Preparation of novel polymer assemblies, "lactosome", composed of poly(L-lactic acid) and poly(sarcosine)

Article abstract of DOI:10.1246/cl.2007.1220

Novel amphiphilic block polymers composed of poly(L-lactic acid) (PLLA) and poly(sarcosine) (PS) were synthesized by successive polymerizations of lactide and sarcosine N-carboxy anhydride (NCA). The degree of polymerization of each block was controlled b

Full text of DOI:10.1246/cl.2007.1220

1220
Chemistry Letters Vol.36, No.10 (2007)
Preparation of Novel Polymer Assemblies, ‘‘Lactosome’’,
Composed of Poly(L-lactic acid) and Poly(sarcosine)
Akira Makino,1 Ryo Yamahara,2 Eiichi Ozeki,2 and Shunsaku Kimuraꢀ1
1Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510
2Technology Research Laboratory, Shimadzu Corporation, Kyoto 619-0237
(Received July 9, 2007; CL-070721; E-mail: shun@scl.kyoto-u.ac.jp)
Novel amphiphilic block polymers composed of poly(L-
Z-Cl
23%
CH3
H2N
H
NH2
N
O
lactic acid) (PLLA) and poly(sarcosine) (PS) were synthesized
by successive polymerizations of lactide and sarcosine N-car-
boxy anhydride (NCA). The degree of polymerization of each
block was controlled by the monomer/initiator ratio in feed.
The block polymers were successfully dispersed in buffer with
taking various morphologies of micelle, lamellar, and vesicle
depending on the hydrophobic/hydrophilic balance of the co-
polymers. The size of the molecular assemblies was in the range
from 20 to 200 nm, which may be available for drug delivery
system (DDS) as nanocarrier.
NH2
2
O
O
3
O
O
∆, Sn(Oct)2
O
CH3
O
H
L-lactide
4
N
O
O
N
H
H
O
CH3
m
5
62%
1) HBr-AcOH/CH2Cl2
2) washed with sat. NaHCO3aq
O
H2N
N
O
CH3
H
O
CH3
m O
Recently, DDS using small particles has attracted much at-
tention in terms of controlled release and targetting.1 Amphiphil-
ic block polymers are known to form various types of molecular
assemblies such as spherical micelles, rod micelles, and vesi-
cles.2 The shape and size of the molecular assemblies are gener-
ally controllable by choosing a proper hydrophobic/hydrophilic
balance of the block polymer. The advantages of using polymers
for the molecular assemblies are the physical stability compared
with liposome made of lipids,3 and their facile size adjustment in
the range from a few ten nm to a hundred nm. In the present
study, biodegradable polymers of poly(L-lactic acid) (PLLA)
and polypeptide were chosen for the components of copolymers,
which may be applicable to DDS as nano-scaled molecular par-
ticles.
A vesicular molecular self-assembly, named peptosome, has
been reported with using amphiphilic polypeptides.4,5 The key
design of the peptosome is a helical peptide segment as the
hydrophobic part. The regular packing of the helices should
contribute to the physical stability of the thin membrane. On
the basis of the concept of peptosome, another helical and
biodegradable polymer of PLLA was used here for preparation
of molecular assemblies. PLLA is well known to form a 310
helical structure.6 Core–shell-type micelle was already prepared
from di- and tri-block polymers composed of PLLA and poly-
(ethylene glycol) (PEG).7 Instead of using PEG, which is
pointed out to cause allergic reactions,8 poly(sarcosine) (PS)
was used here for the hydrophilic segment. Sarcosine is a
naturally occurring amino acid and highly water-soluble. Co-
polymers composed of PLLA and PS are thus considered to be
biodegradable and biocompatible.
O
6
88%
N
H3C
O
-CO2 DMF
7
Sar-NCA
O
H
H
N
O
CH3
CH3
N
N
H
CH3
O
O
n
m
O
8
HO
OH
O
HATU, DIEA DMF
9
O
H
HO
N
O
CH3
N
N
H
CH3
O
n
CH3 m O
1
62% (2 steps)
Scheme 1. Route for the synthesis of PLLA–PS block polymer
1. The yield is shown in the case of Entry 4.
cooled alcohol to remove low molecular weight products.
The polydispersity index (PDI) Mw=Mn values of PLLA were
1.05–1.14. After the deprotection of the Cbz group, the amino
terminal was used as initiator for polymerization of sarcosine
N-carboxy anhydride (NCA) (7). The degree of polymerization
of PS was controlled by choosing a proper ratio of NCA and ini-
tiator in the feed. Finally, the N terminal of PS was endocapped
with glycolic acid (9). All synthetic products were identified by
1H NMR.9
Aqueous dispersion of 1 was prepared by the film rehydra-
tion technique. A solution of polymer 1 (6.0 mg) in chloroform
was evaporated to dryness till a thin film was formed on the
internal surface of a test tube. To the test tube, 10 mM Tris-
HCl buffer (2.0 mL) was added and treated with a bath-type son-
icator. The dispersion was subjected to dynamic light-scattering
(DLS) analysis,10,11 transmission electron microscope (TEM)
observation, and circular dichroism (CD) measurement.12
The composition of the synthetic amphiphilic block poly-
mers 1 and the hydrodynamic diameter of corresponding
molecular assemblies by DLS measurements are summarized
in Table 1.
The synthetic route for the amphiphilic block polymers is
illustrated in Scheme 1. One amino group of 1,2-ethylenedi-
amine (2) was protected by benzyloxycarbonyl (Cbz) group
and the other amino group was utilized as initiator for prepara-
tion of the PLLA block. A mixture of the initiator 3, L-lactide
(4), and 0.2 wt % tin(IV) octanoate as catalyst was heated up
to 120 ꢁC for 12 h. The reaction mixture was washed with ice-
Copyright Ó 2007 The Chemical Society of Japan

Chemistry Letters Vol.36, No.10 (2007)
1221
Table 1. Composition of PLLA–PS block polymers 1 and the
hydrodynamic diameter of their molecular assemblies
PLLA (Entries 5 and 6) formed large lamellar assemblies as
shown by the large diameter from DLS measurements
(Table 1) and the TEM image (Figure 1c). The CD measure-
ments showed positive cotton effects at 210 nm, indicating
helical conformation of the PLLA block.12 However, the molar
residue ellipticity per the lactate unit was decreased to half with
decreasing the length of the PLLA block from 25 to 16 (degree
of polymerization), suggesting the PLLA block taking disor-
dered structure in PLLA16–PS45 but helical structure in
PLLA30–PS143. The cross section area of the PLLA block in
PLLA16–PS45 may be therefore larger than that of the diameter
of the cylindrical shape in PLLA30–PS143, which is considered
as the reason for the change in morphology between the two
copolymers.
Composition of 1a
Yield from
Diameter
/nmb
Polydispersity
index
Entry
3/%
PLLA
PS
1
2
3
4
5
6
30
30
30
25
16
16
50
91
35
41
38
34
6
precipitation
24
—
0.28
0.14
0.05
0.34
0.59
143
150
45
30
52, 204ꢀ
160, 785ꢀ
217, 1092ꢀ
22
28
aThe composition of 1 was determined by 1H NMR spectrometry.
bAsterisks on diameter represent the predominant molecular assembly.
To get information on morphology of PLLA25–PS150 in
buffer, the encapsulation experiment with using a water soluble
pseudo-drug was carried out. FITC-dextran 4000 was used as a
pseudo-drug.12 In the elution profile of the molecular assembly
of PLLA25–PS150 through a size exclusion chromatography
(SEC) showed the coelution of the molecular assembly and
FITC-dextran. Further, the fraction of the molecular assembly
did not loose the encapsulated FITC-dextran upon rechromatog-
raphy on the SEC, showing no leakage or release of FITC
dextran from the molecular assembly. On the contrary, FITC-
dextran was scarcely detected in the fraction of the molecular
assembly of PLLA30–PS143 (micelle) on the SEC. Taken
together, the molecular assembly of PLLA25–PS150 was mainly
vesicle, which is therefore named ‘‘lactosome’’.
Figure 1. Transmission electron micrographs of PLLA–PS mo-
lecular assemblies negatively stained with uranyl acetate: (a)
PLLA30–PS143 (Entry 3), scale bar: 200 nm.; (b) PLLA25–
PS150 (Entry 4), scale bar: 400 nm.; (c) PLLA16–PS45 (Entry
5), scale bar: 500 nm.13
In conclusion, a small change in the PLLA length of the
block polymers influence the morphology of the molecular
assembly. As a result, various morphologies, micelle, lamellar,
and vesicle, can be prepared from PLLA–PS block polymers.
The block polymer PLLA30–PS50 (Entry 1) could not be
dispersed in buffer to generate white precipitates because of
the shortage of the hydrophilic property. With the elongation
of the PS block, PLLA30–PS91 polymer (Entry 2), a stable dis-
persion was obtained but the size distribution was broad (diver-
sity index of 0.28). When the length of the hydrophilic block was
long enough, the size distribution of the molecular assemblies
became uniform to be ca. 30 nm (Entry 3). Figure 1a shows
the TEM image of the dispersion prepared from PLLA30–
PS143. The average diameter of the molecular assembly ana-
lyzed from the TEM image was consistent with that evaluated
from the DLS measurement. On the basis of its size and the
spherical shape, the molecular assembly should be core-shell-
type micelle.
PLLA25–PS150 block polymer (Entry 4) yielded two types
of molecular assemblies with diameters of 52 and 204 nm. The
latter assembly was predominant and considered to be vesicles
from its size, TEM image (Figure 1b) and the encapsulation
experiment described in the following section. It is surprising
that a small decrease of the PLLA block in the length caused
a large difference in morphology of the molecular assembly.
Hydrophobicity, helical content, and rigidity of the PLLA block
should influence the hydration process of the block polymers,
which may be different between these two block polymers to
yield different morphologies. However, the factor determining
the morphology is not straightforward. For example, the molar
residue ellipticity per the lactate unit of PLLA25–PS150 was
stronger by ca. 10% than that of PLLA30–PS143. The helical
structure of the latter may be distorted owing to the large curva-
ture of the micelles. Interestingly, block polymers with a short
This study is a part of joint research, which is focusing on
the development of the basis of technology for establishing
COE for nano-medicine, carried out through Kyoto City
Collaboration of Regional Entities for Advancing Technology
Excellence (CREATE) assigned by Japan Science and Technol-
ogy Agency (JST). We greatly thank Professor Junji Sugiyama
at Kyoto University for the TEM observation.
References and Notes
1
2
3
4
Y. Liu, H. Miyoshi, M. Nakamura, Int. J. Cancer 2007, 120, 2527.
K. Letchford, H. Burt, Eur. J. Pharm. Biopharm. 2007, 65, 259.
H. Bermudez, D. A. Hammer, D. E. Discher, Langmuir 2004, 20, 540.
S. Kimura, D.-H. Kim, J. Sugiyama, Y. Imanishi, Langmuir 1999, 15,
4461.
5
S. Kimura, Y. Muraji, J. Sugiyama, K. Fujita, Y. Imanishi, J. Colloid
Interface Sci. 2000, 222, 265.
6
7
T. Fujiwara, Y. Kimura, Macromol. Biosci. 2002, 2, 11.
Z. Dai, L. Piao, X. Zhang, M. Deng, X. Chen, X. Jing, Colloid Polym.
Sci. 2004, 282, 343.
8
9
P. Dewachter, C. Mouton-Faivre, Allergy 2005, 60, 705.
A typical 1H NMR spectrum of amphiphilic block polymer 1:
PLLA30–PS143, 1H NMR (DMSO): ꢀ 5.2 (30H, q, J ¼ 7:02 Hz,
CH of PLLA), 4.4–3.9 (285H, m, CH2 of PS), 3.0–2.8 (467H, m,
N–CH3 of PS), 2.1 (3H, s, CH3 of acetyl), 1.5 (97H, d, J ¼
7:02 Hz, CH3 of PLLA).
10 S. W. Provencher, Comput. Phys. Commun. 1982, 27, 213.
11 S. W. Provencher, Comput. Phys. Commun. 1982, 27, 229.
12 Supporting Information is available electronically on the CSJ-Journal
Web site, http://www.csj.jp/journals/chem-lett/index.html.
13 Magnified images of Figure 1 are shown in the Supporting Informa-
tion.
Published on the web (Advance View) September 8, 2007; doi:10.1246/cl.2007.1220