Biodegradable thermo-sensitive nanoparticles from poly(L-lactic acid)/poly(ethylene glycol) alternating multi-block copolymer for potential anti-cancer drug carrier

Article abstract of DOI:10.1016/j.ejps.2005.08.012

In order to produce biodegradable thermo-sensitive nanoparticles, alternating multi-block copolymers (MBC) were synthesized by coupling dicarboxylated poly(ethylene glycol) (PEG; M_w 2000) with poly(l-lactic acid) (PLLA)/PEG/PLLA triblock copolymers. Three different multi-block copolymers were synthesized by varying PLLA molecular weight (800 (MBC1), 1600 (MBC2), and 2800 (MBC3)). The MBC formed self-assembled nanoparticles with a unimodal size distribution during a dialysis process. The nanoparticles (NP) had a spherical shape with a size range of 90-330 nm in diameter and critical aggregation concentrations in a range of 5.6-12.6 μg/mL, depending on PLLA length in MBC. The thermo-sensitivity of MBC NP was monitored by the changes in particle size and interior structure as a function of temperature. The particle size slightly decreased as increasing temperature from 37 to 42°C. The interior structure of the NP responded to temperature by altering microviscosity. The microviscosity, measured by the anisotropy (r value) of a fluorescence probe, of MBC1 NP significantly changed with increasing temperature (r = 0.187 at 25°C and 0.216 at 42°C), while MBC2 and MBC3 showed negligible changes in the microviscosity. This indicates that the temperature-dependent interior structure of the NP relied on the portion of PLLA in MBC. The thermo-sensitivity affected to the drug release behavior and cell cytotoxicity. At 42°C, doxorubicin (DOX) loaded MBC1 NP showed enhanced cytotoxicity (～20 times) against Lewis Lung Carcinoma (LLC) cells when compared to that at 37°C.

Full text of DOI:10.1016/j.ejps.2005.08.012

european journal of pharmaceutical sciences 2 7 ( 2 0 0 6 ) 115–122
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ejps
Biodegradable thermo-sensitive nanoparticles from
poly(L-lactic acid)/poly(ethylene glycol) alternating
multi-block copolymer for potential anti-cancer
drug carrier
a
b
c
d,∗
Kun Na , Kwang Hee Lee , Don Haeng Lee , You Han Bae
a
Division of Biotechnology, The Catholic University of Korea, San 43-1 Yokkok 2-dong, Wonmi-gu, Bucheon 420-743, South Korea
School of Chemical Science and Engineering, Inha University, Incheon 402-751, South Korea
Department of Internal Medicine, Inha University, Incheon 402-751, South Korea
b
c
d
Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, 421 Wakara Way, Suite 315,
UT 84108, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to produce biodegradable thermo-sensitive nanoparticles, alternating multi-block
copolymers (MBC) were synthesized by coupling dicarboxylated poly(ethylene glycol) (PEG;
Mw 2000) with poly(l-lactic acid) (PLLA)/PEG/PLLA triblock copolymers. Three different multi-
block copolymers were synthesized by varying PLLA molecular weight (800 (MBC1), 1600
(MBC2), and 2800 (MBC3)). The MBC formed self-assembled nanoparticles with a unimodal
size distribution during a dialysis process. The nanoparticles (NP) had a spherical shape
with a size range of 90–330 nm in diameter and critical aggregation concentrations in a
range of 5.6–12.6 ␮g/mL, depending on PLLA length in MBC. The thermo-sensitivity of MBC
NP was monitored by the changes in particle size and interior structure as a function of
Received 10 May 2005
Received in revised form 19 August
2005
Accepted 24 August 2005
Available online 25 October 2005
Keywords:
Biodegradable polymer
Alternating multi-block copolymers
PEG
◦
temperature. The particle size slightly decreased as increasing temperature from 37 to 42 C.
The interior structure of the NP responded to temperature by altering microviscosity. The
PLLA
microviscosity, measured by the anisotropy (r value) of a fluorescence probe, of MBC1 NP
◦
◦
Thermo-sensitivity
Enhanced drug release
Cytotoxicity
significantly changed with increasing temperature (r = 0.187 at 25 C and 0.216 at 42 C), while
MBC2 and MBC3 showed negligible changes in the microviscosity. This indicates that the
temperature-dependent interior structure of the NP relied on the portion of PLLA in MBC.
◦
The thermo-sensitivity affected to the drug release behavior and cell cytotoxicity. At 42 C,
doxorubicin (DOX) loaded MBC1 NP showed enhanced cytotoxicity (∼20 times) against Lewis
◦
Lung Carcinoma (LLC) cells when compared to that at 37 C.
©
2005 Elsevier B.V. All rights reserved.
1
.
Introduction
(Nishikawa et al., 1996; Na et al., 2003a). The dispersed
hydrophobic domains can be a depot of a water-insoluble
drug and the sea matrix works as a surrounding barrier,
inhibiting the interactions of hydrophobic domains with bio-
components. Recently, new stimuli-sensitive polymers have
Self-assembled nanoparticles (NP) prepared from amphiphilic
polymers form dispersed multiple hydrophobic island
domains in
a hydrophilic zone (sea domain) in a NP
∗
Corresponding author. Tel.: +1 801 585 1518; fax: +1 801 585 3614.
E-mail address: you.bae@m.cc.utah.edu (Y.H. Bae).
0928-0987/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2005.08.012

1
16
european journal of pharmaceutical sciences 2 7 ( 2 0 0 6 ) 115–122
been designed by advanced synthesis technology (Na and
Bae, 2002; Lee et al., 2003) and self-assembled NP prepared
from such polymers have been investigated as an anti-cancer
drug carrier (Lee et al., 2005a,b). Their physical properties,
such as swelling, particle disruption, and/or aggregation,
were altered in response to environmental changes (Na et
al., 2003b, 2004). Such features provide a concept to develop
a better carrier system from which drug release can be
triggered at a solid tumor site, along with altered cellular
interactions.
ature effect on in vitro release of doxorubicin from MBC NP
and on the cytotoxicity against Lewis Lung Carcinoma (LLC)
cells.
2.
Materials and methods
2.1.
Materials
Succinic anhydride, N-dimethyl aminopyridine (DMAP), 1,6-
diphenyl-1,3,5-hexatriene (DPH), dicyclohexyl carbodiimide
(DCC), triethylamine, and stannous octoate were purchased
from Sigma–Aldrich (St. Louis, MO, USA). l-Lactide was puri-
fied by recrystallization from ethyl acetate. PEG (Mw 2000)
was purified by dissolving in methylene chloride, precipitated
Thermo-sensitive polymeric micelles having a hydropho-
bic core and a thermo-sensitive shell for drug delivery was
reported by Okano and co-workers (Chung et al., 1999,
2
000; Kikuchi and Okano, 2002). The shell of the micelles
changed from its hydrophilic nature of fully extended N-
isopropylacrylamide copolymers at body temperature to col-
lapsed hydrophobic polymer at a hyperthermic condition of
◦
diethyl ether and then dried in vacuum at 80 C for 12 h. Methy-
lene chloride was dried over CaH2 and distilled. Doxorubicin
(DOX) was supplied from Aldrich. All other chemicals used
were of reagent grade.
◦
4
2 C. Elevated temperature led to a micelle aggregation and
an enhanced release rate (Chung et al., 1999). This behavior
may produce a synergistic effect of chemotherapy with hyper-
thermic treatment of solid tumors. Soga et al. (2004, 2005)
reported on a new class of biodegradable and thermo-sensitive
polymers, poly(N-(2-hydroxypropyl) methacrylamide lactate).
The polymer showed a lower critical solution tempera-
ture (LCST) in water and the cloud point (CP) was well
2.2.
Synthesis of PLA/PEG/PLA triblock copolymer
PEG was dried by azeotropic distillation in three-necked
flask with magnetic stirring under nitrogen-purged condition.
◦
Then, the flask was immersed in an oil bath at 60 C. A pre-
◦
controlled between 10 and 65 C by the length of lac-
determined amount of l-lactide (lactide/PEG mole ratio in the
tate side group and copolymer composition (Soga et al.,
range from 0.25 to 2) and stannous octoate as a catalyst were
added. The reaction temperature was raised to 110 C and
◦
2
5
004). The polymer formed polymeric micelles of around
0 nm in diameter with a dense core and a hydrophilic
maintained constant for 12 h. The resulting product was pre-
◦
shell.
In this study, poly(ethylene glycol) (PEG)/poly(l-lactic acid)
PLLA) (PEG/PLLA) alternating multi-block copolymers (MBC)
cipitated by diethyl ether at −10 C. The precipitate was filtered
out and dried over P2O5 under reduced pressure (Huh and Bae,
1999; Bae et al., 2000).
(
were used to prepare thermo-sensitive NP. Water-insoluble
MBC based on PEG and PLLA was firstly prepared by the
ring-opening copolymerization of l-lactide/ethylene oxide
using various catalysts including isobutylalumioxane by
Chen et al. (1997). MBC with different block lengths and
molecular weights were successfully synthesized by the
control of reaction conditions, such as temperature, time,
solvent, and catalyst. Bae and co-workers also synthe-
sized PEG/PLLA alternating MBC from the polycondensation
reaction between dicarboxy PLLA and dihydroxy PEG in
the presence of dicyclohexyl carbodiimide and N-dimethyl
aminopyridine as catalysts (Huh and Bae, 1999; Bae et al.,
2.3.
Dicarboxylation of PEG
PEG (4 mmol), succinic anhydride (10 mmol), DMAP (8 mmol),
and triethylamine (8 mmol) were dissolved in dioxane and left
overnight at room temperature. The dioxane was evaporated
in vacuum. The residue was taken up in CCl4, filtered, and
precipitated by diethyl ether.
2.4.
Polycondensation reaction
PEG–COOH (5 mmol) and equimolar PLLA/PEG/PLLA triblock
copolymer were dissolved in anhydrous methylene chloride.
DMAP was added to the solution as a catalyst (1.3 mmol)
and DCC (13 mmol) was added as a coupling agent. The
reaction flask was kept in a dry nitrogen environment at
room temperature while stirring. After 10 min, a white dicy-
clohexylurea (DCU) precipitate formed as a reaction by-
product. Reaction conditions were maintained for 12 h. The
precipitated DCU was filtered off. The filtrate was concen-
trated under reduced pressure and then precipitated by
pouring into a large excess amount of cold diethyl ether
with vigorous stirring. The precipitate was again dissolved
in methylene chloride and re-precipitate was again dis-
solved in methylene chloride and precipitated in the mix-
ture (9:1 in volume fraction) of diethyl ether and methanol
for purification. Various MBCs with different compositions
were prepared using the same procedure. The synthetic
2
000). The synthesized block copolymers have two different
features depending on PLLA length; the block copolymer with
low PLLA length was water-soluble and clouded at a critical
temperature which is associated with a lower critical solution
temperature phenomenon. However, the polymers with a
high PLLA length swelled in water and their optical trans-
parency was influenced by temperature. This observation
can be attributed to an enhanced hydrophobic interaction
between PLLA segments in the polymer chains at elevated
temperatures.
Here, we synthesized water-insoluble PEG/PLLA alternat-
ing multi-block copolymers (PLLA length > 800 Da) using a
modified method from our previous method, and studied
their physicochemical properties, such as particle size, crit-
ical aggregation concentration (CAC), and microviscosity as a
function of temperature. In addition, we investigated temper-

european journal of pharmaceutical sciences 2 7 ( 2 0 0 6 ) 115–122
117
Scheme 1 – Synthesis scheme of PEG/PLLA alternative multi-block copolymer.
routes for PLLA/PEG/PLLA triblock copolymers and coupling
2.7.
Measurement of fluorescence spectroscopy
reaction for PLLA/PEG alternating MBC are illustrated in
Scheme 1.
(pyrene)
−
2
Stock solutions of pyrene (6.0 × 10 M) were prepared in ace-
◦
2
.5.
Preparation of nanoparticles from PEG/PLLA
tone and stored 5 C until used. For the measurement of
alternating multi-block copolymers
steady-state fluorescence spectra, the pyrene solution in ace-
tone was added to distilled water to give a pyrene concen-
−
7
Self-assembled NP were prepared by dialysis method which
produced small particles in size with a narrow size distribu-
tion. A PEG/PLLA alternating MBC (50 mg) was dissolved in
tration of 12.0 × 10 M. The solution was then distilled under
◦
vacuum at 60 C for 1 h to remove acetone from the solution.
The acetone-free pyrene solution was mixed together with
solutions of MBC NP of which the concentration ranged from
3
0 mL of dimethylsulfoxide (DMSO). The solution was stirred
at room temperature and dialyzed against distilled water for
days using a dialysis tube (molecule cut-off 1000) (distilled
−
4
1 × 10
to 1.0 mg/mL. The final concentration of pyrene in
−
6
2
each sample solution was 1.0 × 10 M, which is nearly equal
◦
water was exchanged at intervals of 3 h during the first 24 h).
The solution was filtered with a 0.45-␮m filter to remove pre-
cipitated polymer.
to its solubility in water at 22 C.
2.8.
The measurement of anisotropy value for DPH in
the MBC nanoparticles
2
.6.
Dynamic light scattering (DLS) measurements
−
6
The anisotropy value of DPH (2.1 × 10 M) in the self-
assembled NP was measured by l-format geometry of detec-
tion. Fluorescence anisotropy (r) was calculated from the fol-
lowing relationship:
Dynamic light scattering experiment was performed with an
argon ion laser system (DLS, Malvern Instruments Ltd. Series
4
700) tuned at a wavelength of 488 nm. Each sample was
filtrated through a 0.45-␮m filter directly into a precleaned
0-mm-diameter cylindrical cell. The intensity autocorrela-
s
s
1
(IVV − I ) − G(IVH − I
)
VV
VH
s
r =
(1)
◦
◦
s
tion was measured at a scattering angle (ꢀ) of 90 at 25 C.
The sample concentration was kept at 1 mg/mL. DLS mea-
surements were performed over a time period sufficient to
reach equilibrium, with no evidence of change in size with
time.
(IVV − I ) + 2G(IVH − I
)
VV
VH
where Is is the contribution of scattered light from a sample
solution in the absence of DPH, G (=IVH/IHH) an instrumental
correction factor, and IVV, IVH, IHV, and IHH refer to the resultant

1
18
european journal of pharmaceutical sciences 2 7 ( 2 0 0 6 ) 115–122
emission intensity polarized in the vertical or the horizontal
detection plans (second subindex) when excited with verti-
cally or horizontally polarized light (first subindex) (Lee et
al., 1998; Chang et al., 2000). The excitation wavelength was
3
60 nm, and the emission was measured at 430 nm.
2
.9. Drug loading and release rate measurements
An MBC (MBC1, MBC2, or MBC3) (50 mg) was dissolved in
0 mL of DMSO and 10 mg of DOX was added. The solution
1
was stirred at room temperature and dialyzed against dis-
tilled water for 1 day using a dialysis tube (molecule cut-off
1
000) (distilled water was exchanged at intervals of 3 h). The
Fig. 1 – 1H NMR spectrum of PEG/PLLA alternative
multi-block copolymer in CDCl3.
solution was filtered with a 0.45-␮m filter to remove precip-
itated polymer and drug and then freeze-dried to obtain the
product. To determine the drug loading, a freeze-dried sam-
ple was placed in DMSO, vigorously stirred for 2 h, and then
sonicated for 3 min. The resulting solution was centrifuged at
mers with different PLLA lengths (800, 1600, and 2800) were
obtained. The MBC were synthesized through the polycon-
densation of two bifunctional prepolymers, dicarboxylated
PEG and dihydroxy PLLA/PEG/PLLA triblock copolymers, in the
presence of a catalyst. This synthetic method yielded higher
molecular weight MBC with improved reproducibility than our
previous coupling method (Huh and Bae, 1999) where dicar-
boxy PLLA and dihydroxy PEG were utilized for condensation
reaction. A typical 1H NMR spectrum showed the chemical
2
0,000 × g for 30 min and the supernatant was analyzed using
a UV-spectrophotometer at 490 nm. The DOX loading content
was about 30 wt% for MBC1 nanoparticles, 37 wt% for MBC2,
and 48 wt% for MBC3. The measurement of release rates in
vitro was carried out as follows: 10 mg of drug-loaded nanopar-
ticles was suspended in 1 mL of phosphate buffered saline
(
PBS, pH 7.4), in a dialysis tube. The tube was then introduced
◦
into 15 mL of PBS and stirred at 100 rpm at 37 and 42 C. At
predetermined sampling times, whole medium was removed
and replaced by fresh PBS to maintain a sink conditions. The
amount of DOX in the solution was detected by UV at 365 nm.
Since DOX is light sensitive, all procedures were carried out in
the dark.
composition of MBC1. The peaks of CH and CH in PLLA blocks
3
appeared at 5.2 and 1.6 ppm, respectively, and the peak of main
chain methylene units in PEG blocks was shown at 3.7 ppm
(Fig. 1). The ␣-methylene protons of PEG connected with PLLA
appeared at 4.3 ppm, together with CH protons from PLLA
hydroxyl groups. The result corresponds to other reports on
PEO/PLLA block copolymers (Huh and Bae, 1999; Chen et al.,
1997). Detailed characterization results of synthesized MBCs
are summarized in Table 1.
2
.10. In vitro cytotoxicity measurement of MBC1
nanoparticles loaded with DOX
In vitro cytotoxic activity of MBC1 NP loaded with DOX was
measured using cultured Lewis Lung Carcinoma cells. Cells
MBC NP were prepared by a physical crosslinking between
hydrophobic segments, PLLA, in the polymers during a dialy-
sis process. The physical crosslinking can be accomplished by
several factors, such as hydrophobic interaction, crystalline
microdomains, and chain entanglement (Huh and Bae, 1999).
The yields of the nanoparticles after dialysis and filtration
were over 90%. Their size and size distribution in aqueous
media were measured by DLS. The particles of MBC1 had
a mean diameter below 90 nm with a unimodal size distri-
bution, while those of MBC2 and MBC3 are around 300 nm
(Fig. 2). The longer hydrophobic block length resulted in more
interactions, leading to a larger particle size. The assembling
behavior of MBC in aqueous media was monitored by using
pyrene as a fluorescence probe. At low concentrations of
MBC1 particles, changes in the total fluorescence intensity
and the shift of the (0, 0) band at 334 nm in water are negligi-
ble. As the concentration of the polymer increases, however,
an increase in the total fluorescence intensity and a red shift
of the (0, 0) band are clearly observed. Since pyrene in a
polar environment shows only a weak fluorescence intensity,
the increase in the total fluorescence intensity with the
addition of the polymer indicates that the probe is transferred
from aqueous media to the less polar microdomains of the
interior of self-assembled NP. The (0, 0) band for pyrene at
334 nm shifts to 337 nm on addition of MBC1 (Fig. 3(a)). The
4
were plated at a density of 1 × 10 cells/well and were then
exposed with free DOX, and MBC1 NP loaded with DOX for
◦
3
days at 37 and 42 C, respectively. After drug exposure,
the cells were washed three times with DMEM medium and
00 ␮L of drug-free medium without serum was then added.
5
Chemosensitivity was assessed using the tetrazolium salt
MTT (Sigma, USA) to measure the viability of the cells. The
1
00 ␮L medium containing 20 ␮L of MTT solution was added to
each well. The plates were incubated for an additional 4 h, and
then 100 ␮L of acidic isopropyl alcohol was added to each well,
and the solution was vigorously mixed to solve the reacted
dye. The absorbance of each well read on a microplate reader
at a test wavelength of 570 nm and a reference wavelength
of 630 nm.
3
.
Results and discussion
3
.1.
The physicochemical characterization of MBC
nanoparticles
Triblock copolymers were synthesized by the ring-opening
polymerization of l-lactide in the presence of dihydroxy PEG as
a macroinitiator (Rashkov et al., 1996). Three triblock copoly-

european journal of pharmaceutical sciences 2 7 ( 2 0 0 6 ) 115–122
119
Table 1 – Molecular weights, molecular weight distributions, block ratios, and critical aggregation concentrations of
poly(ethylene glycol) (PEG)/poly(l-lactic acid) (PLLA) alternating multi-block copolymer
Sample
Block length (PEG/PLLA)
Mna
Mwa
MWD
Block ratiob (PEG/PLLA)
CACc (␮g/L)
MBC1
MBC2
MBC3
2000/800
2000/1600
2000/2800
23600
29700
38100
30600
48500
47100
1.3
1.6
1.2
1.06
0.98
1.03
12.6
9.3
5.6
a
Determined by GPC (Waters LC system coupled to a Waters 410 Differential Refractometer using StyragelTM HR Series column at a flow rate
of 1 mL/min; Eluent: tetrahydrofuran (THF); molecular weight standards: polystyrene).
Calculated from 1H NMR.
b
c
Critical aggregation concentration.
critical aggregation concentration, which is the threshold
concentration of self-aggregate formation by intra- and/or
intermolecular association, was determined from the change
of the intensity ratio (I337/I334) of the pyrene in the presence
of the polymers. Fig. 3(b) shows the intensity ratios (I337/I334)
of MBC1, MBC2, and MBC3 as a function of polymer concen-
tration. The CAC values were determined from the crossover
point at low concentrations (Fig. 3(b)). The increase in PLLA
length reduced the CAC values of MBC polymers (Table 1).
3
.2.
Thermo-sensitivity of MBC nanoparticles
The synthesized MBC polymers are water-insoluble at room
temperature and formed self-assembled NP. The thermo-
sensitivity of the NP in water was tested by the measurement
of changes in particle size and microviscosity. The change in
particle size was dependent on the block composition of the
polymer. As temperature increased, all particle size gradually
◦
decreased until 42 C (Fig. 4). However, MBC1 NP increased
◦
its size considerably above 42 C, while the sizes of MBC2
and MBC3 continuously reduced. It indicates that the disin-
◦
tegration of MBC1 NP occurred above 42 C, and the thermo-
sensitivity in the system increased with decreasing the PLLA
block length in the block copolymers. The higher sensitiv-
ity of MBC1 particles at the hyperthermic condition can be
explained by the effect of the PLLA block length. When NP
−
7
Fig. 3 – Excitation spectra of pyrene (6.0 × 10 M) in
distilled water in the presence of MBC1 as a function of
polymer concentration (emission wavelength was 390 nm)
(
a) and the plot of intensity ratio I337/I334 from excitation
spectra vs. log C of MBC nanoparticles (b).
were prepared from the block copolymers, NP with longer
PLLA possesses more rigid hydrophobic inner cores, result-
ing in more stable NP with less thermo-sensitivity. This was
supported by the monitoring of interior structural change as
a function of temperature. The interior structural change of
MBC NP was investigated by the change in the microviscos-
ity by DPH as a function of temperature. The microviscosity
of inner core in the particles can be determined by measuring
the molecular anisotropy as a result of molecular rotational
diffusion. The anisotropy value for DPH increases with core
microviscosity due to restricted rotational motion. The r val-
Fig. 2 – Particle size distributions of MBC nanoparticles at
◦
2
5 C. The values were determined by DLS.

1
20
european journal of pharmaceutical sciences 2 7 ( 2 0 0 6 ) 115–122
Fig. 4 – Changes in particle sizes of MBC nanoparticles as a
function of temperature. MBC1 (᭹), MBC2 (ꢀ), and MBC3 (ꢀ)
nanoparticles (n = 3).
ues of MBC3 nanoparticles showed highest value in the tested
temperature range. It means that MBC3 NP have the most rigid
inner core because high r value implies an enhanced rigid-
ity of the inner cores of the particles. The r value of MBC1
◦
particles significantly increased from 0.187 at 25 C to 0.216
◦
at 42 C, while there was little difference in the anisotropy
of MBC2 and MBC3 particles in the same temperature range
(Fig. 5). These results support that the thermo-sensitivity of
the NP was enhanced by decreasing the PLLA length of the
polymer.
Fig. 6 – DOX release kinetics from MBC nanoparticles at
◦
◦
3
7 C (a) and 42 C (b). MBC1 (᭹), MBC2 (ꢀ), and MBC3 (ꢀ)
nanoparticles (n = 5).
3
.3.
Thermo-responsive DOX release and the
cytotoxicity of MBC1 nanoparticles
DOX release profiles from MBC1, MBC2, and MBC3 NP were
◦
monitored at 37 and 42 C and presented in Fig. 6(a and b).
◦
At 37 C, although the total amount of released DOX for 1 day
from MBC1 reached as high as 43%, there was no noticeable
difference between the nanoparticles (Fig. 6(a)). However, at
◦
4
2 C a noticeably enhanced release rate from MBC1 nanopar-
ticles was observed, while MBC2 and MBC3 NP showed slight
temperature dependency (Fig. 6(b)). After 1 day, total amount
◦
of released DOX at 42 C from MBC1 was approximately two
◦
times higher than that at 37 C. This enhanced DOX release
rate of MBC1 may be induced by the change of interior struc-
ture of the NP.
Fig. 5 – A plot of the anisotropy values for DPH vs. the
temperature of MBC nanoparticles. The excitation
wavelength was 360 nm and the emission was measured at
The in vitro cytotoxicity to LLC cells of MBC1 NP at 37 and
◦
42 C was conducted because of its highest temperature sen-
4
30 nm. MBC1 (᭹), MBC2 (ꢀ), and MBC3 (ꢀ) nanoparticles.
sitivity among tested samples in terms of microviscosity and

european journal of pharmaceutical sciences 2 7 ( 2 0 0 6 ) 115–122
121
Fig. 7 – Photographs of cells exposed to free DOX (1 ␮g/mL) (a), MBC1 nanoparticles (c) and a control experiment without
DOX (e) at 37 C, and with free DOX (1 ␮g/mL) (b), MBC1 nanoparticles (d), and a control experiment without DOX (f) at 42 C
◦
◦
for 3 days. The photographs were taken by a bright field microscope.
Fig. 8 – In vitro cytotoxicity of free DOX (a) and MBC1 nanoparticles containing DOX (b) against Lewis Lung Carcinoma cells,
◦
as a function of drug concentration at 37 and 42 C. Data represent a mean ± S.D., n = 7.

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european journal of pharmaceutical sciences 2 7 ( 2 0 0 6 ) 115–122
drug release rate. In Fig. 7, the photographs of the cells treated
Chung, J.E., Yokoyama, M., Okano, T., 2000. Inner core segment
design for delivery control of thermo-responsive polymeric
micelles. J. Control. Rel. 65, 93–103.
Chung, J.E., Yokoyama, M., Yamato, M., Aoyagi, T., Sakurai, Y.,
Okano, T., 1999. Thermo-responsive drug delivery from
polymeric micelles constructed using block copolymers of
poly(N-isopropylacrylamide) and poly(butylmethacrylate). J.
Control. Rel. 62, 115–127.
with either free DOX (Fig. 7(a and b)) or DOX loaded MBC1
◦
(
Fig. 7(c and d)) for 3 days at 37 and 42 C are shown together
with control (Fig. 7(e and f)) at each temperature. Most cells
exposed to free DOX (1 ␮g/mL) were killed due to DOX cyto-
toxicity at both temperatures. However, the cells with same
amount of DOX in MBC1 NP were observed with different
◦
shapes between two temperatures. The NP at 37 C (Fig. 7(c))
Huh, K.M., Bae, Y.H., 1999. Synthesis and characterization of
poly(ethylene glycol)/poly(l-lactic acid) alternating
multiblock copolymers. Polymer 40, 6147–6155.
◦
demonstrated low cytotoxicity against LLC cells, while at 42 C
it killed the cell almost completely like a free DOX (Fig. 7(d)).
The MTT assay was carried out for quantitative analysis in
the cytotoxicity of the NP. In case of free DOX, there was no
noticeable difference in the cytotoxicity between two temper-
atures at the DOX concentration range of 1 ng/mL to 10 ␮g/mL
Kikuchi, A., Okano, T., 2002. Intelligent thermoresponsive
polymeric stationary phases for aqueous chromatography of
biological compounds. Prog. Polym. Sci. 27, 1165–1193.
Lee, E.S., Na, K., Bae, Y.H., 2005a. Super pH-sensitive
multifunctional polymeric micelle. Nanoletters 5, 325–329.
Lee, E.S., Na, K., Bae, Y.H., 2005b. Doxorubicin loaded smart
polymeric micelles for reversal of resistant MCF-7 tumor. J.
Control. Rel. 103, 405–418.
◦
(
Fig. 8(a)). However, DOX loaded MBC1 at 42 C (50% inhibitory
concentration (IC50); about 0.5 ␮g/mL) showed approximately
◦ ◦
0 times higher cytotoxicity than that at 37 C. IC50 at 37 C
2
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was estimated to be higher than 10 ␮g/mL from Fig. 8(b). It is
interesting to note that the temperature effect on the cytotox-
icity is far more than expected because the released amount
Lee, K.Y., Jo, W.H., Kwon, I.C., Kim, Y.H., Jeong, S.Y., 1998.
Structural determination and interior polarity of
self-aggregates prepared from deoxycholic acid-modified
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◦
of DOX at 42 C for 24 h was less than twice of the amount
◦
at 37 C. One possible explanation for this observation is that
the more hydrophobic surface of the NP induced by temper-
ature may facilitate internalization of the NP in the cells as
noticed with pH-sensitive micelles (Na et al., 2003b) and other
temperature-sensitive micelles (Chung et al., 1999, 2000). The
details of temperature-induced internalization of MBC NP will
be a further subject of investigation.
4
.
Conclusion
The nanoparticles prepared from poly(ethylene glycol)/poly(l-
lactic acid) alternating multi-block copolymer were investi-
gated as an anti-cancer drug carrier which responded to
local temperatures. The sensitivity in the MBC nanoparticles
depended on PLLA length in the polymers. The temperature-
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Pharm. Sci. 18, 165–173.
induced interior structural changes of the nanoparticles at
◦
4
2 C led to accelerated DOX release. The MBC nanoparticle
showed enhanced cytotoxicity against LLC cells. The result
indicated that the thermo-sensitivity of MBC may show the
synergistic effect with local hypothermia for the treatment of
tumor.
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