Synthesis and self-assembly of novel amphiphilic copolymers poly(lactic acid)-block-poly(ascorbyl acrylate)

Article abstract of DOI:10.1002/pola.24840

In this study, a novel type of amphiphilic block copolymers poly(lactic acid)-block-poly(ascorbyl acrylate) (PLA-block-PAAA) with biodegradable poly(lactic acid) as hydrophobic block and poly(ascorbyl acrylate) (PAAA) as hydrophilic block was successfully

Full text of DOI:10.1002/pola.24840

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Synthesis and Self-Assembly of Novel Amphiphilic Copolymers
Poly(lactic acid)-block-Poly(ascorbyl acrylate)
1
2
1
Yanzhai Wang, Junjiao Yang, Jing Yang
1
State Key Laboratory of Chemical Resource, College of Life Science and Technology, Beijing University of Chemical
Technology, Beijing 100029, China
2
College of Science, Beijing University of Chemical Technology, Beijing 100029, China
Correspondence to: J. Yang (E-mail: yangjingbuct@gmail.com)
Received 24 May 2011; accepted 17 June 2011; published online 7 July 2011
DOI: 10.1002/pola.24840
ABSTRACT: In this study, a novel type of amphiphilic block
copolymers poly(lactic acid)-block-poly(ascorbyl acrylate) (PLA-
block-PAAA) with biodegradable poly(lactic acid) as hydropho-
bic block and poly(ascorbyl acrylate) (PAAA) as hydrophilic
block was successfully developed by a combination of ring-
opening polymerization and atom transfer radical polymeriza-
tion, followed by hydrogenation under normal pressure. The
chemical structures of the desired copolymers were character-
bly behavior was monitored by fluorescence-probe technique
and turbidity change using UV–vis spectrometer, and the mor-
phology and size of the nanocarriers via self-assembly were
detected by cryo-transmission electron microscopy and
dynamic light scattering. These polymeric micelles with PAAA
shell extending into the aqueous solution have potential abil-
ities to act as promising nanovehicles for targeting drug deliv-
ery. VC 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym
1
ized by H NMR and gel permeation chromatography. The ther-
Chem 49: 3988–3996, 2011
mal physical properties and crystallinity were investigated by
thermogravimetric analysis, differential scanning calorimetry,
and wide angle X-ray diffraction, respectively. Their self-assem-
KEYWORDS: block copolymers; drug delivery system; self-
assembly
INTRODUCTION Targeting strategies for drug nanocarriers
are currently receiving much attention in nanomedicine due
to their unique advantage of improving the effects of drugs
vated levels of superoxide produced by stromal and/or tu-
8
–10
mor cells.
The studies on neuroprotective role of ascor-
bate and the mechanism of its penetration into the brain
presented the molecular selectivity and physiological disposi-
tion between ascorbate and sodium-dependent vitamin C
1
,2
against various biological models. Besides their outstand-
ing merits, such as enhancing the bioavailability of drugs in
physiological environment and sustaining release of com-
1
1,12
transporters SVCT2.
These biochemical discoveries
3
pounds for long-term therapy, more importantly, the target-
exhibited the significance of ascorbate in the treatment of
tumors and central nervous disorders, meanwhile, also
hinted that it might serve to improve the specificity of over-
all drug delivery system to the pathological sites if ascorbate
as a promising targeting molecule is used to modify drug
carriers.
able nanocarriers can play an active role to increase specifi-
cally cellular delivery, thereby to reduce harmful effects of
4
the loaded drugs on nontargeted cells. In recent years, the
successful exploitation of various endogenous biomolecules
and specific nutrients responsible for tumor tissues and
organs has shed light on the development of drug delivery
Actually, a few studies are available in the literature on AA
as biological target mediating the specific delivery of thera-
peutic or diagnostic molecules. Manfredini et al. reported
that small molecule prodrugs were fabricated by chemical
conjugation of AA with central nervous system (CNS) active
molecules. It was shown that AA endowed such prodrugs
with efficient binding features to epithelial-associated SVCT2
translocator, which benefited to the effective drug release in
5
–7
from passive to active targeting.
Nanocarriers design com-
bining with a good knowledge of biochemical features differ-
ence between normal and tumoral cells would be broadly
investigated as an extremely important way to realize active
targeting strategy in the field of drug delivery.
Ascorbate (L-ascorbic acid, AA) as an essential molecule for
man has been recognized for many years. In recent years, a
great deal of research has focused on the special physiology
of ascorbate. For example, abnormal local accumulation of
ascorbate in tumor tissue has been found as a result of ele-
1
3–15
the vicinity of the site of action.
Although a short biolog-
ical life of such prodrugs in the bloodstream circulation
would be one key barrier to enhance the whole therapeutic
Additional Supporting Information may be found in the online version of this article
V
C
2011 Wiley Periodicals, Inc.
3
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ARTICLE
efficiency, these results highlighted the potential of AA trans-
porter as a new mediator for drug delivery. Taking advantage
of liposome and ascorbate features, Torchilin’s group thoroughly
studied the ability of ascorbate-modified liposome to target and
kill certain types of tumor cells and to potentiate the effect of the
Measurements
The H and C NMR spectra were recorded on a 400 MHz
NMR spectrometer (Bruker, Germany) at room temperature
using deuterated chloroform (CDCl ) or deuterated dimethyl-
sulfoxide (DMSO-d ) as solvent. The chemical shifts were
1
13
3
6
16–18
anticancer drugs delivered by these liposomes,
which sup-
measured against the solvent signals of CDCl or DMSO-d as
3
6
ported that ascorbate-mediated nanocarriers might be particu-
larly effective in targeting cancers. Moreover, this type of lipo-
somes was also investigated in targeting the CNS, and further
exhibited their great potential in the application of specific brain
delivery. Besides, Dur a´ n and coworkers reported that the
nanoparticles capped by ascorbate via nonconjugated bond
showed good antitumoral activity, as well. Among the reported
examples, only one AA was bonded at one end of the molecular
architecture for conducting its targeting property and adjusting
the whole molecular hydrophilic/hydrophobic balance. To the
best of our knowledge, few have been reported on the amphi-
philic polymers with polymeric AA as hydrophilic block.
internal standard. The molecular weights of the polymers
were determined with Waters 515-2410 GPC instrument
equipped with Styragel HT6E-HT5-HT3 chromatographic col-
umn following a guard column and a differential refractive-
index detector. The sample solution was filtered with a
0.45 lm syringe filter before ejection. The measurements
were performed using THF as eluent at a flow rate of 1.0 mL
min at 30 C and a series of narrow polystyrene standards
for the calibration of the columns. UV–vis spectra were
recorded by a Hitachi U-3010 UV–vis spectrometer (Hitachi
High-Technologies, Tokyo, Japan). The fluorescence spectra
were recorded by a Hitachi F-4500 Fluorescence instrument
19
20
ꢀ
1
ꢁ
(
Hitachi High-Technologies) at ambience. Fourier transform
Inspired by particular biological properties of AA, we synthe-
size a class of block copolymers with biodegradable poly(lac-
tic acid) (PLA) as hydrophobic block and poly(ascorbyl acry-
late) (PAAA) as hydrophilic block. When the nanocarriers
formed via the self-assembly of the amphiphilic block copoly-
mers poly(lactic acid)-block-poly(ascorbyl acrylate) (PLA-
block-PAAA) have biodegradable hydrophobic compartment
for encapsulating anticancer drugs, it is expected that poly-
meric AA as hydrophilic shell will exhibit exceptional target-
ing and antitumoral properties. In this work, the fabrication
of novel amphiphilic block copolymers PLA-block-PAAA was
performed by a combination of ring-opening polymerization
infrared (FTIR) spectra were measured on a Nicolet 5700
FTIR spectrometer with 32 accumulations at a resolution of
2
of samples were collected on a Shimadzu XRD-6000 diffrac-
tometer with Cu Ka radiation (40 kV, 30 mA, k ¼ 1.5418 Å).
DLS measurements on polymeric micelles were performed
using a ‘‘Zetaplus’’ zeta potential analyzer (Brookhaven
Instrument) equipped with ZetaPlus Particle Sizing software
and with 35-mW solid state laser operated at a laser light
wavelength of 660 nm. The size measurements were carried
ꢀ
1
.0 cm at room temperature for the samples. WAXD patterns
ꢁ
o
out at 25 C at a scattering angle of 90 .
(
ROP) with atom transfer radical polymerization (ATRP), fol-
The morphological observation of the micelles was carried out
on a Hitachi H800 transmission electron microscopy (TEM;
Hitachi High-Technologies) operated with 100 kV. For sample
preparation, a drop of the filtered sample solution was
dropped on a carbon-coated copper grid, excess solution was
wicked away with filter paper, and the sample was left dried
in air. Samples for cryogenic TEM (cryoTEM) measurement
were prepared as follows: nanosized micellar solution was
loaded onto holey carbon film-supported grids. Vitrobot (pro-
duced by FEI company) was used to make a thin aqueous film
blotting with filter paper and the immediate plunging into liq-
uid ethane. The frozen grids were stored in liquid nitrogen
and transferred to a cryo-transfer holder under liquid nitrogen
lowed by hydrogenation under normal pressure. All of the
intermediates and final products were characterized by nu-
clear magnetic resonance (NMR) and gel permeation chro-
matography (GPC). Their thermal physical properties and
crystallinity were investigated by differential scanning calo-
rimetry (DSC), thermal gravimeter analysis (TGA), and wide
angle X-ray diffraction (WAXD). The amphiphilic block
copolymers can self-assemble into spherical micelles through
the study of dynamic light scattering (DLS) and cryo-trans-
mission electron microscope (cryoTEM).
EXPERIMENTAL
ꢁ
Materials
at ꢂ ꢀ180 C. Images were recorded on a CCD camera (2k ꢃ
2k, Gatan) using a Tecnai 20 field emission gun electron
CuBr purchased from Aldrich was purified by stirring in ace-
tic acid overnight, followed by washing with ethanol and
diethyl ether and dried in vacuum. Triethylamine and meth-
microscope operated at 120 kV with low-dose mode.
ylene dichloride (CH
2
Cl
2
) were dehydrated with KOH and
Synthesis of Monomer 6-Acrylate-2,3-O,O-dibenzyl-L-
ascorbic acid
CaCl overnight and distilled, respectively. Toluene and tetra-
2
hydrofuran (THF) were dried using sodium with benzophe-
none as color indicator. All of the above purified solvent and
reagents were stored in solvent storage flasks before use.
The other reagents such as 2-bromopropionyl bromide, pen-
tamethyldiethylene triamine (PMDETA), and stannous
octoate from Aldrich and AA, ethylene glycol, acryloyl chlo-
ride, benzyl bromide, and CuBr2 from Sinopharm Chemical
The synthesis procedure of monomer 6-acrylate-2,3-O,O-
dibenzyl-L-ascorbic acid (BnAAA) included two steps. The
first step was performed using modified procedure. The mix-
ture of AA (6.0 g, 34.0 mmol), potassium carbonate (11.8 g,
0
85.0 mmol), and N,N -dimethylformamide (DMF) (20 mL)
charged in a 100-mL three-necked round bottom flask was
ꢁ
stirred for an hour at 50 C, followed by the dropwise addi-
(
China) were used as received without further treatment.
tion of benzyl bromide (12.0 g, 70 mmol) in DMF (15 mL).
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After kept stirring for 5 h, the reaction solution was filtered
with celite, and the celite column was rinsed by ethyl ace-
tate. The combined organic phase was washed with distilled
Synthesis of Poly(lactic acid) via ROP
In a typical ROP procedure, a Schlenk flask degassed by three
vacuum-nitrogen cycles was charged with Sn(Oct) (56.3 mg,
2
water several times, and dried with anhydrous MgSO . The
0.14 mmol), initiator 2-hydroxyethyl-a-bromopropionate
(HEBP; 54.7 mg, 0.28 mmol), L-lactide (2.0 g, 13.9 mmol), and
toluene (4.0 mL). The reaction equipment was immersed in
4
concentrated solution was purified by column chromato-
graph with ethyl acetate/hexane (v/v ¼ 1/1) as eluent to
give slight yellow solid product 2,3-O,O-dibenzyl-L-ascorbic
acid (BnAA) in 40% yield (4.8 g).
ꢁ
an oil bath thermostated at 70 C. After predetermined poly-
merization time, the reaction solution was precipitated in the
cold methanol (200 mL) for three times. The polymers were
dried in vacuum at room temperature for 5 h.
1
1
H NMR (400 MHz, CDCl ) d (ppm): 7.19–7.38 (m, 10H,
3
2
C
6
H
5
), 5.03–5.44 (m, 4H, 2CH
hydrogen on C-4), 3.94 (br, 1H, hydrogen on C-5), 3.73–3.78 (m,
H, hydrogen on C-6), 3.39 (br, 1H, OH on C-5), 3.14 (br, 1H, OH
2
Ph), 4.67 (d, J ¼ 4.0 Hz, 1H,
H NMR (400 MHz, CDCl ) d (ppm): 5.15 (m, nH, CH in the
3
2
repeating unit), 4.34–4.37 (m, 6H, CH CHBr, CO CH CH OCO
3
2
2
2
1
3
on C-6). C NMR (100 MHz, CDCl ) d (ppm): 170.2, 157.8,
and CH CHOH), 1.81 (d, J ¼ 4.0 Hz, 3H, CH CHBr), 1.57 (d, J
3
3
3
1
3
1
35.9, 135.3, 129.1, 128.7, 127.8, 121.1, 76.1, 74.1, 73.6, 69.9,
¼ 8.0 Hz, nH, CH in the repeating unit). C NMR (100 MHz,
3
ꢀ
1
ꢀ1
63.4. FTIR (KBr disk): u , 3425 cm (s), uCAH, 2923 cm
CDCl ) d (ppm): 169.7, 69.1, 66.7, 63.1, 62.6, 39.4, 21.4, 20.4,
OH
3
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
ꢀ1
(
m), 2852 cm (s), 1744 cm (m), uC¼C,1655 cm (s).
16.8. FTIR (KBr disk): uCAH, 2955 cm (s), 2924 cm (vs),
852 cm (s), u , 1757 cm (s).
ꢀ
1
ꢀ1
2
C¼O
For the second step, BnAAA preparation was conducted by
the reaction of BnAA (4.8 g, 13.5 mmol), acryloyl chloride
Synthesis of Block Polymers PLA-block-PBnAAA via ATRP
Typically, a Schlenk flask with a magnetic stir bar was
charged with CuBr (10.0 mg, 0.068 mmol), macroinitiator
(
1.5 g, 16.2 mmol) and triethylamine (1.8 g, 17.5 mmol) in
ꢁ
anhydrous CH Cl (20 mL) at 0 C for 5 h. The reaction solu-
2
2
tion was filtered, and the filtrate was washed by distilled
water three times. The concentrated organic mixture was
purified by column chromatograph with the mixture of ethyl
acetate and hexane (v/v ¼ 2/5) as eluent to obtain white
solid product in 54% yield (3.0 g).
2
PLA-Br (396.0 mg, 0.068 mmol) and CuBr (0.8 mg, 0.0034
mmol). The flask was degassed by three vacuum-nitrogen
cycles, and the liquid materials including ligand PMDETA
(14.2 mg, 0.082 mmol), monomer BnAAA (0.7 g, 1.71 mmol)
and toluene (3.0 mL), which were degassed by nitrogen bub-
bling for 20 min before use, were introduced into the reac-
tion flask using syringes under nitrogen atmosphere. The
reaction equipment was further degassed by three freeze-
pump-thaw cycles, and then immersed in a thermostated oil
bath. After predetermined polymerization time, the cooled
down reaction solution was precipitated in cold methanol
(150 mL) three times, and the solid was dried in vacuum at
ambience for 6 h in 41% yield (0.45 g).
1
H NMR (400 MHz, CDCl ), d (ppm): 7.20–7.34 (m, 10H,
3
2
C H ), 6.43 (d, J ¼ 12.0 Hz, 1H, CH ¼¼ CH), 6.14 (q, J ¼ 12.0
6
5
2
Hz, 1H, CH ¼¼ CH), 5.85 (d, J ¼ 12.0 Hz, 1H, CH ¼¼ CH), 5.17
2
2
(
dd, J ¼ 12.0 Hz, 4H, 2PhCH ), 4.69 (d, J ¼ 2.0 Hz, 1H, hydro-
2
gen on C-4 of AA), 4.28–4.42 (m, 2H, hydrogen on C-6 of AA),
.10–4.15 (m, 1H, hydrogen on C-5 of AA), 2.99 (br, 1H, OH
4
1
3
linking with C-5 of AA). C NMR (100 MHz, CDCl
3
), d (ppm):
1
1
3
69.7, 166.0, 157.0, 136.0, 135.4, 131.9, 129.2, 128.8, 127.9,
ꢀ
1
1
21.4, 75.7, 74.1, 73.8, 67.9, 64.9. IR (KBr disk, cm ): uOH
,
H NMR (400 MHz, CDCl ) d (ppm): for PLA block, 5.13–
3
ꢀ
1
ꢀ1
ꢀ1
440 cm (s), uCAH, 2956 cm (s), 2923 cm (vs), 2852
3
5.29 (m, nH, CH), 1.58 (s, 3nH, CH ); for PBnAAA block,
7.10–7.40 [m, 10mH (m representing repeating unit number
ꢀ
1
ꢀ1
ꢀ1
cm (s), u , 1764 cm (s) and 1728 cm (s). Calcd. for
C H O : C, 67.31; H, 5.40%. Found: C, 67.32; H, 5.50%.
C¼O
2
3
22
7
of PBnAAA block), 2C H ], 4.98 (m, 4mH, 2PhCH ), 4.58 (m,
6
5
2
mH, hydrogen on C-4 of AA), 4.07–4.38 (m, 3mH, hydrogen
on C-5 and C-6 of AA), 2.45 (m, mH, CH on the backbone),
Synthesis of Initiator HEBP
In a 100-mL three-necked flask containing ethylene glycol
1
3
1.75–1.97 (m, 2mH, CH
MHz, CDCl ) d (ppm): 175.1, 169.7, 157.3, 136.2, 137.2,
129.0, 128.7, 127.9, 121.5, 76.3, 74.2, 73.6, 69.1, 66.8, 41.2,
on the backbone). C NMR (100
2
(
16.6 g, 26.7 mmol) and triethylamine (2.8 g, 26.7 mmol) in
3
THF (40 mL), 2-bromopropionyl bromide (5.8 g, 26.7 mmol)
in THF (10 mL) was stepwisely added and kept stirring at
ꢀ
1
3
2
7.7, 20.6, 16.8. FTIR (KBr disk): uCAH, 2955 cm
(m),
ꢀ
1
ꢀ1
ꢁ
923 cm (m), u , 1755 cm (s).
0
C for 6 h. After filtration, the filtrate was sequentially
C¼O
ꢀ
1
ꢀ1
washed with 0.01 mol L HCl, 0.01 mol L NaOH, and dis-
tilled water. The combined organic phase was dried over an-
hydrous MgSO4 and concentrated. The crude product was
further purified by column chromatograph with the mixture
of ethyl acetate and hexane (v/v ¼ 1/3) as eluent to obtain
yellow liquid product in 63% yield (3.32 g).
Synthesis of Amphiphilic Block Copolymer PLA-block-
PAAA via Hydrogenation Under Normal Pressure
Palladium on carbon (10%, 63 mg) as catalyst was added
into the polymer solution comprised of poly(lactic acid)-
block-poly(benzyl ascorbyl acrylate) (PLA-block-PBnAAA; 630
mg), anhydrous THF (20 mL) and methanol (15 mL). The
benzyl deprotection was performed under normal H2 pres-
1
H NMR (400 MHz, CDCl ), d (ppm): 4.45 (q, J ¼ 8.0 Hz, 1H,
3
ꢁ
CH CHBr), 4.31 (m, 2H, CH OCO), 3.85 (t, J ¼ 4.0 Hz, 2H,
sure at 30 C for overnight, and then Pd/C catalyst was fil-
3
2
HOCH
2
CH
2
OCO), 2.03 (br, 1H, OH), 1.83 (d, J ¼ 4.0 Hz, 3H,
tered off and washed with methanol. After complete evapora-
tion of the combined filtrate, the solid was redissolved in a
small amount of THF and precipitated in cold ether (50 mL)
to obtain gray white solid in 63% yield (400 mg).
1
3
CH CHBr). C NMR (100 MHz, CDCl ), d (ppm): 170.5, 67.2,
3
3
ꢀ
ꢀ1
1
6
0.6, 39.9, 21.5. FTIR (KBr disk): uOH, 3434 cm (s), uCAH,
ꢀ
1
ꢀ1
2954 cm (m), 2923 cm (m), u , 1736 cm (s).
C¼O
3
990
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SCHEME 1 Synthesis pathway of monomer (A), initiator HEBP (B), and block polymers (C).
1
H NMR (400 MHz, DMSO-d ) d (ppm): for PLA block, 5.19
mated as the cross-point when extrapolating the intensity ra-
6
(
m, nH, CH), 1.45 (s, 3nH, CH ); for PAAA block, 4.64 (m,
tio I338/I333 at low- and high-concentration regions.
3
mH, hydrogen on C-4 of AA), 4.04–4.21 (m, 3mH, hydrogen
on C-5 and C-6 of AA), the main chain signals of PAAA block
Turbidity Measurement and Micelle Formation
ꢀ
1
Turbidity measurements were performed according to the
were covered by DMSO-d . FTIR (KBr disk): uOH, 3438 cm
6
2
1
ꢀ
1
ꢀ1
ꢀ1
published procedures. Deionized water was added at a
(
(
s), uCAH, 2955 cm (m), 2924 cm (m), uC¼O, 1758 cm
s).
ꢀ
1
rate of 5 lL min to the polymer in dioxane or DMSO (0.5
wt %, 2 mL) under slight shaking. After each addition of
water, the solution was left to equilibrate for a while until
the optical intensity was stable. The optical intensity (turbid-
ity) was measured at a wavelength of 600 nm using a quartz
cell (path length: 1 cm) with Hitachi U-3010 UV–vis spec-
trometer. The cycle of water addition, equilibrium, and mea-
surement was continued until the desired amount of water
was achieved (50–70 wt %). The micelle solution was
obtained by dialyzing the mixture against deionized water
Characterization of Thermal Physical Properties
DSC experiments were conducted on a Mettler Toledo. The
polymer sample (8.0–10.0 mg) encapsulated in an aluminum
ꢁ
ꢀ1
pan was run at a scanning rate of 20 C min over a tem-
ꢁ
perature range of 25–190 C with ultra pure nitrogen as
purge gas. Melting points (T ) were evaluated using the top
m
temperatures of the corresponding main peak. Thermogravi-
metric analysis (TGA) was used on a Mettler Toledo TGA,
and the prepared copolymer (4.0–5.0 mg) was scanned at
(
500 mL) for 2 days during which the water was refreshed
ꢁ
ꢀ1
ꢁ
1
0 C min from 30 to 700 C under nitrogen atmosphere
every 4 h.
ꢀ
1
at a flowing rate of 50 mL min . Peak top temperature
Tds) occurring in the differentiated TGA (dTGA) traces was
(
RESULTS AND DISCUSSION
used to assess thermal degradation behavior and stabilities
for the synthesized polymers.
Synthesis and Characterization of PLA-block-PAAA
Among a variety of the reported polymerization methodolo-
gies, ATRP and ROP are two disparate polymerization techni-
ques. ATRP is available to acrylate-series monomers, and
ROP favors for lactone monomers polymerization. A combi-
Critical Micelle Concentration
Critical micelle concentration (CMC) of the amphiphilic block
copolymer was determined by steady-state fluorescence
spectrometry with pyrene as a fluorescent probe. The con-
nation of ATRP and ROP is one efficient strategy to fabricate
–
5
22–26
centration of block polymer was varied from 1.0 ꢃ 10 to
various materials.
As outlined in Scheme 1(C), the syn-
ꢀ
1
2
1
.0 mg mL , and the concentration of pyrene was fixed at
thesis route of PLA-block-PAAA comprises of ROP, ATRP, and
hydrogenation. The initiator HEBP is one key to perform the
combination of ATRP and ROP, and it is also a bridge to link
PLA and PAAA blocks in the resulting polymers. Based on
the preparation of HEBP by the reaction of 2-
–
3
.73 ꢃ 10 lM. The fluorescence spectra were recorded
using Hitachi F-4500 Fluorescence spectrometer with the
emission wavelength of 395 nm, and the excitation fluores-
cence was monitored at 338 and 333 nm. CMC was esti-
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bromopropionyl bromide with ethylene glycol [Scheme 1(b)],
homopolymers PLA with different molecular weights were
carried out in the presence of stannous octoate via ROP, and
the polymerization results are summarized in Supporting In-
formation Table S1. Subsequently, PLA bearing 2-bromopro-
pionyl group initiated the polymerization of monomer BnAAA
(
its preparation route is depicted in Scheme 1(A)) via ATRP to
fabricate a series of diblock copolymers PLA-block-PBnAAA.
The chemical structures of homopolymer PLA and block co-
1
polymer PLA-block-PBnAAA were evidenced by H NMR tech-
1
niques, and the typical H NMR spectra are depicted in Fig-
ure 1. For homopolymer PLA, the proton signals of methine
and methyl on the repeating unit appeared at 5.16 and
1.57 ppm, respectively, and the proton peaks from the initia-
tion moiety [as marked c, d, and e in Fig. 1(A)] were over-
lapped ꢂ4.37 ppm. It is noteworthy that degree of polymer-
ization (DP) for lactide listed in Supporting Information
Table S1 was calculated by comparing the integrals at 5.16
and 1.81 ppm associated with methyl peak (f) in the initia-
tion segment. For PLA-block-PBnAAA, besides the signals of
PLA block appearing at corresponding chemical shift, the
proton (i, h, g) signals on C-4, C-5, and C-6 [marked in
Scheme 1(A)] of AA moiety were found at 4.56, 4.25, and
4
7
(
.06 ppm [Fig. 1(B)], respectively. The peaks at 4.97 and
.18ꢂ7.30 ppm were attributed to the respective methylene
j) and phenyl ring (k, l, m) of benzyl group in the PBnAAA
block. The methine (f) and methylene (e) protons on
PBnAAA backbone were present at 2.42 and 2.00 ppm.
Based on the aforementioned DP of PLA block calculated
1
from H NMR results, DP of PBnAAA block was estimated by
comparing the integrals at 5.16 and 4.56 ppm, as summar-
ized in Table 1. Additionally, GPC analysis for the block poly-
mers PLA-block-PBnAAA revealed unimodal and narrow dis-
tribution (1.25–1.39) without macroinitiator PLA (Fig. 2),
which further substantiated the successful synthesis of struc-
turally well-defined block copolymers PLA-block-PBnAAA.
The hydrogenation to remove benzyl group was performed
using Pd/C as catalyst under normal pressure at room tem-
1
perature. One representative H NMR spectrum of PLA-block-
PAAA is shown in Figure 1(c), in which the peaks associated
with PLA block (d ¼ 5.16 and 1.57 ppm) and AA units (d ¼
4.63, 4.05 and 4.19 ppm) were obviously observed. It is of
great importance that the signals at 7.28 and 4.95 ppm
assigned to phenyl ring and methylene of benzyl group
almost completely disappeared, indicating that the hydroxyl
groups at 2 and 3 positions of AA skeleton were successfully
set free. Because of the presence of deuterated DMSO’s sig-
nal, the methine signals of PAAA backbone were hidden at
1
1
FIGURE 1 (A) H NMR spectrum of homopolymer PLA-3. (B)
H
1
NMR spectrum of PLA-block-PBnAAA (63/10). (C) H NMR spec-
trum of PLA-block-PAAA (63/10).
2.42 ppm. Depending on the ratio change of the integrals for
the phenyl ring (d ¼ 7.28 ppm) and methine of PLA block
resultants PLA-block-PAAA can selectively dissolve in strong
polar solvent such as DMSO or DMF. The solubility changing
before and after hydrogenation indirectly supported the gen-
eration of amphiphilic block copolymers.
(
d ¼ 5.16 ppm) before and after hydrogenation, the meas-
ured hydrogenation efficiency is over 90% for these amphi-
philic block copolymers. The ratio of PLA to PAAA block
estimated by comparing the integrals at 5.16 to 4.63 ppm is
close to the composition ratio of the copolymers before hy-
drogenation, as tabulated in Table 1. Chloroform is a good
solvent for PLA-block-PBnAAA, whereas the hydrogenation
Thermal Physical Properties of PLA-block-PAAA
The thermal stability of the amphiphilic copolymers PLA-
block-PAAA was evaluated using TGA. Figure 3(A) shows the
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TABLE 1 Molecular Characteristics of Block Copolymers PLA-block-PBnAAA and PLA-block-PAAA
PLA-block-PBnAAA
PLA-block-PAAA
Molar ratio
b
c
d
e
DP of
MI
of MI and
Monomer
Temp.
Time
(h)
DPratio
M
n
M
n
M
w
/M
n
Hydrogenation
DPratio
(NMR)
a
ꢁ
f
g
MI
( C)
(NMR)
(NMR)
(GPC)
(GPC)
Efficiency (%)
PLA-1
PLA-2
PLA-3
PLA-3
a
40
44
63
63
1/25
1/40
1/40
1/40
80
70
70
80
15
40
40
40
40/5
7,800
10,400
11,900
13,100
9,700
10,900
13,300
13,500
1.25
1.39
1.26
1.34
90
90
99
97
40/5
44/9
63/7
63/10
44/10
63/7
63/10
e
f
PLA as macroinitiator was tabulated in Supporting Information Table
S1 in detailed.
Obtained by GPC.
Calculated by H NMR results.
1
b
g
DPratio represented polymerization degree ratio of PLA block to
DPratio represented polymerization degree ratio of PLA block to PAAA
1
1
PBnAAA block measured by H NMR.
block measured by H NMR.
c
1
M
M
n
(NMR) represented molecular weight calculated by H NMR results.
n
(GPC) indicated molecular weight determined by GPC
d
measurement.
weight-loss curves for a series of amphiphilic copolymers
compared with homopolymers PLA and PAAA, and the peak
crystalline behavior of PLA-block-PAAA was measured by
WAXD, which was in agreement with DSC results. As shown
in Figure 3(C), homopolymer PAAA presented obvious
top temperature (T s) occurring in the differentiated TGA
d
traces was used to quantitatively assess thermal degradation
behavior. For homopolymer PLA, no obvious weight loss hap-
ꢁ
pened before 200 C and its rapid thermal degradation
ꢁ
occurred at ꢂ257 C. Interestingly, the degradation behavior
of homopolymer PAAA (M
exhibited three stages in the course of measurement [the
n
¼ 9200 and M
w
/M
n
¼ 1.18) was
inset of Fig. 3(A)]. Moreover, when PAAA was heated to
ꢁ
7
00 C, nearly 40% of the initial weight remained. For the
synthesized copolymers PLA-block-PAAA, their T s between
d
ꢁ ꢁ
3
00 and 340 C were 50–80 C higher than homopolymer
PLA, indicating that PAAA introduction is capable of improv-
ing the thermal stability for the overall copolymer. Except
the copolymer PLA-block-PAAA (44/9) (44 and 9 stand for
DP of lactide and ascorbyl acrylate) showing stepwise two-
step thermal degradation, the other copolymers showed only
one-step degradation profiles. For the copolymers PLA-block-
PAAA (63/7) and PLA-block-PAAA (63/10) with identical
PLA length, PLA-block-PAAA (63/10) having long PAAA block
presented slightly higher thermal stability (T value is ꢂ331
d
ꢁ
C). For these four block polymers, more than 20% of overall
ꢁ
weights still remained above 500 C, which was probably
the result of the incomplete degradation of PAAA part and
residual Pd/C.
The melting behavior of the amphiphilic block copolymers
was investigated by DSC, and the heating scans were exhib-
ited in Figure 3(B). Homopolymer PLA-3 had one T
m
at
ꢁ
ꢁ
1
45 C and one T around 66 C which are similar to those
g
2
7
reported previously, whereas homopolymer PAAA (Mn
¼
9
200 and M /Mn ¼ 1.18) did not show obvious T . For
w
m
block copolymers PLA-block-PAAA, they only exhibited Tm
and Tg of PLA block, among which Tm of PLA block was
lower than the corresponding homopolymer, possibly
because of the presence of PAAA blocks confining the melt-
ing behavior of PLA segment. For PLA-block-PAAA (44/9)
and PLA-block-PAAA (63/7), one endothermic peak appeared
FIGURE 2 (A) GPC traces of PLA-3 and PLA-block-PBnAAA (63/
10). (B) GPC traces of PLA-block-PBnAAA (40/5, a), (44/10, b),
(63/7, c), and (63/10, d).
ꢁ
between 60 and 80 C covering T of PLA block, and the rea-
g
son accounting for the peak remained unclear so far. The
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aqueous solution. CMC determination was carried out for
these copolymers by the use of fluorescence probe tech-
2
9–32
nique.
The characteristic shift of pyrene excitation spec-
tra from 333 to 338 nm indicating pyrene partition into the
hydrophobic core of the micelle was utilized to determine
the CMC of the block copolymers in aqueous solution. Figure
4(A) shows the intensity ratio of I338/I333 in the pyrene exci-
tation spectra as a function of the logarithm of the copoly-
mer concentrations. A negligible change in the intensity ratio
of I338/I333 was detected at the low-concentration range,
whereas the intensity ratio exhibited a substantial increase
with the copolymer concentration increasing, indicating that
the pyrene molecules incorporated into the hydrophobic
region. The I338/I333 versus log C plot presented one sigmoid
curve; therefore, the CMC value was determined from the
cross-over point in the low-concentration range. The CMCs
ꢀ
1
of the copolymers ranged between 13 and 28 mg L
in
Table 2. Moreover, for these amphiphilic polymers having
FIGURE 3 (A) TGA curves of homopolymers PLA, PAAA, and block
polymers PLA-block-PAAA. (B) DSC heating curves of PLA (a),
PAAA (b), PLA-block-PAAA (40/5, c), PLA-block-PAAA (44/9, d), PLA-
block-PAAA (63/7, e), and PLA-block-PAAA (63/10, f). (C) WAXD pat-
ꢁ
terns of PLA, PAAA, and PLA-block-PAAA (63/10) at 25 C.
amorphous phase, and PLA-block-PAAA (63/10) have only
two crystalline peaks appearing at 2y ¼ 16.6ꢁ and 19.0 ,
corresponding to (200)/(110) and (203) reflections of PLA
block, respectively. Other block copolymers had identical
WAXD results (not shown).
ꢁ
FIGURE 4 (A) Plots of I338/I333 ratio of pyrene excitation spectra
in water as a function of PLA-block-PAAA (44/9) concentration
2
8
ꢁ
at 25 C. (B) Turbidity (optical density) curves of the amphi-
philic polymers solution as a function of the amount of deion-
ized water added to the polymer solution. The turbidity values
for water addition lower than 10 wt % are close to zero. The
curves of PLA-block-PAAA (40/5), (63/10), and (63/7) are shifted
for clarity.
Self-Assembly of Amphiphilic Block Copolymers
PLA-block-PAAA in Aqueous Solution
The amphiphilicity of the copolymers PLA-block-PAAA pro-
vides one good possibility to form assembly aggregates in
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TABLE 2 Solution Properties of Polymeric Micelles Formed
From PLA-bock-PAAAs
size component about 300 nm, and their mean hydrody-
namic diameters were ꢂ216 nm (Table 2). In theory, the
contour length of the polymer PLA-block-PAAA (63/7) is cal-
culated to be ꢂ32 nm, with the PLA segment estimated to
be ꢂ30 nm and the PAAA moiety estimated to be ꢂ1.8 nm
(it is assumed that carbon–carbon single bond length is
about 1.54 Å, and carbon–oxygen single bond length is
ꢂ1.40 Å). However, the mean particle size in the DLS mea-
surement is much larger than that calculated, indicating that
the observed spherical nanoparticles are probably micellar
DLS
a
CMC
Mean
b
c
Amphiphilic Copolymers
(mg/L)
Diameter (nm)
PDI
PLA-block-PAAA(40/5)
PLA-block-PAAA(44/9)
PLA-block-PAAA(63/7)
PLA-block-PAAA(63/10)
13.4
25.6
21.1
27.2
180.9
135.6
216.5
42.2
0.148
0.209
0.171
0.157
3
3,34
aggregates.
The similar observations were performed as
shown in Supporting Information Figures S5 and S6. The mi-
cellar sizes in the majority for all of these polymers are
below 150 nm, particularly for PLA-block-PAAA (44/9) and
PLA-block-PAAA (63/10), which have potential to increase
the accumulation possibility of these nanocarriers at patho-
a
b
c
ꢁ
Measured at 25 C.
ꢁ
The particle average dimension determined by DLS at 25 C.
PDI meant polydispersity index of particle dimension measured by
DLS.
3
5
logical sites.
identical PLA block length, the effect of shorter hydrophilic
block is evident in the lower CMC value, indicating a greater
driving force for the self-assembly in aqueous solution.
Dioxane and DMSO are good nonselective solvents for PLA
and PAAA, whereas water is a good selective solvent for
PAAA but is poor for PLA. Therefore, the addition of water
into the amphiphilic copolymers in dioxane would induce
2
1
the self-assembly of amphiphilic polymer PLA-block-PAAA.
On the progressive addition of water into the corresponding
polymer solution at the concentration of 0.5 wt %, the tur-
bidity diagrams of the copolymers PLA-block-PAAA were
obtained using UV–vis spectrometer. As shown in Figure
4(B), the turbidity curves exhibited one jump on the gradual
addition of water, followed by the turbidity values reaching a
plateau, which is probably associated with the formation of
the nanoscaled aggregates. The polymers having long PLA
blocks, such as PLA-block-PAAA (63/7) and (63/10), showed
relatively fast and obvious jump when water content reached
10% of the whole mixture, whereas the polymers bearing
short PLA blocks did not exhibit certain nanoaggregation
behavior until water content was over 40%. These results
further suggest the amphiphilic polymers bearing long
hydrophobic block have stronger driving force of self-assem-
bly in aqueous solution. The final turbid mixtures were
transferred into the dialysis bag (cut-off molecular weight
3500) and extensively dialyzed against deionized water for
complete removal of organic solvent. The morphology and
size distributions of the copolymer aggregates were investi-
gated by TEM observation and DLS, respectively.
All of the samples were not filtered before TEM and DLS
measurements so that the pristine state of the dissolved co-
polymer was analyzed in this study. The morphology of the
polymeric aggregates was examined by cryoTEM. Figure 5(A)
presents one representative cryoTEM pattern for the copoly-
mer PLA-block-PAAA (63/7), in which the aggregates exhib-
ited the closely spherical polymeric micelles with diameter
range of 100–150 nm. The micellar size of PLA-block-PAAA
(
63/7) was further determined by DLS [Fig. 5(B)]. These
FIGURE 5 (A) CyroTEM image of PLA-block-PAAA (63/7).
(B) DLS graph for micellar size distribution of PLA-block-PAAA
(63/7).
polymeric micelles had characteristic bimodal distributions
with a small size component around 120 nm and a large
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CONCLUSIONS
12 Cioffi, N.; Losito, I.; Terzano, R.; Zambonin, C. G. Analyst
2
000, 125, 2244–2248.
Based on the reported special physiological function of AA
and combined with drug delivery nanotechnology, one type
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copolymers PLA-block-PAAA with biodegradable PLA as
hydrophobic block and potential targeting PAAA as hydro-
philic block was explored for the first time. The synthesis of
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ROP of lactide, ATRP of BnAAA, and hydrogenation with Pd/
C as catalyst under normal pressure. The CMC values of the
copolymers measured by fluorescence probe techniques
were in the range of 13 and 28 mg L . These amphiphilic
polymers self-assembled to form spherical micellar aggre-
gates with average size in the range of 40–220 nm, which is
suitable to the circulation in vivo. This study explored the
possibility of polymeric AA as hydrophilic block in the
amphiphilic copolymers, and this class of nanocarriers could
be used as a selective drug delivery to facilitate the potential
therapeutic for the treatment of some brain-associated dis-
eases and tumor cure. Further investigation on their biocom-
patibility and targetability is underway in our laboratory.
1
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