissolv-
ing 20 mg of PMMA-b-P(NIPAAm-co-NAS) copolymer in 40 mL of

C. Chang et al. / International Journal of Pharmaceutics 420 (2011) 333–340
335
distilled water at 20 ◦C. The SCL micelle was fabricated by adding
0.5 mg of ethylenediamine into the above micelle solution for cross-
linking. After reaction for 24 h, the mixture solution was transferred
into a dialysis tube and dialyzed against distilled water for 24 h to
remove any unreacted ethylenediamine and byproduct NHS result-
ing from the aminolysis of NAS.
Table 1
Molecular weight of PMMA and PMMA-b-P(NIPAAm-co-NAS) determined by
SEC–MALLS.
Polymer
Mn bara
Mw barb
PDI (Mw bar/Mn bar)
PMMA
3,300
32,800
6,000
48,500
1.82
1.48
PMMA-b-P(NIPAAm-co-NAS)
ꢀ
ꢀ
a
Mn bar =
Mw bar =
NiMi/
Ni
ꢀ
ꢀ
ꢀ
ꢀ
2.9. Transmission electron microscopy (TEM) observation
b
WiMi/
Wi =
NiMi2
/
NiMi
The sample was prepared by dipping a carbon film coated cop-
per grid into the uncross-linked and cross-linked micelle aqueous
solutions (500 mg/L). After the deposition of particles, the residue
of sample solution was blotted away with a strip of filter paper.
The samples were further stained with 0.2% phosphatetungstic
acid aqueous solution, and dried in air prior to TEM observa-
tion on a JEM-100CX II instrument operating at an acceleration
voltage of 80 kV. For DMF suspension, the sample was prepared
in the same way. The specimens at high temperature were pre-
pared according to our previous study (Chang et al., 2009). Briefly,
a drop of micelle suspension preheated at 40 ◦C using a water
bath was placed onto a carbon film coated copper grid, which
was then placed in a baking oven thermostated at 40 ◦C for quick
drying.
fresh medium after each sampling. The total amount of prednisone
acetate loaded in the micelles was calculated as the summation
of cumulative amount of drug released from the micelles and the
amount of drug remained in the micelles after release. The amount
of prednisone acetate released from micelles was measured using
UV absorbance at 242 nm (Wei et al., 2007; Zou et al., 2007; Li et al.,
2007). To determine the amount of drug retained in micelle, the
micelle suspension after drug release was freeze-dried, and then
dissolved in DMF and analyzed by UV absorbance at 270 nm (Chang
et al., 2009). The entrapment efficiency (EE) and drug loading (DL)
capacity were calculated based on the following formulas,
mass of drug loaded in micelles
EE =
DL =
× 100%,
× 100%.
mass of drug fed initially
2.10. Size distribution measurements
mass of drug loaded in micelles
mass of micelles
Zetasizer Nano ZS (Malvern Instruments) was used to deter-
mine the average size and size distribution of uncross-linked and
cross-linked micelles at different temperatures. The micelle solu-
tion (800 mg/L) was passed through a 0.45 ␮m pore-sized syringe
filter prior to measurements.
3. Results and discussion
3.1. Synthesis of PMMA-b-P(NIPAAm-co-NAS) copolymer
2.11. LCST
The order of constructing the blocks of a block copolymer
can be very important in the RAFT polymerization. The pri-
mary rule that should follow is the propagating radical for the
first synthesized block must be a good homolytic leaving group
with respect to that of the second block, thus in the synthesis
of a methacrylates–styrene or methacrylates–acrylamide diblock
copolymer, the methacrylate block should be prepared first. The
styrene or acrylamide propagating radicals are very poor leav-
ing groups as compared with methacrylates propagating radicals.
Herein, PMMA-b-P(NIPAAm-co-NAS) diblock copolymer was syn-
thesized by a two-step procedure, i.e., the synthesis of PMMA by
RAFT polymerization using CPA as a CTA, followed by the synthesis
of PMMA-b-P(NIPAAm-co-NAS) diblock copolymer via RAFT poly-
merization using PMMA as a macro-CTA. The detailed synthesis
route is illustrated in Scheme 1.
The optical absorbance of uncross-linked and cross-linked
micelle solutions (800 mg/L) at various temperatures was mea-
sured at 500 nm using a Lambda Bio40 UV–Vis spectrometer
(Perkin-Elmer). Sample cells were thermostated in a circulator bath
at different temperatures from 23 to 49 ◦C prior to the measure-
ments, and the heating rate was set at 0.1 ◦C min−1. The LCST was
defined as the temperature producing a half increase of the total
increase in optical absorbance.
2.12. Drug loading and in vitro drug release
Lyophilized PMMA-b-P(NIPAAm-co-NAS) copolymer (25 mg)
and prednisone acetate (5 mg) were dissolved in 2 mL of DMF,
which was then added into 50 mL of distilled water under stir-
ring. The mixture solution was placed into a dialysis tube and
dialyzed against 1 L of distilled water for 24 h to obtain the drug-
loaded uncross-linked micelles. The distilled water was renewed
every 8 h to remove the unloaded free drug and DMF, and then the
solution in the dialysis tube was freeze-dried. Thereafter, 20 mg of
drug-loaded uncross-linked micelle was re-dispersed in 10 mL of
distilled water. The solution was equipartitioned into two parts,
and each was placed into a dialysis tube followed by immersing
the tube into the PBS (pH = 7.4, I = 0.1) at 20 ◦C (labeled as A-1) and
45 ◦C (labeled as A-2), respectively.
Drug-loaded SCL micelles were prepared in the same way as that
of drug-loaded uncross-linked micelles mentioned above, except
for the addition of ethylenediamine (0.5 mg) as the cross-linker into
the micelle solution and further stirring for 24 h prior to dialysis.
The two samples for drug release study of SCL micelles at 20 ◦C and
45 ◦C were labeled as B-1 and B-2, respectively.
1H NMR was utilized to characterize the structure of the as-
prepared copolymers. As shown in Fig. 1A, the characteristic
peaks of MMA units (1.0–2.0 ppm (a + b, –CH2C(CH3)–, 3.6 ppm (c,
–COOCH3)) are readily visible in the 1H NMR spectrum of PMMA
using CDCl3 as a solvent. In comparison to Fig. 1A, all the character-
istic peaks of NIPAAm (5.9–7.0 (d, –NH–), 4.0 ppm (e, –CH(CH3)2),
1.0 ppm (f, –CH(CH3)2)), and NAS (2.9 ppm (g –CH2–CH2–)) units
appear in the 1H NMR spectrum of purified PMMA-b-P(NIPAAm-
co-NAS) (Fig. 1B), confirming the successful synthesis of the target
diblock copolymer. The molar ratio of NIPAAm and NAS units in
P(NIPAAm-co-NAS) block was calculated to be 15:1 based on the
integral ratio of resonance signals at 4.0 ppm (e) and 2.9 ppm (g).
SEC–MALLS analysis was used to determine the molecular
weight and distribution of the copolymers. It can be seen from
Table 1 that Mw bar of PMMA-b-P(NIPAAm-co-NAS) (48,500) is
larger than that of PMMA (6000). The SEC chromatograms in Fig. 2
demonstrate that both synthesized polymers are unimodal. Most
importantly, in comparison to the SEC trace obtained for the PMMA
precursor (Fig. 2A), there is a clear shift to the higher molecular
The volume of PBS for drug release study was 10 mL, which
was withdrawn periodically and held constant by adding 10 mL of

336
C. Chang et al. / International Journal of Pharmaceutics 420 (2011) 333–340
Scheme 1. Synthesis of PMMA-b-P(NIPAAm-co-NAS) diblock copolymer.
Fig. 1. 1H NMR spectra of (A) PMMA, (B) PMMA-b-P(NIPAAm-co-NAS), and (C) lyophilized SCL micelles in CDCl3.
weight for the PMMA-b-P(NIPAAm-co-NAS) (Fig. 2B) diblock
copolymer, indicating a high reactivity of PMMA as a macro-CTA
and a successful preparation of the target PMMA-b-P(NIPAAm-co-
NAS) diblock copolymers by consecutive RAFT techniques.
The degree of polymerization (DP) of MMA units was deter-
mined to be 60, based on the molecular weight of PMMA block
(6000). Thereafter, the DP of NIPAAm and NAS units was calcu-
lated to be 330 and 22 respectively, based on the molar ratio
(15:1) obtained from 1H NMR analysis and the molecular weight
of P(NIPAAm-co-NAS) block (48,500). Thus the diblock copolymer
was labeled as PMMA60-b-P(NIPAAm330-co-NAS22). It should be
pointed out that the actual composition of the copolymer can be
calculated based on either the integrity ratio in the NMR spectra
or the absolute molecular weight determined by SEC–MALLS. Both
methods were used in our research, however, the results obtained
from molecular weight were closer to the molar feed ratio of the
copolymer, therefore we, herein, only reported the calculation of
the real composition using the data from molecular weight. The
same method has been used in our recent publication (Chang et al.,
2011) as well.
3.2. SCL micelle formation
In this study, SCL micelles were designed and prepared to
enhance the micellar stability and increase the drug loading capac-
ity as well as achieve the sustained drug release. Uncross-linked
micelles were fabricated simultaneously to make a control. The
schematic illustration of SCL micelle formation and proposed
thermo-induced drug release behavior from SCL micelle are pre-
sented in Fig. 3.
1H NMR spectrum of lyophilized SCL micelle in CDCl3 (Fig. 1C)
was employed to confirm the occurrence of cross-linking reaction
in the micellar shell. Upon comparison of part B and C in Fig. 1,
we observe the significant attenuation of characteristic signal (g)
of NAS units, which is feasibly attributed to the transformation of
Fig. 2. SEC traces of (A) PMMA and (B) PMMA-b-P (NIPAAm-co-NAS).

C. Chang et al. / International Journal of Pharmaceutics 420 (2011) 333–340
337
Fig. 5. Thermo-sensitive behavior of aqueous solution of (A) uncross-linked micelles
and (B) SCL micelles.
results were also reported in our previous studies (Chang et al.,
2009; Wei et al., 2008b).
3.3. Thermo-responsive properties of uncross-linked and
cross-linked micelles
Fig. 3. Schematic illustration of the formation of SCL micelles and temperature
controlled drug release from SCL micelles in aqueous solution.
The optical absorbance of uncross-linked micelle and SCL
micelle solution as a function of temperature was examined to con-
firm their thermo-responsive properties. As presented in Fig. 5,
the uncross-linked PMMA-b-P(NIPAAm-co-NAS) micelle under-
goes a change in the thermoresponsive P(NIPAAm-co-NAS) shell
at a temperature around 31 ◦C. Generally, in the case of a ran-
dom copolymer of NIPAAm with hydrophobic comonomer, the
LCST shifts to a lower temperature because incorporation of the
hydrophobic comonomer favors chain aggregation, resulting in a
decrease of the LCST (Polozova and Winnik, 1999; Koga et al.,
2001). Since NAS is hydrophobic, the P(NIPAAm-co-NAS) copoly-
mer exhibits a lower LCST than that (33 ◦C) of the PNIPAAm
homopolymer. Additionally, it can be seen from Fig. 5 that the
response rate of the LCST behavior for PMMA-b-P(NIPAAm-co-
NAS) micelle remains fast, indicating that the introduction of
an appropriate amount of NAS comonomer into the PNIPAAm
chain as the cross-linking sites results in P(NIPAAm-co-NAS)
copolymer with unaltered response rate, albeit a small decrease
in the LCST value with respect to the PNIPAAm homopoly-
mer.
activated ester group in NAS unit to the amide linkage after cross-
linking by ethylenediamine.
The lyophilized SCL micelles were further dissolved in organic
solvent to prove sufficient cross-linking. Fig. 4A displays the TEM
image obtained for the SCL micelles in DMF, in which the particles
exhibit well-defined spherical shape with diameter ranging from
90 to 140 nm. The results convincingly show that the cross-linked
structure of the micelle remains intact after transfer into organic
solvent, in which the assembly would be destroyed. It turns out that
cross-linking of micellar shell is effective and the micellar structure
is locked by cross-linking of the hydrophilic shell layer. Dynamic
light scattering (DLS) reveals that the average size of SCL micelles in
DMF is 255 nm (Fig. 4B). The difference between the sizes measured
by DLS and visualized by TEM is due to the fact that the dimension
determined by the DLS is based on the swollen micelles in solution,
while the size observed by TEM is for the dried particles. Similar
Fig. 4. TEM micropicture (A) and size distribution (B) of SCL micelles in DMF.

338
C. Chang et al. / International Journal of Pharmaceutics 420 (2011) 333–340
Fig. 6. TEM micropictures and size distributions of (A and D) uncross-linked micelles at 20 ◦C, SCL micelles at (B and E) 20 ◦C and (C and F) 40 ◦C.
However, a significantly increased LCST at 40.8 ◦C is recorded
temperature alteration is coincident with the TEM observation pre-
sented in Fig. 6.
for the SCL micelles, which feasibly results from the following
two reasons, 1) due to the incorporation of ethylenediamine as
the cross-linker into the micellar structure, the neighboring ther-
mosensitive P(NIPAAm-co-NAS) chains in the shell are diluted and
interrupted; as a result, the temperature sensitivity of the shell is
a little weakened, namely, from Fig. 5 it is apparent that the tem-
perature response rate of the SCL micelle becomes slightly slower
as compared with that of the uncross-linked micelle, and the rela-
tively retarded response rate accounts for an elevated LCST for the
SCL micelle; 2) the hydrophobic activated ester group of NAS con-
verts to the hydrophilic amide linkage after the formation of SCL
micelles, leading to the increase of LCST as well.
Variation of morphology and dimension at different tempera-
tures were investigated to demonstrate the temperature sensitivity
of uncross-linked and cross-linked micelles. As presented in Fig. 6,
both uncross-linked and cross-linked micelles are well-dispersed
with regularly spherical shape at 20 ◦C, and their diameter is
estimated to be 80–130 nm and 110–180 nm, indicating that cross-
linking of micellar shell has no obvious effect on the morphology
of particles. Instead of deformation and aggregation, the shrink-
age of SCL micelles takes place at 40 ◦C, leading to the observation
of smaller nanoparticles with 70–130 nm in diameter (Fig. 6).
Although the SCL micelles tend to form aggregates via hydrophobic
interactions at the elevated temperature, the existence of cross-
linked structure in micellar shell prevents such tendency and
stabilizes the micelles, as a result, SCL micelles can maintain the
spherical shape even at the high temperature. The average parti-
cle size observed by TEM is in close agreement with the results
determined by particle-size analyser as depicted in Fig. 6.
3.4. In vitro drug loading and drug release
Prednisone acetate (molecular weight: 400.47, melting point:
235–242 ◦C) is an anti-inflammatory drug with rather low solubil-
ity in aqueous phase. It can be easily dissolved in many organic
solvents, such as DMF and CHCl3 (Wei et al., 2006a). In order to
improve its solubility in aqueous medium and to enhance its thera-
peutic efficiency, prednisone acetate was chosen as a model drug to
be encapsulated into the hydrophobic cores of the micelles in this
study. The drug-loaded uncross-linked and cross-linked micelles
were both prepared by a classical dialysis technique to investigate
the difference of their drug release characteristics. The EE and DL
were calculated to be 30.7% and 5.9% for uncross-linked micelles,
and 27.3% and 5.5% for SCL micelles, respectively.
As shown in Fig. 8, the drug release profiles of uncross-
linked and cross-linked micelles are close to each other at 20 ◦C,
The average size of uncross-linked and cross-linked micelles in
aqueous media was further measured as a function of tempera-
ture by DLS. As shown in Fig. 7, the average size of uncross-linked
micelles decreases obviously from 25 to 37 ◦C, suggesting the
shrinkage of micellar shell resulting from the collapse of P(NIPAAm-
co-NAS) chains. In the case of SCL micelles, slower and steadier
decrease of size is observed over the whole recorded temperature
range, confirming the apparent stability of cross-linked structure
even at high temperatures. The change of micelle diameter against
Fig. 7. The average sizes of (A) uncross-linked and (B) SCL micelles against temper-
ature change from 25 to 37 ◦C.

C. Chang et al. / International Journal of Pharmaceutics 420 (2011) 333–340
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uncross-linked micelles at (A-1) 20 ◦C and (A-2) 45 ◦C, SCL micelles at (B-1) 20 ◦
and (B-2) 45 ◦C.
C
i.e., the cumulative drug release is 36.1% and 32.9% within
120 h, respectively, indicating that cross-linking affects little the
release behaviors at low temperature. However, when tempera-
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release than SCL micelles, which seems plausibly attributed to the
deformation and aggregation of uncross-linked micelles at high
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4. Conclusion
Thermo-responsive diblock copolymer PMMA-b-P(NIPAAm-co-
NAS) was synthesized by successive RAFT polymerization and
SCL micelles were further fabricated using ethylenediamine as a
di-functional cross-linker. Due to the cross-linked structure, the
resultant SCL micelles were able to maintain well-defined spher-
ical shape in organic solvent as well as at high temperature.
Most importantly, the SCL micelles exhibited more sustained drug
release behavior than uncross-linked micelles at high temperature.
Based on the results, the SCL micelles developed herein will have
great potential as novel smart carriers for controlled drug release.
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Acknowledgements
The authors are grateful to the financial support from the
Ministry of Science and Technology of China (2011CB606202,
2009CB930300), Trans (New)-Century Training Programme Foun-
dation for the Talents from the Ministry of Education of
China and Natural Science Foundation of Hubei Province, China
(2009CDA024).
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