Effect of block lengths on the association behavior of poly(l-lactic acid)/poly(ethylene glycol) (PLA-PEG-PLA) micelles in aqueous solution

Article abstract of DOI:10.1007/s13738-013-0319-0

A series of poly(l-lactic acid)/poly(ethylene glycol) triblock copolymers with a PLA-PEG-PLA architecture were synthesized by a ring-opening polymerization (ROP) process. The copolymers were characterized by ¹H NMR and GPC. The total number average molecular weights were in the range of 4,700-50,000, whereas the degrees of polymerization of the PLA and PEG blocks varied from 15 to 359 and from 68 to 136, respectively. The self-association of these copolymers in aqueous environment was studied by emission fluorescence spectroscopy of anilinonaphthalene probe and the critical association concentration (CAC) of the copolymers was measured. It was found that the micellization process of these copolymers was mainly determined by the length of the hydrophobic LA block, while the length of the hydrophilic PEG block had little effect. Furthermore, the low CAC values of the copolymers suggest that the copolymers form stable supramolecular structures in aqueous solutions.

Full text of DOI:10.1007/s13738-013-0319-0

J IRAN CHEM SOC (2014) 11:467–470
DOI 10.1007/s13738-013-0319-0
ORIGINAL PAPER
Effect of block lengths on the association behavior of poly(L-lactic
acid)/poly(ethylene glycol) (PLA–PEG–PLA) micelles in aqueous
solution
Pouneh S. Pourhosseini
Ali A. Saboury Farhood Najafi
•
•
Reza Amani
•
•
Mohammad Imani
Received: 8 April 2013 / Accepted: 17 July 2013 / Published online: 17 August 2013
Ó Iranian Chemical Society 2013
Abstract A series of poly(L-lactic acid)/poly(ethylene gly-
col) triblock copolymers with a PLA–PEG–PLA architecture
were synthesized by a ring-opening polymerization (ROP)
Introduction
Biodegradable diblock copolymers of poly(ethylene glycol)
(PEG) and poly(lactic acid) (PLA–PEG), and their corre-
sponding triblocks are widely used for fabricating nanopar-
ticles (NPs) as drug delivery carriers. The amphiphilic
character of these copolymers enables the formation of
micelles in water through a self-assembly process: the
hydrophobic PLA blocks aggregate to form a core reservoir
for hydrophobic drugs and the hydrophilic PEG segments
serve as the shell projecting into the aqueous environment
[1–3]. In recent years, most research has focused on the drug
encapsulation and release behavior [4]. In contrast, only a
few studies have been performed on the micellization prop-
erties of PLA/PEG block copolymers [2, 5]. It is known that
the micelle assembly is controlled by the relative molecular
weight of the hydrophobic and hydrophilic blocks. However,
not much is known about how the ratio of hydrophobic and
hydrophilic blocks influences the micellization behavior of
copolymers. In this study, a series of PLA–PEG–PLA tri-
block copolymers with various block lengths and molecular
weights were synthesized. The characterization of the
copolymers, the formation of micelles and their critical
association concentration (CAC) in aqueous solutions are
reported. The objective of this study is to elucidate the effect
of block lengths on the CAC values of the micelles, which is
of great interest for applications in the field of drug delivery.
1
process. The copolymers were characterized by H NMR and
GPC. The total number average molecular weights were in
the range of 4,700–50,000, whereas the degrees of polymer-
ization of the PLA and PEG blocks varied from 15 to 359 and
from 68 to 136, respectively. The self-association of these
copolymers in aqueous environment was studied by emission
fluorescence spectroscopy of anilinonaphthalene probe and
the critical association concentration (CAC) of the copoly-
mers was measured. It was found that the micellization pro-
cess of these copolymers was mainly determined bythe length
of the hydrophobic LA block, while the length of the hydro-
philic PEG block had little effect. Furthermore, the low CAC
values of the copolymers suggest that the copolymers form
stable supramolecular structures in aqueous solutions.
Keywords Poly(lactic acid) Á Triblock copolymer Á
Block length Á Polymeric micelles
P. S. Pourhosseini (&) Á R. Amani Á A. A. Saboury
Institute of Biochemistry and Biophysics, University of Tehran,
Tehran 1417614411, Iran
e-mail: pourhosseini@hotmail.com
A. A. Saboury
Center of Excellence in Biothermodynamics, University
of Tehran, Tehran 1417614411, Iran
Experimental
F. Najafi
Department of Resin and Additives, Institute for Color Science
and Technology, Tehran 1668814811, Iran
Polymer synthesis and characterization
M. Imani
Iran-Polymer and Petrochemical Institute,
Tehran 115/14965, Iran
PEG was dried at 40 °C in vacuum overnight prior to use.
Toluene was dried by storing with metal sodium in a bottle.
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J IRAN CHEM SOC (2014) 11:467–470
Scheme 1 Synthesis of
L-lactide and PLA–PEG–PLA
triblock copolymers
Synthesis of L-lactide
stirred for 30 min at room temperature (RT), and then soni-
cated 15 min to obtain a homogenized dispersion. Desired
concentrationswere preparedbydilution of the stocksolutions.
L-Lactide was synthesized from L-lactic acid, using antimony
trioxide (Sb O ) as a catalyst. The reaction was composed of
2
3
Solvent injection
two steps: (a) oligomerization at 180–200 °C, and (b) depo-
lymerization and then dimerization at 250 °C. The produced
L-lactide was recrystallized three times from ethyl acetate and
dried in vacuum at 40 °C overnight before use (Scheme 1).
A solution of P copolymer in DMF (15 mg/mL) was added
5
dropwise to stirring buffer (PBS) to produce 0.3 mg/mL
dispersion. The micellar dispersion was further stirred and
then sonicated. Dilution of this stock solution was performed
with diluting buffer, containing the same amount of DMF as
the stock solution.
Synthesis of PLA–PEG–PLA triblock copolymers
Triblock copolymers were synthesized by a ring-opening
polymerization (ROP) process [3]. In brief, a given amount of
purified L-lactide was charged into a 100 mL round bottle flask
containing pre-dried PEG, with the protection of nitrogen.
Stannous octoate dissolved in dry toluene was transferred into
the flask [Stannous octoate/lactide, 0.15 % (w/w)]. The mix-
ture solution was heated to 130 °C and allowed to reflux for
Critical association concentration (CAC)
ANS was used as a hydrophobic fluorescent probe. A given
amount of the ANS stock solution in PBS (2 mM) was added
to each of a series of the copolymer dispersions, followed by
adding different amounts of buffer. The samples obtained,
12 h (Scheme 1). The products were purified by repeated dis-
-4
containing a fixed concentration (1.0 9 10 M) of ANS and
varying concentrations (0.003–2.8 mg/mL; P5: 0.0005–0.28
mg/mL) of the copolymer, were stirred for 2 h at RT to
achieve equilibration. Fluorescence measurements were per-
formed at RT. Each spectrum was scanned three times to
acquire the final fluorescence emission spectra.
solution into dichloromethane and precipitated by cold ethanol.
The isolated polymers were dried at 40 °C under vacuum for
24 h. Various PLA–PEG–PLA triblock copolymers with dif-
ferent compositions were prepared using the same procedure.
1
The copolymers characterized by GPC and H NMR.
Micellar dispersions
Results and discussion
Direct dissolution
Copolymer characterization
Stock solutions were prepared directly by dissolving a given
amount of copolymer (P , P , P , and P ) in phosphate-
1
Figure 1 shows the typical H NMR spectrum of P . The
1
2
3
4
3
buffered saline (PBS), pH 7.4 (10–15 mg/mL). Samples were
peaks of CH and CH in PLA blocks appear at 1.6 and
3
1
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J IRAN CHEM SOC (2014) 11:467–470
469
5
.2 ppm, respectively, and the peak of main chain methy-
CAC determination
lene units in PEG blocks is shown at 3.7 ppm. The a-
methylene protons of PEG connected with PLA is clearly
present at 4.3 ppm, indicating that the copolymerization of
LA and PEG really takes place. The result corresponds to
other reports on PLA/PEG block copolymers [3–5]. GPC
measurements show monomodal molecular weight distri-
butions, implying that no homopolymeric fractions of both
PLA and PEG are present in products (not shown). Table 1
summarizes the molecular characteristics of triblock
copolymers. It can be seen that copolymers with different
molecular weights and compositions can be obtained by
copolymerizing LA and PEG under controlled conditions.
The number average molecular weight of the copolymers
fell into the range of 4,700–50,000. The polydispersity
indices (PDI = M /M ) of the copolymers P , P , and P
4
One of the usual ways to measure the critical association
concentration (CAC) of copolymers is by the fluorescence
probe technique. Here, ANS was employed as a fluorescent
probe. Changes in ANS fluorescence on micelle formation
are used to establish the CAC, which is a measure of the
stability of the associates [2]. Typical emission spectra of
ANS in aqueous solutions of P copolymer at various
3
concentrations are shown in Fig. 2a. The two noteworthy
features of these spectra are that the fluorescence intensity
shows a marked enhancement with increasing copolymer
concentration, and that there is a progressive shift in the
emission maximum toward the blue, from about
532–488 nm. These changes are due to the transfer of ANS
molecules from the aqueous environment to the hydro-
phobic parts of the micellar structures and thus provide
information on the location of ANS probe in the system [6].
Figure 2b shows the fluorescence intensity at kmax as a
w
n
2
3
are inferior to 1.4. It indicates the narrow molecular weight
distribution of these copolymers, which is a critical factor
for forming nanoparticles with similar structures.
function of P copolymer concentration. It is observed that
3
the intensity has a negligible change below a certain con-
centration and increases substantially thereafter. The CAC
is taken as the intersection of the two straight lines drawn
through the points at the lower copolymer concentrations,
lying on a nearly horizontal line, and the points on the
rapidly rising part of the plot. This indicates a CAC of
about 20 lM. Table 1 lists the CAC values of the five
triblocks. The copolymers have low CAC values, which
indicate a good stability of the micelles in aqueous solu-
tions. The values also vary with block lengths. Figure 3
shows the logarithmic CAC values versus the total LA
monomer units in a polymer chain. The data points are
2
basically on the straight line (R = 0.95). The longer the
LA block length, the smaller the CAC is.
Similar studies have been performed on some surfac-
tants in aqueous solutions. They have reported that both
blocks could influence the CAC value with the hydropho-
bic block having a strong negative effect and the hydro-
philic block having only a minor positive effect [7]. In our
case, a comparison of the data points of copolymer
1
Fig. 1 H NMR spectrum of P
3
copolymer, in CDCl
3
Table 1 Molecular characteristics of PLA–PEG–PLA triblock copolymers and their CAC values in aqueous solutions
1
6
Number
M
nPEG
H NMR
GPC
CAC 9 10
mol/L)
(
a
Block ratio (LA/EO) in product
Copolymer
M
n
w n
M /M
P
1
P
2
P
3
P
4
P
5
a
3,000
3,000
4,000
6,000
6,000
0.44
1.91
2.90
0.54
5.28
LA15–EO68–LA15
LA65–EO68–LA65
LA132–EO91–LA132
LA37–EO136–LA37
LA359–EO136–LA359
4,750
12,406
22,994
14,540
49,631
1.7
150.3
42.3
20
1.26
1.18
1.33
1.77
46.5
2
Copolymers are denoted by lactic acid–ethylene oxide–lactic acid followed by the degrees of polymerization of the blocks
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J IRAN CHEM SOC (2014) 11:467–470
Fig. 2 a Fluorescence spectra of ANS in PBS with increasing concentrations of P
1
3
copolymer (arrow direction). Excitation and emission slits:
0.0 nm, scan rate: 600 nm/min, kex = 370 nm, 25 °C. b Plot of the fluorescence intensity at kmax versus the P
3
concentration
technique. The results show that the length of the hydro-
phobic end LA blocks is the dominant factor on the CAC
values of the micelles. Increasing the LA block length
enhances the hydrophobicity of the copolymer chains.
Thus, they can self-assemble more easily to form micelles,
leading to lower CAC values. The effect of the EO block
length is insignificant. Furthermore, the results show that
the copolymers have low CAC values indicating the
desirable stability of the micelles.
Acknowledgments The authors thank the Iran National Science
Foundation (INSF) and Research Council of University of Tehran for
their financial support.
Fig. 3 Plot of logarithmic CAC values of PLA–PEG–PLA triblock
copolymers in aqueous solution versus number of LA units in a
polymer chain at 25 °C. The data points were labeled as L
n m n
E L .
References
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1
2
3
4
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1
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(
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6
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Conclusion
The self-assembly of PLA–PEG–PLA triblock copolymers
in aqueous solutions was studied by the fluorescence probe
1
23