Journal of Controlled Release 51 (1998) 169–178

Clonazepam release from core-shell type nanoparticles in vitro

Young-Il Jeong a , Jae-Bok Cheon a , Sung-Ho Kim b , Jae-Woon Nah c , Young-Moo Lee d ,
e f a,
Yong-Kiel Sung , Toshihiro Akaike , Chong-Su Cho *
a
Department of Polymer Engineering, Chonnam National University, Kwangju 500 -757, Korea
b
College of Pharmacy, Chosun University, Kwangju 501 -759, Korea
c
Department of Polymer Science and Engineering, Sunchon National University, Sunchon 540 -742, Korea
d
Department of Industrial Chemistry, Hanyang University, Seoul 133 -791, Korea
e
Department of Chemistry, Dongguk University, Seoul 100 -715, Korea
f
Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Midoriku, Yokohama 227, Japan

Received 27 August 1996; received in revised form 21 July 1997; accepted 6 August 1997

Abstract

Block copolymers consisting of poly(g-benzyl L-glutamate) (PBLG) as the hydrophobic block and poly(ethylene oxide)
(PEO) as the hydrophilic block were synthesized and characterized. Core-shell type nanoparticles of the block copolymers
(abbreviated as GE) were prepared by the diafiltration method. The particle size diameter obtained by dynamic light
scattering of GE-1 (PBLG content: 60.5 mol %), GE-2 (PBLG content: 40.0 mol %), GE-3 (PBLG content: 12.4 mol %)
copolymer was 309.96160.9, 251.96220.6 and 200.56177.1 nm, respectively. The shape of the nanoparticles by SEM or
TEM was almost spherical. The critical micelle concentration of the block copolymers obtained by fluorescence spectroscopy
was dependent on the chain length of hydrophobic PBLG. The micelle structure of the copolymer nanoparticle was very
stable against sodium dodecyl sulfate. Clonazepam (CZ) was loaded onto the core part of the nanoparticle as the crystalline
state. Release of CZ from the nanoparticles in vitro was dependent on the drug loading contents and PBLG chain length.
 1998 Elsevier Science B.V.

Keywords: Clonazepam release; Core / shell nanoparticles; In vitro studies

1. Introduction short blood circulation, structural fragility, lower

Novel drug delivery systems have been proposed
for effectively targeted drug delivery, such as poly-
meric micelles, surface-modified particles, liposomes
and nanoparticles [1–7]. Many problems still exist
such as biodistribution of drugs, drug solubility,
undesirable side effects, rapid clearance by re-
ticuloendothelial system (RES), thermal instability,
*Corresponding author. Tel.: 182 62 520 7078; fax: 182 62
522 5264; e-mail: chocs@orion.chonnam.ac.kr
loading efficiency, etc.
Ringsdorf et al. first reported that polymeric
micelles can be used to achieve the sustained release
of drug from the polymer-drug conjugate [1]. Re-
cently, diblock copolymers composed of poly(aspar-
tic acid) or poly(b-benzyl-L-aspartate) as the hydro-
phobic part and poly(ethylene glycol) as the hydro-
philic part were used to prepare polymeric micelles
by Kataoka et al. [8–10]. Also, Z. Hruska et al.
reported the synthesis of poly(g-benzyl L-glutamate)
(PBLG) and poly(ethylene oxide) (PEO) diblock

0168-3659 / 98 / $19.00  1998 Elsevier Science B.V. All rights reserved.

PII S0168-3659( 97 )00163-6
170 Y.-I. Jeong et al. / Journal of Controlled Release 51 (1998) 169 – 178
copolymers and micelle formation in water [11].
Diblock copolymers generally exhibit surfactant
behaviour [12] and form micelles due to am-
phiphilicity. Generally, polymeric micelles have a
hydrophobic core and a hydrophilic outer shell.
Hydrophobic segments form the inner-core of the
structure, which acts as a drug incorporation site,
especially for hydrophobic drugs. Hydrophobic drugs
may be easily physically entrapped within the inner-
core of the polymeric carriers by hydrophobic inter-
actions [13]. Hydrophilic blocks form a hydrated
outer shell which may cloak the hydrophobic core to
avoid its being quickly uptaken by the RES and more
active clearing organs such as liver, spleen, lungs
and kidneys and then increase blood circulation
times of nanoparticles. Predominant characteristics
of this system have been reported such as reduced
toxic side effects of anticancer drugs by micelle
formation with block copolymer and selective target-
ing, solubilization of hydrophobic drugs, stable
storage, long blood circulation, favourable biodistri-
bution and lower interactions with RES [14,15].
Thus, this micelle type polymeric carrier may be an
appropriate vehicle for drug delivery.
In this study, we have synthesized PBLG / PEO
diblock copolymers and core-shell type nanoparticles
were prepared by the diafiltration method [4]. PBLG
is more hydrophobic than poly(b-benzyl L-aspartate)
(PBLA) [16], and is biodegradable [17]. PEO is the
hydrophilic block, is a non-toxic and non-immuno-
genic water-soluble polymer and is known to prevent
interactions with proteins [18]. Clonazepam (CZ) as
a model drug is an anticonvulsant benzodiazepine,
which is efficacious for the treatment of panic
disorder and has considerably hydrophobic character
(water solubility ,14.66 mg drug / ml) [19,20]. Also,
we have investigated physicochemical states of their
nanoparticles and drug release from nanoparticles in
vitro. It may be expected that core-shell type
nanoparticles are applicable to the passive-targetable
drug carriers.
2. Materials and methods
2.1. Materials
PBLG / PEO (abbreviated as GE) block copoly-
mers were prepared by polymerization of g-benzyl
L-glutamate N-carboxyanhydride (g-BLG NCA)
initiated with mono amine-terminated PEO kindly
supplied by Nippon Oil and Fat Co. (M.W.512,000
g / mol) in a methylene dichloride solution by the
method described previously [10,21–23].
Clonazepam (CZ) was obtained from ROCHE,
Switzerland. Dimethylformamide (DMF) as reagent
grade was used without further purification.
2.2. Preparation of CZ loaded nanoparticles by
diafiltration method
The formation of core-shell type nanoparticles and
drug loading procedure [13] were carried out by a
diafiltration method. Briefly, 20 mg of PBLG / PEO
diblock copolymer was dissolved in 10 ml of DMF
and subsequently 20–80 mg of CZ was added. And
then the solution was stirred at room temperature and
solubilized entirely. To form core-shell type
nanoparticles and remove free drug, the solution was
dialysed using molecular cut-off 12 000 g / mol
dialysis tube against 1.0 l33 of distilled water for 3
h and then distilled water exchange at intervals of
3|4 h during 24 h. Then, the solution was freeze-
dried.
For measurement of drug loading content, freeze-
dried samples of GE nanoparticles were suspended
into methanol and vigorously stirred for 2 h and
sonicated for 15 min. The resulting solution was
centrifuged with 12 000 g for 20 min and supernatant
was taken for measurement of drug concentration
using UV spectrophotometer (Shimadzu UV-1201) at
306 nm.
2.3. Measurement of fluorescence spectroscopy
To investigate the fluorescence spectroscopy
characteristics, GE block copolymer solutions with-
out drug were prepared as follows: 20 mg of GE
block copolymer was dissolved in 10 ml of DMF and
dialysed up to 24 h as in the method described
above. Resultant solution was adjusted to the various
concentrations of block copolymers.
To estimate the critical micelle concentrations
(CMC) of block copolymers, CZ [24] or pyrene
[25–28] was used as a hydrophobic probe. CZ /
chloroform stock solution was prepared to the con-
 Y.-I. Jeong et al. / Journal of Controlled Release 51 (1998) 169 – 178
centration of 1 mg CZ / ml CHCl 3 and 0.1 ml of
stock solution was dropped very slowly to the 10 ml
of various concentrations of block copolymer solu-
tions with mixing (final concentration of CZ: 0.01
mg / ml). To remove solvent, solutions were vigor-
ously stirred for 3 h at room temperature. Emission
spectra were measured by fluorometer (Shimadzu
RF-5000) (excitation wavelength of 306 nm). Excita-
tion and emission bandwidths were 5.0 nm and 5.0
nm, respectively.
Critical micelle concentrations (CMC) of the GE
block copolymers were estimated to prove the po-
tential of micelle formation by the measurement of
fluorescence spectroscopy (JASCO FP 777 spec-
trofluorometer, Japan Spectroscopic Co. Ltd., Tokyo,
Japan) using pyrene as a probe. To obtain sample
solutions, a known amount of pyrene in acetone was
added to each of a series of 20 ml vials and the
acetone evaporated. The final concentration of
pyrene was 6.0310 27 M. 10 ml of various con-
centrations of block copolymer solutions was added
to each vial and then heated for 3 h at 658C to
equilibrate the pyrene and the micelles, and left to
cool overnight at room temperature. Emission wave-
length was 390 nm for excitation spectra. Excitation
and emission bandwidths were 3 nm and 1.5 nm,
respectively. Deoxygenation procedure was per-
formed by nitrogen gas bubbling.
2.4. Differential scanning calorimetry
measurements
The melting temperatures of CZ itself and that in
nanoparticles were measured with a PL DSC-30
(PL-Thermal Sci., UK). The measurements were
carried out in the range of 208C to 3508C under
nitrogen at a scanning rate of 108C / min.
2.5. Scanning electron microscope ( SEM) and
transmission electron microscope ( TEM)
measurements
The morphology of the nanoparticles was ob-
served using a SEM (JEOL, JSM 5400, JAPAN). A
drop of the nanoparticle suspension was placed on a
graphite surface. After freeze-drying, the sample was
coated with gold / palladium using an Ion Sputter
171
(JEOL, JFC-1100). Coating was provided at 20 mA
for 4 min. Observation was performed at 25 kV.
A drop of nanoparticles suspension in ethanol was
placed on a carbon film coated on a copper grid for
TEM. The specimen on the copper grid was not
stained. Observation was done at 80 kV in a JEOL
JEM-2000 FX P.
2.6. Dynamic light scattering ( DLS) measurements
DLS was measured with a S4700 (Malvern instru-
ments, UK) with an argon laser beam at a wave-
length of 488 nm at 208C. The scattering angle of
908 was used. A nanoparticle solution prepared by
diafiltration method was used for DLS measurement
(concentration: 0.1% w / v) and measured without
filtering.
2.7. In vitro release studies
The release experiment was carried out as fol-
lowed: CZ loaded nanoparticles and 1 ml phosphate
buffered saline (PBS, 0.15 M and pH 7.4) were put
into a dialysis tube (MWCO: 12 000) and then the
dialysis tube was introduced into vial with 10 ml
PBS with 40 mmol / , sodium dodecyl sulphate
(PBS–SDS) (sink condition) [6] and the media was
stirred at 100 rpm at 378C. At specific time intervals,
medium was taken and replaced with fresh PBS.
The concentration of the released CZ into PBS–
SDS was determined by UV spectrophotometer
(Shimadzu UV-1201) at 306 nm (´ : 10 500
M 21 cm 21 ).
3. Results and discussion
Block copolymers were prepared by polymeri-
zation of g-BLG NCA initiated by the mono amine-
terminated PEO in methylene chloride as per the
method previously reported [21]. It may be assumed
that the polymerization mechanism is the primary-
amine mechanism in which the initiator amine
undergoes a nucleophilic addition to the C-5 carbox-
yl group of the NCA, as reported by Goodman et al.
[29]. The copolymer composition and the molecular
weight were estimated from peak intensities of the
172 Y.-I. Jeong et al. / Journal of Controlled Release 51 (1998) 169 – 178

methylene proton signal of the PBLG block and the of the hydrophobic environments. A plot of I370 /I348
methylene proton signal of the PEO block in the 1 H vs. log c in Fig. 1(b) was obtained from data of Fig.
NMR spectrum. Assuming that all the amine groups 1(a) and showed small vibrational change at low
of PEO participate in the polymerization, the num- concentration, extreme and logarithmic increase at
ber-average molecular weights, Mn of the copolymer high concentration. Then the crossover region be-
can be calculated from the copolymer composition tween small vibrational changes in low concentration
and the molecular weight of PEO chains. Com- extreme and logarithmic increase region were at-
positions and molecular weights of the copolymers tained to estimate CMC values of GE block co-
are listed in Table 1. polymer. The CMC of this polymer, as determined
Since core-shell type nanoparticles based on block by fluorescence probe technique using CZ as a
copolymer exhibit micellar behaviour as reported hydrophobic probe, is 7.42310 27 mol. The CMC
elsewhere [13,28,30], fluorescence spectroscopy was values for the core-shell type nanoparticles according
used to examine the CMC of the CZ-loaded to the composition of the copolymers are shown in
nanoparticles. The characteristic peak of emission Table 2. The estimation values of CMC for the
spectra of CZ itself in aqueous solutions appeared at polymers decreased with increasing PBLG chain
ca. 348 nm and a broad band between 400 nm and length as the hydrophobic part in the block co-
500 nm, probably due to the existence of an excimer
of CZ (not shown).
The emission spectrum of CZ with various con-
centrations of GE-3 for the observations of associa-
tion behaviour GE block copolymer and CZ in the
aqueous system are shown in Fig. 1(a). It was found
that the total fluorescence intensity increased and
another peak at ca. 370 nm appeared above the
concentration of 0.01 mg / ml for the GE-3, reflecting
a change in the vibrational structure of CZ monomer
emission upon association of GE-3. It is thought that
CZ was preferentially solubilized into the nanoparti-
cles composed of core-shell structure when CZ and
PBLG were introduced into aqueous phase from a
good solvent for both species. The core-shell type
nanoparticles were formed as the solvent is removed
[13]. The PBLG acts as a reservoir to incorporate CZ
for its hydrophobic core. The ratio of I370 /I348 is
sensitive to the polarity of the environment and its
plot with the polymer concentration enables us to
detect the concentration of the onset of the nanoparti-
cle formation, the CMC, that implies the formation

Table 1
Characterization of PBLG / PEO diblock copolymer
Sample Contents of monomer-
ic units in mol % Mn
PBLG PEO
Fig. 1. Fluorescence emission spectra of CZ / GE-3 against con-
GE-1 60.5 39.5 103 700
centration of GE-3 in distilled water (excitation wavelength: 306
GE-2 40.0 60.0 51,800
nm) (a) and plots of the intensity ratio I370 /I348 from CZ emission
GE-3 12.4 87.6 20,400
spectra vs. log c of the GE-3 copolymer in distilled water (b).
M.W. of PEO: 12,000.
 Y.-I. Jeong et al. / Journal of Controlled Release 51 (1998) 169 – 178 173

Table 2
The estimation of CMC values for the diblock copolymer
Sample PBLG content CMC (mol)
in mol % CZ Pyrene
28
GE-1 60.5 4.62310 1.95310 28
GE-2 40.0 2.73310 27 4.18310 28
GE-3 12.4 7.42310 27 1.32310 27

polymers. It is thought that the nanoparticle with a

core-shell structure can be easily formed with an
increase of hydrophobic components.
The possibility of preferential partitioning of CZ
into hydrophobic domains is not clear. Therefore, we
attempted to compare the micropolarities between
CZ and pyrene in the nanoparticles. It has already
been reported (use of the hydrophobic probe pyrene),
the emission characteristics of which change with the
onset of association of the block copolymer [26].
Fig. 2(a) shows the fluorescence spectra of pyrene
in the various concentration of GE-3 block copoly-
mers. This result indicated that red shift of pyrene in
the excitation spectrum was observed with an in-
crease of concentration of GE-3 block copolymer. It
is thought that pyrene was preferentially partitioned
into the PBLG segment as the hydrophobic core as
the similar tendency of polystyrene (PS) / PEO dib-
lock copolymer reported by Wilhelm et al. [26]. The
intensity ratio of I339 /I333 against polymer concen-
tration in the pyrene excitation spectra was plotted in
Fig. 2(b). This result indicated that the ratio is almost
flat at the low concentration extreme and rapidly
increases at the high concentration. Log c values
shown as arrows in Fig. 2(b) are taken from the
intersection of the tangent to the curve at the
inflection with the horizontal tangent through the
points at low polymer concentrations. We define the Fig. 2. Fluorescence excitation spectra of pyrene / GE-3 against
value of 0.0027 g / l as the CMC of the GE-3 block concentration of GE-3 in distilled water (emission wavelength:
copolymer. The CMC values for the block copoly- 390.0 nm) (a) and plots of the intensity ratio I339 /I333 from pyrene
excitation spectra vs. log c of the GE-3 copolymer in distilled
mers obtained by CZ and pyrene were shown in water (b).
Table 2. As shown in Table 2, the tendency of CMC
decrease with an increase of PBLG chain length is
same for both. But all CMC values against PBLG 3.1. Morphology and size distribution of GE
content obtained with CZ were larger than those nanoparticles
obtained with pyrene. It is thought that higher water
solubility of CZ (14.66 mg / ml) than pyrene (0.12 Fig. 3(a) shows SEM photographs of GE-3. The
mg / ml) induces less sensitivity to the formation of shape of the nanoparticles was mostly spherical and
hydrophobic environment. the sizes were ranged about 50 nm|500 nm in
174 Y.-I. Jeong et al. / Journal of Controlled Release 51 (1998) 169 – 178
Fig. 3. Scanning electron microscopy photograph (a) and transmis-
sion electron microscopy photograph (b) of GE-3 nanoparticles.
diameter. TEM observations of GE-3 nanoparticles
was also shown in Fig. 3(b). Their appearances were
spherical shapes and ranged about 50 nm|200 nm
which is similar to the SEM results. By close
observation of TEM photographs, interestingly, it
was found that bright and dark images were seen in
the nanoparticles, demonstrating that the block co-
polymer showed core-shell type nanoparticles. It
seems that the dark part should be assigned to the
core of the hydrophobic PBLG and the bright part
should be assigned to the shell of the hydrophilic
PEO. It can be said that PBLG-PEO diblock co-
polymeric nanoparticles prepared by diafiltration
Fig. 4. Particle size distribution of GE-3 nanoparticles by dynamic
light scattering.
method has potential of self-assembly to hydropho-
bic-core and hydrophilic-outershell structure.
Fig. 4 shows particle size distribution of GE-3
nanoparticle by dynamic light scattering (DLS). In a
different way with SEM and TEM observations, the
results of DLS of GE-3 copolymer nanoparticles
revealed a relatively large diameter, i.e. particle size
of GE-3 copolymer was 200.56177.1 nm in number
average. The particle size distribution against PBLG
chain length are shown in Table 3. The longer the
PBLG chain length, the bigger the particle size.
These results indicated that the particle sizes were
dependent on the PBLG chain length. In a different
way with number and volume average, particle size
of intensity average was exhibited as trimodal of
GE-1 and bimodal of GE-2 and GE-3, respectively,
although a relatively small portion of larger particles
were present (as shown in Table 3). Usually, the
nanoparticles as polymeric micelles should be small
and have narrow size distribution [31]. But at this
moment, the nature of the larger particle size is still
unclear. We consider several possibilities, (1) the
individual micelles are further associated by the
hydrophobic–hydrophobic interactions between ex-
posed cores [32], (2) multilayer structure with
alternating concentric layers of solvated and undis-
solved blocks [31], (3) secondary aggregates with
time due to the weak steric stabilization of PEO
chains [33], (4) a mixture of micelles and a consider-
able amount of secondary aggregates [34].
 Y.-I. Jeong et al. / Journal of Controlled Release 51 (1998) 169 – 178 175

Table 3
Particle size distribution of nanoparticles against PBLG chain length
Sample PBLG content Particle size (nm)
in mol % Number average Volume average Intensity average
GE-1 60.5 309.96160.9 362.66155.5 231.5625.7 (41.9%)
370.86199.9 (50.6%)
724.36155.3 (7.5%)
GE-2 40.0 251.96220.6 298.46149.7 242.36183.8 (90.6%)
423.8651.4 (9.4%)
GE-3 12.4 200.56177.1 277.06202.2 219.66198.0 (89.5%)
433.26146.8 (10.5%)

3.2. Drug release study in vitro days). Then, we decided to add SDS into the release
medium. But the effect of SDS on the nanoparticle
First of all, effect of sodium dodecyl sulphate structure during the release study should be checked
(SDS) on the stability of block copolymer micelles in more detail.
was tested before in vitro release study since SDS The CZ release from the nanoparticles against
generally affected the micelle stability [13,30]. When
SDS was added to the aqueous solution of CZ / GE-3,
fluorescence intensity at ca. 370 nm as shown in Fig.
5 decreased, indicating the partial disruption of the
micelles of the GE-3. In contrast, fluorescence
intensity of the frozen CZ disappeared with the
addition of SDS, indicating strong interactions be-
tween CZ and SDS.
CZ release from the nanoparticles was performed
in vitro without adding SDS. It was found that CZ
was released very slowly (About 40 wt % of CZ
from GE-3 nanoparticles was released within 70

Fig. 6. CZ release from nanoparticles against PBLG chain length

Fig. 5. Fluorescence emission spectra of CZ and CZ / GE-3 with (drug loading contents: GE-1: 37.8 wt %, GE-2: 35.2 wt %, GE-3:
and without SDS (20 mg / ml). Excitation wavelength: 306 nm. 32.8 wt %) (a) and drug loading content in GE-3 nanoparticles (b).
176 Y.-I. Jeong et al. / Journal of Controlled Release 51 (1998) 169 – 178
Fig. 7. Differential scanning calorimetry thermograms of GE-3 nanoparticles (CZ loading content: 32.8 wt %).
PBLG chain length was shown in Fig. 6(a). It was
found that longer PBLG chain length, the slower the
drug release. These results may be due to hydro-
phobic interaction between hydrophobic domain of
polymer and drug. Also, the more hydrophobic
domain of polymer should lead to the stronger
hydrophobic interaction.
Fig. 6(b) shows CZ release from nanoparticles
against drug loading content. This results indicated
that the more the drug content, the slower the drug
release. At lower loading, CZ is present as a
dispersed state in the core segment whereas a
crystallization of drug in the PBLG core occurs at
higher loading. These results were supported by
thermal analysis as shown in Fig. 7. CZ itself melts
at 238.98C whereas melting point of CZ for the
CZ-loaded nanoparticles is 222.98C, indicating that
parts of the entrapped drug exist in crystalline form.
As reported elsewhere [7], the crystallized drug
should be more slowly dissolved and diffused into
the outer aqueous phase. As shown in Fig. 6(a) and
(b), CZ release rate was observed almost as zero-
order, indicating that control of drug release kinetics
can be achieved by hydrophobic content of diblock
copolymer and drug loading content.
In conclusion, the diblock copolymer based on
PBLG as the hydrophobic part and PEO as the
hydrophilic one was synthesized and characterized.
Through the use of fluorescent probes, it suggests
that PBLG / PEO diblock copolymers may associate
in water to form core-shell type nanoparticles and the
CMC values of the block copolymers decreased with
increasing PBLG chain length in the block copoly-
mers. Nanoparticles are spherically shaped from the
results of SEM and TEM observations. From DLS
study, size of nanoparticles of GE-3 copolymer was
200.56177.1 nm. CZ release rate was slower in
longer PBLG chain length and more CZ loading than
short PBLG chain length and lower loading of CZ
due to the hydrophobic interaction between PBLG
domain and CZ. Melting point of CZ for the CZ-
loaded nanoparticles was decreased due to crys-
tallization of CZ in core segments of the nanoparti-
cles.
Acknowledgements
This study was supported by the Korea Science
Engineering and Technology Foundation (Grant No.
95-0300-16-3).
 Y.-I. Jeong et al. / Journal of Controlled Release 51 (1998) 169 – 178
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