Synthesis and character of novel polycarbonate for constructing biodegradable multi-stimuli responsive delivery system

Article abstract of DOI:10.1002/pola.28243

Here, we design a novel triple-stimuli-sensitive graft copolymer assembly which responds to the changes in temperature, reducing agent, and light. The graft copolymer consists of thermo-responsive tetraethylene glycolyl poly(trimethylene carbonate) (P(MTC-4EG)) as backbone and light-sensitive poly(2-nitrobenzyl methacrylate) (PNBM) as side chain linked by an intervening disulfide bond. In aqueous solution, the polymer can self-assemble into micelle with thermo-sensitive shell (P(MTC-4EG)), light-sensitive core (PNBM), and disulfide linker. The assemblies in response to stimuli were revealed by dynamic light scatting (DLS) and transmission electron microscopy (TEM). The drug release behaviors of Nile Red (NR)-loaded carriers were also valued with stimuli from temperature, reducing agent, and light.

Full text of DOI:10.1002/pola.28243

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Synthesis and Character of Novel Polycarbonate for Constructing
Biodegradable Multi-Stimuli Responsive Delivery System
Mengmeng Xie, Lin Yu, Zhao Li, Zhen Zheng, Xinling Wang
State Key Laboratory of Metal Matrix Composites, School of Chemistry and Chemical Engineering, Shanghai Jiao Tong
University, Shanghai 200240, People’s Republic of China
Correspondence to: X. Wang (E-mail: xlwang@sjtu.edu.cn)
Received 9 May 2016; accepted 21 July 2016; published online 00 Month 2016
DOI: 10.1002/pola.28243
ABSTRACT: Here, we design a novel triple-stimuli-sensitive graft
copolymer assembly which responds to the changes in temper-
ature, reducing agent, and light. The graft copolymer consists
of thermo-responsive tetraethylene glycolyl poly(trimethylene
carbonate) (P(MTC-4EG)) as backbone and light-sensitive
poly(2-nitrobenzyl methacrylate) (PNBM) as side chain linked
by an intervening disulfide bond. In aqueous solution, the
polymer can self-assemble into micelle with thermo-sensitive
shell (P(MTC-4EG)), light-sensitive core (PNBM), and disulfide
linker. The assemblies in response to stimuli were revealed by
dynamic light scatting (DLS) and transmission electron micros-
copy (TEM). The drug release behaviors of Nile Red (NR)-load-
ed carriers were also valued with stimuli from temperature,
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reducing agent, and light.
2016 Wiley Periodicals, Inc. J.
Polym. Sci., Part A: Polym. Chem. 2016, 00, 000–000
KEYWORDS: controlled release; graft copolymer; multi-stimuli
responsive; nanocarrier; polycarbonate
particular, oligo(polyethylene glycol)-based polymers which
show high biocompatibility and well-responsive property are
widely used to construct thermo-responsive carriers.14,17–19
Meanwhile, the photo-responsive-system can undergo photo-
chemical cleavage reactions or isomeric rearrangements that
cause the structure change of micelle to release its cargo.
What is more, the drug release can be realized in remote
and spatiotemporal way by easily tuning the wavelength,
energy, and the site of irradiation.20,21
INTRODUCTION In the past decades, nanocarriers show sig-
nificant applications in drug delivery system. They can
endow the medicines with higher stability in blood circula-
tion and enhance ability of cellular uptake which improve
therapeutic efficacy and reduce side effects.1–8 To further
increase the intended therapeutic efficacy, various stimulate
groups were introduced into these systems to make them
undergo variations and thus release the cargos in response
to external stimuli. These systems can further minimize the
drug leakage in blood circulation and enhance the drug
release within tumor tissues.9,10 Among these stimuli, reduc-
tion stimulus is widely applied. The concentration of gluta-
thione (GSH) varies from 2–20 lm in extracelluar
environment to 2–10 mM in intracellular compartment, the
high concentration of GSH in cell can cut the disulfide in car-
rier. Therefore, the drug release can be easily achieved with-
out additional outside stimuli.11,12 In addition, for stimuli
from outside, thermo and light irritation was popular due to
their easy controllability and practical advantages.13–16 The
thermo-responsive carrier experiences the dehydration of
outer shell above the lower critical solution temperature
(LCST) of the polymer, which leads the structure change and
facilitates the drug release. On the other hand, the relative
hydrophobic shell can enhance micelle–cell interaction, thus
increasing the cellular uptake of the nanoparticles. In
Recently, synthetic degradable polymers, aliphatic polycarbon-
ates, are gaining a great attention in constructing nanocarriers.
Polycarbonates possess the merits of biodegradation, absence
of acidic degradation compounds and easy achievement of
functionalization.22–24 To construct the functional polycarbon-
ates, the introduction of functional pendant into the cyclic car-
bonate monomers (MCC) with further ring opening
polymerization (ROP) is the common choice.24 However, due to
the incompatible of some functional groups with ROP process,
further modifications following the polymerization are often
required.25 With the methods, various functional polycarbon-
ates were constructed and applied as drug carriers. Specially,
polycarbonate carriers with thermo, reduction, and light
stimuli-responsive groups were designed and showed well
applications in drug delivery system. For example, thermo-
responsive polycarbonate carrier with oligo(polyethylene
Additional Supporting Information may be found in the online version of this article.
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EXPERIMENTAL
Materials
5-Methyl-5-propargyloxycarbonyl-1,3-dioxan-2-one (MPC), 2-
Nitrobrnzyl methacrylate (NBM), and the thiourea catalyst
(TU) were synthesized according to the previous meth-
ods.29–31 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU, 98%,
Adams), 2,2-dimethoxypropane (DMP, 98%, Sigma Aldrich),
N,N,N0,N00,N00-pentamethyldiethylenetriamine (PMDETA, 98%,
Adams), cuprous bromide (Cu(I)Br, 99%, Sigma Aldrich),
sodium hydride (NaH, 60% dispersion in mineral oil, Sigma
Aldrich), 3-hydroxymethyl-3-methyloxetane (98%, Adams),
hydrobromic acid (HBr, 48% in water, Adams), potassium
carbonate (K2CO3, 99%, Adams), 2,2-bis(hydroxymethyl) pro-
pionic acid (bisMPA, 98%, Sigma Aldrich), 4-nitrophenyl
chloroformate (NPC, 96%, Sigma Aldrich), Bromoisobutyryl
bromide (BIBB, 98%, Sigma Aldrich), 2-hydroxyethyl disul-
fide (90%, Alfa), and triphosgene (98%, Sigma Aldrich) were
used as received. Dichloromethane (DCM), benzyl alcohol
and pyridine were distilled over calcium hydride.
Tetrahydrofuran was distilled with potassium before use.
Methyl Tetraglycol (4EG) was purchased from Sigma Aldrich
and dried by azeotropic distillation in toluene. All other sol-
vents and regents were obtained from Sinoreagent and used
without purification.
SCHEME 1 Illustration of structure changes of polycarbonate
micelle under the stimuli of temperature, redox, and light. [Col-
or figure can be viewed in the online issue, which is available
at wileyonlinelibrary.com.]
glycol) reported by Kim et al. showed enhanced drug delivery
rate and cellular uptake when the carrier was heated above
the LCST.26 Liu et al. created a series of redox-responsive car-
riers from polycarbonate–drug conjugates which were synthe-
sized from ROP of prodrug monomers consisting of cyclic
carbonate and drug with disulfide linker.27 Yan et al. prepared
Measurements
1H NMR spectra of monomers and polymers were measured
by a BrukerAvance III 400 MHz NMR spectrometer operating
at 400 MHz.
a
kind of MCC containing
a
protected thiol group.
Gel permeation chromatography (GPC) measurements were
performed on Tosoh HLC-8320 GPC instrument by using
DMF with LiBr (1 g/L) as the eluent at 45 8C relative to pol-
ymethyl methacrylate as standard.
Polymerization and further deprotection gave the polycarbon-
ate thiol group which was used to build redox-responsive carri-
er.28 In addition, a modified MCC derivative of 5-methyl-5-(2-
nitro-benzoxycarbonyl)21,3-dioxan-2-one (MNC) was created
by Xie et al. to design light-responsive polycarbonate.29 Howev-
er, many of these polycarbonate carriers were in response to
single or two stimuli, polycarbonate-based carrier with multi-
stimuli was still rarely reported. Multi-responsive carriers can
provide a platform to fine tune the drug release profile in
response to independent signal stimulus and combined multi-
ple stimuli which can better meet the demand for
bioapplications.9,10
The turbidity of material solution was measured by UV/Vis
turbidity measurement. The Perkin Elmer Lambda 35 UV/
Vis spectrometer was equipped with a temperature regulat-
ing equipment. The transmittance of solution at 500 nm was
recorded with heat at a rate of 1 8C/min. All solutions were
filtered with 0.45 lm filter to remove the dust before
measurements.
The size of the micelle was measured by Malvern Zetasizer
Nano S90, and all the solutions were also filtered with 0.45
lm filter before use. The morphologies of the micelles before
and after stimuli were observed with a JEOL JEM-2100
transmission electron microscopy (TEM) with an acceleration
voltage of 200 kV. Samples were prepared by depositing the
micelle solutions (1 mg/mL) onto copper grids coated with
carbon and then allowing water to evaporate from the grids
at room temperature (RT) over night.
To construct the desired multi stimuli-sensitive polycarbon-
ate with thermo, reduction, and light stimuli-responsive
property, the methods that combine the polymerization of
functional MCC and the following modifications were used. It
was designed with thermo-responsive (P(MTC-4EG)) as back-
bone, light-sensitive hydrophobic PNBM as side chain, and
redox-sensitive disulfide bond as linker. The amphiphilic
polycarbonate can self-assemble into micelle in water with
the capacity of responding to various stimuli which are pre-
sented in Scheme 1. The assemblies in response to stimuli
were monitored by DLS and TEM. NR was used as a model
hydrophobic drug and whose release behaviors under the
stimuli of light, temperature and reducing agent were further
valued.
Synthesis of N3ssBr (Scheme 2)
2-Hydroxyethyl disulfide (10 g, 64.8 mmol) and triethyl-
amine (9 mL, 64.8 mmol) were codissolved in dry THF
(100 mL) and cooled to 0 8C. A solution of NPC (11.8 g, 58.3
mmol) in 40 mL THF was added dropwise into the solution
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allowed to warm to RT and stirred for 4 h. 100 mL deionized
H2O was added into the clear solution which was extracted
with 90 mL Et2O three times. Finally, the combined organic
phase was dried with anhydrous Na2SO4 and evaporated to
afford Diol-Br (10.3 g, yield 90%).
Diol-Br (10.3 g, 56.3 mmol) was dissolved in 80 mL acetone.
The solution was cooled to 0 8C followed by slow addition of
DMP (9.4 g, 90.1 mmol). The mixture was allowed to RT and
stirred for 4 h. Then, NaHCO3 was added to stop the reaction
and the reaction mixture was concentrated. Thereafter, the
concentrated mixture was added with CH2Cl2 and followed
by washing sequentially with saturated NaHCO3 solution
(3 3 150 mL) and brine (3 3 100 mL). The resulting organ-
ic phase was dried over Na2SO4. After filtration and evapora-
tion, DMP-Diol-Br (11.8 g, yield 94%) was achieved as pale
yellow oil.
SCHEME 2 Synthetic routes for the redox-sensitive disulfide
linker.
Under a nitrogen atmosphere, NaH (1.7 g, 72.2 mmol) was
slowly added into 80 mL dry DMF solution containing 4EG
(14.3 g, 68.8 mmol). After stir at RT for 3 h, DMP-Diol-Br
(11.8 g, 52.9 mmol) was added into the solution and the
reaction was kept at 50 8C for 12 h. Then, DMF was evapo-
rated under reduced pressure distillation, followed by addi-
tion of CH2Cl2 and sequentially washed with saturated
NaHCO3 (3 3 150 mL) and NaCl (3 3 150 mL) solution. The
organic phrase was dried with anhydrous Na2SO4 and con-
centrated to get the desired product DMP-Diol-4EG (15.4 g,
yield 83%).
over 30 min. Subsequently, the mixture was stirred at RT for
another 6 h. The resulted white precipitant was removed by
filtration and the filtrate was concentrated with rotary evap-
oration. Then, the concentrated solution was charged with
150 mL CH2Cl2, washed sequentially with saturated NaHCO3
solution (3 3 150 mL) and brine (3 3 100). The organic
layer was collected, dried with anhydrous Na2SO4 and con-
centrated to get the crude product. Finally, the crude product
was purified by column chromatography with hexane/ethyl
acetate (3:1) to get the desired material NPCssOH (8.7 g,
yield 42%).
CF3COOH (10.0 g, 87.8 mmol) was added dropwise into the
ice cold methanol solution of DMP-Diol-4EG (15.4 g, 43.9
mmol). After stir at RT for 12 h, concentration of the solu-
tion gave the product Diol-4EG (13.1 g, yield 96%).
2-Azido-1-ethylamine (3.5 g, 41.0 mmol) in 20 mL THF was
added dropwise into the THF solution (50 mL) of NPCssOH
(8.7g, 27.3 mmol) which was left to stir at RT for 4 h. Then
the solvent was removed by rotary evaporation. The
obtained residue was dissolved in 100 mL CH2Cl2, which
was washed with saturated NaHCO3 solution (3 3 100 mL),
brine (3 3 100 mL), and dried with anhydrous Na2SO4 over
night. After removing the solvent, the resulted crude product
was purified by column chromatography with hexane/ethyl
acetate to achieve N3ssOH (6.0 g, yield 83%).
Triphosgene (6.3 g, 21.1 mmol) in CH2Cl2 (25 mL) was
added dropwise into a mixture of Diol-4EG (13.1g, 42.1
mmol) and pyridine (20.0 g, 253.6 mmol) in 100 mL anhy-
drous CH2Cl2 at 284 8C over a period of 1 h, and the mix-
ture was stirred at RT for 3 h. Thereafter, the saturated
aqueous NH4Cl was added into the mixture to quench the
reaction which was washed sequentially with 1M aqueous
HCl (3 3 150 mL), saturated aqueous NaHCO3 (3 3
150 mL), and brine (2 3 150 mL). Finally, the organic phase
was dried over Na2SO4 and concentrated to get the crude
product which was further purified by column
N3ssOH (6.0 g, 22.7 mmol) and triethylamine (4.6 g, 45.4
mmol) were dissolved in 70 mL dry THF in a round bottom
flask. This flask was immersed into ice bath and BIBB (7.8 g,
34.1 mmol) in 20 mL THF was added slowly into the flask.
Then, the flask was allowed to stir at RT for 6 h. The solvent
was removed and 100 mL CH2Cl2 was added. The solution
was washed with saturated NaHCO3 solution (3 3 100 mL)
and brine (3 3 100 mL). Finally, the concentrated mixture
was purified by column chromatography with hexane/ethyl
acetate to obtain N3ssBr (7.3 g, yield 78%) (Supporting
Information Fig. S1).
Synthesis of MTC-4EG (Scheme 3)
HBr (48% aqueous solution, 20.9 mL) was dropped slowly
into the ice cold THF containing 3-hydroxymethyl-3-
methyloxetane (6.35 g, 62.3 mmol). The reaction was then
SCHEME 3 Synthetic routes for monomer MTC-4EG.
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chromatography with hexane/ethyl acetate (1:4) to yield the
desired monomer MTC-4EG (7.3 g, yield 46%) as pale yellow
oil (Supporting Information Fig. S2).
solutions were recorded with a fluorescence spectrophotom-
eter. An emission wavelength at 372 nm was used and the
excitation spectra from 300 nm to 350 nm were collected.
Synthesis of Multi-Sensitive Polycarbonate
Biocompatibility of Micelle
P[(MTC-4EG)-co-(MTC-ss-PNBM)]
The cytotoxicity assessment of P[(MTC-4EG)-co-(MTC-ss-
PNBM)] micelle was achieved by MTT assay. L929 cells were
seeded in 96-well plates at a density of 1 3 104 per well
with DMEM (containing 10% FBS) and incubated for 24 h
(37 8C with 5% CO2). Thereafter, the culture medium was
replaced by fresh media containing micelles (the concentra-
tions were 0.02, 0.05, 0.1, 0.2, 0.4, 0.7, 1 mg/mL). After incu-
bation for 48 h, the medium was removed and replaced by
0.2 mL DMEM solution containing MTT (0.5 mg/mL) for 4 h
incubation. Finally, the medium was completely removed, fol-
lowed by replenishing DMSO (0.2 mL) into each well and
shook for 3 min, The absorption of each well at 570 nm was
measured with a microplate reader. Cells cultured in DMEM
medium containing 10% FBS (with no micelles) were used
as blank controls. The relative cell viability was achieved by
comparing the absorbance of the well containing materials
to that of the blank control.
Polymerization of P[(MTC-4EG)-co-(MPC)] was achieved by
ROP catalyzed by TU/DBU. In brief, for P[(MTC-4EG)45-co-
(MPC)4], under a nitrogen atmosphere, MTC-4EG (1.1 g, 3
mmol), initiator benzyl alcohol (5.4 mg, 0.05 mmol), and
catalysis TU/DBU (55.4 mg/15.3 mg, 0.15 mmol/0.1 mmol)
were sequentially added into the dry CH2Cl2. After stir at RT
for 6 h, MPC (39.6 mg, 0.2 mmol) in CH2Cl2 was added.
Three hours later, benzoic acid was added to stop the reac-
tion and themixture was poured into hexane/diethyl ether
(4/6). The product was recovered by centrifugation and
dried under vacuum (620 mg).
P[(MTC-4EG)45-co-(MPC)4] (620 mg), N3ssBr (124.5 mg, 0.3
mmol), and PMDETA (5.2 mg, 0.03 mmol) were codissolved
in THF and the solution was degassed through freeze–vacu-
um–thaw cycles for three times. Afterward, in glovebox,
Cu(I)Br (4.3 mg, 0.03 mmol) was introduced into the solu-
tion at 35 8C to start the reaction, After 6 h, the mixture
solution was diluted with THF and passed through an alumi-
na column to remove the copper catalysis. Finally, the result-
ing solution was concentrated and precipitated into hexane/
diethyl ether (1/9). The product was collected with centrifu-
gation and dried in a vacuum oven (540 mg).
In Vitro Release of Nile Red from Micelle in
Different pH Condition and Under Stimuli
of UV, GSH, and Temperature
Nile Red-loaded micelles were fabricated by a dialysis method.
Briefly, 1 mg NR and 10 mg polycarbonate were codissolved
in 1 mL DMF and stirred for 2 h. Then 2 mL PBS was added
dropwise into the solution which was further purified by dia-
lyzing against water. Finally, the unloaded NR was removed
by filtering the solution over 0.45 lm syringe filter and the
resulted NR-loaded micelle solution was stored at 4 8C.
P[(MTC-4EG)-co-(MTC-ss-Br)] (540 mg), NBM(1.7 g, 7.8
mmol), and PMDETA (19.0 mg, 0.11 mmol) were added to
round flask containing dry THF. After three times of freeze–
vacuum–thaw cycles, Cu(I)Br (15.8 mg, 0.11 mmol) was
introduced into the solution which was kept at 65 8C for 2 h.
The reaction was then diluted with THF and passed through
an alumina column to remove the copper catalysis. The elu-
ent was concentrated by rotary evaporation and precipitated
into diethyl ether. Finally, the precipitation was collected and
dried to afford the multi-sensitive polycarbonate (810 mg).
To study the release behavior at different pH, 5 mL micelles
(1 mg/mL) with NR were incubated in different buffer solu-
tion at 25 8C. For the controlled release with redox stimulus,
5 mL micelles (1 mg/mL) with NR in 10 mM pH 7.4 PBS
were incubated with different concentrations of GSH in
water bath at 25 8C. For the controlled release under UV
stimulus, 5 mL micelle (1 mg/mL) with NR in 10 mM pH 7.4
PBS was put under a high-pressure mercury lamp (wave-
length: 365 nm, intensity: 50 mW/cm2) in water bath at 25
8C. For the controlled release in different temperature in
aqueous solution, 5 mL micelle solution (1 mg/mL) with NR
was kept at different temperature. At scheduled time, absor-
bance changes of micelle suspensions were recorded.
Preparation of the Multi-Sensitive Micelle
The multi-sensitive micelle was prepared by dialysis method.
In brief, 10 mg P[(MTC-4EG)-co-(MTC-ss-Br)] was dissolved
in 1 mL DMF, followed by adding 2 mL pH 7.4 PBS with
fierce agitation. Then, DMF was removed by dialyzing against
PBS with a cellulose membrane (Mw cutoff: 3500) for 2
days. The buffer was changed six times a day. Finally, the
dialyzed solution was filtered with 0.45 lm filter, giving the
solution of P[(MTC-4EG)-co-(MTC-ss-Br)] micelle.
RESULTS AND DISCUSSION
Synthesis of the Multi-Responsive Polycarbonate
Determination of the Critical Micelle Concentration
(CMC) Value of Micelle
Ring opening polymerization (ROP) catalyzed by metal-free
organo-catalyst showed great control in synthesizing polycar-
bonate and its derivatives with relatively narrow polydisper-
sity and high conversion rate. As show in Scheme 4, the
polycarbonate backbone was achieved by a two-step ROP
with sequential addition of TMC-4EG and MPC at 25 8C with
DBU/TU as catalyst. The ring structures of the monomers
showed splitting spectral pattern around 4.2 ppm and 4.5
The CMC value of micelle was determined by using pyrene
as a fluorescence probe. In brief, the micelle solutions at var-
ious concentrations were added to the containers with pyr-
ene and the concentration of pyrene in each solution was set
as 6 3 1027 M. Then, these samples were equilibrated at RT
for 12 h. The excitation spectra of pyrene in micelle
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SCHEME 4 Synthetic routes for polycarbonate P[(MTC-4EG)-co-(MTC-ss-PNBM)].
ppm in 1H NMR spectrum (Supporting Information Fig. S2).
After polymerization, the appearance of peaks at 4.1 ppm
and 4.3 ppm confirmed the successful achievement of
P[(MTC-4EG)-co-(MPC)]. By integrating the protons from the
initiator versus the methylene protons of each block, the
polymerization degrees of two blocks were easily calculated.
P[(MTC-4EG)-co-(MPC)]s with different molar ratios of MTC-
4EG and MPC were prepared and shown in Table 1. The
resulting polycarbonates were further characterized by GPC.
The discrepancy of Mn values between 1H NMR and GPC
measurements was on account to the difference in hydrody-
namic volume of the brush polymer and the polymethyl
methacrylate used as standard.32
complex protection and deprotection processes.28 Herein, the
macroinitiator P[(MTC-4EG)-co-(MTC-ss-Br)] with disulfide
bond was conveniently got by click reaction between N3ssBr
containing azide and alkynyl functionalized polymer P[(MTC-
4EG)-co-(MPC)]. The structure of the macroinitiator was
firstly determined by FTIR (Supporting Information Fig. S3),
the peak at 1640 cm21 was assigned to the amide bonds in
functional side chain ssBr. Further evidence of successful
conjugation of N3ssBr to polycarbonate backbone was veri-
fied by 1H NMR, the presence of characteristic peaks at 1.9,
2.9, 3, 4.2, 4.4 ppm (methyl and methylene protons in
N3ssBr), and the complete shift of the resonance at 4.8 ppm
(the methylene protons adjacent to alkynyl group) to 5.3
ppm proved 100% conversion of azide to triazole. GPC anal-
ysis of the polymer (Fig. 2) revealed a shift to lower reten-
tion time while behaving the molecular weight distribution
similar to that of the unmodified P[(MTC-4EG)-co-(MPC)].
Polycarbonates with side chains linked by disulfide have
been widely prepared and the most common method was
polymerizing the MTC monomers with disulfide group.33
However, high efficient catalyst pair DBU/TU was forbidden
in these polymerizations for the cleavage of the disulfide
bond by strong basicity of DBU.34 The commonly used cata-
lysts in these systems (such as p-toluenesulfonic acid, stan-
nous octoate) could only get the oligomer of MTC-4EG. The
other possibility is using MTC with protected thiol groups,
but obtaining the final products was troublesome with
The macroinitiator P[(MTC-4EG)-co-(MTC-ss-Br)] was finally
used to initiate NBM to form the amphiphilic graft copoly-
mer P[(MTC-4EG)-co-(MTC-ss-PNBM)]. The product was
characterized by FTIR and 1H NMR (Supporting Information
Fig. S3 and Fig. 1). New bands at 1529 and 1350 cm21 (the
characteristic peaks of nitrobenzene) emerged in FTIR
TABLE 1 Molecular Characteristics of P[(MTC-4EG)-co-(MPC)], P[(MTC-4EG)-co-(MTC-ss-Br)], and P[(MTC-4EG)-co-(MTC-ss-PNBM)]
Feed Molar Ratio
I: MTC-4EG: MPC
Resulted Molar Ratio
I: MTC-4EG: MPCa
Mna
Mnb
Mw/
Mnb
LCST
Polymer
(kDa)
(kDa)
(8C)
P[(MTC-4EG)46-co-(MPC)11
]
1:60:12
1:60:8
1:60:4
–
1:46:11
1:42:7
1:45:4
17.6
15.5
15.9
17.6
35.3
7.3
6.9
6.8
7.8
13.1
1.35
1.47
1.48
1.42
1.92
33.0
40.2
43.6
37.5
39.0
65P[(MTC-4EG)42-co-(MPC)7]
P[(MTC-4EG)45-co-(MPC)4]
P[(MTC-4EG)45-co-(MTC-ss-Br)4]
P[(MTC-4EG)45-co-(MTC-ss-PNBM20)4]
–
a
b
The results were calculated according to 1H NMR results.
The results were got from GPC measurements.
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FIGURE 1 1H NMR spectra of polycarbonates (A) P[(MTC-4EG)-co-(MPC)] in CDCl3, (B) P[(MTC-4EG)-co-(MTC-ss-Br)] in d6-DMSO,
and (C) P[(MTC-4EG)-co-(MTC-ss-PNBM)] in CDCl3. [Color figure can be viewed in the online issue, which is available at wileyonli-
nelibrary.com.]
spectrum of P[(MTC-4EG)-co-(MTC-ss-PNBM)] indicated the
successful initiation of NBM from the macroinitiator. The
new peaks in the 1H NMR spectrum further revealed the
composition of the polycarbonate. The DP of PNBM was cal-
culated from the integral values of the peaks from 7.4 to 8.0
ppm (peak 12) and the peaks at 3.3 ppm (peak i and
peak g). The GPC measurement in Figure 2 also confirmed
the successfully growing PNBM chains from polycarbonate
backbones. To avoid the possible gelation of the material due
to the multifunctional nature of the macroinitiator, the con-
version was kept below 30%. However, the polydispersity
index of the synthesized copolymer P[(MTC-4EG)-co-(MTC-
ss-PNBM)] was still rather board (Fig. 2). Therefore, during
the polymerization, the slight crosslinking might still happen
which was unavoidable.
Properties of Polymeric Micelles
The CMC of amphiphilic P[(MTC-4EG)45-co-(MTC-ss-
PNBM20)4] was determined by a fluorescence probe method.
To obtain the CMC of the polycarbonate, micelles loaded
with pyrene were adjusted to a series of concentrations and
the fluorescence excitation intensities were record. The plot
of the values of I337/I334 versus the log of the concentrations
of the micellar solutions was achieved. In Figure 3, the CMC
value of P[(MTC-4EG)45-co-(MTC-ss-PNBM20)4] micelle was
0.032 mg/mL.
FIGURE 3 Plot of I337/I334 ratio of pyrene excitation spectra in
pH 7.4 PBS as a function of the concentrations of P[(MTC-
4EG)45-co-(MTC-ss-PNBM20)4]. [Color figure can be viewed in
the online issue, which is available at wileyonlinelibrary.com.]
FIGURE 2 GPC traces of polycarbonates. [Color figure can be
viewed in the online issue, which is available at wileyonlineli-
brary.com.]
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P[(MTC-4EG)46-co-(MPC)11] and P[(MTC-4EG)42-co-(MPC)7]
showed LCST values around 33.0 8C and 40.2 8C. It is com-
mon that thermo-responsive polymer with hydrophobic
block would decrease the LCST. However, given the vast
decrease in the LCST in this system, we deduced that trans-
esterification might happen during the polymerization, the
insertion of hydrophobic MPC monomer would greatly affect
the hydrophility of P(MTC-nEG) and thus caused the
phenomena.
P[(MTC-4EG)45-co-(MPC)4] was further introduced with
N3ssBr by click reaction. The resulting macroinitiator
P[(MTC-4EG)45-co-(MTC-ss-Br)4] showed LCST value at 37.5
8C, which was obviously lower than that of P[(MTC-4EG)45
-
co-(MPC)4]. The result of 37.5 8C should be ascribed to the
increased hydrophobic effect caused by N3ssBr group.
Interestingly, the final product P[(MTC-4EG)45-co-(MTC-ss-
PNBM20)4] with longer hydrophobic side chains behaved
higher LCST than that of P[(MTC-4EG)45-co-(MPC)4]. It might
be due to the fact that the P[(MTC-4EG)45-co-(MTC-ss-
PNBM20)4] solution used for measurement was in a state of
self-assembly in water, the hydrophilic P(MTC-4EG) chains
totally covered the hydrophobic PNBM20 chains which made
PNBM20 have little influence on the hydrophilicity of the
total nanoparticle and even alleviated the influence from
N3ssBr. The LCST of P[(MTC-4EG)45-co-(MTC-ss-PNBM20)4]
was around 39 8C, which was slightly higher than the physio-
logical temperature that made it as a good candidate in ther-
motherapy field.
FIGURE 4 Biocompatibility of polymeric micelles with L929
cells after 48 h incubation. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
The biocompatibility of micelles to L929 cells was assessed
by MTT assays. In Figure 4, the results showed that the poly-
meric micelle has no obvious cytotoxicity to L929. Therefore,
the polycarbonate micelle was suitable for drug carriers.
It was well known that polycarbonate underwent degrada-
tion under variable pH conditions. Therefore, the stability of
micelle was evaluated at different pH by DLS. In Supporting
Information Figure S4, obvious increase in micellar size at
pH 11 was observed, which was due to the destruction of
micelle structure caused by the fast degradation of carbonic
ester bond at base condition. As comparison, in acid condi-
tion, the size change was feeble. The micelle swelled slightly
at pH 3.6. In addition, the micellar size kept intact for 1
week storage around neutral pH (pH 5.0, 7.4, and 8.9).
Moreover, NR release studies were further carried to evalu-
ate the degradation behavior of polycarbonate carriers at dif-
ferent pH (Supporting Information Fig. S5). Notably, at pH
11, obvious release was observed due to the degradation of
polycarbonate backbone which was in accordance with DLS
result. However, at other pH value, the release amount was
slight over 1 week, which further proved the intact of
micelle structure.
Morphological Changes of the Micelle Under Stimulation
with Temperature, Light, and Reductant
The stimuli-responsive properties of P[(MTC-4EG)45-co-
(MTC-ss-PNBM20)4] assemblies at pH 7.4 10 mM PBS in
response to temperature, light, and redox potential were
investigated by DLS and TEM. The DLS dates of assemblies
on various stimuli are shown in Figure 6. In pH 7.4 10 mM
Temperature-Responsive Behavior of Copolymers
Polycarbonate with different units of OEG side chains is a
kind of novel biodegradable temperature-responsive polymer.
It can undergo a reversible hydration and dehydration pro-
cesses in water at a certain temperature which was defined
as LCST. The light transmittance of P[(MTC-4EG)-co-(MPC)],
P[(MTC-4EG)-co-(MTC-ss-Br)], and P[(MTC-4EG)-co-(MTC-ss-
PNBM)] in water against temperature are depicted in Figure
5. Akashi and coworkers have systematically studied the
LCST behavior of P(MTC-nEG) homopolymers, the LCST of
P(MTC-4EG) with Mn 5 2300 was around 70 8C.35 However,
copolymerizing P(MTC-nEG) with hydrophobic PMPC chain
obviously decreased the LCST of resulting copolymers
(Fig. 5). The LCST of P[(MTC-4EG)45-co-(MPC)4] was found
to be about 43.6 8C. With longer length of PMPC chains,
FIGURE 5 Turbidity measurements at 500 nm for the determi-
nation of LCST for P[(MTC-4EG)-co-(MPC)], P[(MTC-4EG)-co-
(MTC-ss-Br)], and P[(MTC-4EG)-co-(MTC-ss-PNBM)]. The con-
centration for each sample was set at 2 mg/mL. [Color figure
can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
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approached the desired LCST (39 8C), the hydrophilic
P(MTC-4EG) partly dehydrated which caused the aggregation
of micelles into large clusters (Fig. 6 and Supporting Infor-
mation Fig. S4). Interestingly, compared with the slight size
change in water, a much more significant increase in hydro-
dynamic size was observed in PBS with heating (Supporting
Information Fig. S5). In the case of P[(MTC-4EG)45-co-
(MPC)4] and P[(MTC-4EG)45-co-(MTC-ss-Br)4], the difference
also appeared (Supporting Information Fig. S5). It might be
due to the reason that the ion could replace the water mole-
cule around the polymer chain which made easier dehydra-
tion of PEG chain. Therefore, larger aggregation was formed
around the LCST. When the micelle solution was exposed to
UV light, the color of the solution turned to light brown due
to the release of nitrobenzyl alcohol (Supporting Information
Fig. S6). The decline of the size of the assemblies was
observed with DLS, which was attributed to the photolysis of
nitrobenzyl ester that leaded to the conversion of hydropho-
bic PNBM segments to hydrophilic poly(methylacrylic acid)
chains which caused the destruction of the micelle structure.
Otherwise, the redox-induced disruption of micelle structure
was also valued. The free thiol groups in GSH could cut the
disulfides between the hydrophobic core and hydrophilic
shell. With no coverage of hydrophilic shell, the hydrophobic
FIGURE 6 The DLS curves of the assemblies of P[(MTC-4EG)45
-
co-(MTC-ss-PNBM20)4] in pH 7.4 10 mM PBS under the stimuli.
The data under the stimulation of GSH were not shown for the
large size of precipitant which beyond the measuring range.
[Color figure can be viewed in the online issue, which is avail-
able at wileyonlinelibrary.com.]
PBS, P[(MTC-4EG)45-co-(MTC-ss-PNBM20)4] self-assembled
into micelle which was in a diameter of 18 nm with
PDI 5 0.235. In the heating process, as the temperature
FIGURE 7 TEM images of the amphiphilic P[(MTC-4EG)45-co-(MTC-ss-PNBM20)4] assemblies in pH 7.4 10 mM PBS (A) before stim-
uli and under the stimuli of (B) heating at 45 8C in PBS, (C) 10 mM GSH for 24 h, and (D) UV irradiation.
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FIGURE 8 Cumulative release profiles of NR from the micelles with (A) UV light irradiation, (B) different concentrations of GSH,
and (C) at different temperature. All the measurements were performed in triplicate. [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]
PNBM core was no longer stable in PBS and the sediment of
large aggregates was appeared.
micelle incubated at 41 8C (above LCST) showed faster
release rate than that at either 25 8C or 36 8C (Fig. 8(C)).
The phenomenon was caused by two facts. One is the faster
diffusion of NR at higher temperature. The other is when the
temperature is above LCST (41 8C), the outer P(MTC-4EG)
collapsed and induced the transformation of micelle struc-
ture, which further accelerated the release rate of NR. Based
on the above results, the controlled release of hydrophobic
molecule from the carriers could be achieved under the
stimuli of UV, reducing agent, and at different temperature.
The morphological changes of P[(MTC-4EG)45-co-(MTC-ss-
PNBM20)4] micelles on stimuli were further investigated
with TEM images (Fig. 7). Compared with the results from
DLS, the particles in TEM showed smaller diameter. It was
due to the shrinkage of particles in dry state for TEM obser-
vation. In the same tendency as the DLS results, there was
an obvious increase in diameters of micelles with response
to temperature and redox potential. With heating, the partly
dehydration of P(MTC-4EG) was happened which caused the
aggregation of nanoparticle. Therefore, the larger cluster
with broad size distribution was observed. The loose and
irregular sharp was observed when treated with GSH. In
addition, upon UV light irritation, smaller nanostructure was
observed. All the results were in accordance with that from
DLS. The obvious disruptions of P[(MTC-4EG)45-co-(MTC-ss-
PNBM20)4] micelles were achieved upon stimuli.
CONCLUSIONS
Redox-, temperature- and light-responsive amphiphilic poly-
carbonate P[(MTC-4EG)-co-(MTC-ss-PNBM)] was synthesized
by three steps: (i) ROP of MTC-4EG and MPC monomers, (ii)
further click reaction to achieve macroinitiator P[(MTC-4EG)-
co-(MTC-ss-Br)], and (iii) ATRP of NBM from macroinitiator.
The resulted polycarbonate consisted of thermo-sensitive
P(MTC-4EG) backbone and light-sensitive side chain PNBM
which were linked by redox-responsive disulfide bond. The
polycarbonate could self-assemble in aqueous solution and
its morphology could be altered by triple stimuli which
endowed the micelle with fine tuned drug delivery behavior
under stimuli. With these characters, the multi-responsive
nanocarrier could show potential application in the field of
biomedicine for controlled release of drugs.
Controlled Release of NR Under Stimuli
from UV, GSH, and at Different Temperature
To assess the controlled release behaviors of encapsulated
drug under stimuli. Here, a model hydrophobic molecule, NR,
was used. When it was released from the micelle into aque-
ous solution, a decrease in fluorescence is observed. The
changes in absorbance of NR at 560 nm were recorded.
Figure 8(A) showed the responsive release profile with UV.
Compared with the negligible release amount of NR with no
stimulus, there was a fast release on UV irradiation. Under
the UV lamp at the intensity of 50 mW/cm2, it was found
that 40% of NR was released within 10 min and over 70%
of the loaded NR was released after irradiation for 40 min.
The fast release rate was due to the disappearance of hydro-
phobic core caused by the photolysis of nitrobenzyl ester.
Figure 8(B) showed the release profiles of NR from micelles
at different concentrations of GSH in 10 mM pH 7.4 PBS.
Without GSH, no significant release was observed (only
10%) for 30 h. However, drastic release rateappeared with
addition of GSH, nearly 50% of NR for 15 h with 5mM GSH,
55% of NR for only 6 h with 10mM GSH. The free thiol
groups at GSH could cut the disulfide bonds in micelle which
disrupted the micelle structure and thus enhanced the
release rate of the encapsulation. In addition, The NR-loaded
ACKNOWLEDGMENTS
This work is financially supported by National Natural Science
Foundation of China (21274085) and Shanghai Leading Aca-
demic Discipline Project (no. B202).
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