RAFT aqueous dispersion polymerization yields poly(ethylene glycol)-based diblock copolymer nano-objects with predictable single phase morphologies

Article abstract of DOI:10.1021/ja410593n

A poly(ethylene glycol) (PEG) macromolecular chain transfer agent (macro-CTA) is prepared in high yield (>95%) with 97% dithiobenzoate chain-end functionality in a three-step synthesis starting from a monohydroxy PEG₁₁₃ precursor. This PEG

Full text of DOI:10.1021/ja410593n

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RAFT Aqueous Dispersion Polymerization Yields
Poly(ethylene glycol)-Based Diblock Copolymer
Nano-Objects with Predictable Single Phase Morphologies
Nicholas J. Warren,* Oleksandr O. Mykhaylyk, Daniel Mahmood, Anthony J. Ryan, and Steven P . Armes*
Department of Chemistry, University of Sheffield, Brook Hill, Sheffield S3 7HF, United Kingdom
*
S Supporting Information
ABSTRACT: A poly(ethylene glycol) (PEG) macromolecular
chain transfer agent (macro-CTA) is prepared in high yield
(
>95%) with 97% dithiobenzoate chain-end functionality in a
three-step synthesis starting from a monohydroxy PEG113
precursor. This PEG113-dithiobenzoate is then used for the
reversible addition−fragmentation chain transfer (RAFT) aqueous
dispersion polymerization of 2-hydroxypropyl methacrylate
(
HPMA). Polymerizations conducted under optimized conditions
1
at 50 °C led to high conversions as judged by H NMR
spectroscopy and relatively low diblock copolymer polydispersities
(
M /M < 1.25) as judged by GPC. The latter technique also indicated good blocking efficiencies, since there was minimal PEG
w
n
1
1
3
macro-CTA contamination. Systematic variation of the mean degree of polymerization of the core-forming PHPMA block allowed
PEG -PHPMA diblock copolymer spheres, worms, or vesicles to be prepared at up to 17.5% w/w solids, as judged by dynamic light
113
x
scattering and transmission electron microscopy studies. Small-angle X-ray scattering (SAXS) analysis revealed that more exotic
oligolamellar vesicles were observed at 20% w/w solids when targeting highly asymmetric diblock compositions. Detailed analysis of
SAXS curves indicated that the mean number of membranes per oligolamellar vesicle is approximately three. A PEG -PHPMA phase
113
x
diagram was constructed to enable the reproducible targeting of pure phases, as opposed to mixed morphologies (e.g., spheres plus
worms or worms plus vesicles). This new RAFT PISA formulation is expected to be important for the rational and efficient synthesis of a
wide range of biocompatible, thermo-responsive PEGylated diblock copolymer nano-objects for various biomedical applications.
INTRODUCTION
available ABA triblock copolymers comprising two outer PEG ‘A’
blocks and a central poly(propylene glycol) ‘B’ block. These
copolymers exhibit thermo-responsive behavior and undergo
spontaneous self-assembly in aqueous solution to form various
nanostructures depending on the copolymer concentration,
■
It is well-known that conjugation of a hydrophilic poly(ethylene
glycol) (PEG; also known as poly(ethylene oxide) or PEO)
chain to a biologically relevant molecule typically enhances its
1
,2
water solubility and hydrolytic stability. In the case of proteins,
17−21
temperature, salt concentration, and relative block lengths.
this so-called ‘PEGylation’ approach can also significantly reduce
1
−4
Various types of micellar gels can be obtained with tunable
immunogenicity.
Moreover, PEGylation can also extend
17
rheological properties. Moreover, it has been shown that these
copolymers usually exhibit low cytotoxicities and can also bind to
in vivo circulation lifetimes for important biomolecules such as
insulin, allowing a lower dose frequency and hence greater
1
8,22
5
,6
cell membranes.
patient compliance. These attractive properties have led to the
recent development of a number of FDA-approved PEGylated
It is well-known that amphiphilic block copolymers can self-
assemble in a suitable selective solvent to produce cylindrical/
worm-like micelles, toroids, tubes, or vesicles, in addition to the
1
,5
therapeutic entities. Various PEGylation strategies have been
developed, such as the site-specific attachment of PEG chains to
proteins via disulfide linkages
11,23−27
6
−8
more common spherical micelles.
This behavior is
or direct polymerization of
broadly analogous to that of small molecule surfactants, where
oligo(ethylene glycol) methacrylate from specific surface sites on
3
the precise nanostructure is dictated by the packing parameter,
proteins.
2
4,25,28,29
P.
The value of P depends on the relative volume
There are many examples of PEG-based amphiphilic diblock
copolymers for drug delivery applications, where the hydro-
phobic core-forming block may comprise poly(L-lysine) (PLL),
poly(lactic acid) (PLA), or poly(ε-caprolactone) (PCL).
PCL and PLA both undergo slow hydrolytic degradation,
fractions of the hydrophilic and hydrophobic blocks and the
interfacial energy associated with the diblock junction. In
contrast to small molecule amphiphiles, the rate of exchange of
unimers between colloidal aggregates and individual diblock
copolymer chains can be negligible, leading to a range of
9
−15
9
,10
while disulfide bonds can be introduced to produce block
copolymer nano-objects that only degrade under reductive
conditions, such as those found within the cell cytoplasm.
Pluronics (sometimes called Poloxamers) are commercially
16
Received: October 16, 2013
Published: December 19, 2013
©
2013 American Chemical Society
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Article
3
0
kinetically frozen structures. Since the pioneering work by
Discher and co-workers, anisotropic (or worm-like) block
copolymer nano-objects have attracted considerable interest
due to their qualitatively different circulation times and cell entry
Scheme 1. Synthesis of (a) a Well-Defined PEG-Based Macro-
CTA Suitable for RAFT Polymerizations and (b) Various
PEG113-PHPMA Diblock Copolymer Nano-Objects Produced
x
via RAFT Aqueous Dispersion Polymerization of HPMA Using
the Principle of Polymerization-Induced Self-Assembly (PISA)
1
1,12
behavior compared to isotropic nanoparticles.
Traditionally, diblock copolymers have been synthesized using
controlled/living polymerization techniques in a good solvent for
both blocks. After purification, the desired self-assembly is
usually achieved by postpolymerization processing, which is
31
typically conducted in highly dilute solution (<1%). Recent
synthetic advances in polymerization-induced self-assembly
(
PISA) now allow spherical micelles, worm-like micelles or
vesicles composed of well-defined AB diblock (or ABC triblock)
copolymers to be prepared directly in concentrated aqueous
solution via either RAFT dispersion or emulsion polymer-
32−42
ization.
In particular, we have reported robust formulations
comprising thermo-responsive PHPMA chains as the hydrophobic
core-forming block and either poly(2-methacryloyloxy)ethyl
36
phosphorylcholine (PMPC) or poly(glycerol monomethacrylate)
33,35,41,42
(PGMA)
as the hydrophilic stabilizer block. In each case
detailed phase diagrams were constructed for a fixed degree of
polymerization of the stabilizer block, using the degree of
polymerization of the core-forming block and the diblock copolymer
concentration as variables. This approach enables the desired nano-
emphasizing that the RAFT dithiobenzoate end-group is
attached to the PEG chain via an amide bond, which is rather
more resistant to hydrolytic degradation under physiological
conditions than the ester bond linkage that is more typically
34,36
object morphology to be targeted consistently.
Moreover, if a
relatively short stabilizer block is utilized, a morphological evolution
from spheres to worms to vesicles can be observed during such PISA
syntheses, which is driven by the reduction in molecular curvature as
52
reported in the literature.
33
the core-forming PHPMA block grows.
The RAFT aqueous dispersion polymerization of HPMA using
this PEG113 macro-CTA has been optimized with respect to the
solution temperature and the total solids concentration so as to
achieve high conversions, low polydispersities, and good blocking
efficiencies for the resulting diblock copolymer nano-objects. This
enabled construction of a detailed phase diagram that allows the
reproducible targeting of pure copolymer morphologies. In addition
to the expected spheres, worms and vesicles, oligolamellar vesicles
are obtained for the first time using PISA, and this phase is
characterized together with a complete chain of self-assembled
structural morphologies using transmission electron microscopy
(TEM) and small-angle X-ray scattering (SAXS).
It should be emphasized that, unlike traditional equilibrium
phase diagrams, these phase diagrams contain kinetically trapped
morphologies as well as thermodynamically stable morphologies.
The former are frustrated spheres prepared at relatively low
copolymer concentrations, whereas the latter are obtained at
high copolymer concentrations. It has been suggested to us that
‘
state diagrams’ may be a more appropriate term. However,
strictly speaking, state diagrams only refer to solids, liquids, and
gases, rather than the various copolymer morphologies that are of
interest here. Thus in the present work we continue to use the
phrase ‘phase diagram’ with the above caveat.
As far as we are aware, almost all previous PISA formulations
have used macromolecular chain transfer agents (macro-CTAs)
RESULTS AND DISCUSSION
■
3
4,36,37,39,43−45
derived from various vinyl monomers.
Moreover,
First, a mono-aminated PEG intermediate was prepared in 83%
yield via a two-step synthesis starting from the monohydroxy-
capped PEG precursor according to a previously reported
the very few reports of the use of PEG macro-CTAs in
heterogeneous media have often achieved only relatively low
RAFT end-group functionality (∼85−90%), which inevitably
53
literature protocol. The mean degree of amination was deter-
47−50
1
reduces the blocking efficiency.
Herein, a commercially
mined to be 98% using H NMR spectroscopy (Figure S1a).
available near-monodisperse monohydroxy-terminated
PEG precursor is efficiently converted into a well-defined PEG-
dithiobenzoate macro-CTA via a high-yielding multistep synthesis
involving a monoaminated PEG113 intermediate (see Schemes 1
and S1). This macro-CTA is subsequently utilized to synthesize a
wide range of well-defined diblock copolymer nano-objects via the
RAFT aqueous dispersion polymerization of HPMA.
Then the N-hydroxysuccinimide activated ester of 4-cyano-4-
(phenylcarbonothioylthio) pentanoic acid (CPADB) was
conjugated to the terminal primary amine group on the
PEG113 chain. It is well-documented that direct carbodiimide
coupling only produces PEG macro-CTAs with relatively low
54
blocking efficiencies. However, previous studies suggested that
activating the carboxylic acid provides a highly efficient means of
conjugating (macro)molecules to primary amine-functional
One advantage of such a PEG-based macro-CTA is that the
same mean degree of polymerization (in this case 113 ethylene
glycol units) can be consistently obtained. This is not necessarily
the case for the various (meth)acrylic RAFT macro-CTAs that
5
5
PEG. Thus the succinimide-modified cyanopentanoate
dithiobenzoate (SCPDB) CTA was reacted with the PEG -NH
113
2
precursor to yield the PEG113-DB macro-CTA. After
33−46,51
have been reported by both ourselves and others.
Indeed,
purification, the extent of end-group modification was
1
it is relatively difficult to prepare the latter macro-CTAs with a
specific desired mean degree of polymerization since such
syntheses are usually terminated at intermediate conversions to
ensure high chain-end fidelity. It is perhaps also worth
determined by H NMR spectroscopy (see Figure S2). Three
distinct proton signals attributed to the RAFT end-group were
visible: (i) the aromatic signals at δ 7.3−7.8 ppm (see peaks i and j
in Figure S1b); (ii) the four methylene protons at δ 2.3−2.7 ppm
1
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see peaks f and g) assigned to the pentanoic acid end-group; (iii)
Article
(
polymerization (DP) of 200 for the core-forming PHPMA block.
Initially, such syntheses were performed at 70 °C, as previously
reported by Armes and co-workers for methacrylic-based macro-
CTAs. However, although H NMR studies indicated that very
high HPMA conversions were achieved (data not shown), some
macroscopic precipitation was also observed. This is presumably
due to the reduced aqueous solubility of the PEG chains at this
reaction temperature. Moreover, THF GPC studies of the
copolymer product indicated a relatively high polydispersity
(M /M = 1.53) and a prominent low molecular weight shoulder,
the three methyl protons adjacent to the nitrile group at δ 1.8 ppm
(
see peak h). Comparison of the integrated intensities of each of
3
3
1
thesethree signals tothat ofthe ethylene glycol protonsin thePEG
chain at δ 3.0−4.0 ppm indicated a mean degree of dithiobenzoate
functionalization of 93 ± 9%. We also used visible absorption
spectroscopy as a complementary method for determining the
degree of end-group functionalization. A calibration plot was
prepared from a series of methanolic solutions of CPADB; this
small molecule closely resembles the RAFT end-group that is
conjugated to the PEG chain, which is a prerequisite for reliable
57
w
n
indicating a relatively poor blocking efficiency (see Figure 1). In the
56
end-group analysis using this technique. The visible absorption
spectrum recorded for PEG -DB (see Figure S3a) is almost
light of these observations, a further series of PEG -PHPMA
113
200
syntheses were conducted over various reaction temperatures
ranging between 45 and 60 °C. An appropriate radical initiator was
selected in order to maintain a sufficient radical flux at each
temperature. Thus 2,2′-azobis(2-methylpropionamide) dihydro-
chloride (AIBA) was used for copolymer syntheses performed at
55 or 60 °C, whereas 2,2′-azobis[2-(2-imidazolin-2-yl)propane
dihydrochloride (AIPD) was employed for syntheses conducted at
45 or 50 °C. In all cases, more than 99% HPMA conversion was
achieved.
1
13
identical to that of the CPADB spectrum, with a peak maximum at
516 nm being observed in both cases. From the CPADB
calibration plot (see Figure S3b), a molar extinction coefficient of
∼
−1
−1
1
04 800 ± 640 L mol cm was calculated. Assuming anidentical
molar extinction coefficient for the terminal dithiobenzoate groups
on the PEG -DB chains, the mean degree of functionalization for
113
this RAFT macro-CTA was calculated to be 97 ± 2%. Given that
1
visible absorption spectroscopy is more sensitive than H NMR
spectroscopy, this value was considered to be more accurate than
that indicated by H NMR analysis. It should be noted that the
THF GPC studies indicated that the 50 °C synthesis produced
1
the most well-defined PEG -PHPMA diblock copolymer, as
113
200
3
−7% nonfunctionalized PEG impurity indicated by end-group
judged by its relatively low polydispersity and minimal homopol-
ymer contamination. The latter observation confirms the high mean
degree of functionalization of the PEG -DB macro-CTA chains
analysis may result from a small amount of PEG secondary
2
26
amine during the reaction with ammonia. This is consistent with
the GPC chromatogram for the PEG113 macro-CTA shown in
Figure 1, which contains a minor high molecular weight
component at 15.4 min.
113
indicated by the visible absorption spectroscopy studies. Thus this
optimized RAFT aqueous dispersion polymerization formulation
(50 °C; AIPD initiator; [PEG -DB]/[AIPD] = 3.0) was selected
113
for all subsequent diblock copolymer syntheses described in this
study. A kinetic plot for the polymerization of HPMA (Figure 2,
Figure 2. Kinetics of polymerization of HPMA via RAFT aqueous
dispersion polymerization at 50 °Cand10%w/wsolids. APEG -PHPMA
Figure 1. Gel permeation chromatograms obtained for PEG113-PHPMA200
diblock copolymer syntheses conducted at various temperatures using
RAFT aqueous dispersion polymerization at 10% w/w solids at a
113
300
diblock copolymer was targeted using a [PEG113-DB]/[AIPD] molar
ratio of 3.0. The semilogarithmic plot shows three regimes: an initial
induction period up to 50 min, a slow rate of solution polymerization
between 50 and 60 and 120 min, and a relatively fast rate of
polymerization after 120 min. The latter time corresponds to the onset
of micellar nucleation, which occurs at an HPMA conversion of around
[
PEG113-DB]/[initiator] molar ratio of 3.0. Suitable free radical
initiators were selected for a given reaction temperature: ACVA was
used at 70 °C, AIBA was employed at 55 and 60 °C, and AIPD was
utilized at 50 and 45 °C (see main text for details).
4
5%. The inset shows the GPC curves obtained for a subset of these
kinetic data, indicating a linear evolution of M and a gradual reduction
n
in copolymer polydispersity with monomer conversion.
RAFT AQUEOUS DISPERSION POLYMERIZATION
OF HPMA
To assess the efficiency of this new PEG113-DB RAFT agent, the
RAFT aqueous dispersion polymerization of HPMA was
conducted at 10% w/w solids, targeting a mean degree of
■
target DP = 300) at 50 °C using the PEG113 macro-CTA was
obtained by periodic sampling of the reaction solution. The
semilogarithmic plot in Figure 2 clearly shows three distinct regimes.
1
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There is an initial induction period of ∼50 min, which is commonly
58−61
observed for RAFT polymerizations.
The second regime,
which occurs between 60 and 120 min, corresponds to the onset of
polymerization and the formation of molecularly dissolved diblock
33
copolymer chains. In the third regime, there is a dramatic increase
in the rate of polymerization, which corresponds to the onset of
micellar nucleation. At this point, unreacted HPMA monomer
enters the micelle cores to solvate the hydrophobic PHPMA chains,
which produces a high local monomer concentration and hence the
33
observed rate enhancement. Interpolation of the green and blue
lines indicates that micellar nucleation occurs at around 110 min,
which corresponds to 45% HPMA conversion for this particular
formulation. This intermediate conversion corresponds to a mean
degree of polymerization of 135 for the core-forming PHPMA
block, which is somewhat higher than the critical DP of
∼
92 previously reported by Blanazs et al. for a PGMA47 macro-
CTA utilized to polymerize HPMA (target DP = 200) at 70 °C.
33
Presumably, the growing PHPMA chains are more effectively
solubilized by the relatively long hydrophilic PEG113 macro-CTA in
the aqueous reaction solution. However, other parameters cannot
be ignored. For example, the lower polymerization temperature of
Figure 3. Gel permeation chromatograms (THF eluent, refractive index
detector) obtained for a series of PEG113-PHPMA diblock copolymers
x
synthesized at 50 °C and 10% w/w solids, where x is systematically
varied from 100 to 400. Calibration was achieved using a series of near-
monodisperse poly(methyl methacrylate) standards.
5
0 °C used in the present work is known to affect the water
63
solubility of the HPMA monomer. GPC analysis (see inset in
Figure 2) confirms that the evolution of M with HPMA monomer
n
conversion is linear, and the polydispersity of the final diblock
copolymer is relatively low (M /M = 1.22). These characteristics
worms. TEM images obtained for PHPMA DPs of 200−220
indicated a pure worm phase. These dispersions formed soft,
reasonably transparent gels, presumably because multiple inter-
w
n
are typical for a pseudo-living radical polymerization and indicate
that good control is maintained under dispersion polymerization
conditions, despite the relatively low CTA/initiator molar ratio used
42,67
worm contacts lead to a percolation network.
In contrast,
58−61
PEG -PHPMA formed a mixed phase consisting of worms,
113
250
in these RAFT syntheses.
It is also noteworthy that the non-
vesicles, and some ‘jellyfish’ structures, which have previously been
zero y-intercept is simply due to the PEG113 macro-CTA, which has
observed as important intermediates for the worm-to-vesicle
an apparent M of around 8000 (vs poly(methyl methacrylate)
n
33
transition. A pure vesicle phase was obtained when the target DP
calibration standards) when analyzed using THF GPC. The effect of
increasing the target DP of the core-forming PHPMA block from
of the PHPMA block exceeded 250, with DLS intensity-average
diameters of either 359 nm (PDI = 0.05) or 377 nm (PDI = 0.07)
being observed for DPs of 300 or 400, respectively. To further
investigate the RAFT aqueous dispersion polymerization of
HPMA at 50 °C, a detailed phase diagram was established by
100 to 400 was systematically investigated by preparing a series of
PEG -PHPMA copolymers at a fixed concentration of 10% w/w
113
x
solids. In each case more than 99% HPMA conversion was
attained, as judged by the eventual disappearance of the vinyl signals
1
utilizing the same systematic approach adopted for related PISA
at δ 6.2−6.6 in the H NMR spectra (Figure S1c). For target DPs of
34,36,43,44
formulations.
This phase diagram is shown in Figure 4,
1
00−220, GPC studies indicated that RAFT control was
maintained, with a systematic increase in M and relatively low
n
polydispersities (M /M = 1.16−1.19) being observed (Figure 3).
w
n
However, a significant increase in polydispersity was observed
when targeting DPs of 300 or 400 (M /M = 1.39 and 1.78,
w
n
3
3,36,63
respectively). This problem is well-documented
and is
actually due to the presence of a small amount (<0.3 mol %) of
dimethacrylate in the HPMA monomer, rather than loss of
RAFT control. This impurity inevitably leads to light branching
64−66
as longer PHPMA chains are targeted.
Visual inspection of the final aqueous dispersions indicated
progressively greater turbidity as the DP of the PHPMA core-
forming block was increased, suggesting systematic variation in
the size and/or morphology of the colloidal PEG113-PHPMAx
nano-objects prepared under these conditions. This was
confirmed by DLS and TEM studies (see Table S1). A mono-
modal distribution with an intensity-average diameter of 26 nm
(
polydispersity, PDI = 0.06) was recorded for PEG -PHPMA ,
113 100
which suggested the formation of near-monodisperse spherical
micelles. For PEG -PHPMA , there was an increase in both the
mean diameter and polydispersity (38 nm, PDI = 0.29), suggesting
a possible change in copolymer morphology due to the greater
volume fraction of the hydrophobic block. This was confirmed by
TEM studies, which revealed a mixture of spheres and short
Figure 4. Phase diagram elucidated for the RAFT aqueous dispersion
polymerization of HPMA at 50 °C using a PEG113-DB macro-CTA
based on combined TEM and DLS studies. Phase regions consist of
spheres (S), worms (W), unilamellar vesicles (ULV), oligolamellar
vesicles (OLV), ill-defined lamellae (L), jellyfish (J), mixed phases (M),
and a precipitate phase (P).
113
180
1
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with some representative TEM images for the various pure phases
being depicted in Figure 5. For concentrations up to 17.5% w/w,
Figure 5. Representative TEM images obtained for PEG113-PHPMAx
diblock copolymers (where x = 150, 200, or 400): (a) spheres prepared
at 10% w/w; (b) worms prepared at 7.5% w/w; (c) vesicles prepared at
7
1
.5% w/w; (d) spheres prepared at 15% w/w; (e) worms prepared at
5% w/w; and (f) vesicles prepared at 15% w/w.
Figure 6. Schematic formation of the oligolamellar vesicles obtained
from PISA syntheses of PEG113-PHPMA conducted at 20% w/w solids
x
and the unilamellar vesicles formed when the PISA syntheses are
the phase diagram resembles those previously reported for
PHPMA-based PISA formulations. At the lowest concentration
investigated (5.0% w/w), only mixed phases were observed.
Presumably, this simply reflects the reduced probability of inter-
sphere fusion under these conditions, which is considered to be the
conducted at lower copolymer concentrations.
(
see Figures 7a, 7b), respectively. However, a remarkable range of
diblock copolymer nano-objects was observed when targeting
higher PHPMA DPs. For example, a mixed phase containing flat
lamellar disks interlinked by long worms was observed for the
33
first step in the generation of higher order morphologies. TEM
studies on PEG -PHPMA nano-objects prepared at between 7.5
113
x
PEG -PHPMA dispersion (Figure 7c). Similar morphologies
and 17.5% w/w confirm the presence of pure sphere, worm, and
vesicle phases. Pure spheres are typically observed at PHPMA DPs
of 150 or less. Pure worm phases are observed at DPs between 180
and 220, but this upper limit is reduced to 200 at higher copolymer
concentrations (15% w/w). The relatively narrow worm phase
window is almost encompassed by two mixed phases: spheres plus
worms and worms plus vesicles. Conventional unilamellar (i.e.,
single-walled) vesicles are generally observed above DP 250. In
addition to these various structures, some ‘jellyfish’ are observed;
this is a known intermediate in the morphological transformation of
113
180
have been previously reported by Jain and Bates for postpolymeriza-
68
tion processed amphiphilic PEG-based diblock copolymers. For a
PHPMA DP of 220, a viscous gel was formed that contained a wide
range of exotic structures (see Figure 7d). As well as worms and
some vesicles, many ill-defined lamellae and jellyfish were also
observed. The presence of these exotic nanostructures suggests that
the preferred molecular curvature of the copolymer chains is
intermediate between that for cylindrical and that for flat lamellar
sheets. Presumably, this parameter approaches a relatively narrow
range whereby the energy penalty required to form the edges of
large lamellar sheets is less than the energy required for these sheet
33
worms into vesicles (see Figure 6). THF GPC studies conducted
on these copolymers indicate diblock copolymer chains of relatively
low polydispersity for PHPMA DPs below 300 (see Table S1).
When syntheses were conducted at 20% w/w solids, GPC
polydispersities ranged from 1.32 to 1.84 for target DPs of 100 to
69,70
to wrap up to form vesicles.
TEM studies indicate that further
increasing the PHPMA DP up to 250−300 (Figure 7e) yields
mostly large ‘vesicles within vesicles’ (a.k.a., oligolamellar vesicles,
OLVs) and some nanotubes. These aqueous dispersions also
formed opaque gels, which is most likely due to close packing of the
lamellae and/or vesicles.
2
50, which are generally somewhat higher than those observed for
the same copolymer compositions prepared at 10% w/w (M /M =
w
n
1
.17−1.32). Moreover, M /M values of 3.8 and 18.7 were obtained
w
n
when targeting PHPMA DPs of 300 and 400, respectively. These
polydispersities are much higher than those observed for the
equivalent diblock copolymers prepared at lower concentrations
SAXS ANALYSIS
■
Scattering techniques, in particular SAXS, is an ideal in situ
integral method for assessing the morphologies of diblock
copolymer nanoparticles. Since the TEM images of the
dispersions synthesized at 20% w/w reveal morphologies that
have not previously been observed in such PISA syntheses (see
Figures 4 and 7a−e), statistically robust SAXS measurements
were conducted to exclude possible artifacts resulting from the
TEM sample preparation. Unlike TEM, SAXS is conducted on
aqueous dispersions and is averaged over many millions of
nanoparticles. Since a wide range of diblock copolymer
morphologies (spheres, worms, stacked lamellae, and both
unilamellar and oligolamellar vesicles) were observed for the
dispersions synthesized at 20% w/w (Figure 4), various
(
e.g., M /M = 1.32 and 1.86 respectively at 15% w/w) and cannot
w n
be solely attributed to the dimethacrylate impurities in HPMA
monomer. This was confirmed by conducting a PEG -PHPMA
113
300
synthesis at 20% w/w using purified HPMA monomer (column
chromatography reduces the level of dimethacrylate impurity
33
substantially ), since the copolymer polydispersity could only be
reduced to 1.49 (Figure S5). This suggests some loss of
RAFT control under these conditions. In principle, the relatively
high viscosity of the reaction mixture may retard diffusion of the
macro-CTA, but further studies are clearly warranted. For the
2
0% w/w copolymer syntheses, PHPMA DPs of 100 and 150
produced spheres and a mixed phase of spheres and short worms
1
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Figure 7. Representative TEM images obtained for the various PEG113-PHPMA diblock copolymer nano-objects produced using a PISA formulation
x
for the RAFT dispersion polymerization of HPMA at 20% w/w solids. (a) PEG113-PHPMA100; (b) PEG113-PHPMA150; (c) PEG113-PHPMA180; (d)
PEG113-PHPMA220 jellyfish and stacked lamellae (black arrows); (e) PEG113-PHPMA300 oligolamellar vesicles. (f) SAXS patterns (symbols) and data
fits (solid lines) obtained for the respective dilute (1.0% w/w) copolymer dispersions. Fitting curves for three populations used for the PEG113
PHPMA220 SAXS data (blue circles) analysis are also shown: worms (black dashed line), oligolamellar structures (red dashed line), and Gaussian chain
blue dashed line) The upper green circles SAXS pattern shows the thermally-induced fusion of a 1.0% w/w dispersion of PEG113-PHPMA100 spheres to
-
(
form short worms after being exposed to the synchrotron X-ray beam for 1 s.
structural models had to be used to analyze the SAXS data. Both
form and structure factors for these models are discussed below.
SAXS scattering patterns (see Figure 7f) were consistent with the
corresponding TEM images (see Figure 7a−e). Systematic
1
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Journal of the American Chemical Society
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Table 1. Structural Parameters Obtained from SAXS Data Fitting: Cross-Section for Spheres (2R ), Worms (2R ), and Vesicle
ss
sw
Membranes (R ), the Worm Kuhn Length (b), the Worm Contour Length (L), the Mean Number of Stacks in the Oligolamellar
sv
Vesicles (N), and the Mean Stacking Separation in the Oligolamellar Vesicles (d)
#
DP
morphology
cross-section, nm
σ, nm*
b, nm
L, nm
N
d, nm
1
1
2
00
00
20
spheres
27.4(1)
28.8(1)
30.9(1)
17.5(2)
19.0(1)
3.2(1)
3.4(1)
4.0(1)
2.3(3)
2.5(1)
−
−
28(2)
−
−
−
−
−
2
−
−
−
61(1)
spheres, spherical dimers, trimers
worms
173(12)
ULV + OLV
−
−
−
−
3
00
ULV + OLV
3
49(1)
*
#
σ is a standard deviation of the cross-section parameter normal distribution. Numbers in brackets represent errors for the fitted parameters.
variation of the mean degree of polymerization of the core-forming
PHPMA block leads to an evolution in diblock copolymer
morphology. Thus PEG -PHPMA produced SAXS patterns
corona block is 41.8 nm (113 × 0.37). If the PEG Kuhn length is
76
1.0 nm, then the radius of gyration of an unperturbed
0.5
PEG chain is (41.8/6) = 2.6 nm. It was also found that
spheres (which represent individual micelles) dominate the
PEG113-PHPMA100 aqueous dispersion (relative volume fraction,
1
13
100
corresponding to mainly spherical micelles (Figure 7f, see lowest
SAXS curve). However, in view of the TEM images obtained for this
dispersion (see Figure 7a,b) and our previous work suggesting dimer
k ∼ 0.8) with only a small population of dimers and trimers
1
42
formation, minor populations of spherical dimers and trimers
were also included. In addition, this is justified by the fact that the
SAXS pattern at low q deviates slightly from a zero slope, which is
characteristic for spheres, suggesting a presence of an elongated
structural morphology. Because the X-ray scattering length
(total volume fraction, k + k ∼ 0.2).
2
3
This diblock copolymer morphology is temperature-sensitive:
increasing the solution temperature can cause an order−order
transition to form worm-like micelles. The normal X-ray
exposure and data acquisition time were 100 ms per frame.
Under these conditions, no changes in diblock morphology were
observed. However, continuous exposure of the diblock
copolymer dispersion to the synchrotron X-ray beam for just
1 s (i.e., 10 frames) caused local heating and induced discernible
changesinthe SAXS patterns. Theslope of the curve at low q tends
toward −1, indicating transformation from a spherical morphology
into more anisotropic structures. Fitting the SAXS pattern
obtained after 1 s of X-ray exposure indicated that the proportion
of dimers and trimers increased significantly (k + k = 0.7). The
10
−2
densities of the copolymer blocks (ξPHPMA = 11.11 × 10 cm ,
1
0
−2
core block of the micelles, and ξPEG = 11.37 × 10 cm , corona
block of the micelles) are similar, it was found that the SAXS
patterns of the PEG -PHPMA spherical micelles could be
1
13
100
fitted reasonably well by a relatively simple model based on a
sphere form factor (Figure 7f, lower curve). SAXS analyses using a
71,72
more sophisticated micelle model,
validating this approach, are
given in the Supporting Information, Figure S6. The total
scattering intensity of a mixture of spherical unimers, dimers,
and trimers, I, can be expressed as
2
3
sphere diameter is also slightly increased from 27.4 to 28.8 nm
Table 1), presumablyowing tothermal expansion. The worm-like
(
micelles are described as flexible chains (cylinders) with a circular
cross-section and a uniform scattering length density, for which the
scattering intensity, I, can be expressed as
3
2
2
I = k Φ (qR ) + Φ (qR ) ∑ nk S (q) + c F(q, R )
1
ss
ss
n n
c c
g
n=2
(1)
I = Pworm(q, L, b)P
(q, Rsw)
cs_worm
(3)
where n is the number of spheres forming unimers (n = 1),
dimers (n = 2) or trimers (n = 3), and k is the volume fraction of
where b is the Kuhn length of the worms, L is the mean contour
length of the worms, and Rsw is their cross-section radius. The
n
3
each nano-object in the sample, ∑
k = 1. Φ(qR ) =
n =
1
n
ss
cross-section form factor is given by P
= [2J (qR )/
cs_worm 1 sw
3
3
[sin(qR ) − qR cos(qR )]/(qR ) is the form factor amplitude
2
ss
ss
ss
ss
(qR )] , where J (qR ) is the first-order Bessel function of the
sw 1 sw
of a sphere of radius R . The second term in eq 1 represents the
ss
first kind. A complete expression for the form factor of self-
form factor for spherical dimers and trimers, where S (q) can be
n
avoiding chains, P (q, L, b), can be found either in the SASfit
worm
73
77
78
obtained using the Debye equation:
software manual or in the original paper.
Scattering patterns corresponding to unilamellar and
n−1
n
2
n
sin(qrij)
oligolamellar vesicles (ULV and OLV) were fitted using a
S (q) = 1 + ∑ ∑
n
79,80
qrij
model for weakly ordered membrane stacks
implemented in
i=1 j=i+1
(2)
77
SASfit. The scattering intensity, I(q), is expressed as
and the intersphere separation distances are expressed as
2
I = Pcs_layer(q, R )S(q)/q + c F(q, R )
sv
c c
g
(4)
r = r = 2R , and r = 4R . The background scattering of the
12
23
ss
13
ss
PEG corona block is modeled using the Debye function,
where Rsv is the cross-section of the vesicle membrane. The
2
2
2
2
4 4
F (q,R ) = 2[exp(−q R ) − 1 + q R ]/(q R ). The radius of
c
g
g
g
g
form factor for this membrane is given by P
(q, R ) =
cs_layer
sv
gyration of the corona block is R and c is the relative
2
g
c
[sin(qR /2)/(qR /2)] and the structure factor, S(q), for
sv sv
80
concentration of the corona block. Thus five parameters (Rss,
R , c , k , and k ) are used to fit the SAXS data. Programming
stacked membranes is described using paracrystalline theory:
g
c
2
3
7
4
N−1
tools within the Irena SAS Igor Pro macros were used for
2
2 2
S(q) = N + N + 2
∑
(N − k)cos(kqd)exp(−k q Δ /2)
u
model fitting.
k=1
The fitted R was about 2 nm, which is close to the calculated
g
value. Assuming that the projected contour length of an ethylene
glycol repeat unit is 0.37 nm (estimated from the crystal structure
of PEG homopolymer), the contour length of the PEG113
where N is the mean number of stacks, d is the mean stacking
separation, Δ is the stacking disorder parameter, and N is the
u
75
number of uncorrelated scattering layers. Paracrystalline theory
1
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Journal of the American Chemical Society
Article
does not include any contribution from uncorrelated scattering,
the final copolymer morphology that is obtained at 20% solids.
This is consistent with the corresponding TEM images. A
PEG113-PHPMA400 diblock copolymer synthesis conducted at
20% w/w merely produced a viscous macroscopic precipitate
with little or no TEM evidence for the formation of any nano-
objects. Thus this latter diblock composition most likely
represents an effective upper limit for the PEG113-PHPMAx
formulation whereby the copolymer chains do not have sufficient
mobility to undergo well-controlled self-assembly on the time
scale of the relatively fast RAFT polymerization.
thus N accounts for an additional diffuse background. Its origin
u
can be attributed to membrane layers with strong lattice defects
or unilamellar vesicles. The background scattering of the corona
block in eq 4 was modeled similarly to eq 1 using the Debye
function, F (q, R ). Thus only three fitting parameters (b, L, and
c
g
Rsw) for the worm-like micelles and seven fitting parameters
R , N, d, Δ, N , c and R ) for the ULV and OLV structures are
(
sv
u
c
g
required to describe their SAXS patterns. No global parameters
such as direct beam intensity, normalization coefficient, geometry
of the measurements, or X-ray beam polarization param-
eters are included in the intensity expressions (eqs 1, 3, and 4).
Size polydispersity of the copolymer morphologies was
determined assuming a normal distribution of the cross-section
parameters in all SAXS models (R , R , and R ). Reasonably
Higher order structures such as lamellae or close-packed
cylinders are well-known for concentrated aqueous solutions of
Pluronic-type diblock copolymers and other block copoly-
1
7,18,83−85
mers.
However, as far as we are aware, the present study
is the first example of such structures being formed during a PISA
synthesis. These results suggest that PISA formulations offer
considerable potential for the convenient and efficient
generation of block copolymer-based nanostructured fluids.
ss
sw
sv
good fits to the SAXS patterns recorded for the worm-like micelles
and vesicles can be obtained, even though only simplified structural
models are utilized (Figure 7f). The PEG -PHPMA diblock
113
220
copolymer is best described as a mixture of worm-like micelles and
stacked membrane layers and so represents an intermediate
structure in the evolution from worm-like micelles to vesicles, see
TEM image shown in Figure 7d. No diblock copolymer
composition could be identified at the highest concentration
EFFECT OF TEMPERATURE ON NANO-OBJECT
■
MORPHOLOGY
42,62
There is literature precedent
to suggest that these PEG113-
PHPMAx nano-objects should undergo thermo-responsive
behavior, thus temperature-dependent rheology studies were
undertaken. A 10% w/w aqueous dispersion of PEG113
PHPMA220 formed a viscous liquid at 25 °C, which became a gel
on cooling to 12 °C (see Figure 8). Further cooling causes
(
(
20% w/w solids) that enabled a pure worm phase to be obtained
see Figure 4). Thus structural parameters for the worms could only
-
be determined from the mixed-phase dispersion. In total, 14
parameters (four parameters for the worms, including R standard
sw
deviation, eight parameters for the stacked membrane layers,
including R standard deviation, and two parameters describing the
sv
relative volume fractions of these phases) were required to fit the
SAXS data (Figure 7f, blue circles). The fitting model was initially
constructed using results obtained for the pure phase of stacked
membrane layers (OLV + ULV, Figure 7f, red circles) and micellar
spherical dimers and trimers (generated by local heating during an
X-ray exposure of 1 s, Figure 7f, upper green circles). The
parameters corresponding to different structural morphologies were
allowed to vary alternately during subsequent fittings. The worm
Kuhn length is comparable to their cross-section (Table 1), which
suggests that these diblock copolymer worms are quite flexible. The
worm contour length is about 6 times that of the Kuhn length.
However, the contour length L is likely to be longer, as its value is
usually affected by the structure factor originating from worm
81
packing. No structure factor was included in the worm model
eq 3), because this would require the concentration dependence of
(
the apparent molar mass of the worms; this is not possible since no
pure worms phase can be obtained at 20% w/w solids (see Figure 4).
SAXS data obtained for PEG113-PHPMA300 with a relatively long
PHPMA core block can be satisfactorily fitted using the stacked
membrane model, eq 4 (Figure 7f), suggesting that the
transformation of worm-like micelles into vesicles is complete
during synthesis. The mean number of stacks, N, is higher
compared to the PEG113-PHPMA220 intermediate morphology,
while the mean stacking separation d is lower (Table 1). There is
an obvious correlation between the cross-section of the diblock
morphologies and the degree of polymerization of the PHPMA
block. The former parameter increases from spheres to worms to
vesicles (in the latter case the cross-section is reduced by
approximately a factor of 2 compared to spheres and worms,
presumably due to substantial interdigitation of the PHPMA
Figure 8. Storage (G′) and loss (G″) moduli recorded for a 10% w/w
PEG1 -PHPMA worm gel undergoing a temperature cycle between
13
220
25 and 4 °C (measurements were conducted at 1.0% strain and an
−1
angular frequency of 1.0 rad s ).
degelation, producing a transparent free-flowing liquid. Surprisingly,
this transformation was irreversible: the dispersion remained a fluid
during the heating cycle. Degelation was confirmed by frequency-
dependent rheology studies (see Figure S7): G′ exceeds G″ over a
wide frequency range at 12 °C, whereas G″ is greater than G′ at
4 °C. Moreover, a strain sweep confirms the viscoelastic properties
of the gel formed at 12 °C. A second thermal cycle between 25 and
11 °C was conducted (see Figure 8). In contrast, this thermal cycle
proved to be fully reversible. The relatively weak gel (G′ ∼ 10 Pa)
formed at 25 °C became a significantly stronger gel (G′ ∼ 65 Pa) at
11 °C, before returning to its original state at 25 °C. TEM studies
8
2
chains). In summary, SAXS characterization of aqueous
dispersions of PEG-PHPMA diblock copolymer nano-objects
confirm that, for a fixed degree of polymerization of the PEG
block, the degree of polymerization of the PHPMA block dictates
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Journal of the American Chemical Society
Article
8
7
suggest a subtle change in morphology from short worms (and
much more favorable for intracellular delivery applications. An
important advance over these earlier studies is the ability to induce a
relatively rapid worm-to-vesicle transition at copolymer concen-
trations of up to 20% w/w, rather than merely for highly dilute
68
loops) to relatively long worms, which are more efficient gelators
see Figure S8). Similar thermo-responsive behavior was also
observed for the PEG -PHPMA vesicles, which underwent a
(
113
300
63
vesicle-to-sphere transition on cooling to 2 °C overnight. By staining
a cold TEM grid with the cold sample, the resulting spheres could be
imaged by TEM, and DLS experiments reported a mean spherical
diameter of 40 nm (Figure 9a). On incubating at 50 °C for 24 h, these
dispersions. This means that the change in copolymer morphology
profoundly affects the physical form of the dispersion, i.e., whether it is
agel orafluid. Moreover, the relatively high copolymer concentration
should in principle facilitate efficient vesicle encapsulation. If this is
correct, this thermal transition could provide a highly attractive
mechanism for loading PEG-PHPMA vesicles with protein or
antibody payloads for biomedical applications. In this context, it is
noteworthy that the temperature is not so high as to cause
denaturation of the payload.
CONCLUSIONS
■
Well-defined PEG-based RAFT agents with high degrees of
functionalization can be readily prepared by reacting mono-
aminated PEG chains with the activated ester of a commercially
available carboxylic acid-functionalized RAFT agent. The resulting
PEG macro-CTA can be used for the RAFT aqueous dispersion
polymerization of HPMA at 50°C. This relatively low temperature
ispreferred in viewof thelimitedsolubilityof thePEG macro-CTA
in hot aqueous solution. An optimized formulation using an AIPD
initiator (PEG macro-CTA/AIPD = 3.0) afforded diblock
copolymers with relatively low polydispersities and high blocking
efficiencies, as judged by GPC analysis. High monomer
conversions (>95%) were achieved in all cases and both TEM
and DLS studies indicated that polymerization-induced self-
assembly led to the generation of various PEGylated nano-objects,
including spherical micelles, worms, or vesicles, depending on the
precise synthesis conditions. A systematic approach enabled the
elucidation of a detailed phase diagram for this new PEG -
1
13
PHPMA diblock copolymer formulation. Interestingly, high
x
copolymer concentrations (>17.5% w/w) and relatively long
PHPMA blocks produced a remarkable range of nanostructures,
including the first observation of oligolamellar vesicles formed
during a PISA synthesis. SAXS studies allowed characterization of
this new phase as well as some complex intermediate structures. In
particular, detailed SAXS analysis indicates an average of three
membranes per oligolamellar vesicle. SAXS also confirms the
Figure 9. Effect of carrying out a temperature cycle on a 10% w/w
dispersion of PEG113-PHPMA300 vesicles. TEM images obtained for (a)
spherical micelles after cooling in ice for 1 h; (b) smaller vesicles after
warming to 50 °C for 24 h; (c) large, polydisperse vesicles initially
obtained after synthesis; (d) DLS size distributions obtained for the
respective samples after dilution to 0.1% w/w; (e) centrifuge tube
containing sedimented PEG113-PHPMA300 vesicles loaded with
fluorescently poly[(2-methacryloyloxy)ethyl phosphorylcholine].
thermo-responsive nature of these PEG113-PHPMA nano-
x
objects, since partial fusion of spherical micelles can be
induced by local heating caused by the synchrotron X-ray
beam. Moreover, the PEG113-PHPMA gels formed at above
x
spheres transform back into vesicles that are somewhat smaller and
less polydisperse than the original vesicles (compare Figure 9b,c).
Again, this TEM observation is confirmed by DLS studies (see
Figure 9d), where the hydrodynamic diameter is reduced from
10% w/w solids exhibit interesting rheological properties:
unlike the previously reported PGMA-PHPMA formulation,
the gel strength increases at lower temperatures, with a
critical gelation temperature being observed on gradual
cooling. This is presumably because branched structures and
perforated bilayers are converted into linear worms. Sub-
sequent warming to 40 °C causes a worm-to-vesicle transition,
which results in degelation. Cooling of PEG113-PHPMA300
vesicles to 2 °C overnight induces a vesicle-to-sphere transition.
Intriguingly, smaller, lower polydispersity vesicles are produced
on warming to 50 °C. This thermal transition indicates a
convenient loading mechanism for the in situ encapsulation
of proteins or antibodies, which in turn suggests that such
PEGylated nano-objects may have biomedical applications in
the context of intracellular delivery. Finally, we note that the
optimized RAFT aqueous dispersion polymerization protocol
described herein offers a convenient and cost-effective synthetic
route, since no postpolymerization processing or purification is
required other than dialysis.
4
50 nm (PDI = 0.30) to 120 nm (PDI = 0.09). This thermal cycle was
utilized to encapsulate a water-soluble rhodamine 6G-labeledpoly-
86
((2-methacryloyloxy)ethyl phosphoryl-choline) [PMPC] as a
model water-soluble macromolecule. The vesicles could be
sedimented by centrifugation, which enabled free (nonencapsulated)
PMPC chains to be removed via decantation of successive
supernatants. After the sixth centrifugation−redispersion cycle, UV
irradiation confirmed that the vesicle sediment was fluorescent,
whereas the supernatant was nonfluorescent (see Figure 9e). This
indicates retention of the encapsulated PMPC chains and successful
removal of the free PMPC chains. This thermal transition
demonstrates a very promising strategy for relatively efficient
encapsulation of macromolecules and/or nanoparticles within the
aqueous lumen of the vesicles at reasonably high concentrations.
Moreover, the reduction in vesicle dimensions to around 100 nm is
1
031
dx.doi.org/10.1021/ja410593n | J. Am. Chem. Soc. 2014, 136, 1023−1033

Journal of the American Chemical Society
Article
(
16) Chen, W.; Zheng, M.; Meng, F.; Cheng, R.; Deng, C.; Feijen, J.;
ASSOCIATED CONTENT
Supporting Information
Full experimental section and further characterization details for
the monoaminated PEG113 precursor, the PEG113 macro-CTA,
the SCPDB CTA, PEG113-PHPMA block copolymers, and
■
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AUTHOR INFORMATION
■
Corresponding Authors
n.warren@sheffield.ac.uk
s.p.armes@sheffield.ac.uk
8
(
9
(
Notes
The authors declare no competing financial interest.
(
2
ACKNOWLEDGMENTS
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Svetomir Tzokov is thanked for carbon coating TEM grids. The
authors are grateful to ESRF for providing beamtime. Diego
Pontoni (PSCM) and the personnel of ID02 station is thanked
for help with SAXS experiments. EPSRC is thanked for a
Platform grant (EP/J007846/1) to support NJW. SPA thanks
the European Research Council for an ERC Advanced
Investigator grant (PISA 320372).
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