Novel amphiphilic diblock copolymers bearing acid-labile oxazolidine moieties: Synthesis, self-assembly and responsive behavior in aqueous solution

Article abstract of DOI:10.1016/j.polymer.2011.02.022

A novel oxazolidine based acid-labile monomer N-acryloyl-2,2-dimethyl-1,3- oxazolidine (ADMO) was synthesized and polymerized by reversible addition fragmentation chain transfer (RAFT) polymerization using poly(ethylene glycol) based chain transfer agent (PEG-CTA). The diblock copolymers PEG-b-PADMO were composed of hydrophilic PEG with fixed length and hydrophobic PADMO with different lengths, which formed core-shell micelles in water. Morphologies and sizes of micelles were obtained by transmission electron microscopy (TEM) and dynamic light scattering (DLS), which showed that the shapes of polymeric aggregates developed from small spherical micelles, worm-like micelles to larger size of vesicles, as the length of PADMO increased. The hydrolysis kinetics of the micelles was studied using ¹H NMR, DLS and release of loaded Nile Red dye, whose rate strongly depended on pH and micellar structure. It led to the disruption of polymeric micelles and concomitant release of the guest molecules, due to the transformation of hydrophobic PADMO into hydrophilic poly(2-hydroxyethyl acrylamide) (PHEAM).

Full text of DOI:10.1016/j.polymer.2011.02.022

Polymer 52 (2011) 1755e1765
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Novel amphiphilic diblock copolymers bearing acid-labile oxazolidine moieties:
Synthesis, self-assembly and responsive behavior in aqueous solution
,
,
a
Qianling Cui a b, Feipeng Wu a , Erjian Wang
*
a Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, PR China
b Graduate University of Chinese Academy of Sciences, Beijing 100049, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel oxazolidine based acid-labile monomer N-acryloyl-2,2-dimethyl-1,3-oxazolidine (ADMO) was
synthesized and polymerized by reversible addition fragmentation chain transfer (RAFT) polymerization
using poly(ethylene glycol) based chain transfer agent (PEG-CTA). The diblock copolymers PEG-b-PADMO
were composed of hydrophilic PEG with fixed length and hydrophobic PADMO with different lengths,
which formed core-shell micelles in water. Morphologies and sizes of micelles were obtained by
transmission electron microscopy (TEM) and dynamic light scattering (DLS), which showed that the
shapes of polymeric aggregates developed from small spherical micelles, worm-like micelles to larger
size of vesicles, as the length of PADMO increased. The hydrolysis kinetics of the micelles was studied
using 1H NMR, DLS and release of loaded Nile Red dye, whose rate strongly depended on pH and micellar
structure. It led to the disruption of polymeric micelles and concomitant release of the guest molecules,
due to the transformation of hydrophobic PADMO into hydrophilic poly(2-hydroxyethyl acrylamide)
(PHEAM).
Received 27 October 2010
Received in revised form
30 December 2010
Accepted 16 February 2011
Available online 23 February 2011
Keywords:
Amphiphilic diblock copolymers
pH-responsivity
RAFT polymerization
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
or chemical properties in response to external stimuli such as pH
[13], temperature [14,15], ionic strength [16], light [17], redox
Stimuli-responsive polymers have attracted growing interests in
recent years, due to their potential applications in fields such as
catalysis, drug and gene delivery, separation and purification of
biomolecules and biosensor etc [1e3]. Amphiphilic block copoly-
merswith stimuli-responsiveelementsare ofparticularimportance.
These copolymers self-assemble in solution into various ordered
structures such as spherical micelles, worm-like or cylindrical
micelles, and vesicles, controlled by the ratio of the block lengths,
polymer concentration, temperature, additives and other factors
[4e8]. Polymeric micelles have several advantages, such as unique
core-shell architecture, which provides interiors that can non-
covalently encapsulate guest molecules. The micelles, with average
size typically between 20 and 100 nm, can accumulate the drug at
target sites via an enhanced permeation and retention (EPR) effect
[9]. In addition, the polymeric micelles are easily prepared, and
provide design flexibility for controlling nanostructure and func-
tionality [10e12]. Based on this concept, many stimuli-responsive
polymeric micelle systems have been designed and prepared based
on amphiphilic block copolymers, which can change their physical
potential [18,19] and other environmental variables.
The pH-sensitive polymers are among the most important
stimuli-responsive materials. They are of particular interest for
biomedical and biotechnological applications, since there are
numerous pH gradients existing in both normal and pathological
states, which provide a potential localized trigger for the release of
drugs from acid-sensitive carriers [20]. According to the difference
ofresponsemechanism, pH-sensitive polymers can be classified into
two types. Typical pH-sensitive polymers contain reversible ioniz-
able segments such as polyacids and polybases [21,22], whose
properties change reversibly by protonation or deprotonation as the
pH of the surroundings changes. Another strategy for preparation of
pH-sensitive polymers is introducing acid-labile linkages or moie-
ties such as acetals [23e30], orthoesters [31e35], hydrazones
[36e38], anhydrides [39] and others [40,41] into the polymer main
or side chain. Acid-catalyzed cleavage of these linkers induces
disruption of the micelles or vesicles and consequential release of
loaded guests. Kataoka et al. [42,43] prepared polymeric micelles by
attaching doxorubicin (DOX) to amino acid units of PEG-b-P(Asp) via
hydrazone linkages. Park and coworkers [44] reported a block
copolymer (PEG-b-PLLA) with terminal conjugated DOX through
a hydrazone or cis-acotinyl bond. These acid-sensitive micelles had
much faster release of free DOX at acid pHs than at neutral pH.
* Corresponding author. Tel.: þ86 10 82543569; fax: þ86 10 82543491.
E-mail address: fpwu@mail.ipc.ac.cn (F. Wu).
0032-3861/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.polymer.2011.02.022

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Q. Cui et al. / Polymer 52 (2011) 1755e1765
The acetals and orthoesters are two kinds of widely used acid-
2. Experimental section
sensitive linkages for pH-responsive micelles or other nanoparticles,
whose hydrolysis are generally proportional to the hydronium ion
concentration and the products are diol and ketal [45]. Their hydro-
lysis rates can be designed and controlled by the degree of substitu-
tion and substituent structure [46]. Fréchet and coworkers [23]
provided an approach to acid-sensitive micelles by attaching
hydrophobes tooneblock ofdiblock copolymersvia anacetallinkage.
Since common aliphatic acetals generally hydrolyze slowly, the
introduction of benzylidene acetals with electron-donating methoxy
groups in the ortho and para positions accelerated the hydrolysis rate,
which increased rapidly as the pH changed from 7.4 to 5.0. However,
the aromatic hydrolysis product is not a biocompatible and biode-
gradable material. This problem has been avoided in several studies
[47e49]. For example, Li and coworkers [47] prepared the new
monomer N-(2,2-dimethyl-1,3-dioxan-5-yl) (meth)acrylamide and
corresponding acid-sensitive polymers containing pendant cyclic
acetal groups. The degradation products in that case were neutral
diols and acetals, thus eliminating the inflammation problems, but
the hydrolysis rate of this six-membered cyclic acetal was quite slow.
It was known that cyclic orthoesters were hydrolyzed about two
ordersofmagnitudefasterthancyclicacetals [46]. Forthatreason, Li’s
group also prepared polymers or diblock copolymers bearing six-
membered cyclic orthoester groups [48e50], which were found to
hydrolyze mainly through two paths, including endocyclic and
exocyclic cleavage, giving rise to a mixture of side chain structures.
Heller [51] and Tang [52] reported another type of pH-responsive
nanoparticles assembled from amphiphilic diblock copolymers
bearing a hydrophobic five-membered cyclic orthoester as pendant
groups. Hydrolysis of these orthoester moieties followed a distinct
exocyclic mechanism and produced water soluble polymeric formate
and a small molecule carbonyl compound. Although the hydrolysis
rate of cyclic orthoesters was faster, the acidic degradation products
frequently inhibited their in vivo application. To develop a new type
of acid-labile linkage that is environment friendly and biocompatible
is still an important task for chemists.
2.1. Materials
2-Hydroxyethyl acrylamide (HEAM, Sigma Aldrich), 2,2-dime-
thoxypropane (DMP, Alfa Aesar), poly(ethylene glycol) mono-
methyl ether (PEG, Mn ¼ 2010 g molꢀ1, BASF), Nile Red (NR, Alfa
Aesar) were used without further purification. p-Toluenesulfonic
acid monohydrate (p-TSA), N,N0-dicyclohexyl carbodiimide (DCC),
and 4-dimethylaminopyridine (DMAP) were purchased from
Aladdin Reagent Company (Shanghai, China). 4-Cyanopentanoic
acid dithiobenzoate (CPDB) was synthesized according to the
literature procedure [57]. 2,20-Azobis(isobutyronitrile) (AIBN) was
recrystallized twice from ethanol and stored in the refrigerator.
Tetrahydrofuran (THF) was purified by distillation from sodium
with benzophenone, and 1,4-dioxane was distilled over CaH2 prior
to use. DCl (20% in D2O solution) and other deuterated solvents
(99.8% purity) were purchased from Beijing Seaskybio Company.
Other solvents and reagents were purchased from Beijing Chemical
Reagent Company, and used as received. All the aqueous solutions
were prepared using deionized water.
2.2. Synthesis of N-acryloyl-2,2-dimethyl-1,3-oxazolidine (ADMO)
monomer
2-Hydroxyethyl acrylamide (14.4 g, 0.12 mol), 2,2-dimethox-
ypropane (18.4 mL, 0.15 mol), and p-TSA (0.09 g, 0.48 mmol) were
dissolved in 250 mL anhydrous THF. The reaction mixture was
stirred at ambient temperature for 24 h. After removing most part
of the solvent by a rotary evaporator, the residue was purified by
silica gel column chromatography using hexane/ethyl acetate (4/1,
v/v) as the eluent, to afford clear colorless oil and the yield was
w46%. 1H NMR (CDCl3,
-NeCH2e), 4.02 (t, 2H, eCH2eOe), 5.67 (dd, 2H, CH2] CHe), 6.38
(d, 4H, CH2] CHe). 13C NMR (CDCl3,
, ppm): 24.3 (eC(CH3)2), 46.0
(NeCH2eCH2e), 63.0 (eCH2eCH2eOe), 94.7 (eC(CH3)2), 127.7
(CH2] CHe), 129.6 (CH2] CHe), 162.2 (eC]O). HR-MS (ESI): calcd
for C8H13NO2 [M þ Na]þ: 155.08385, found: 178.08426.
d, ppm): 1.61 (s, 6H, -C(CH3)2), 3.68 (t, 2H,
d
Oxazolidines are highly pH-sensitive molecules that can be
regarded as cyclic acetal analogs with one oxygen atom replaced by
nitrogen, and have been widely used for protection of
b-amino
alcohols for asymmetric synthesis [53] and used as prodrug forms
[54]. These heterocycles have been found to undergo a facile and
complete hydrolysis in a wide pH range, producing the correspond-
2.3. Synthesis of poly(ethylene glycol)-based chain transfer agent
(PEG-CTA)
ing carbonyl compound and
capacity of the oxazolidine can be adjusted by introducing functional
groups on the nitrogen atom [56]. Since -amino alcohols and/or
carbonyl-containing compounds are widely found in the biological
and pharmaceutical sciences, this category of oxazolidines could be
used as another kind of excellent acid-labile group, in addition to
cyclic acetals and orthoesters, to construct novel pH-sensitive poly-
meric micelles. To the best of our knowledge, there have been no
reports on polymeric micelles involving the oxazolidines.
In this work, the oxazolidine based monomer N-acryloyl-2,2-
dimethyl-1,3-oxazolidine (ADMO) was synthesized as well as its
homopolymer (PADMO), which was readily hydrolyzed in mildly
acidic media, producing the parent compound HEAM (or PHEAM),
while stable in neutral and basic aqueous solution. Based on the
acid-labile oxazolidine type monomer (ADMO), a series of novel
pH-responsive amphiphilic diblock copolymers PEG-b-PADMO was
designed and synthesized by RAFT polymerization. The copolymers
were composed of hydrophilic PEG blocks and polyacrylamide
segments of varying length with pendant oxazolidine moieties. The
micellization and stimuli-responsive behavior of the copolymers in
aqueous solution were studied using 1H NMR spectroscopy, DLS,
TEM measurements, fluorescence probe technique and release of
encapsulated Nile Red dye.
b
-amino alcohol [55]. The hydrolysis
PEG-CTA was synthesized according to the literature procedure
[58]. PEG (13 g, 6.5 mmol), CPDB (2.3 g, 8.2 mmol), and a trace of 4-
dimethylaminopyridine (DMAP) were dissolved in 50 mL CH2Cl2
solution. Thismixturewasadded bya CH2Cl2 solution (50 mL) of N,N0-
dicyclohexyl carbodiimide (DCC) (2.8 g,13.7 mmol) over 30 min. After
stirred for 20 h in oil bath at 40 ꢁC, the mixture was filtrated to
remove dicyclohexylurea. The solvent was removed, and the
residue was purified by silica gel column chromatography using
CHCl3 as the eluent, followed by a mixture of CHCl3 and CH3OH (95/5,
v/v). The product was dried under vacuum at room temperature
for 24 h, obtained as red solid with a yield 52.3%. 1H NMR (CDCl3,
b
d, ppm): 1.93 (s, 3H, CH3eC-CN), 2.55 (m, 2H, eCH2eCH2eCeCN),
2.75(m, 2H, eCH2eCH2eC-CN), 3.37 (s, 3H, PEGeOCH3), 3.45e3.75
(m, PEG backbone), 3.81 (t, 2H, eCH2eCH2eOOCe), 4.27 (t, 2H,
eCH2eCH2eOOCe), 7.39e7.89 (5H, eC6H5).
2.4. Synthesis of poly(ethylene glycol)-block-poly(N-acryloyl-2,2-
dimethyl-1,3-oxazolidine) (PEG-b-PADMO)
As shown in Scheme 3, diblock copolymers were synthesized
via reversible addition fragmentation chain transfer polymeriza-
tion (RAFT) using PEG-CTA as macro RAFT agent. To obtain diblock
copolymers with different PADMO block lengths, the molar ratio

Q. Cui et al. / Polymer 52 (2011) 1755e1765
1757
of monomer to CTA was varied at 20, 30, 50, and 100 respectively.
2.8. Determination of critical aggregates concentration (CAC)
As an example, for synthesis of copolymer with PADMO length of
30 units, ADMO (0.7 g, 4.5 mmol), PEG-CTA (0.375 g, 0.15 mmol)
and AIBN (2.5 mg, 0.015 mmol) were dissolved in 5 mL 1,4-
dioxane. The solution was deoxygenated by purging with nitrogen
gas for 30 min. Polymerization was carried out in water bath at
70 ꢁC for 24 h, and terminated by ice bath and exposure to air. The
polymer was isolated by three times re-precipitations from THF to
hexane and dried under vacuum at room temperature for 24 h,
Critical aggregates concentrations (CAC) of the copolymers were
estimated by a fluorescence spectroscopic method using pyrene as
the fluorescence probe. 200
mL of pyrene in methanol solution
(5.0 ꢂ 10ꢀ5 mol Lꢀ1) was added into a series of 10 mL volumetric
flasks, and the solvent was then evaporated completely. A series of
copolymer aqueous solutions at different concentrations ranging
from 1.0 ꢂ 10ꢀ4 to 1.0 mg mLꢀ1 were added to the bottles, whereas
obtained as a pink powder. 1H NMR (CDCl3,
d, ppm): 1.55 (eC
the finalconcentration of pyrene in each flaskwas 1.0 ꢂ10ꢀ6 mol Lꢀ1
.
(CH3)2), 1.10e3.00 (e(CH2eCH)e), 3.38 (PEG-OCH3), 5.61 (PEG),
3.00e4.40 (eNeCH2eCH2eOe), 7.39e7.89 (eC6H5).
The excitation spectra of pyrene were obtained on a Hitachi F-4500
fluorescence spectrometer, from 300 to 350 nm with the emission
wavelength at 383 nm. The value of CAC was determined from the
plots of the intensity at 338 and 333 nm against the polymer
concentration.
2.5. Synthesis of homopolymer poly(N-acryloyl-2,2-dimethyl-1,3-
oxazolidine) (PADMO)
Homopolymer PADMO was prepared by traditional radical
polymerization as follows: ADMO (0.78 g, 5 mmol), AIBN (1.6 mg,
0.01 mmol) were dissolved in 10 mL 1,4-dioxane. The solution was
deoxygenated by purging with nitrogen gas for 30 min. Polymer-
ization was carried out in water bath at 60 ꢁC for 12 h. The
polymer was subsequently purified by precipitation into hexane
2.9. Determination of the hydrolysis rate
For the measurement of the monomer, ADMO was dissolved in
0.5 mL D2O, and pH was adjusted to 3.0, 5.0, and 7.4, by addition of
20 mL of 5.0 M pH 3.0, 5.0 acetate buffer or 7.4 phosphate buffer. For
pH 2.0, the solution was adjusted by 5 M HCl. The 1H NMR spectra
were recorded at different time points. For the polymer, the
micelles solution in D2O with a concentration 5 mg mLꢀ1 was
and obtained as a white powder. 1H NMR (CDCl3,
d, ppm): 1.55 (eC
(CH3)2), 1.10e2.05 (e(CH2eCH)e), 2.05e3.00 (e(CH2eCH)e),
3.15e4.30 (eNeCH2eCH2eOe).
added by 50 m
L DCl (20% in D2O solution), and measured by 1H NMR
at predetermined time.
2.6. Characterization methods
1H NMR (400 MHz) and 13C NMR (400 MHz) spectra were
recorded on a Bruker DPX-400 NMR spectrometer in the deuter-
ated solvents at 25 ꢁC, using tetramethylsilane (TMS) as an internal
standard.
The high resolution mass spectrometer (HR-MS, ESI) analyses
were carried out on a Bruker apex IV FT mass spectrometer.
The molecular weights and molecular weight distributions of
polymer were estimated by gel permeation chromatography (GPC)
on equipment composed of a Waters 1525 binary HPLC pump,
a Waters 2414 refractive index detector, and three Waters Styragel
columns. THF was used as eluent at a flow rate of 1.0 mL minꢀ1, and
linear polystyrene standard was used for calibration.
Transmission electron microscopy measurements (TEM) were
conducted on a JEOL JEM-2100 transmission electron microscope
with an accelerating voltage of 200 kV. The sample was prepared
by placing a drop of the copolymer solution onto a carbon-
coated copper grid (400 mesh), staining with 1% (w/v) uranyl
acetate solution, and allowing the sample to dry in air before
measurement.
Average hydrodynamic radius and distributions of the aggre-
gates formed in polymer aqueous solution were measured by
DynaPro NanoStar instrument (Wyatt Technology), with a HeeNe
2.10. Encapsulation and release of Nile Red
200
mL
of Nile Red in dichloromethane solution
(5.0 ꢂ 10ꢀ5 mol Lꢀ1) was added into a 10 mL volumetric flask, and
the solvent was then evaporated completely. The polymer aqueous
solution (0.2 mg mLꢀ1) was added to the bottle, whereas the final
concentration of Nile Red was 1.0 ꢂ 10ꢀ6 mol Lꢀ1. The solution was
placed overnight to reach the equilibrium. 2 mL solution was
adjusted to pH 3.0, 5.0, and 7.4, by addition of 50 mL of 5.0 M pH 3.0,
5.0 acetate buffer or pH 7.4 phosphate buffer. The samples were
incubated at 37 ꢁC and the fluorescence intensity was measured at
the desired time points. The excited wavelength was set at 550 nm,
and slit widths were maintained at 5.0 nm.
3. Results and discussion
3.1. Synthesis and characterization of acid-labile monomer ADMO
The novel acid-labile monomer N-acryloyl-2,2-dimethyl-1,3-
oxazolidine (ADMO) was synthesized from 2-hydroxyethyl acryl-
amide (HEAM) and 2,2-dimethoxypropane (DMP) in the presence
of p-toluenesulfonic acid (p-TSA) as catalyst, involving condensa-
tion and cyclization (Scheme 2a). The new monomer was stable at
room temperature and easily soluble in common organic solvents
and water. The chemical structure and configuration of ADMO was
confirmed by 1H NMR, 13C NMR spectroscopy and HR-MS (ESI)
analysis.
laser (
l
¼ 658 nm) operated at 10 mW. The measurements were
made at the scattering angle
purified by Millipore filters with 0.45
q
¼ 90ꢁ at 25 ꢁC, and the samples were
mm pore size before the
measurements.
2.7. Preparation of aqueous micelle solutions
From the 1H NMR spectrum of ADMO, the difference in the
doublet peaks at 1.55e1.61 ppm in D2O and CDCl3 solutions of
ADMO indicated that the monomer comprised diastereomeric
mixtures of rotational isomers, as shown in Fig. 1a and Fig. S1.
However, the trans isomer Z was predominantly excess over the cis
isomer E, in which the unfavorable steric interactions existed,
especially the dimethyl substitution created an even greater
affection. Indeed, solvent effect was evident as illustrated in 1H
NMR spectra by the higher Z isomer populations in D2O than in
CDCl3 [59]. Since this work did not focus on the stereochemistry of
The micelle solutions of the block copolymers were prepared by
dissolving the polymers in water directly except sample E45A88
,
which was treated by dialysis method. The sample E45A88 was first
dissolved in THF, and water was then added slowly into the solution
to induce the formation of micelles. The solution was stirred for 2 h
at room temperature before being placed in a dialysis bag (MW
cutoff 3500 Da) for dialysis against water for three days (water was
frequently refreshed).

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Q. Cui et al. / Polymer 52 (2011) 1755e1765
the monomer and the polymer, these aspects were not further
investigated.
Since the oxazolidines exhibit n(N) / s
*(CeO) electron delo-
calization and are sensitive to a wide range of pHs, electron with-
drawing or electron-donating groups attached to the nitrogen atom
would be expected to affect the reactivity of the five-membered
ring. It has been proved that when the nitrogen lone pair is
conjugated with a carbonyl group (n(N) /
p(C]O) electron
delocalization) as in N-acryloyl oxazolidines, both hydrolysis and
reductive ringeopening reactions become much more difficult, as
a consequence of a concomitant decrease of oxygen basicity and
increase of the intra-cyclic CeO bond strength. Thus their ring-
eopening reactions require the presence of a strong oxophilic
catalyst (proton or Lewis acid) [56]. The monomer ADMO, con-
taining an N-substituted carbonyl moiety, was found by thin layer
chromatography (TLC) and 1H NMR spectroscopy to undergo facile
hydrolysis under acidic catalysis while being stable in neutral or
basic medium for a long time.
Fig. 1 shows 1H NMR spectra of ADMO before and after addition of
DCl to the D2O solution of the monomer. The peak at 1.55 ppm,
assigned to the dimethyl group protons of ADMO, significantly
Fig. 2. Hydrolysis kinetics of acid-labile monomer ADMO at different pHs at 25 ꢁC.
reduced after 20 mL DCl was added, and disappeared after 0.5 h.
lives were calculated from the slopes of the linear plots. At pH 5.0,
hydrolysis proceeded very slowly. The hydrolysis rate increased
rapidly with decreasing pH, with half-life 44 h at pH 3.0 and 4.5 h at
pH 2.0.
Concomitantly, a new peak at 2.10 ppm, assigned to the byproduct
acetone, increased rapidly with time. According to previous studies,
oxazolidine hydrolysis occurs in two stages: reversible ring opening
through CeO bond cleavage to give a cationic Schiff base species,
followed by hydrolysis of this intermediate to give the b-aminoalcohol
and the carbonyl component [54]. Thus, the hydrolysis mechanism of
ADMO monomer can be described as shown in Scheme 2b.
The kinetics of hydrolysis of ADMO was further studied in
aqueous solution at different pH values at 25 ꢁC using 1H NMR
spectroscopy. The degree of hydrolysis was estimated by moni-
toring the change of the ratio of intensities of the signal at 2.10 ppm,
which was ascribed to the released acetone protons, to the signal at
1.55 ppm of the dimethyl protons of oxazolidine group. As seen in
Fig. 2, the hydrolysis of ADMO displayed strict first order kinetics
and strong pH dependence. The hydrolysis rate constants and half-
ADMO monomer, being an acrylamide derivative, was found to
easily undergo free radical polymerization. The homopolymer
PADMO was soluble in common organic solvents including acetone,
tetrahydrofuran, ethanol, methanol, ethyl acetate, chloroform,
toluene, 1,4-dioxane, dimethylsulfoxide and N,N-dimethylforma-
mide, but insoluble in hexane and water. At low pH, the homo-
polymer was readily converted from hydrophobic PADMO to
hydrophilic PHEAM (Scheme 1) via hydrolysis of oxazolidine
pendant groups, displaying the expected effective acid stimuli-
responsiveness. Thus PADMO showed similar pH-responsive
behavior to that of acid-sensitive polymers containing cyclic
orthoesters [51,52] and acetals [23].
Fig. 1. 1H NMR spectra of ADMO monomer before and after addition of DCl to its solution in D2O. (a) 0 min, (b) 5 min, and (c) 0.5 h.

Q. Cui et al. / Polymer 52 (2011) 1755e1765
1759
Scheme 1. Schematic representation of the functional aggregation behavior of the pH-responsive diblock copolymer PEG-b-PADMO.
3.2. Synthesis and characterization of diblock copolymers
formulation as follows [61,63]: Mn(theor) ¥ (Ntheor ꢂ MADMO ꢂ [M]0/
[CTA]0) þ MCTA, where Ntheor is the targeted degree of polymeri-
zation, [M]0 is the initial concentration of monomer, MADMO is the
molecular weight of ADMO monomer, [CTA]0 is the initial
concentration of PEG-CTA, and MCTA is the molecular weight of
PEG-CTA. Considering the high conversion (>95%) for ADMO
polymerization in the given experiment condition, which was
determined by 1H NMR method (shown in Supplementary data
Fig. S8). It is reasonable to calculate Mn(theor) within the permit error
range on the assumption of complete conversion (Ntheor ¼ 100%).
The theoretical molecular weight Mn(theor) for different copolymer
samples are listed in Table 1.
Diblock copolymers PEG-b-PADMO with well-defined struc-
tures, consisting of hydrophilic PEG part and hydrophobic PADMO
part, were prepared via RAFT controlled radical polymerization of
functional monomer ADMO using PEG-based macromolecular
chain transfer agent (PEG-CTA) and initiator AIBN, as shown in
Scheme 3. PEG-CTA was synthesized via esterification of the
terminal hydroxyl group of poly(ethylene glycol) monomethyl
ether (Mn ¼ 2010 g molꢀ1,) with 4-cyanopentanoic acid dithio-
benzoate (CPDB), a chain transfer agent commonly used in RAFT
polymerization of (methyl)acrylamide monomers [57,60], in the
presence of DCC and DMAP catalysts. The structure of the product
was supported by 1H NMR spectra, in accordance with that previ-
ously reported [58]. The RAFT functionality of PEG-CTA was calcu-
lated to be 95% based on the data of 1H NMR spectra, and the
impurity may include the residual PEG and dead products from side
reactions [61,62].
A series of PEG-b-PADMO diblock copolymers with PADMO
blocks of varying length and PEG blocks with fixed length were
obtained as listed in Table 1, using different feed ratios of initial
concentration of monomer to CTA ([M]0/[CTA]0), ranging from 20,
30, 50 to 100 respectively, at a constant ratio ([CTA]0/[I]0) of 10:1.
The polymers obtained were soluble in common organic solvents
except hexane, and soluble in water (except for sample E45A88).
The molecular weights of the copolymers were determined by
theoretical and experimental methods. The synthesis details and
characterization data for various PEG-b-PADMO diblock copoly-
mers are summarized in Table 1. The targeted number average
molecular weight Mn(theor) was calculated based on a simplified
From the 1H NMR spectra of the block copolymers in CDCl3, Mn
was obtained by comparing the integrated signals of the
(NMR)
protons of the dimethyl groups plus polyacrylamide backbones at
1.30e3.00 ppm, with those of the signals at 3.10e4.50 ppm corre-
sponding to the sum of PEG (3.45e3.75 ppm) and methylene
protons on the five-membered ring of PADMO. The numbers of
ADMO repeating units in the different length diblock copolymer
samples were estimated to be 18, 28, 47 and 88, close to the feed
compositions. The corresponding copolymers are denoted as
E45A18, E45A28, E45A47, and E45A88, where E and A represents PEG
and PADMO block respectively. The Mn(NMR) values (Table 1) for the
block copolymers were in fair agreement with the Mn(theor) values.
As an alternative method, molecular weights were also esti-
mated by GPC. Fig. 3 shows GPC elution curves for PEG-CTA and the
diblock copolymers. As polymer growth increased with the [M]0/
[CTA]0 ratio, the peak of the corresponding copolymer shifted
toward lower retention time (higher molecular weight). All GPC
traces presented a monomodal, narrow molar mass distributions
Scheme 2. Synthetic pathway (a) and hydrolysis mechanism (b) for the monomer ADMO.

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Q. Cui et al. / Polymer 52 (2011) 1755e1765
Scheme 3. Synthesis routes of PEG-CTA and diblock copolymer PEG-b-PADMO.
with polydispersity indexes (Mw/Mn) from 1.13 to 1.19, demon-
strating efficient polymerization control with the RAFT agent PEG-
CTA. It should be noted that the GPC traces for the copolymers
exhibited a small shoulder on the low molar mass side, which is
corresponding to the position of PEG-CTA curve, suggesting minor
contamination of the original macro RAFT agent. Since the RAFT
functionality of PEG-CTA synthesized was determined to be 95% as
indicated above, the residual PEG and dead products derived from
side reaction as impurities may be brought in the copolymer
products [61,62], which have not been removed completely by re-
precipitation. As a result, it can be seen that a shoulder was found at
the low Mw side of GPC curves for various copolymers. Considering
the very small content of PEG-based contaminants, their influence
on the assembly behaviors in water of the copolymers should be
not of importance, which was proved by experiment (see
comparison study in Supplementary data Fig. S9).
to acetone-d6 the intensity of the sharp proton signals of the
PADMO hydrophobic block was greatly reduced and broadened
(Fig. 4b), and almost completely disappeared in neat D2O (Fig. 4c),
while the intensity of the PEG signal remained constant. These
observations can be accounted for by the protons of PADMO chains
becoming less mobile and less solvated in hydrophobic domains
that formed in water. Thus we postulate the formation of micelle-
like aggregates in water, with PADMO forming the compact
hydrophobic core and PEG forming the solvated hydrophilic corona,
as depicted schematically in Scheme 1.
The critical aggregation concentration (CAC) of the diblock
copolymers PEG-b-PADMO was determined using pyrene as a fluo-
rescent probe [64,65], whose fluorescent properties are sensitive
primarily to the polarity of the microenvironment in which it is
located. The ratio of intensity of 338 nm to 333 nm in the excitation
spectrum would increase sharply if the polymers spontaneously
aggregate, followed by pyrene probe transfer from water into the
hydrophobic domains of the aggregates [66]. Plots of I338/I333 versus
copolymer concentration are displayed in Fig. 5. The I338/I333 ratio
remained almost constant in the low concentration region, and
increased significantly upon further increasing the polymer
concentration, indicating that the molecularly dissolved polymer
chains began to associate into micelles and pyrene was partitioned
into the less polar core. The CAC for PEG-b-PADMO copolymers
with different compositions, determined from these plots are
given in Table 1. The CAC values are comparable to those of other
amphiphilic diblock copolymers such as PHPMAmDL-b-PEG
The relatively small polydispersity indexes and the good
correlation between the experimentally determined molar masses
and the values obtained theoretically suggest that the polymeri-
zation proceeded in a more controlled fashion in the experimental
conditions described above [61].
3.3. Aggregation behavior of the diblock copolymers
The amphiphilic PEG-b-PADMO copolymers readily self-
assembled into micelle-like aggregates in water, and remained
transparent solution except in the case of E45A88.
1H NMR spec-
(0.15e0.015 mg mLꢀ1) [67] and PLA-b-PEG (0.035e0.0025 mg mLꢀ1
)
troscopy and a fluorescence probe technique were used to establish
whether the copolymers were in the form of single molecules in
water or self-associated into micelles with diameter smaller than
the wavelength of visible light. Fig. 4 presents 1H NMR spectra of
the diblock copolymer E45A28 in deuterated acetone, D2O and an
acetone-d6/D2O mixture. Fig. 4a shows that the characteristic
resonance signals of the dimethyl groups of PADMO (1.55 ppm) and
PEG (about 3.60 ppm) were visible in acetone-d6. As D2O was added
[68], which should make the PEG-b-PADMO copolymers useful for
pharmaceutical applications. As expected, the CAC values were
significantly dependent on the length of the core-forming PADMO
hydrophobic block. As the PADMO block length grew from E45A18 to
E45A88, the CAC values decreased about two orders of magnitude,
indicating sufficient hydrophobicity of the PADMO block to form
micelles at very low concentration.
Table 1
Polymerization conditions and properties of diblock copolymers.
Mn(theor) ꢂ 10ꢀ4
Mn(NMR) ꢂ 10ꢀ4
Mn(GPC) ꢂ 10ꢀ4
Mw(GPC) ꢂ 10ꢀ4
PDI (Mw/Mn)d
CAC
a
Sample
[M]0 /[CTA]0
b
c
d
d
e
(g molꢀ1
)
(g molꢀ1
)
(g molꢀ1
)
(g molꢀ1
)
(mg mLꢀ1
)
E45A18
E45A28
E45A47
20
30
50
0.54
0.69
1.00
1.78
0.51
0.66
0.96
1.59
0.47
0.57
0.81
1.34
0.53
0.65
0.92
1.61
1.13
1.14
1.13
1.19
0.120
0.065
0.029
0.007
E
45A88
100
a
[M]0/[CTA]0 is the ratio of initial concentration of monomer to PEG-CTA, while the ratio of concentration of PEG-CTA to initiator was kept at a constant value ([CTA]0/
[I]0 ¼ 10). Polymerization was performed in 1,4-dioxane at 70 ꢁC for 24 h.
b
Calculated from the equation, assuming that the reaction went to the completion.
c
Determined from 1H NMR spectra.
d
Determined by GPC in THF, relative to linear polystyrene.
The value of critical aggregates concentration (CAC) was estimated by a fluorescence spectroscopic method using pyrene as the fluorescence probe.
e

Q. Cui et al. / Polymer 52 (2011) 1755e1765
1761
47 nm (for maximal chain stretching) for E45A88, taking the length
of a CeC bond as 0.154 nm, and CeO bond as 0.143 nm respectively.
Thus, it was thought that the polymer E45A88 may aggregate into
shapes such as cylinders or vesicles, rather than spherical micelles.
The morphologies of the aggregates from the different block
copolymers PEG-b-PADMO were visualized by TEM. Fig. 7 shows that
the aggregates formed by E45A28 were spherical. E45A47 coexists as
spherical and worm-like micelles, in accordance with the bimodal
distribution found by DLS. The clear bilayer structure for E45A88
demonstrated formation of vesicles with radius about 100 nm. The
difference in the diameters obtained by TEM and DLS was most
probably due to shrinkage of the PEG shell upon drying, because DLS
measurement was conducted in aqueous solution while TEM obser-
vation was on the collapsed state after evaporation of water. Conse-
quently, the micellization behavior of the diblock PEG-b-PADMO
copolymers clearly demonstrated that the small spherical micelles of
E45A28 in aqueous solution transformed into worm-like micelles of
E45A47 and into larger vesicles for E45A88, as the hydrophobic block
length increased. These findings are in accordance with the general
view that micelle morphology tends to change from spherical through
rod-like or worm-like to a bilayer structure or vesicle, with increasing
length of hydrophobic block, as for PS-b-PAA [69] and PLA-b-PEG [70].
It was of interest to find that the aqueous micelle solution of
E45A47, at concentration higher than 2 mg mLꢀ1, was transparent at
refrigerator (about 4 ꢁC) and became cloudy at room temperature.
This behavior was not observed for either the copolymers (E45A18
and E45A28), with shorter hydrophobic blocks or for E45A88 with
a longer hydrophobic block, under the same conditions. This
observation suggests that this type of block copolymer, with suit-
able composition, may exhibit thermally induced phase transition.
This issue will be investigated elsewhere.
Fig. 3. GPC curves of PEG-CTA and the diblock copolymers PEG-b-PADMO obtained by
RAFT polymerization at various [M]0/[CTA]0 ratios.
3.4. Sizes and morphologies
The hydrodynamic radius and distributions of the PEG-b-
PADMO copolymers were measured in water by DLS at 1 mg mLꢀ1
concentration, much higher than the CAC to ensure micelle
formation. Fig. 6 shows a clear increase of particle size with
increasing length of the PADMO block, and narrow size distribu-
tion. The copolymers E45A18 and E45A28 with shorter hydrophobic
blocks showed similar behavior with a monomodal distribution
and average hydrodynamic radius about 10 and 11 nm, respectively.
E45A47 showed a bimodal distribution corresponding to average
hydrodynamic radius 16 and 90 nm, indicating coexistence of two
different aggregates, while E45A88, with the longest hydrophobic
3.5. pH-responsive properties of diblock copolymer micelles
block, showed
a monomodal distribution with radius about
129 nm. According to the calculation method in the literature [61],
the theoretical maximum value of the radius of spherical micelles is
The PEG-b-PADMO diblock copolymers bearing acid-sensitive
oxazolidine pendant groups were stable in neutral and basic
Fig. 4. 1H NMR spectra of polymer E45A28 dissolved in (a) acetone-d6; (b) mixture of acetone-d6/D2O (2/5 v/v); (c) D2O.

1762
Q. Cui et al. / Polymer 52 (2011) 1755e1765
Fig. 5. I338/I333 ratio for pyrene as a function of the logarithmic concentration of diblock copolymers.
conditions but easily hydrolyzed in acidic media. Upon hydrolytic
cleavage of the five-membered ring, the hydrophobic part of the
PADMO block is converted to hydrophilic poly(2-hydroxyethyl
acrylamide) (PHEAM), thus causing disruption of the micelles.
To confirm hydrolytic cleavage of the oxazolidine groups of the
copolymer in acidmedium, an aqueous solution ofcopolymerE45A88
(1 mg mLꢀ1) was treated with 0.1 mol Lꢀ1 HCl at room temperature
for 3 days. The micelle solution turned from cloudy to transparent,
indicating dissociation. The resulting solution was dialyzed in
a dialysis bag (MW cutoff 3500 Da) against water for three days,
to remove small molecules and byproducts. After lyophilization,
the macromolecular hydrolysis product was obtained as white
powder that was readily soluble in water. 1H NMR spectra of solu-
tions in DMSO-d6 of the copolymer before hydrolysis and the
macromolecular product after complete hydrolysis, are shown in
Fig. 8. The characteristic signals (1.55 ppm) of dimethyl groups of
PADMO disappeared after hydrolysis, and new signals appeared at
7.50e8.00 and 4.50e5.00 ppm; these signals were assigned to the
protons of imide and hydroxyl groups of PHEAM [71]. This result
verified that the ADMO pendant groups were indeed accessible to
hydrolysis byacid catalysis. As a result, the amphiphilic diblock PEG-
b-PADMO copolymer was converted to the double hydrophilic
diblock copolymer PEG-b-PHEAM in the acid environment, giving
rise to concomitant dissociation of the micelles.
The size change of polymeric micelles in response to PADMO
block hydrolysis was followed by DLS measurement. Fig. 9 shows
the distribution of hydrodynamic radius of the micelles of copol-
ymer E45A28 in pH 3.0 acetate buffer before and after hydrolysis.
The micelles displayed a monomodal distribution prior to hydro-
lysis, with apparent hydrodynamic radius about 11 nm. The size of
the micelles slowly decreased with hydrolysis time. Initially, the
average size of the micelles increased to approximately 17 nm after
1 day: as a result of partial hydrolysis of the pendant oxazolidine
groups, the micelle cores became increasingly hydrophilic and
absorbed more water, leading to swelling of the micelles. As
hydrolytic cleavage progressed, micelle disruption occurred via
swelling and disintegration or dissociation, and a bimodal distri-
bution appeared after 4 days. The new peak at about 2 nm can be
attributed to single polymer chains, because the molecular weight
of copolymer E45A28 was relatively small (about 6900 g molꢀ1) [72].
Finally, after one week there was only one peak at about 1 nm. The
reason was most probably the shrinking of the polymer coil by
intramolecular hydrogen bonding between the amide and hydroxyl
moieties as well as PEG units in the resulting PEG-b-PHEAM
copolymer, because the HEAM units provided hydrogen for H-
bonding (see Scheme 1). Similar results have been found for several
polymers, including poly(hydroxyethyl methacrylate) [73,74] and
polymethyacrylamide derivatives [49].
Fig. 6. Hydrodynamic radius distribution measured by DLS for the aqueous solutions
of the four diblock copolymers. Cp ¼ 1 mg mLꢀ1
.

Q. Cui et al. / Polymer 52 (2011) 1755e1765
1763
Fig. 7. TEM images of the aggregates formed by the diblock copolymers: (a) E45A28; (b) E45A47; (c) E45A88. The scale bars are 100 nm.
rate, hydrolysis of the oxazolidine groups of the PEG-b-PADMO
copolymers was studied for the micelles of each copolymer in D2O
in the presence of DCl by monitoring, using 1H NMR, the acetone
generated from hydrolysis of the ADMO groups of the copolymer.
The concentration of each block copolymer used for these experi-
ments was 5 mg mLꢀ1, ensuring the formation of micelles. Fig. 10
shows typical 1H NMR spectra for hydrolysis of copolymer E45A28
.
The characteristic signal of acetone (hydrolysis product) was found
at 2.10 ppm and increased gradually with reaction time. Thus the
extent of hydrolysis was calculated by comparing the acetone peak
at 2.10 ppm with the peak at 3.38 ppm of terminal methyl protons
of PEG, which did not change in the course of hydrolysis and was
used as an internal standard. The kinetic data for each polymer are
shown in Fig. 11.
The rate of hydrolysis was clearly dependent on the core-
forming hydrophobic block length of PADMO, in the order
E45A18 > E45A28 > E45A47. Since hydrolysis of oxazolidine moieties
of copolymer took place within the micelle core, micelles with
larger size and compact structure provided less accessibility of
hydronium ions to the inner hydrophobic core of the micelles, thus
strongly retarding hydrolytic cleavage of oxazolidine side groups.
Fig. 8. 1H NMR spectra of DMSO-d6 solutions of (a) copolymer E45A88 before hydrolysis
and (b) the product after complete hydrolysis.
3.6. pH-responsive release of Nile Red
The hydrolysis rates of micelles are commonly influenced by
several factors. In addition to pH, temperature and ionic strength,
the aggregate size and structure may play an important role that
will strongly affect access of hydronium ions. The data from DLS
given in section above provide the relevant information that the
sizes of the micelles of PEG-b-PADMO copolymer increased from 10
to 129 nm as the length of the PADMO block increased from E45A18
to E45A88. To verify the micelle size and structure effect on reaction
The acid-sensitive destabilization of the micelles, which was
expected to trigger release of a hydrophobic payload, was investi-
gated using Nile Red (NR) as model substrate. NR is a hydrophobic
Fig. 9. Micelle hydrodynamic radius distributions at different hydrolysis times for
Fig. 10. 1H NMR spectra at different reaction times for hydrolysis of polymer E45A28
in D2O.
E45A28 at pH 3.0, at 37 ꢁC. Cp ¼ 1 mg mLꢀ1
.

1764
Q. Cui et al. / Polymer 52 (2011) 1755e1765
monomer N-acryloyl-2,2-dimethyl-1,3-oxazolidine (ADMO).
A
series of novel pH-responsive diblock copolymers composed of fixed
hydrophilic PEG block and various lengths of hydrophobic PADMO
block were prepared by RAFT polymerization. Themolecular weights
obtained by 1H NMR and GPC were close to the targeted theoretical
data, suggesting the polymerization followed a controllable mecha-
nism. The amphiphilic PEG-b-PADMO block copolymers prepared by
RAFT polymerization easily self-assemble into micelle-like aggre-
gatesinwater, withcompact PADMO cores andsolvatedPEGcoronas,
at polymer concentrations above the CAC. The sizes and shapes of the
polymeric micelles can be altered from spheres through worm-like
structures to vesicles by increasing the PADMO block length. Under
acid hydrolysis, the hydrophobic pendant oxazolidine groups are
converted to water soluble b-hydroxyl amine derivative, leading to
conversion of the amphiphilic PEG-b-PADMO copolymer to the
double hydrophilic diblock copolymer PEG-b-PHEAM, followed by
micelle disruption and release of loaded Nile Red. The hydrolysis rate
of the copolymer micelles, demonstrated by 1H NMR spectra, DLS,
and fluorescence emission measurements of Nile Red, increases
remarkably at acid pH and decreases with PADMO block length, due
to the relatively large size and compact structure of the polymer
micelles. Thus by utilizing the acid-labile PADMO segment as func-
tionalbuildingblock, it would be interesting to explore novelkinds of
stimuli-responsivepolymer materials andpotentialbiotechnological
applications. The other functionalities of oxazolidine based polymer
materials, such as temperature responsive behavior, are currently
under investigation in our group.
Fig. 11. Release of acetone as a function of hydrolysis time for polymer E45A18, E45A28
,
E45A47 in aqueous solutions, based on the data from 1H NMR spectra.
Appendix. Supplementary data
Supplementary data associated with this article can be found,
free of charge, in the online version at doi:10.1016/j.polymer.2011.
02.022.
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