Amphiphilic amino acid copolymers as stabilizers for the preparation of nanocrystal dispersion

Article abstract of DOI:10.1016/j.ejps.2004.12.010

The recent advance of particle size engineering in nanometer ranges has widened the formulation opportunities of relatively water-insoluble drugs. However, the 'nanoformulation' suffers from a lack of systematic understanding about the requirements of polymeric stabilizers. Furthermore, the polymers that can be used for the preparation of nanocrystals are so limited that finding a proper stabilizer for a given formulation is often difficult. In this study, amino acid copolymers whose properties can systematically be tailored are developed, and their morphological and compositional effects are investigated. Copolymers containing lysine (K) as their hydrophilic segments, and phenylalanine (F) or leucine (L) as their hydrophobic segments successfully produce stable nanocrystals (200-300 nm) in water, while copolymers of K and alanine (A) could not generate nanosized particles. Not the morphology but the hydrophobicity of copolymers seems to be a critical parameter in the preparation of drug nanocrystals by wet comminution. The effective stabilization performance of copolymers requires the hydrophobic moiety content to be higher than 15 mol%. Comminution for only 5 min is long enough for nanocrystal preparation, and the crystallinity of drug is found intact after the processing.

Full text of DOI:10.1016/j.ejps.2004.12.010

European Journal of Pharmaceutical Sciences 24 (2005) 441–449
Amphiphilic amino acid copolymers as stabilizers for
the preparation of nanocrystal dispersion
Jonghwi Leea, 1, Soo-Jeong Lee , Ji-Yeun Choi , Ji Youn Yoo , Cheol-Hee Ahn
b, ∗
b
a
a
a
Department of Nano Science and Engineering, Biomaterials Research Group, Sejong University, Kwang-Jin-Gu, Gun-Ja-Dong 98, Seoul, South Korea
Research Institute of Advanced Materials (RIAM), Hyperstructured Organic Materials Research Center (HOMRC), School of Materials Science and
Engineering, Seoul National University, San 56-1, Shilim-dong, Gwanak-Ku, Seoul 151-744, South Korea
b
Received 20 August 2004; received in revised form 22 December 2004; accepted 27 December 2004
Abstract
The recent advance of particle size engineering in nanometer ranges has widened the formulation opportunities of relatively water-insoluble
drugs. However, the ‘nanoformulation’ suffers from a lack of systematic understanding about the requirements of polymeric stabilizers.
Furthermore, the polymers that can be used for the preparation of nanocrystals are so limited that finding a proper stabilizer for a given
formulation is often difficult. In this study, amino acid copolymers whose properties can systematically be tailored are developed, and their
morphological and compositional effects are investigated. Copolymers containing lysine (K) as their hydrophilic segments, and phenylalanine
(F) or leucine (L) as their hydrophobic segments successfully produce stable nanocrystals (200–300 nm) in water, while copolymers of K
and alanine (A) could not generate nanosized particles. Not the morphology but the hydrophobicity of copolymers seems to be a critical
parameter in the preparation of drug nanocrystals by wet comminution. The effective stabilization performance of copolymers requires the
hydrophobic moiety content to be higher than 15 mol%. Comminution for only 5 min is long enough for nanocrystal preparation, and the
crystallinity of drug is found intact after the processing.
©
2005 Elsevier B.V. All rights reserved.
Keywords: Nanoparticles; Nanocrystals; Particle size; Insoluble drug; Dispersion; Particle engineering
1
. Introduction
The technology of particle size reduction to nanome-
ters has significantly advanced over the last several decades.
Comminution, high pressure homogenization, impinging jet,
electrospraying, liquid-based methods, and supercritical fluid
processes are the technologies that are currently available or
being actively developed for nanoparticle preparation (Lee
et al., 2000; Serajuddin, 1999; Liversidge and Conzentino,
Reducing the particle size of an active pharmaceutical
ingredient (API) has opened new formulation opportunities
in various dosage forms. Particularly, the bioavailability of
relatively insoluble drugs, which is often limited by poor
dissolution rates, benefits from the development of the
‘nanoformulation’ techniques (Lee et al., 2000; Serajuddin,
1
1
995; Liversidge and Cundy, 1995; Merisko-Liversidge et al.,
996; Zheng and Bosch, 1997; Yamada et al., 1999; Grau
1
999). Based on the Noyes–Whitney equation, which sup-
ports the linear dependence of the dissolution rate on the sur-
face area, increasing surface area by reducing particle sizes
from microns to nanometers leads to one or more order(s) of
magnitude increase in the dissolution rate (Lee et al., 2000).
et al., 2000; Lee, 2003). Methods to prepare drug nanoparti-
cles can be categorized into two approaches, i.e., thermody-
namic and kinetic approach. In the approaches, energy inputs
or surface stabilization compensate the dramatic increase in
surface energy (extra Gibbs free energy) accompanying with
particle size reduction (Lee et al., 2000; Serajuddin, 1999;
Liversidge and Conzentino, 1995; Lee, 2004). In most pro-
cessing, both the approaches are employed together.
∗
Corresponding author. Fax: +82 2 883 8197.
E-mail address: chahn@snu.ac.kr (C.-H. Ahn).
Present address: Chemical Engineering, Chung Ang University, 221
1
Heuksuk-Dong, Dongjak-Ku, Seoul 156-756, South Korea.
0
928-0987/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2004.12.010

4
42
J. Lee et al. / European Journal of Pharmaceutical Sciences 24 (2005) 441–449
Stabilizers usually cover the surface of nanoparticles pro-
a few polymers generally recognized as safe and effective
(GRAS) can be employed (Liversidge and Conzentino, 1995;
Liversidge and Cundy, 1995; Merisko-Liversidge et al., 1996;
Zheng and Bosch, 1997; Yamada et al., 1999; Grau et al.,
2000; Lee, 2003). Finding a proper stabilizer among the cur-
rently available polymers is the leading key in the successful
nanoformulation of a drug.
ducing ionic or steric stabilization (Ploehn and Russel, 1990;
Evertsson and Nilsson, 1997; Berglund et al., 2003a, b).
Nanocrystal preparation by wet comminution uses polymeric
stabilizers, which adsorb onto the surfaces of drug nanocrys-
tals and provide steric stabilization effect (Evertsson and
Nilsson, 1997; Berglund et al., 2003a, b). The adsorbed chain
molecules on surface have ceaseless thermal motion, result-
ing in dynamically rough surface preventing coalescence by
repulsive entropic force. Polymer steric stabilization is good
for the stability of drugs during processing since it does not
usually destroy the crystal structure of drug particles unlike
the conventional surfactants of small molecular weights such
as sodium lauryl sulfate. The surfactants tend to form mi-
celles containing a small number of dissolved drug molecules
Although numerous nanocrystal formulations are under
active development worldwide, efforts for developing a new
class of materials for the stabilization of nanocrystals are still
insufficient. To resolve the current limitations of nanocrystal
processing, novel polymeric stabilizers should have flexibil-
ity in engineering their properties from synthetic point of
view. Considering biocompatibility and diversity in the se-
lection of monomers, amino acid copolymers are excellent
candidates for this purpose (Szabo et al., 2002; Santosoa et
al., 2002; Nowak et al., 2002; Hern a´ ndez and Klok, 2003;
Deming, 1997a, b, 2002; Chiang and Yeh, 2003; Iizuka et
al., 1993). Their biocompatibility has been proved as can be
found elsewhere (Szabo et al., 2002; Santosoa et al., 2002;
Nowak et al., 2002; Hern a´ ndez and Klok, 2003; Deming,
1997a, b, 2002; Chiang and Yeh, 2003; Iizuka et al., 1993).
More than 20 amino acids and their derivatives are available
for polymer construction. Depending on the kind of amino
acids and their compositions, the chemical and physical prop-
erties of the copolymers can conveniently be adjusted for a
specific nanoformulation. The wide range of hydrophobic-
ity of amino acids imparts control over the systematic varia-
tion of hydrophobicity to resulting copolymers (Szabo et al.,
2002; Santosoa et al., 2002; Nowak et al., 2002; Hern a´ ndez
and Klok, 2003; Deming, 1997a, b, 2002; Chiang and Yeh,
2003; Iizuka et al., 1993).
More importantly, tailoring amino acid copolymers will
provide systematic understanding of the effect of their chain
composition and morphology. This will be our initial step to
reveal what are the critical physical properties of polymers
for wet comminution processes. In this study, random and
block copolymers based on lysine (K), phenylalanine (F),
leucine (L), and alanine (A) were prepared via ring open-
ing polymerization and were used for nanocrystal prepara-
tion (Kricheldorf, 1987; Deming, 1997a, b, 2000; Van Dijk-
Wolthuis et al., 1997; Daly and Poche, 1988). The effects of
their composition and morphology were systematically in-
vestigated. The amino acids, K, F, L, and A, were chosen
because they have quite different hydrophobicities, and so
the effects of hydrophobicity on nanocrystal processing can
easily be compared. Naproxen was chosen as a typical water
insoluble drug for easy comparison with the previous works
on it (Liversidge and Conzentino, 1995; Lee, 2003).
(
Adamson and Gast, 1997). For establishing successful stabi-
lization within a reasonable processing time, strong and fast
adsorption at full coverage and a long time scale for desorp-
tion are necessary (Evertsson and Nilsson, 1997; Berglund
et al., 2003a, b) in addition to steric repulsion.
When a polymeric stabilizer is used in a wet comminu-
tion process, its physical adsorption onto the surface of drug
particles and subsequent steric stabilization are important
processes. However, they are not simple processes. Poly-
mer diffusion, convection, adsorption, and desorption can oc-
cur simultaneously with the mechanical fracturing of crystal
(
1
1
Liversidge and Conzentino, 1995; Liversidge and Cundy,
995; Merisko-Liversidge et al., 1996; Zheng and Bosch,
997; Yamada et al., 1999; Grau et al., 2000; Lee, 2003;
Evertsson and Nilsson, 1997; Berglund et al., 2003a). Crystal
surfaces freshly generated by fracturing will attract polymer
chains if the entropy loss by physical adsorption is smaller
than the related enthalpy gain. Thus, the physical and chem-
ical properties of the crystal surfaces and the solution be-
haviors of polymers are important. The mechanism of frac-
turing and subsequent stabilization of particles in the wet
comminution process remains largely unknown. To the best
of our knowledge, the critical physical properties of polymers
for the effective wet comminution have never been system-
atically examined yet, while the micelle forming properties
of polymers have intensively been investigated (Ploehn and
Russel, 1990; Evertsson and Nilsson, 1997; Berglund et al.,
2
003a, b; Torchilin, 2000). In industry, a proper polymeric
stabilizer is chosen for a specific formulation by a simple
trial-and error approach (Liversidge and Conzentino, 1995;
Liversidge and Cundy, 1995; Merisko-Liversidge et al., 1996;
Zheng and Bosch, 1997; Yamada et al., 1999; Grau et al.,
000; Lee, 2003).
It is unrealistic to find a single polymer that properly works
2
for all different wet comminution processes. Different formu-
lation imposes different requirements on polymeric stabiliz-
ers. Furthermore, differences in the surface characteristics of
drug crystals would require different properties of polymers.
However, the properties of polymers critical in nanocrys-
tal processing cannot easily be tuned for a specific formu-
lation, due to the limited number of available polymers. Only
2
. Materials and methods
2.1. Materials
Triphosgene (98.0%), n-hexylamine (99.0%), hydrogen
bromide (30 wt.% solution in acetic acid), and l-alanine

J. Lee et al. / European Journal of Pharmaceutical Sciences 24 (2005) 441–449
443
␧
were purchased from Aldrich. N -carbobenzyloxy-l-lysine
(
drides (NCAs). The ring opening polymerization of ␣-amino
acid N-carboxy anhydrides (NCAs) is a fast and efficient
route for the synthesis of polypeptides (Kricheldorf, 1987;
Deming, 1997a, b, 2000; Van Dijk-Wolthuis et al., 1997; Daly
and Poche, 1988). The lysine-based copolymers were synthe-
sizedbyringopeningpolymerizationviatheaminoacidNCA
using amino acids and triphosgene (Van Dijk-Wolthuis et al.,
1997; Daly and Poche, 1988).
␧
N -CBZ-l-lys) was commercially available from Bachem
AG. l-Phenylalanine and l-leucine were purchased from
TCI America. Diethylamine (98.0%) was obtained from
Yakuri Pure Chemical Co. and N,N -dimethylformamide
ꢀ
(
99.5%) from Junsei. Tetrahydrofuran (THF, 99.0%),
n-hexane (95.0%), and diethyl ether (97.0%) were purchased
from Daejung Chemicals and Metals Co. THF, n-hexane
and diethyl ether were refluxed over Na and freshly distilled
before use. Diethylamine and n-hexylamine were distilled
from CaH2 and stored over molecular sieves. All other
chemicals were used as received.
As an API for wet comminution, Naproxen (GMP) pro-
duced by Merck & Co. was used. The physical properties of
crystalline Naproxen particles (purity > 95%) listed in Table 1
are the basic material parameters for this study. The particle
morphology is also shown in a SEM micrograph, which is
equant (cubic), plate-like or columnar. Hydroxypropyl cellu-
lose (HPC, JP) was obtained from Nisso. Its average molecu-
lar weight was 60 kg/mol, and surface energy was 45 mN/m.
Yittria-stabilized zirconium beads (performance ceramics,
␧
N -CBZ-l-lysine, l-phenylalanine, l-leucine, and l-
alanine were suspended in anhydrous THF (10 wt.%), and
◦
the reaction temperature was increased to 55 C. A calcu-
lated amount of triphosgene, dissolved in anhydrous THF,
was added dropwise into the reaction mixture and the
suspended mixture became a clear solution as NCA was
formed after a certain reaction time. To ensure the ring
formation, the reaction mixture was vigorously stirred for
an additional 1 h. The reaction mixture was condensed
and poured to a 10-fold excess amount of anhydrous n-
hexane to precipitate amino acid NCAs. The amino acid
NCAs were obtained by filtration and dried in vacuum
1
for 48 h. The amino acid NCAs were characterized by H
0
.8 mm diameter) and HPLC-grade water from Aldrich were
NMR.
used without any further purification.
2.3. Synthesis of amino acid copolymers
2
.2. Synthesis of amino acid N-carboxy anhydrides
(NCAs)
Calculated amounts of amino acid NCAs were placed in
an 100 ml two-neck round bottom flask and dissolved in an-
hydrous DMF (10 wt.%) in nitrogen atmosphere (Van Dijk-
Wolthuis et al., 1997). Freshly distilled n-hexylamine, previ-
␧
N -CBZ-l-lysine, l-phenylalanine, l-leucine, and l-
alanine were converted to corresponding N-carboxy anhy-
Table 1
Properties of active pharmaceutical ingredient (API), Naproxen particles
API
Naproxen
Molecular weight
Particle size distribution
230.266 g/mol (C14H14O3)
Bimodal
Particle size (␮m)
Sonication
Mean
0s
37
30s
40
60s
46
120s
38
300s
15
D10
D50
D90
Contact angle
True densities (g/cm )
Surface area (m /g)
3.9
13.7
0.9
13.3
117.5
0.8
16.3
129.4
0.8
8.1
111.6
0.9
0.9
60.0
109.1
◦
◦
62 with water, 13 with ethanol
3
1.243
2.02 ± 0.13
2
Particle morphology: SEM micrograph

4
44
J. Lee et al. / European Journal of Pharmaceutical Sciences 24 (2005) 441–449
ously diluted in anhydrous DMF, was added into the solution
to initiate ring-opening polymerization. In case of l-lysine
and l-leucine copolymerization, diethylamine was used as an
initiator for the measurement of copolymer molecular weight
For the atomic force microscopy (AFM), a PSIA XE-100
with TESP or TESP7 etched single crystal silicone probes
was used (20–100 N/m force constant and 200–400 kHz res-
onance frequency). The surfaces of the dry compacts were
scanned in a tapping mode at ambient conditions.
1
by H NMR.
The polymerization was continued for 72 h at room tem-
perature to make sure of the consumption of all the NCA
monomers. The resulting slightly viscous solution was pre-
cipitated in 20-fold excess water. The precipitate was filtered
and dried in vacuum overnight. The molecular weights of
3. Results and discussion
3.1. Preparation of amino acid copolymers
1
copolymers before deprotection were measured by H NMR.
The protection groups of copolymers (CBZ groups in l-
lysine side chains) were removed using HBr (Deming, 2000).
Copolymers with protection groups were dissolved in 30%
HBr/glacial acetic acid solution (20 ml/g). As deprotection
reaction went on, carbon dioxide evolved out and the copoly-
mer rapidly precipitated from the solution. After 30 min, a
Amino acids were successfully copolymerized into block
andrandomcopolymers(Fig. 1). Theirdesignationsandbasic
properties are given in Table 2. Random copolymers of K and
F, K and L, and K and A were designated as K-r-F, K-r-L,
and K-r-A, respectively. For block copolymers, the letter ‘b’
was used in the place of the letter ‘r’, e.g., K-b-F.
1
0-fold excess amount of anhydrous diethyl ether was added
The molecular weights, degrees of polymerization (DP),
and compositions of copolymers were measured by H NMR.
1
to the reaction mixture. The precipitate was filtered and
washed with diethyl ether. After drying, the deprotected poly-
mer was dissolved again in deionized water, transferred to a
pre-swollen dialysis membrane (MWCO = 500) and dialyzed
against deionized water for 2 days. Poly(amino acid) copoly-
mers for wet comminution were obtained by freeze drying
for 3 days. The compositions of comonomers and the degree
Thedegreeofpolymerizationcontrolledbytheinitialconcen-
tration ratio of the NCA monomers to that of initiator, was
calculated based on the integration ratio of a characteristic
peak from the initiator to a peak from the amino acids in the
repeating units. In the cases of polymerization initiated by n-
hexylamine (DMSO-D ), the peak from initiator at 0.83 ppm
6
1
of polymerization were characterized by H NMR.
(t, 3H, RNCH2(CH2) CH3) was compared to the peak at
5
4
.99 ppm in the l-lysine side groups with a CBZ protection
group (s, 2H, ArCH2O), 7.17 ppm in l-phenylalanine (m,
H, ArH), and 1.36 ppm in l-alanine (m, 3H, CHCH3). In the
2
.4. Wet comminution
5
diethylamine-initiated polymerization (DMSO-D ), the peak
6
API particles were mixed in a 1.3 ml bottle with dis-
at 1.11 ppm (s, 6H, RN(CH2CH3)2) was compared to that
at 4.99 ppm in l-lysine (s, 2H, ArCH2O) and 0.80–0.90 ppm
in l-leucine (m, 6H, CH(CH3)2). The results are summarized
in Table 2. In the n-hexylamine-initiated polymerization, the
DP was almost equal to initial monomer-to-initiator ratio.
However, the DP of diethylamine-initiated copolymers was
about twice as high as that expected from initial concentration
ratio, which follows the same trend reported previously (Van
Dijk-Wolthuis et al., 1997). The amino acid compositions of
copolymers were successfully controlled by the initial con-
centration ratio of amino acid NCAs.
tilled water, polymeric stabilizer (1.33 wt.%) and polystyrene
beads (500 ␮m, 0.354 g). Then, high speed shaking of
4
800 rpm at room temperature produced a nanosuspension
TM
with a Mini-Beadbeater
(Biospec Products). The weight
of the slurry (API + HPC + water) was 3.75 g, and the con-
centration of API in water was 8 wt.%. The concentrations of
HPC mother liquid were 0.38, 0.77, 1.54, and 3.08 wt.%. Un-
less otherwise specified, the comminution time was 30 min.
2
.5. Characterizations after comminution
The particle size distribution was measured in water us-
3.2. Types of amino acids
ing an Electrophoretic Light Scattering Spectrophotometer,
◦
ELS-8000 (Otsuka, 632.8 nm He–Ne laser (10 mW), 25 C,
Wet comminution in the presence of polymeric stabilizers
can produce effective size reduction (Lee et al., 2000; Sera-
juddin, 1999; Liversidge and Conzentino, 1995; Liversidge
andCundy, 1995;Merisko-Liversidgeetal., 1996;Zhengand
Bosch, 1997; Yamada et al., 1999; Grau et al., 2000; Lee,
2003). Polymer stabilizers tend to adsorb onto the surface
of hydrophobic drug, since they have relatively hydrophobic
moieties. Polymer chains anchored onto the surface provide
steric or ionic stabilization depending on the characteristics
of polymers (Ploehn and Russel, 1990). Without polymer
adsorption, stabilization cannot occur, and so no dispersed
nanoparticles result from wet comminution. The mechanical
relative refractive index = 1.3313, Marquadt analysis method,
00 repeated tests). The sonication power of the particle size
1
analyzer was 70 W (39 kHz). For the differential scanning
calorimetry (DSC) experiments, solid powders were prepared
by centrifuging wet-milled suspensions at 17,000 rpm for 1 h
using a Micro 17R centrifuge (Hanil).
DSC experiments were performed using a DSC-7 (Perkin
Elmer) in nitrogen atmosphere. The samples were scanned
from 50 to 200 C at a heating rate of 5 C/min, and the first
heating scans were reported to measure the melting points
and heat of fusion of nanocrystals.
◦
◦

J. Lee et al. / European Journal of Pharmaceutical Sciences 24 (2005) 441–449
445
Fig. 1. Reaction schemes of amino acid copolymers.
Table 2
Designations and properties of amino acid copolymers
Amino acid copolymers:
designations
Architecture (random or
block copolymer)
K content (mol%)
Molecular weight, Mw
Degree of polymerisation,
DP
K-r-F-1
K-r-F-2
K-r-F-3
K-r-F-4
K-r-F-5
K-r-F-6
K-r-F-7
K-r-F-8
K-r-F-9
K-r-F-10
K-r-F-11
K-r-F-12
K-r-F-13
K-b-F-1
K-b-F-2
K-b-F-3
K-r-L-1
K-r-A-1
Random
Random
Random
Random
Random
Random
Random
Random
Random
Random
Random
Random
Random
Block
100
100
89
88
84
83
81
77
73
68
60
60
50
66
62
60
78
79
4800
8400
9200
38
66
70
102
96
204
77
13300
12600
26800
10100
6300
13300
14000
15200
17500
14700
7500
48
100
105
112
129
107
56
186
35
105
110
Block
Block
Random
Random
25200
4800
13100
12900

4
46
J. Lee et al. / European Journal of Pharmaceutical Sciences 24 (2005) 441–449
Fig. 2. Mean particle size changes of various copolymer systems as a func-
tion of comminution time.
Fig. 3. Size distribution curves of drug particles measured by quasi-elastic
light scattering method.
fracture energy input of comminution itself cannot produce
stable nanoparticles since it cannot overcome and compen-
sate the surface energy of nanoparticles.
A longer comminution time than 5 min was not necessary
to further decrease mean particle size (Fig. 2). The particle
sizes seem to reach their steady state values within 5 min
[particle size before processing = 15–46 ␮m (Table 1)]. This
efficient processing time indicates that the mechanical frac-
turing of drug particles into several hundred nanometers and
their stabilization by nearby copolymers can be completed
in a short time period. The results support that the rate of
polymer diffusion (under convection) and adsorption onto
the surface of the hydrophobic drug is fast enough to stabi-
lize particles within 5 min. Fig. 2 shows that K-r-F and K-r-L
are likely to adsorb onto the surfaces within 5 min, but K-r-A
does not significantly adsorb even in a processing time longer
than 5 min. This result is in contrast to our previous study us-
ing common cellulose type polymers such as hydroxyl propyl
cellulose (HPC) (Lee, 2003). Due to the viscose nature of
cellulose polymers, processing using HPC seems to take a
longer time. However, a direct comparison between the two
sets of data is difficult because of differences in processing
equipment, preparation scale, etc.
Fig. 3 shows the typical particle size distributions of three
suspension systems, K-r-F, K-r-L, and K-r-A. The distribu-
tion curves show broad ranges of particle size produced by
wet comminution. K-r-A system shows distinct bimodal dis-
tribution in bigger size ranges than the other two systems.
Its distribution curve above micron is similar to that of drug
particles as received (Table 1), and the particles should be un-
stable. Therefore, the K-r-A system seems to have physical
adsorption process not enough to produce stable dispersions
of nanocrystals.
The hydrophilic moiety of copolymers is K segment. Be-
cause of its hydrophilic nature, the segment will tend to lo-
cate itself toward water instead of hydrophobic drug surface.
Thus, it can provide effective steric or ionic stabilization for
drug nanoparticles. However, like the conventional surfac-
tants of small molecular weights, it may excessively help the
dissolution of hydrophobic drugs, resulting in the destruction
of crystalline structures. The destruction produces micelle-
like structures in liquid formulations. This possible series of
events will be examined in the stability study of nanocrystals
below.
While K was used as the hydrophilic moiety of copoly-
mers, three other amino acids, F, L, and A, were used as
hydrophobic moieties. The use of different amino acids im-
parts a different hydrophobicity to amino acid copolymers. If
all other factors such as the K content, polymer morphology,
etc., remain the same, the hydrophobicity of copolymers in-
creases in the order of K-r-A, K-r-L, and K-r-F (Black and
Mould, 1991). The hydrophobicity of hydrophobic moiety
varies with 0.616 (A), 0.943 (L), and 1.000 (F) (according to
the hydrophobicity scale of Black and Mould (1991)). The
difference in the hydrophobicity of copolymers is the net
changes in the driving force of the absorption of the copoly-
mers onto hydrophobic drug surfaces. Desorption is also re-
lated with the hydrophobicity of copolymers.
Fig. 2 shows the effect of the hydrophobicity of consti-
tuting amino acids. The effect of hydrophobicity is distinct.
K-r-A systems show significantly higher mean particle sizes
(usually above micron) than the other K-r-L and K-r-F sys-
3.3. Content of hydrophobic moiety
tems (near 200–300 nm). The differences between K-r-L and
K-r-F systems are not statistically significant, although only
K-r-F systems can have specific ␲–␲ interactions between
the benzene rings of F and Naproxen (Adamson and Gast,
As the content of hydrophobic moieties increases, the
adsorption of copolymers is expected to be enhanced. It is
an interesting and valuable subject to figure out the mini-
mum content of hydrophobic amino acids required to trigger
1
997).

J. Lee et al. / European Journal of Pharmaceutical Sciences 24 (2005) 441–449
447
Fig. 4. Mean particle sizes of drug nanocrystals vs. K content in copolymers.
The nanocrystals were prepared in the presence of amino acid copolymers
of various K contents.
Fig. 5. Atomic force micrograph of drug nanoparticles prepared with using
amino acid copolymers (K-b-F-1).
the adsorption of copolymers. Additionally, the morphology
of copolymers, i.e., the distribution of hydrophobic moiety
along a polymer chain, could be an important factor. To esti-
mate the effect of the morphology, block and random copoly-
mers having the same mole fraction of hydrophobic amino
acids are compared.
steric repulsion is known to depend on molecular weight, the
variation in molecular weight may not be large enough to
identify its effect. Yet, it is clear from the successful prepara-
tion of nanoparticles shown in Fig. 4 that the polymer chains
of 5000–25,000 g/mol are long enough to provide steric re-
pulsion (Adamson and Gast, 1997).
Fig. 4 shows the effect of hydrophobic amino acid con-
tent on nanoparticle formation. A statistically meaningful re-
sult is that when the hydrophobic content is above 15 mol%
It should be mentioned that the differences among the
three data points above 500 nm (Fig. 4) may not be signifi-
cant, particularly due to the effect of sonication. Since these
suspensions were not found thermodynamically and kineti-
cally stable, their mean particle size strongly depended on
the history of stirring and sonication. However, the wide size
range might be related to a possible physical ion pairing be-
tween K and the acid functional groups of Naproxen. If any,
the ion pairing can lead the K chain segments to partially
adsorb onto the hydrophobic surfaces of Naproxen.
(K content < 85 mol% in Fig. 4), the mean particle size
can be reduced to below 400 nm. Below 15 mol% (K con-
tent > 85 mol%), it spans a wide size range of 500–1500 nm.
Above 15 mol%, a significant change in mean particle sizes
is not observed. Therefore, hydrophobic moiety of 15 mol%
is enough for copolymer chains to attach onto the surface
of drug particles and maintain stable physical adsorption
(
Ploehn and Russel, 1990; Evertsson and Nilsson, 1997;
Berglund et al., 2003a, b). (They were sable up to at least
0 days as will be seen in Fig. 7.) Their desorption kinetics
The nanoparticles prepared in the presence of amino acid
copolymers were visualized using atomic force microscopy
(AFM) and a typical micrograph is provided in Fig. 5. Parti-
cles were dried on a silicone wafer for microscopy study, and
particle aggregation during the drying process was inevitable.
In aqueous conditions, the surface energy of particles is coun-
terbalanced by steric or ionic stabilization. However, in dried
conditions, polymer chains solidify themselves and their sta-
bilization can be no longer effective, resulting in significant
aggregation as can be found in the previous study (Lee, 2003).
Indeed, both single particles and aggregates can be found in
the micrographs. They are not typically spherical, indicating
their crystalline nature that can be confirmed in the thermal
analyses discussed below.
3
should be long enough for successful nanosuspension prepa-
ration.
In random copolymers, the hydrophobic amino acids are
randomly distributed, but in diblock copolymers, they are
localized in one segment. As a consequence, multiple at-
tachments of polymer chains onto hydrophobic surface could
be easier in random copolymer adsorptions than in diblock
copolymer cases. However, the mean particle size results of
Fig. 4 do not reflect the possible differences in the chain
conformations of adsorbed copolymers. The morphology of
copolymers, diblock or random copolymer chains, is not a
determining factor in controlling particle size reduction.
The effect of molecular weight was not critical either (the
details are not given in Fig. 4). The variation in molecular
weight (Table 2) was intentionally generated to investigate its
effect, but a systematic increase or decrease in particle size
with an increase in molecular weight was not found. Since
3.4. Stability of nanoparticles
Retaining the crystallinity of drug particles during pro-
cessing is beneficial to the stability of drugs. Wet comminu-
tion could sometimes significantly damage the crystallinity

4
48
J. Lee et al. / European Journal of Pharmaceutical Sciences 24 (2005) 441–449
Fig. 7. Plot of mean particle size vs. time shows the physical stability of
drugnanosuspensionsatroomtemperatureafterwetcomminution. Wetcom-
minution times are given in parentheses.
Fig. 6. DSC thermograms of drug nanocrystals stabilized by amino
acid copolymers show the crystallinity of drug: (A) drugs as received,
ꢀHf = 128 J/g; (B) nanoparticles with K-b-F-1, ꢀHf = 117 J/g; (C) nanopar-
ticles with K-r-F-13, ꢀHf = 127 J/g.
Hydrophobicity mainly influences the physical adsorption
of polymers onto hydrophobic drug surfaces, while chain
morphology mainly affects the adsorption conformation and
resulting steric repulsion of polymers. As a result, this study
indicates that the physical adsorption of polymers is the pri-
mary determining factor in the effective wet comminution
and steric stabilization of drug nanocrystals. In fact, wet com-
minution continually provides mechanical fracture energy to
drug crystals and creates fresh fracture surfaces (or cleav-
ages), which will tend to fuse together unless quickly stabi-
lized. Actual size reduction by fracturing could occur only
with the fast adsorption of enough polymeric stabilizers onto
the surfaces. The fast physical adsorption primarily requires
a certain degree of hydrophobicity. This understanding can
guide us to the future development of polymeric stabilizers
for wet comminution processes, although it still needs further
verification.
The amphiphilic copolymers in solid dispersion systems
behave differently from those in emulsion systems (Ploehn
and Russel, 1990; Adamson and Gast, 1997). In emul-
sions, they can form unique structures depending on the
concentration of amphiphilic copolymers and their thermo-
dynamic driving forces (Ploehn and Russel, 1990; Adamson
and Gast, 1997). For example, critical micelle concentra-
tion depends on the concentration and hydrophobicity of
copolymers. However, the preparation of drug nanocrystal
dispersion depends on the fracture process of drug crystals
and subsequent stabilization.
of particles, and so possible changes in the crystallinity of
the drugs by wet comminution were checked by DSC. Fig. 6
shows that the crystallinity of the drugs was not significantly
changed by wet comminution. In the figure, changes in heat
of fusion and melting temperature were almost negligible.
Caution needs to be taken in the generalization of this result
to other pharmaceutical active ingredients, since each drug
crystal has a different physical stability.
After the preparation of nanoparticles by wet comminu-
tion with the aid of polymers, nanoparticles could slowly
aggregate back into microparticles with a desorption process
of polymers. Also, crystal growth via the Osward ripening
(Adamson and Gast, 1997) due to the dissolution of drug
particles can trigger significant changes in particle size. To
investigate the physical stability of prepared nanoparticles
in water, particle size was measured at an appropriate time
interval up to 30 days at room temperature. Fig. 7 provides
the typical data showing that there is no significant change
in the mean particle sizes of nanosuspensions during the ex-
amined period of time. They seem to be stable, which opens
the possibility in the application of the presented formulation
process to an actual pharmaceutical unit operation.
3
.5. Chain architecture
In this study, both the type of amino acids and their com-
position are the major factors determining particle size reduc-
tion. The hydrophobicity of polymers depends on the two fac-
tors. Thus, it is the case that the hydrophobicity of polymers
is more important than the chain morphologies of polymers
such as block and random copolymers. Interestingly, the par-
ticle size results of Figs. 2 and 4 show that the relationship
between hydrophobicity and mean particle size may not be
linear.
4. Conclusion
Amphiphilic amino acid copolymers having molecular
weights of 5000–25,000 g/mol were found to be able to stabi-
lize drug nanocrystals during wet comminution. K was used
as the hydrophilic moiety of the copolymers, and A, L, and F

J. Lee et al. / European Journal of Pharmaceutical Sciences 24 (2005) 441–449
449
were employed as the hydrophobic moieties. In the case of A
as the hydrophobic moiety, successful size reduction down to
few hundred nanometers was not observed. Copolymers of K
and the other two hydrophobic amino acids, i.e., F andL, were
found to be efficient in nanoparticle formation by triggering
stable polymer adsorption onto the hydrophobic surfaces of
drugs for dispersion stabilization. For successful polymer ad-
sorption and particle size reduction, the mole fraction of the
hydrophobic moieties needed to be at least 15 mol%. The
morphology of copolymers was not an important factor in
determining particle size reduction. Once wet comminution
in the presence of a proper amino acid copolymer produced
drug nanocrystals, their particle size were found to be stable
up to 30 days without significant aggregation. From these
results, the hydrophobicity of polymer is a critical physical
property, and amphiphilic amino acid copolymers seem to be
an effective class of materials for pharmaceutical formulation
of drug nanocrystals.
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Institute of Industrial Technology Evaluation and Planning
(project no. 10010884) is gratefully acknowledged. Ahn
thanks the Hyperstructured Organic Materials Research Cen-
ter (HOMRC) for their financial support.
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