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Cite this: DOI: 10.1039/c0xx00000x
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Thiol-responsive block copolymer nanocarriers exhibiting tunable
release with morphology changes
Qian Zhang,a Samuel Aleksanian,a Seung Man Nohb and Jung Kwon Oh*a
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
New thiol-responsive nanocarriers of amphiphilic block copolymers consisting of a pendent disulfidelabeled methacrylate polymer block (PHMssEt) and a hydrophilic poly(ethylene oxide) (PEO) block were
reported. These well-controlled block copolymers were synthesized by atom transfer radical
polymerization (ATRP) of a new pendent disulfide-functionalized methacrylate (HMssEt) in the presence
of PEO-Br macroinitiator. Due to its amphiphilic nature, the PEO-b-PHMssEt with narrow molecular
weight distribution self-assembled in aqueous solution to form monomodal micellar aggregates with
PHMssEt cores surrounded with hydrophilic PEO coronas. In response to thiols, the disulfide linkages
were cleaved, and thus self-assembled micelles were either converted to core-crosslinked micelles or
destabilized to further disintegrate, depending on the amount of added thiols. Such change in morphology
led to tunable release of encapsulated model drugs in aqueous solutions.
Introduction
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In recent years aqueous micellar aggregates of well-defined
amphiphilic block copolymers (ABPs) exhibiting stimuliresponsive (bio)degradation (SRD) have been extensively
explored due to their tremendous potential as multifunctional
drug delivery nanocarriers.[1-4] Nanoparticles exhibiting SRD
show great promise as nanotherapeutics due to their controllable
and tunable response to external triggers. This enables the
enhanced release of encapsulated therapeutics into targeted cells
while facilitating the removal of empty vehicles after the release.
Furthermore, SRD has been utilized to tune the morphologies of
self-assembled nanostructures.[5-6] Of external stimuli typically
including oxidative,[7] enzymatic reaction,[8] low pH,[9-13] and
light,[14-15] reductive reaction employing disulfide-thiol platform
is particularly promising because disulfide linkages are cleaved to
the corresponding thiols in response to thiols or reducing
agents.[16] In biological systems, glutathione (GSH, a tripeptide
containing cysteine) which plays an important role in cellular
metabolism is found at millimolar concentrations in cells,[17-18]
and at further elevated levels in cancer cells.[19]
a
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Department of Chemistry and Biochemistry and Center for Nanoscience
Research (CENR), Concordia University, Montreal, Quebec, Canada
H4B 1R6; E-mail: john.oh@concordia.ca
b
PPG Industries Korea , Cheonan 330-912 & Department of Chemical
and Biological Engineering, Korea University, Seoul 136-713, Republic
of Korea
†Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/b000000x/
This journal is © The Royal Society of Chemistry [year]
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A variety of thiol-responsive degradable ABPs and their
nanosized assemblies have been reported.[20-21] For example,
mono-cleavable micelles having single disulfide bonds in the
middle of symmetric triblock copolymers[22-24] and sheddable
micelles having single disulfides at the junctions of diblock
copolymers[25-36] have been explored. These micelles are
characterized as block copolymers having single disulfides along
the main polymer chains. Multi-cleavable micelles are also
designed with block copolymers having disulfides positioned in
the hydrophobic blocks as having multiple repeats or groups
along the main chains. Previous reports from our research
group[37-38] and others[39-41] have shown that these backbone
multi-cleavable micelles exhibit rapid and controlled release of
encapsulated biomolecules including fluorescent dyes and
anticancer drugs through main chain degradation mechanisms.
Furthermore, the degradation can be tuned with varying densities
of disulfides[42-43] and degrees of crystallinity.[44]
Another promising class of multi-cleavable micelles employs
block or copolymers having pendent disulfide linkages as side
chains. The design of these pendent multi-cleavable micelles is
advantageous in that they can be converted to core-crosslinked
micelles or nanogels with disulfide crosslinks through intra and
inter-chain thiol-disulfide polyexchange reactions in response to
reductive reactions. Such core-crosslinking provides enhanced
colloidal stability as well as prevents premature release of
encapsulated drugs during the circulation in the body.[45-46] A
further advantage of utilizing such reversible redox reaction is
that the newly-formed disulfide crosslinks are cleaved in the
presence
of
excess
thiols.
Such
change
in
hydrophobic/hydrophilic balance causes crosslinked micelles to
dissociate, thus enhancing the release of encapsulated drugs in
targeted cells. Several crosslinked nanogels labeled with pendent
[journal], [year], [vol], 00–00 | 1
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Materials and instrumentation
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No DTT
3,3'-Dithiodipropionic acid (ss-DCOOH), 2-hydroxyethyl
methacrylate (HEMA), N,N-dicyclohexyl carbodiimide (DCC),
4-dimethylaminopyridine (DMAP), copper(I) bromide (CuBr,
>99.99%),
N,N,N′,N′′,N′′-pentamethyldiethylenetriamine
(PMDETA, >98.0%), methanol (MeOH), and ethanol (EtOH)
from Aldrich and DL-dithiothreitol (DTT, 99%) from Acros
Organics were purchased and used as received. A water-soluble
macroinitiator, PEO-functionalized bromoisobutyrate (PEO5000Br, EO units DP  113), was synthesized using a reported
procedure.[55] Poly(ethylene glycol) monomethyl ether
(PEO5000-OH, Aldrich, 20 g, 4 mmol) reacted with 2-bromo-2methylpropionic acid (98%, Acros, 1 g, 6.0 mmol) in the
presence of DCC (0.9 g, 4.4 mmol), and a catalytic amount of
DMAP in CH2Cl2 (150 mL). The product was mixed with THF,
DTT < 1eq
DTT > 1eq
ss
Thiol-triggered
degradation
HS
ss
In DMF
Experimental
ss
SH
SH
ss
ss
PEO-b-PHMssEt
Model
drug
( )
Highly-branched or
crosslinked polymers
PEO-b-PHMSH
Aqueous
micellization
SH
In water
SH
ss
20
In this work, we have developed new thiol-responsive
nanocarriers based on well-controlled ABPs consisting of a
pendent disulfide-labeled methacrylate polymer block and a
hydrophilic poly(ethylene oxide) (PEO) block, exhibiting tunable
release of encapsulated model drugs with change in morphology
as a result of thiol-triggered degradation. The reductivelycleavable ABPs here were synthesized by atom transfer radical
polymerization (ATRP), a successful living/controlled radical
polymerization, of a new pendent disulfide-functionalized
methacrylate (HMssEt) in the presence of PEO-Br macroinitiator,
yielding PEO-b-PHMssEt (Scheme 1). PEO is a biocompatible
polymer with low toxicity that is well known to prevent
nonspecific protein adsorption, and has been FDA-approved for
clinical use. [53-54] The PHMssEt block is designed to be
responsive to thiols in hydrophobic micellar cores. Thus, in
response to different amounts of thiols, self-assembled micelles
with spherical morphology were either converted to corecrosslinked micelles or destabilized to further disintegrate. Such
ss
15
Scheme 1. Synthesis of well-defined PEO-b-PHMssEt ABP by ATRP initiated
with PEO-Br macroinitiator.
ss
10
change in morphology (here, core-crosslinked micelles or
destabilization-driven aggregates) led to tunable release of
encapsulated model drugs in aqueous solution (Scheme 2).
ss
5
disulfides have been developed. These nanogels are mostly based
on random copolymers prepared through post-modification of
dextrans with lipoic acids[47-48] and radical copolymerization of
pendent disulfide-labeled methacrylates.[49-51] However, only few
reports describe the synthesis of block copolymers and pendent
multi-cleavable micelles of our interests. An example includes
well-controlled ABPs consisting of poly(N-(2-hydroxypropyl)
methacrylamide)
and
poly(2-(pyridyldisulfide)ethyl
methacrylate) prepared by reversible addition fragmentation
chain transfer (RAFT) polymerization.[52] Further, there are still
needs to design new types of degradable micelles for better
understanding of structure-property relationship between
morphological variance and stimuli-responsive degradation.
SH
SH
SH
SH
Micelles
Core-crosslinked
micelles
PEO-b-PHMSH
Scheme 2. Degradation of PEO-b-PHMssEt and change in morphology of self-assembled micellar aggregates in response to different amounts of DTT, leading tunable
release of encapsulated model drugs.
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This journal is © The Royal Society of Chemistry [year]
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isolated from dispersion by vacuum filtration, and then dried in a
vacuum oven at 30 C for 12 h. In this way, residues such as
excess DCC, DMAP, and dicyclohexyl urea as a by-product
could be removed from the final product.
1H-NMR spectra were recorded using a 500 MHz Varian
spectrometer. The CDCl3 singlet at 7.26 ppm and DMSO-d6
quintet at 2.5 ppm were selected as the reference standard.
Spectral features are tabulated in the following order: chemical
shift (ppm); multiplicity (s - singlet, d - doublet, t – triplet, m complex multiple); number of protons; position of protons.
Molecular weight and molecular weight distribution were
determined by gel permeation chromatography (GPC). The first,
a Viscotek GPC, was equipped with a VE1122 pump and a
refractive index (RI) detector, two PolyAnalytik columns (PAS103L and 106L) were used with THF as an eluent at 30 C at a
flow rate of 1 mL/min. The second was an Agilent GPC with a
1260 Infinity Isocratic Pump and a RI detector, two Agilent
columns (PLgel mixed-D and mixed-C) were used with DMF
containing 0.1 mol% LiBr as an eluent at 50 C at a flow rate of 1
mL/min. Linear polystyrene (PSt) and poly(methyl methacrylate)
(PMMA) standards from Fluka were used for calibration.
Aliquots of polymer samples were dissolved in either THF or
DMF/0.1 mol% LiBr. The clear solutions were filtered using a
0.25 m PTFE filter to remove any THF-insoluble species. A
drop of anisole was added as a flow rate marker. Monomer
conversion was determined using 1H-NMR in CDCl3.
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Synthesis and purification of PEO-b-PHMssEt using ATRP
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Synthesis of HMssEt methacrylate bearing a pendent
disulfide linkage
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In the first step to synthesize HMss-COOH, a clear solution of
DCC (18.0 g, 87.2 mmol) in THF (35 mL) was drop wise added
into a mixture consisting of HEMA (10.5 g, 80.9 mmol), ssDCOOH (70.0 g, 332.9 mmol), DMAP (0.88 g, 7.2 mmol), and
THF (700 mL) in an ice bath for 40 min under vigorous stirring.
The resulting mixture was kept at 0 C for 30 min, and then
stirred at room temperature for 12 hrs. The formed solids
(dicyclohexyl urea) as by-products were removed by a vacuum
filtration and the organic solvents were removed using a rotary
evaporator at 40 C. The resulting residues were mixed with
chloroform (280 mL) and solids (presumably unreacted ssDCOOH) were removed by vacuum filtration. Chloroform was
removed by rotary evaporation to form residues containing
HMss-COOH.
In the second step, DCC (20.0 g, 96.9 mmol) dissolved in
chloroform (30 mL) was added drop wise to a solution consisting
of the oily residues (synthesized in the first step), DMAP (0.12 g,
0.98 mmol), EtOH (19 g, 412.4 mmol), and chloroform (80 mL)
in an ice bath. The resulting mixture was kept in the ice bath for
20 min and then stirred at room temperature for 12 hrs. The
formed solids (dicyclohexyl urea) were removed by vacuum
filtration. The organic solution was washed with 0.2 M aqueous
HCl solution (75 mL) and aqueous saturated NaHCO3 solution
(75 mL) twice, and then dried over MgSO4. Solvents were
removed by rotary evaporation and the product was purified by
silica column chromatography with a mixture of ethyl aceta
te/hexane (1/9 v/v). The product was collected as the second of
the total two bands off a silica gel column. The product was
isolated by evaporation of solvents in reduced pressure to form an
This journal is © The Royal Society of Chemistry [year]
oily residue (HMssEt). Yield = 11.9 g (42%). Rf = 0.36 on silica
(3/7 ethyl acetate/hexane). 1H-NMR (CDCl3, ppm) 6.12 (s, 1H,
H2C=C-), 5.59 (s, 1H, H2C=C-), 4.35 (s, 4H, C(O)OCH2CH2O(O)C-), 4.15 (q, 2H,-C(O)OCH2CH3), 2,91 (t,
4H, -CH2SSCH2-), 2.76 (t, 2H, -O(O)CCH2CH2SS-), 2.71 (t, 2H,
-SSCH2CH2C(O)O-), 1.94 (s, 3H, H2C=C(CH3)-), 1.26 (t, 3H, C(O)OCH2CH3). 13C-NMR (CDCl3, ppm): 171.5, 171.3, 166.9,
135.8, 126.0, 62.4, 62.2, 60.7, 34.1, 33.9, 33.2, 32.9, 18.2, 14.1.
Mass calculated for (C14H22O6S2Na+): 373.07555. Found:
373.07554.
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A procedure for ATRP of HMssEt catalyzed with
CuBr/PMDETA complex in the presence of PEO-Br
macroinitiator was carried out in anisole at 50 C. PEO-Br (0.30
g, 0.058 mmol), HMssEt (1.2 g, 3.42 mmol), PMDETA (4.5 mg,
0.026 mmol), and anisole (4.0 mL) were mixed in a 25 mL
Schlenk flask. The resulting mixture was deoxygenated by three
freeze-pump-thaw cycles. The reaction flask was filled with
nitrogen and then CuBr (2.9 mg, 0.02 mmol) was added to the
frozen solution quickly. The flask was closed, evacuated with
vacuum and backfilled with nitrogen three times. The mixture
was thawed and then the flask was immersed in an oil bath
preheated at 50 C to start polymerization. Aliquots were
withdrawn at different time intervals to analyze molecular weight
by GPC and conversion by 1H-NMR. Polymerization was
stopped by cooling and exposing the reaction mixture to air. For
purification, the as-synthesized polymer solution was passed
through a basic alumina column to remove the copper complex,
and then solvents were removed by rotary evaporation. The
products were precipitated from hexane three times, and then
dried in vacuum oven at room temperature for 18 hrs.
Determination of critical micellar concentration (CMC) by
tensiometry
An aliquot of the purified, dried PEO-b-PHMssEt (15 mg) was
dissolved in THF (2.5 mL). The clear polymer solution was dropwise added into deionized water (15 mL). The resulting
dispersion was kept under stirring overnight to remove THF,
allowing for colloidally stable micellar dispersion at a
concentration of 1.0 mg/mL. Then, aliquots of the aqueous stock
solution were diluted with different amounts of deionized water,
forming a series of aqueous PEO-b-PHMssEt solutions at various
concentrations ranging from 10-6 to 1.0 mg/mL. A tensiometer
was used to measure the pressure (mN/m) of the solutions as
follows; an aliquot of each solution (600 L) was carefully placed
on each well and equilibrated before measurements. The
tensiometer was calibrated using air and water.
Aqueous micellization of PEO-b-PHMssEt
105
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For the preparation of micellar aggregates in aqueous solutions,
deionized water was added drop-wise into clear polymer
solutions consisting of aliquots of the purified, dried PEO-bPHMssEt dissolved in THF (2.5 mL). The resulting dispersions
were kept under stirring for 24 hrs to remove THF. For a micellar
dispersion at 1 mg/mL, PEO-b-PHMssEt (15 mg) and water (15
mL) were used.
Journal Name, [year], [vol], 00–00 | 3
Transmission Electron Microscopy (TEM)
a
b
5
TEM images were taken using a Philips CM200 HR-TEM,
operated at 200 kV electrons and equipped with thermionic LaB6
cathode filament, anti-contamination cold finger, Genesis EDAX
system, and AMT V600 2k X2K CCD camera. The point-to-point
resolution and the line resolution of the machine are 0.24 nm and
0.17 nm, respectively. To prepare specimens, the micellar
dispersion at 1 mg/mL was dropped onto the TEM copper grids
(400 mesh, carbon coated). The grids were dried in air.
c
eh
f
c
d
b
15
Thiol-responsive degradation of PEO-b-PHMssEt in DMF
20
25
Aliquots of micellar dispersion (1 mg/mL, 2 mL) were mixed
with different amounts of DTT under stirring overnight. An
aliquot was taken for DLS measurements. For DLS
measurements in DMF, an aliquot of the mixture of aqueous
micelles with and without DTT (100 L) was mixed with DMF
(900 L). For GPC measurements, water was evaporated by
rotary evaporation, and the residual polymers were dissolved in
DMF/0.1 mole% LiBr.
Thiol-triggered release of NR from NR-loaded micelles
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NR-loaded micelles were prepared as follows; a stock solution of
NR in THF (5 mg/mL, 316 L), PEO-b-PHMssEt (15.8 mg), and
THF (2.5 mL) were mixed with water (15 mL). After the
resulting mixture was stirred for 24 hrs to remove THF, free NR
molecules were removed by filtration using a 0.45µm PES filter.
The final concentration of PEO-b-PHMssEt was adjusted to be
1.0 mg/mL. The sample was then divided into three equivalent
aliquots (3 mL each) in 20 mL vials. To two aliquots were added
DTT (0.2 mg, 0.2 equivalents to disulfides, 0.2 mM) and DTT
(4.95 mg, 5 equivalents to disulfides, 5 mM) and another aliquot
was used as control without DTT under stirring. Their
fluorescence spectra (ex = 480 nm) were measured at different
time intervals and fluorescence intensity at 610 nm was
monitored.
a
a
A stock solution of PEO-b-PHMssEt at 10 mg/mL was prepared
by mixing PEO-b-PHMssEt (100 mg) with DMF (10 mL). Then,
an aliquot of the stock solution (2 mL) was mixed with different
amounts of DTT. The mixtures were stirred for 12 hrs to analyze
molecular weight of PEO-b-PHMssEt by Agilent GPC/DMF and
size and size distribution by DLS.
Thiol-responsive degradation of micellar aggregates in
aqueous solution
j
7
6
e
h
i
5
4
3
2
1
0
Chemical shift/ppm
Fig. 1 Synthetic scheme and 1H-NMR spectrum of a new methacrylate bearing a
pendent disulfide linkage HMssEt in CDCl3.
55
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g). The 1H-NMR results, combined with those of 13C-NMR and
high resolution mass spectroscopies, confirm the structure of
HMssEt.
With successful synthesis and characterization of HMssEt,
ATRP of HMssEt catalyzed with CuBr/PMDETA in the presence
PEO-Br macroinitiator was carried out in anisole at 50 C. ATRP
conditions include [HMssEt]0/[PEO-Br]0/[CuBr]0/[PMDETA]0 =
60/1/0.35/0.45 and HMssEt/anisole = 0.4/1 wt/wt. For kinetic
studies, aliquots were taken to determine monomer conversion
using 1H-NMR and molecular weight and molecular weight
distribution using GPC. Fig. S1a shows the first-order kinetic
plot, reaching over 70% conversion in over 4 hrs. Such fast
polymerization is presumably attributed to high concentration of
active centers. However, molecular weight increased linearly with
PEO-Br
Mn = 8,400 g/mol
Mw/Mn = 1.03
3.0
3.5
PEO-b-PHMssEt
Mn = 21,700 g/mol
Mw/Mn = 1.09
4.0
4.5
5.0
5.5
log Mn (g/mol)
a
Results and Discussion
g
f
g
10
j
i
d
b
e
c
g
i
k k
d
EO protons
j
h
f
Synthesis of PEO-b-PHMssEt and kinetic studies
45
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First, a new methacrylate monomer bearing a pendent disulfide
linkage (HMssEt) was synthesized in two steps. As described in
Fig. 1 (upper), HEMA reacted with excess ss-DCOOH in THF
through a carbodiimide coupling reaction, resulting in HMssCOOH intermediate. Without purification, the intermediate
reacted with excess EtOH, and then column chromatography was
used to purify HMssEt at 43% yield. As seen in Fig. 1 (lower),
1H-NMR shows typical peaks: two singlets at 5.6 and 6.2 ppm
corresponding to vinyl protons (a) and triplets at 2.9 ppm
corresponding to two methylene protons adjacent to disulfide (f,
4 | Journal Name, [year], [vol], 00–00
f
g
c
i
j
x
x
d
5
k
e h
EO
a
4
3
2
b
1
0
Chemical shift/ppm
Fig. 2 For well-controlled PEO113-ss-PHMssEt42, GPC trace, compared with PEOBr macroinitiator (a) and 1H-NMR spectrum in CDCl3 (b). ATRP conditions:
[HMssEt]0/[PEO-Br]0/[CuBr]0/[PMDETA]0 = 60/1/0.35/0.45, HMssEt/anisole =
0.4/1 wt/wt. x denotes a trace of THF.
This journal is © The Royal Society of Chemistry [year]
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PEO-b-PHMssEt is amphiphilic and can form micellar aggregates
consisting of a hydrophobic PHMssEt core containing pendent
disulfide linkages surrounded with hydrophilic PEO coronas. To
determine critical micellar concentration (CMC) of PEO-bPHMssEt using tensiometry, a series of aqueous solutions of
various concentrations of PEO-b-PHMssEt ranging from 10-6 to
0.1 mg/mL were prepared. As seen in Fig. S3, the pressure does
not change at its lower concentration; however with an increasing
concentration of PEO-b-PHMssEt the surface pressure began to
increase, until a sharp rise around the CMC. From the two
equations obtained by fitting each data set to a linear relationship,
the CMC of PEO-b-PHMssEt was determined to be 49 g/mL.
Aqueous micellization through self-assembly of PEO-bPHMssEt using a solvent-evaporation method yielded colloidally
stable micellar aggregates in aqueous solution. The size and
morphology of degradable micelles were then examined using
dynamic light scattering (DLS) and transmission electron
microscopy (TEM) at a micellar concentration of 1.0 mg/mL;
well above the CMC (95 g/mL) (Fig. 3). DLS results indicate a
a)
25
hydrodynamic diameter = 50.9 ± 0.5 nm with a monomodal
distribution. TEM images indicate spherical micelles with
average diameter = 40.1 ± 6.7 nm, which is smaller than the size
determined by DLS. The difference in micellar sizes between
DLS and TEM can be attributed to the dehydrated state of the
micelles.[56] These results suggest the formation of spherical
monomodal micelles having PHMssEt cores surrounded with
PEO coronas in water.
Thiol-responsive cleavage of pendent disulfide linkages of
PEO-b-PHMssEt in DMF
45
Aqueous micellization of well-defined PEO-b-PHMssEt
50
The well-controlled PEO113-b-PHMssEt42 block copolymer
contains pendent disulfide linkages (42 disulfides in each
polymer chain). These disulfide linkages can be cleaved through
thiol-disulfide exchange reactions in response to DTT (a thiol) in
DMF as homogeneous media. Aliquots of PEO-b-PHMssEt were
mixed with DTT whose amounts are defined as the mole
No DTT
Mn = 25,000 g/mol
Mn = 63,000 g/mol
Mn = 17,000 g/mol
DTT
0.02 eq
Mn = 18,000 g/mol
Mn = 112,500 g/mol
0.5 eq
Mn = 21,700 g/mol
2.5 eq
4.0
4.5
5.0
5.5
log Mn (g/mol)
Fig. 4 GPC traces of PEO-b-PHMssEt in the absence and presence of different
amounts of DTT defined as mole equivalent ratio of DTT/disulfide in DMF.
Molecular weight data were obtained using a GPC with DMF as an eluent.
20
Dav = 50.9
% Volume
5
conversion and molecular weight distribution remained narrow
with Mw/Mn < 1.1. Furthermore, the GPC traces evolved to high
molecular weight region over the course of the polymerization
(Fig. S1b). These results suggest that ATRP of HMssEt
proceeded in a living fashion.
The as-synthesized polymer solutions were purified by passing
through a basic aluminum oxide column to remove Cu species
and precipitating from hexane to remove unreacted monomers,
yielding PEO-b-PHMssEt with Mn = 21,700 g/mol and Mw/Mn
=1.1 (Fig. 2a). 1H-NMR allows to determine the degree of
polymerization (DP) of PHMssEt block to be 42 from integral
ratios of peaks (d/EO, note the DP of PEO = 113), thus
suggesting the synthesis of well-defined PEO113-b-PHMssEt42
block copolymer (Fig. 2b).
0.5 nm
No DTT
15
D = 5.7 nm
10
DTT
5
0
10
100
1000
D = 9.1 nm
0.02 eq
Diameter (nm)
D = 10.5 nm
b)
0.5 eq
D = 4.3 nm
2.5 eq
1
10
100
Diameter (nm)
100 nm
Fig. 3 DLS diagram (a) and TEM image (b) of self-assembled PEO-b-PHMssEt
micellar aggregates. Scale bar = 100 nm.
This journal is © The Royal Society of Chemistry [year]
Fig. 5 DLS diagrams of PEO-b-PHMssEt in the absence and presence of different
amounts of DTT defined as mole equivalent ratio of DTT/disulfide in DMF.
Journal Name, [year], [vol], 00–00 | 5
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coronas. Here, the response of PHMssEt blocks having pendent
disulfide linkages in micellar cores to the different amounts of
DTT in aqueous solutions was investigated. Fig. 6 show DLS and
GPC results of micellar aggregates at different DTT
concentrations. In the absence of DTT, the micellar aggregates
had a monomodal distribution of both size and molecular weight.
In the presence of 0.2 mole equivalent DTT, the DLS diagram
shows a bimodal size distribution with a small population of large
aggregates (d > 1 m). The formation of such large aggregates
could be attributed to destabilization of micelles caused by the
cleavage of pendent disulfide linkages in micellar cores. Similar
to the results in DMF, GPC traces of PEO-b-PHMssEt as micellar
aggregates after the removal of water also show a bimodal
distribution with an occurrence of highly branched or crosslinked
polymers with Mn  887,000 g/mol. These high molecular weight
species could be formed by thiol-disulfide polyexchange
reactions in micellar cores. To further examine the formation of
crosslinked micelles in the presence of 0.2 mole equivalent DTT,
the aqueous micellar dispersion with and without 0.2 mole
equivalent DTT were mixed with DMF, a good solvent to both
PEO and PHMssEt blocks. As seen in Fig. S4, DLS results
indicates the diameter  20 nm for the micelles with 0.2 mole
equivalent DTT due to the occurrence of core-crosslinking, while
< 3 nm for the micelles without DTT. These DLS and GPC
results suggest the formation of mainly crosslinked micelles in
the presence of <1.0 mole equivalent DTT.
When the DTT increased to 5 mole equivalent, the size
distribution became multimodal with high populations of large
aggregates. Such destabilization of micelles is attributed to
change in polarity upon the cleavage of pendent disulfides. The
resulting PEO-b-PHMSH is less hydrophilic, not causing the
a)
25
Mn=25 kg/mol
20
% Volume
10
50
15
10
5
0
b)
15
Mn=29.8 kg/mol
(67%)
10
% Volume
5
equivalent ratios of DTT/disulfides. Fig. 4 shows GPC traces in
DMF mixed with and without different amounts of DTT after 12
hrs. In the absence of DTT, PEO-b-HMssEt had Mn = 25,000
g/mol with monomodal molecular weight distribution, using GPC
with DMF as an eluent. Note that this value is somewhat larger
than that (Mn = 21,700 g/mol) determined by GPC with THF as
eluent (Fig. 2). In the presence of DTT as the mole equivalent
ratio of DTT/disulfide = 0.02/1, the GPC trace of the degraded
PEO-b-PHMssEt became bimodal, having both low molecular
weight species with Mn 17,000 g/mol and high molecular weight
species with Mn  63,000 g/mol. When the DTT/disulfide ratio
was increased to 0.5/1, the bimodal GPC trace had low molecular
weight species with Mn 18,000 g/mol, which is similar to that
with the DTT/disulfide = 0.02/1, and high molecular weight
species with Mn  112,500 g/mol, which is significantly larger
than that with the DTT/disulfide = 0.02/1. It should be noted that
significant amounts of extremely branched polymers and
crosslinked gels could be removed during filtration of polymer
solutions prior to injection into GPC. When the DTT/disulfide
ratio further increased to 2.5/1, the GPC trace of the degraded
PEO-b-PHMssEt shows a monomodal molecular weight
distribution with Mn = 21,700 g/mol, which is little bit smaller
than that of PEO-b-PHMssEt in the absence of DTT (Mn =
25,000 g/mol). DLS measurements of the same mixtures in DMF
were taken and are shown in Fig. 5. The diameter increased from
5.7 nm to 9-11 nm with DTT/disulfide <1/1, while it decreased to
4.2 nm in the presence of excess DTT (DTT/disulfide = 2.5/1).
The cleavage of pendent disulfide linkages in response to thiols
generates two thiols: polymeric pendent thiols (i.e. PHMSH
units) and small molecular weight thiols (Scheme 3). Because of
co-existence with disulfides in the same or different polymer
chains, the resulting thiols, particularly polymeric pendent thiols,
can trigger intermolecular (in the different chains) or
intramolecular (in the same chains) thiol-disulfide polyexchange
reactions. When DTT/disulfide was <1/1, these reactions result in
highly branched or crosslinked polymers with higher molecular
weight (>50,000 g/mol) through the formation of new disulfide
crosslinks, causing the size to increase. However, with excess
DTT (DTT/disulfide >1/1), most disulfide linkages even
including new disulfide crosslinks formed through intermolecular
or intramolecular thiol-disulfide polyexchange reactions are
cleaved, resulting in predominantly PEO-b-PHMSH containing
pendent thiols, leading to decreasing sizes.
Mn=887 kg/mol
(33%)
5
0
c)
15
Mn=21 kg/mol
(95%)
% Volume
12
Scheme 3. Degradation of PEO-b-PHMssEt to corresponding thiols including
polymeric pendent thiol (PEO-b-PHMSH) and small molecular weight thiol in
response to excess DTT in DMF.
9
6
Mn=136 kg/mol
(5%)
3
0
1
10
100
1000
Hydrodynamic diameter (nm)
45
Thiol-responsive change in
aggregates in aqueous solution
morphologies
of
micellar
Self-assembled micellar aggregates of PEO-b-PHMssEt consist
of hydrophobic PHMssEt cores surrounded with hydrophilic PEO
6 | Journal Name, [year], [vol], 00–00
10000
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
log Mn (g/mol)
Fig. 6 DLS diagrams in water (left) and GPC traces in DMF (right) for micellar
aggregates of PEO-b-PHMssEt in the absence (a) and presence of different
amounts of DTT defined as mole equivalent ratio of DTT/disulfide = 0.2/1 (b)
and 5/1 (c). Molecular weight data were obtained using a GPC with DMF as an
eluent.
This journal is © The Royal Society of Chemistry [year]
5
10
micelles to completely dissociate, but leading to aggregation to
large particles.[47] The GPC trace shows a bimodal distribution;
however, the major population consists of low molecular weight
species with Mn = 21,000 g/mol, which are the cleaved PEO-bPHMSH having pendent thiols. A small population of <5 wt%
high molecular weight species is presumably attributed to the
unexpected oxidation of pendent thiols in oxygen present during
evaporation to remove water for GPC analysis. Overall, these
results suggest the disintegration of micelles upon the cleavage of
significant amounts of disulfide linkages in the presence of 5
mole equivalent DTT (excess DTT).
45
50
Tunable release of Nile Red with morphology change of
micelles in response to thiol
20
25
30
35
40
We then further investigated the release of Nile Red (NR), a
hydrophobic model drug, from NR-loaded micelles in the
presence of different amounts of DTT in water. We employed a
method to qualitatively characterize the release kinetics of NR
from micelles reported in literature.[57-58] This method,
monitoring the change in fluorescence (FL) of NR over time, is
based on the phenomenon that the FL of NR is intense when NR
is entrapped in the hydrophobic core of micelles, but it is
significantly lower in water due to either low solubility or
quenching of fluorescence by water molecules. In our
experiments, NR-loaded micellar dispersion at 1.0 mg/mL with
an average diameter = 56.4 ± 0.6 nm by DLS was prepared by the
solvent evaporation method (see DLS diagrams in Fig. S5). Each
dispersion was then divided into three samples: one without DTT
(as control) and others with 0.2 (0.2 mM) and 5 mole (5 mM)
equivalent DTT. Fig. S6 shows the overlaid fluorescence spectra
of NR in the two mixtures and Fig. 7 shows the evolution of
normalized FL intensity at em = 610 nm over time. In the
absence of DTT, no significant change in FL intensity was
observed, suggesting neither release nor photobleaching of a
significant amount of NR from micelles and that NR is confined
in small micellar cores. In the presence of 0.2 equivalent DTT
(DTT/disulfide <1/1), a slight decrease in FL intensity was
observed, compared to that without DTT. Due to the formation of
core-crosslinked micelles through thiol-disulfide polyexchange
reactions, a significant amount of NR is still confined in micellar
cores. However, little NR could be leaked from micellar cores
55
Conclusions
60
65
70
75
80
1.0
Normalized FL intensity
15
due to destabilization of micelles (evidenced by a small
population of large aggregates in Fig 6b), which are presumably
caused by dynamic equilibrium between micelles and unimers as
well as generation of hydrophilic small molecular weight thiols
that could escape from micelles. When excess DTT (5 mole
equivalents) was added, the FL intensity gradually decreased over
48 hrs. Such FL decrease is attributed to enhanced release of NR
from hydrophobic micellar cores to aqueous solution as a result
of thiol-induced degradation of micelles by cleavage of
disulfides. Because of the low solubility of NR in water, the
resulting micellar dispersion containing released free NR became
turbid. These significant results indicate that the change in
morphologies of micellar aggregates of PEO-b-PHMssEt in the
presence of different amounts of DTT could tune the release of
encapsulated model drugs in aqueous solutions.
A new methacrylate bearing pendent disulfide linkages was
synthesized and incorporated into a hydrophobic block of novel
thiol-triggered degradable ABPs having a PEO hydrophilic block
using ATRP. The polymerization of HMssEt in the presence of
PEO-Br macroinitiator proceed in a living manner as evidenced
by linear increase in molecular weight over conversion, narrow
molecular weight distribution as low as Mw/Mn < 1.1, and
continuous evolution of molecular weight distribution to higher
molecular weight region. These well-defined PEO-b-PHMssEt
copolymers self-assembled to form monomodal, spherical
micellar aggregates in aqueous solutions, confirmed by DLS and
TEM measurements, at above the CMC of 95 g/mL determined
by tensiometry.
In response to thiols, pendent disulfide linkages were cleaved
to the corresponding thiols: polymeric pendent thiols and small
molecular weight thiols. Such thiol-responsive degradation
caused the change in morphologies. Crosslinked micelles of
highly branched or crosslinked polymers with newly-formed
disulfide bonds were formed through thiol-disulfide
polyexchange reactions in the presence of <1 equivalent DTT to
disulfides, while micelles were disintegrated through
destabilization in the presence of >1 equivalent DTT. Such
change in morphology tuned the release of encapsulated model
drugs. These significant results suggest that thiol-responsive
PEO-b-PHMssEt micelles can offer versatility in multifunctional
drug delivery.
Acknowledgements
0.9
85
0.8
0.7
control
0.2 eq DTT
5 eq DTT
0.6
90
0.5
0
10
20
30
40
50
60
70
80
This work is supported from NSERC Discovery Grant, Canada
Research Chair (CRC) Award, and partially Advanced
Technology Center program (10032218) in the Korean Ministry
of Knowledge Economy. JKO is entitled Tier II CRC in
Nanobioscience as well as a member of Centre Québécois sur les
Matériaux Fonctionnels (CQMF) funded by FQRNT.
Notes and references
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Time (hrs)
[2]
Fig. 7 Release profile of NR from NR-loaded PEO-b-PHMssEt micellar
aggregates with and without DTT in aqueous solutions.
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