Synthesis of high drug loading, reactive oxygen species and esterase dual-responsive polymeric micelles for drug delivery

Article abstract of DOI:10.1039/c8ra09770d

Stimulus-responsive, controlled-release systems are of great importance in medical science and have drawn significant research attention, leading to the development of many stimulus-responsive materials over the past few decades. However, these materials

Full text of DOI:10.1039/c8ra09770d

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Synthesis of high drug loading, reactive oxygen
species and esterase dual-responsive polymeric
micelles for drug delivery†
Cite this: RSC Adv., 2019, 9, 2371
Nan Wang,a Xiao-Chuan Chen,b Ruo-Lin Ding,c Xian-Ling Yang,a Jun Li,a
a
a
b
*
*
Xiao-Qi Yu,
Kun Li
and Xi Wei
Stimulus-responsive, controlled-release systems are of great importance in medical science and have
drawn significant research attention, leading to the development of many stimulus-responsive materials
over the past few decades. However, these materials are mainly designed to respond to external stimuli
and ignore the key problem of the amount of drug loading. In this study, exploiting the synergistic effect
of boronic esters and N-isopropylacrylamide (NIPAM) pendant, we present a copolymer as an ROS and
esterase dual-stimulus responsive drug delivery system that has a drug loading of up to 6.99 wt% and an
entrapment efficiency of 76.9%. This copolymer can successfully self-assemble into polymer micelles in
water with a narrow distribution. Additionally, the measured CMC hinted at the good stability of the
polymeric micelles in water solution, ensuing long circulation time in the body. This strategy for
increasing the drug loading on the basis of stimulus response opens up a new avenue for the
development of drug delivery systems.
Received 28th November 2018
Accepted 7th January 2019
DOI: 10.1039/c8ra09770d
rsc.li/rsc-advances
morphology is controlled by the regulation of the pH.6 Sumerlin
et al. prepared pH- or sugar-responsive block copolymers via
RAFT polymerization.7 Sumerlin's group also combined pH-
Introduction
In recent decades, multifunctional polymeric materials have
attracted growing interest due to their wide range of applica-
tions, especially in the biomedical eld.1 These polymeric
materials achieve better control of architecture2 and function-
ality via the copolymerization of different monomers. Among
the numerous polymerization methods, reversible addition-
fragmentation chain transfer (RAFT) polymerization has
proven to be an excellent and convenient route to block co-
polymers via the use of the desired RAFT agents.3 Meanwhile,
by selectively incorporating certain functional groups, desired
amphiphilic block copolymers or responsive materials can be
synthesized using RAFT methods. In aqueous solutions,
amphiphilic block copolymers can self-assemble into a variety
of different nanostructures, including spherical micelles,
cylindrical micelles, and drug-delivery vesicles.4 These smart
materials show mono-, dual- or multi-responsive abilities and
can efficiently response to external stimuli.5 For example, Qin
and sugar-responsiveness with the thermo-responsiveness of
NIPAM to prepare block copolymers.1 However, biological
applications have not been explored in their studies. And
according to literature, esterase are expressed in numerous
tissues.8 And a series of esterase-responsive probes have been
designed.9 In 2016, Shen's group taking advantage of the high
esterase activity in HeLa cancer cells to design a esterase-
responsive charge-reversal polymer, whose polyplexes had
a selective gene expression in the cancer cells high in ester-
ases.10 Generally, although pH, glucose-sensitive or multiple-
sensitivity boronic acid block polymers have already been re-
ported, high drug loading, ROS and esterase-responsive mate-
rials for drug delivery have not been synthesized or investigated
to date.
Herein, we constructed a di-block copolymer system con-
taining boronic esters and N-isopropyl acrylamide via RAFT
methods that offers genuine synergies between the esterase and
ROS stimuli. Arylboronic esters could be oxidized by ROS (e.g.,
H2O2)11 and then undergo rearrangement to unmask the
modied group, and the ester bonds could be hydrolysed by
esterase.12 Hydrogen peroxide (H2O2) is one of the most
important ROS and plays critical roles in a number of physio-
logical and pathological processes.13 This reactive strategy has
been usually applied to design small-molecule-based uores-
cent probes in recent years.14 In addition to dual-stimulus
responsiveness, phenylboronic acid, pinacol ester pendent
et al. prepared
a boronic acid block copolymer whose
aKey Laboratory of Green Chemistry and Technology, Ministry of Education, College of
Chemistry, Sichuan University, Chengdu, China 610064. E-mail: xqyu@scu.edu.cn
bOperative Dentistry and Endodontics, Guanghua School of Stomatology, Affiliated
Stomatological Hospital, Guangdong Province Key Laboratory of Stomatology, Sun
Yat-sen University, Guangzhou, Guangdong, China 510055. E-mail: weixi@mail.
sysu.edu.cn
cWest China College of Stomatology, Sichuan University, Chengdu, China 610064
† Electronic supplementary information (ESI) available: 1H NMR and 13C NMR
spectrums of intermediates and polymer. See DOI: 10.1039/c8ra09770d
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Table 1 Basic characterization of the polymeric micelles
can function as an electron acceptor that affords donor–
acceptor coordination with doxorubicin to obtain micelles with
a high drug-loading ability.15 This kind of co-polymer can self-
assemble into novel polymeric micelles that show low cytotox-
icity, low critical micelle concentration, high drug-loading and
dual response to external stimuli. Moreover, in vitro drug-
release studies revealed that this polymeric system can serve
as a novel controlled-release system (Scheme 1).
a
a
a
a
Sample Mn
Mw
Mz
Mw/Mn Sizeb (nm)
PDIb
P2
P3
7393 8768
8220 10 641 13 623 1.29
10 436 1.19
148.6 ꢃ 0.36 0.135 ꢃ 0.01
64.84 ꢃ 0.22 0.154 ꢃ 0.001
a
b
Determined by GPC against a poly(ethylene glycol) standard. The
size and PdI (polydispersity index) of the micelles were determined by
DLS.
Anti-HER2 Affibody was purchased from Affibody (Solna,
Sweden).
Experimental
1H NMR and 13C NMR spectra were measured using a Bruker
AM 400 NMR spectrometer. Proton chemical shis of the NMR
spectra were given in ppm relative to the internal TMS reference
(1H, 0.00 ppm). ESI-HRMS spectral data were recorded using
a Finnigan LCQDECA mass spectrometer. Fluorescence emis-
sion spectra were obtained using a Hitachi F-7000 spectrometer
at 298 K. Gel permeation chromatography (GPC) was carried to
conrm the polymer molecular weights and their distributions
using a Waters HPLC system equipped with a model 1515 iso-
cratic pump, a 717 plus autosampler, and a 2424 refractive
index (RI) detector with Waters Styragel® HT3 and HT4
columns in series. The eluting solvent was THF at a ow rate of
1.0 mL minꢁ1 at 45 ꢂC. The retention times were calibrated
against a poly(ethylene glycol) standard with a molecular weight
range of 600–80 000 Da. Transmission electron microscopy
(TEM, Hitachi H-600) at an acceleration voltage of 100 kV was
performed to investigate the micelle morphology. Dynamic
Materials and methods
Acryloyl chloride (96%), carbon disulphide (AR), 1-dodeca-
nethiol (99.5%), methyl-trioctylammonium chloride (97%),
poly(ethylene glycol) (Mn ꢀ 2000), triethylamine (>99%), N,N0-
dicyclohexyl carbodiimide (99%), 4-(dimethyl amino)-pyridine
(99%), N-isopropylacrylamide (98%), doxorubicin hydrochlo-
ride (DOX-HCl) (98%), and 2,20-azobis(2-methyl-propionitrile)
(98%), were purchased from Aladdin Reagent (Shanghai,
China), and esterase was purchased from Sigma Aldrich.
Sodium hydroxide, 4-hydroxymethylphenylboronic acid, and
pinacol ester (98%) were purchased from Energy Chemical
Reagent (Shanghai, China). The N-isopropylacrylamide was
recrystallized from n-hexane three times prior to use, and 2,20-
azobis(2-methylpropionitrile) was recrystallized from ethanol
three times to afford a white acicular crystal. Dichloromethane
(DCM), 1,4-dioxane, acetone, and trichloromethane (CHCl3)
were dried and distilled rst with purication. HeLa and HL-
7702 cells were obtained from Shanghai Institute of Biochem-
istry and Cell Biochemistry and Cell Biology, Chinese Academy
of Science. Dulbecco's modied Eagle's medium (DMEM),
Roswell Park Memorial Institute medium 1640 (RPMI 1640), 3-
(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul-
phophenyl)-2H tetrazolium (MTS), and foetal bovine serum
(FBS) were provided by Gibco (Lige Technologies, Switzerland).
ꢂ
light scattering (DLS) measurements were carried out at 25 C
using a Zetasizer Nano-ZS90 system from Malvern Instruments
equipped with a 633 nm HeNe laser using backscattering
detection with a xed detector angle of 90ꢂ. Confocal lasing
scanning microscopic (CLSM) images of single-photo were ob-
tained using LSM 780 (Zeiss) and Nikon A1R MP+, and FTIR
spectra were recorded using
a
Shimadzu FTIR-4200
Scheme 1 Self-assembly properties and stimuli-responsive release properties of mPEG2000-PNIPAM-co-PBAPE (P3).
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acetone (6.054 g, 103.5 mmol) was added, and during this time,
the mixture turned red. Ten minutes later, chloroform
(10.688 g, 90 mmol) was added in one portion, followed by the
dropwise addition of a 50% sodium hydroxide solution (24 g,
300 mmol) over 30 min. The reaction mixture was stirred
overnight, and 90 mL of water was added, followed by 15 mL of
concentrated HCl to acidify the aqueous solution. Nitrogen gas
was purged through the reactor with vigorous stirring to assist
the evaporation of acetone. The solid was collected through
ltering and then was dissolved in 150 mL of 2-propanol. The
undissolved solid was ltered off, and the rest of the 2-propanol
solution was concentrated and dried, and the resulting solid
was recrystallized from n-hexane to obtain 12.4 g of yellow
crystalline solid (yield: 57%). 1H NMR (400 MHz, CDCl3) d 3.31–
3.25 (t, 2H), 1.72 (s, 6H), 1.70–1.63 (m, 2H), 1.25 (m, 18H), 0.88
(t, J ¼ 6.9 Hz, 3H). 13C NMR (101 MHz, CDCl3) d 220.8, 178.4,
55.5, 37.0, 31.9, 29.6, 29.5, 29.4, 29.3, 29.1, 29.0, 27.8, 25.2, 22.7,
14.1. HRMS (ESI): m/z: calcd for C17H32O2S3Na: 387.1462 [M +
Na]+; found: 387.1460.
Fig. 1 The IR spectrum of mPEG2000-PNIPAM (P2) and mPEG2000
PNIPAM-co-PBAPE (P3) (a and b). TEM images and particle size
distributions of P3 (c) and DOX-loaded P3 (d).
-
Synthesis of the marco-CTA-COOH (P1). The CTA-COOH was
conjugated on the pendant of mPEG2000-OH through an ester-
ication reaction.17 Briey, mPEG2000-OH (1.9397 mmol) and
DMAP (1.4218 g, 11.6382 mmol) were dissolved in anhydrous
DCM (20 mL) and placed in a 50 mL round-bottom ask in an
ice bath, followed by the addition of DCC (0.2397 g, 1.1638
mmol) with a magnetic stirring bar, and CTA-COOH (0.7073 g,
1.9397 mmol) was dissolved into anhydrous DCM and dropped
slowly into the ask. The reaction was carried out for 2 h in an
ice bath and allowed to reach room temperature for another
24 h with stirring. Then, the white precipitate was ltered out,
and the remaining ltrate of the P1 was precipitated in cold
diethyl ether (40 mL) twice in order to remove the unreacted
small molecule compounds. Aer purication and lyophilisa-
tion, the macro-CTA-COOH (P1) was obtained as a light yellow
solid (4.10 g). 1H NMR (400 MHz, CDCl3) d 4.28–4.21 (t, 2H),
3.28–3.21 (t, 2H), 1.69 (s, 6H), 1.24 (m, 18H), 0.88 (t, J ¼ 11.6,
5.0 Hz, 3H. Additionally, the synthesis of P1 was also conrmed
by matrix-assisted laser desorption ionization time-of-ight
mass spectrometry (MALDI-TOF MS), and the matrix was
Fig. 2 (a) Release profiles of DOX from P3 micelles at different
conditions (black: blank; red: 10 mM H2O2; blue: 10 mM H2O2 + 30 mg
mLꢁ1 esterase). (b–d) TEM images of DOX-loaded P3, DOX-loaded P3
with 10 mM H2O2; DOX-loaded P3 with 10 mM H2O2 and 30 mg mLꢁ1
esterase.
prepared at a concentration of 10 mg mLꢁ1
.
Synthesis of the mPEG2000-PNIPAM (P2). The polymer
mPEG2000-PNIPAM (P2) was synthesized by reversible addition-
fragmentation chain transfer polymerization.18 Briey, N-iso-
propylacrylamide (1.107 g, 9.78 mmol) was initially dissolved in
20 mL of 1,4-dixone in a Schlenk tube under ultrasonication,
followed by dissolving macro-CTA-COOH (1.2 g, 0.51 mmol),
and AIBN (2 mg) into this mixture. Aer three freeze–pump–
spectrometer. Ultrapure grade 10ꢄ phosphate-buffered saline
(PBS) buffer with pH ¼ 7.4 was purchased from rst BASE
Singapore. Milli-Q water was supplied by Milli-Q Plus System
(Millipore Corporation, Bedford, MA).
Synthetic procedures and characterization
Synthesis of the CTA-COOH. First, a small-molecule-based thaw cycles, the polymerization was allowed to proceed at 70 ꢂC
RAFT agent was synthesized according to the reported for 24 hours. Then, the polymerization was quenched by cooling
method.16 Generally, a three-neck ask was cooled below 10 ꢂC the system, and the resulting solution was precipitated with
with a cold circulating pump while removing the air contained excess amount of cold diethyl ether three times. The precipi-
in the ask with nitrogen. Then, 1-dodecanethiol (12.11 g, 59.85 tated copolymer was collected by ltration and was subjected to
mmol), acetone (28.86 g, 496.90 mmol), and Aliquot 336 (tri- dialysis against ultrapure water for 48 h. A white powder was
caprylylmethylammonium chloride, 0.97 g, 2.41 mmol) were nally obtained with a yield of 2.04 g aer freeze drying.
added, followed by the dropwise addition of a sodium hydroxide
Synthesis of the B1-AC. Acrylate-ended B1 (B1-AC) was
solution (50 wt%, 5.03 g). The reaction mixture was stirred for synthesized according to the references with a few modica-
another 15 min before carbon disulphide (4.563 g, 60 mmol) in tions.19 In brief, 4-hydroxymethylphenylboronic acid, pinacol
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Fig. 3 Cell imaging (14 mg mLꢁ1) in HeLa cells: P3/DOX (A)-P3/DOX (R); (1 mg mLꢁ1) in HeLa cells: DOX-HCl (A)-DOX-HCl (R); (1 mg mLꢁ1) in HeLa
cells: DOX (A)-DOX (R); column (A, D, G, J, M, P) bright field, column (B, E, H, K, N, Q) 488 nm excitations, column (C, F, I, L, O, R) merged image,
column (A, B, C) 2 h, column (D, E, F) 4 h, column (G, H, I) 8 h, column (J, K, L) 12 h, column (M, N, O) 24 h, column (P, Q, R) 48 h, (scale bar ¼ 10
mm).
petroleum ether (1 : 20) as the eluent to afford a white solid
(1.428 g, 67%).1H NMR (400 MHz, CDCl3) d 7.81 (d, J ¼ 8.0 Hz,
2H), 7.38 (d, J ¼ 8.0 Hz, 2H), 6.45 (dd, J ¼ 17.3, 1.4 Hz, 1H), 6.17
(dd, J ¼ 17.3, 10.4 Hz, 1H), 5.86 (dd, J ¼ 10.4, 1.4 Hz, 1H), 5.21 (s,
2H), 1.34 (s, 12H). 13C NMR (101 MHz, CDCl3) d 166.1, 139.0,
135.1, 131.3, 128.4, 127.4, 84.0, 66.3, 25.0. HRMS (ESI): m/z:
calcd for C16H21BO4 Na: 311.1431 [M + Na]+; found: 311.1426.
Synthesis of the mPEG2000-PNIPAM-co-PBAPE (P3). The
Fig. 4 In vitro cytotoxicity of (a) P3 in both HeLa and HL-7702 cell; (b)
DOX-loaded P3 in HeLa cells.
synthesis steps for obtaining P3 were very similar to those used
to obtain P2. Briey, B1-AC (1.107 g, 9.78 mmol) was initially
dissolved in 1,4-dixone (20 mL) in a Schlenk tube under ultra-
sonication, followed by dissolving P2 (1.2 g, 0.51 mmol), and
AIBN (2 mg) into this mixture. Aer three freeze–pump–thaw
ester monomer (7.39 mmol) was dissolved into anhydrous DCM
at 20% solids concentration in
a Schlenk ask. TEA
cycles, the polymerization was allowed to proceed at 70 ꢂC for 24
hours. Then, the polymerization was quenched by cooling the
system, and the resulting solution was precipitated with excess
cold diethyl ether three times. The precipitated copolymer was
collected by ltration and was subjected to dialysis against
ultrapure water for 48 h. A white powder was nally obtained
with a yield of 1.04 g aer freeze drying.
(11.09 mmol, 1.5 eq.) was added with gentle stirring, and the
ask was immersed in an ice bath, followed by the dropwise
addition of acryloyl chloride (11.09 mmol, 1.5 eq.) for 10 min.
The reaction was carried out for 2 h in an ice bath, and the
reaction mixture was allowed to reach room temperature for
further 24 h with stirring. Aer washing with dilute HCl solu-
tion three times and precipitating, the solids were dried under
vacuum for 48 h, and then the crude product was further
puried by column chromatography by using ethyl acetate/
IR-characterization. The chemical structures of the as-
synthesized P2 and P3 were further analysed using a Shi-
madzu FTIR-4200 spectrometer.
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Preparation of polymeric micelles
weight of loaded drug
LCð%Þ ¼
ꢄ 100%
(1)
(2)
weight of drug loaded micelle
Preparation of polymeric micelles P3. Micelles of P3 were
prepared using the dialysis method. The polymers (30 mg) were
dissolved in 6 mL DMF. Then, 5 mL deionized water was added
to the solution under gentle stirring. Aer stirring for 2 h at
room temperature, the solution was transferred into a dialysis
membrane (MWCO 2000 Da) and dialyzed for 48 h against
deionized water. The outer phase was replaced with fresh
deionized water every 6 h. The resulting solution was nally
lyophilized. The preparation method of micelles P2 is very
similar to that for P3, and specic experimental steps are shown
in ESI.†
Preparation of DOX-loaded polymeric micelles P3. Here, we
identied a common anticancer drug, doxorubicin (DOX), as
a model drug, and the water-insoluble DOX was encapsulated
into P3 micelles by adding 1.5 mL of 2 mg mLꢁ1 DOX in DMF to
6 mL of P3 solution in DMF (5 mg mLꢁ1), followed by stirring at
room temperature for two hours in absolute darkness condition
to stabilize the system, and then, the resulting solution was
placed in a dialysis membrane (MWCO 2000 Da) and dialyzed
for 48 h against deionized water, with constant stirring and
frequent water changes. Then, the solution was ltered through
a 0.45 mm syringe lter, and dynamic light scattering (DLS)
measurements were conducted at 25 ꢂC. The preparation
method of DOX-loaded polymeric micelles P2 is very similar to
that for P3, and specic experimental steps are shown in ESI.†
weight of loaded drug
EEð%Þ ¼
ꢄ 100%
weight of drug in feed
In vitro drug release experiments. The release test of the
polymeric micelle nanocarrier platform was determined by the
dialysis method (MW: cut-off: 3.5–5 kDa) at 37 ꢂC. In short,
dialysis membrane carrying 2.0 mL of DOX-loaded polymeric
micelle (P3/DOX) were immersed in 20 mL of PBS solution
(0 mM H2O2, 10 mM H2O2, 10 mM H2O2 and 30 mg mLꢁ1
esterase, 0.2 mg mLꢁ1) in a conical ask in the shaking incu-
bator with the stirring speed of 100 rpm while the bulk poly-
meric micelle immersed in the PBS solution (0 mM H2O2 and no
esterase, 0.2 mg mLꢁ1) under the same conditions were used as
control groups. At predetermined intervals, aliquots of media
(1.0 mL) were taken and quantied by measuring at 500 nm
with the Hitachi F-7000 spectrometer. Throughout the entire
process, the total volume was kept constant.
In vitro drug release experiments
HeLa cells (or HL-7702 cells) were cultured in Dulbecco's
modied Eagle medium (DMEM) (or in Roswell Park Memorial
Institute (RPMI) 1640 Medium (1640)) containing 10% foetal
bovine serum and 1% antibioticꢂ–antimycotic (penicillin–strep-
tomycin, 10 000 U mLꢁ1) at 37 C in a 5% CO2/95% air incu-
bator, and the medium was replenished every other day.
Critical micelle concentrations determination
The critical micelle concentration (CMC) was determined using
Nile Red as the uorescence probe. The block copolymer
Cell imaging experiments
concentration varied from 1.0 ꢄ 10ꢁ6 mg mLꢁ1 to 0.2 mg mLꢁ1
,
For uorescence imaging, cells (4 ꢄ 103/well) were passed on
a 6-well plate and incubated for 48 h. Prior to the staining
experiment, the cells were washed twice with physiological
saline, incubated with 14 mg Lꢁ1 DOX-loaded polymeric micelle
P3/DOX for different times (2 h, 4 h, 8 h, 12 h, 24 and 48 h) at
37 ꢂC, and then were washed twice with physiological saline.
The confocal uorescent images were captured with an excita-
tion light at 488 nm. At the same time, DOX and DOX-HCl under
the same conditions were used as control groups (1 mg Lꢁ1).
and the Nile Red concentration was xed at 1.0 ꢄ 10ꢁ6 M. The
uorescence spectra were recorded using a Hitachi F-7000
spectrometer at 298 K. Both the emission and excitation slit
widths were 5 nm. The samples were excited at 560 nm, and the
emission spectra were recorded from 580 to 800 nm. The
emission uorescence values, I631.6 at 631.6 nm was used for the
subsequent calculations. The CMC was determined from the
plots of the I631.6 versus the logarithm of the polymer concen-
tration using the intersection of the linear regression lines as
the CMC value.
In vitro cytotoxicity assay
TEM and DLS characterization
The materials' toxicity towards HeLa cells was determined by
The micelle solutions of P3 and DOX-loaded P3 at a concentra- MTS Cell Proliferation Colorimetric Assay Kit following the
tion of 0.5 mg mLꢁ1 were prepared as described above. To procedures described in the literature. Approximately 104 cells
further study the formation of the micelles, their morphologies per well were seeded in 96-well plates and cultured overnight for
and sizes were also characterized by TEM and DLS 70–80% cell conuence. The medium was replaced with 100 mL
measurements.
of fresh medium with different concentration of the materials,
Entrapment efficiency and drug loading capacity determi- to which 100 mL complexes were added at 200 mL. Aer 48 hours,
nation. The standard curve for the determination of the free 100 mL of 20% MTS solution in PBS was replaced with the old
DOX content was measured by the uorescence method with medium in each well for additional 0.5 h incubation. The
a Hitachi F-7000 spectrometer. The concentration of free DOX metabolic activity of the probe-treated cells was expressed
was calculated by the standard curve (Fig. S8†). The encapsu- relative to untreated cell controls taken as 100% metabolic
lation efficiency and loading of free DOX were obtained by the activity. The cytotoxicity study towards HL-7702 cells was
following formulas.20
carried out following the same procedure.
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solution owing to their low CMC values, even aer extreme
dilution by the larger volume of systemic circulation in vivo.
Furthermore, as shown in Fig. 1(c and d), TEM character-
ization reveals that most of the polymer particles were spher-
ical, further supporting the conclusion that polymeric micelles
were formed. The results of the DLS measurements are also
presented in Table 1. The results showed that the P3 spherical
micelles had average diameters of 60–70 nm whereas the DOX-
loaded P3 had the average diameters of 100–110 nm. These
observations provided clear evidence not only for the micelli-
zation of P3 in water, similar to the results of the CMC analysis
did, but also for their rather uniform morphology and size.
Results and discussion
Synthesis and characterization
P1 was synthesized via esterication. As shown in Fig. S1,† the
reversible addition-fragmentation chain transfer (RAFT) agent
CTA-COOH was graed with mPEG2000-OH by the esterication
reaction using DCC and DMAP as the coupling agent and
catalyst, respectively. Fig. S2† shows the matrix-assisted laser
desorption ionization time-of-ight mass spectrum (MALDI-
TOF MS) of P1. The MALDI-TOF spectrum of P1 has system-
atic peaks centred at ꢀ1990 m/z, extending from 1400 to 2500 m/
z. The difference in the m/z values between the neighbouring
peaks is 44.036, corresponding to the molar mass of the
mPEG2000 unit. Then, the monoblock polymer P2 and diblock
polymer P3 were successfully synthesized by using the macro-
molecular chain transfer reagent P1. P2, and P3 were synthe-
sized and detected, as shown in Table 1. We can also calculate
the degree of polymerization of polymer P2 and P3 according to
the results of NMR. Fig. S3† shows the particle size distributions
of P2 micelles and we carried out the thermal responsive
properties of materials P2 as it shown in Fig. S4 and S5† shows
the macro change of P2 temperature sensitivity. In addition,
Fig. S6† also examines the temperature-sensitive properties of
P3. In the present study, P1 was graed as the water-soluble part
by conjugating it to the N-isopropylacrylamide segment, and
then the polymer was also modied by boronic acid, pinacol
ester on the side chain introduced to obtain ROS sensitivity and
increase the drug loading.
Entrapment efficiency and drug loading capacity
determination
The entrapment efficiency and loading capacity of free DOX of
P3 were calculated by the standard curve, according to the
following equation. Entrapment efficiency ¼ 76.9%; loading
capacity ¼ 6.99 wt%. We also investigated the entrapment
efficiency and loading capacity of P2, and the data were shown
in Fig. S9.†
In vitro release of DOX from prodrug DOX-loaded P3
In vitro drug release performances of the DOX-loaded P3 under
physiological conditions (PBS, pH 7.4, control), oxidation
environment only (PBS, 10 mM H2O2) and both oxidation and
esterase (PBS, 10 mM H2O2 with 30 mg mLꢁ1 esterase) condi-
tions were investigated, as shown in Fig. 2. The drug release rate
was signicantly inuenced by both H2O2 and esterase,
providing an additional indication of the dual-stimulus-
sensitivity of DOX-loaded P3. At blank conditions, the prodrug
DOX-loaded P3 show weak release, and the cumulative release
amount of DOX was only 30% for 54 h, resulting from the stable
and compact DOX-loaded P3 at normal physiological condi-
tions. In 10 mM H2O2, the cumulated release of 54 h drugs was
30% higher than that in the blank group, as shown in Fig. 2(a),
and the TEM image of the release uid presented in Fig. 2(c)
shows that these micelles were adhesive and some particles are
smaller than those observed in Fig. 2(b). In contrast, in the
presence of both H2O2 and esterase, the drug release rate was
accelerated obviously, and the cumulative release amount
reached 90%, and as shown in Fig. 2(a), these micelles were
completely destroyed as observed from the TEM image pre-
sented in Fig. 2(d). Furthermore, as shown in Fig. S10 and Table
S1,† we investigated the particle size changes of P3 under
different in vitro conditions. Compared to P3, the size of the
particles increased with the addition of H2O2 and the addition
of both esterase and H2O2. Meanwhile, we investigated the in
vitro release in the presence of different esterase concentra-
IR-characterization
As shown in Fig. 1, in the P2 spectrum, the peaks at 3465, 3420,
and 3338 cmꢁ1 correspond to the N–H stretching vibration of
the –NH– group, whereas the weak band at 3035 cmꢁ1 is due to
the aromatic C–H stretching vibration, the peaks at 2960–
2850 cmꢁ1 correspond to the alkane C–H vibration, the band at
1645 cmꢁ1 is due to the C]O stretching vibration in amide I
bonds, and the peaks at 1543 cmꢁ1 can be ascribed to the N–H
stretching vibrations of the amide II bonds. The peaks at 1389
and 1365 cmꢁ1 are induced by the deformed vibration absorp-
tion peak of the two methyl in –CH (CH3)2–, while the two peaks
at 1170 and 1130 cmꢁ1 are associated with the C–O stretching
mode in the ester and the absorption peak of the PEG ether
bond at 1060 cmꢁ1 covers the C]S bond absorption. These
characteristic peaks prove that the N-isopropylacrylamide
monomer was successfully polymerized onto the polymer chain.
Additionally, in the P3 spectrum, the strong peaks at 833–
810 cmꢁ1 are derived from 1,4-two substituted benzene, while
the peak at 1730 cmꢁ1 is ascribe to the overtone bands.
Characterization of polymeric micelles P3 and DOX-loaded P3
Because the amphiphilic polymer P3 can self-assemble into tions, and as shown in Fig. S11(a),† it was found that compared
micelles in aqueous solutions, to the best of our knowledge, the to the control group, the amount of the release did not increase
micelles can self-assemble above a certain threshold concen- signicantly; this is in line with the TEM observations shown in
tration, leading to a rapid increase in the emission intensity of Fig. S11(b).† This may be due be to the rearrangement of ROS-
Nile Red, as shown in Fig. S7.† The measured CMC values sensitive DOX-loaded P3 pendant and the cleavage of the
hinted at the good stability of these polymeric micelles in esterase-sensitive main chain on the copolymer.
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viability obtained using the MTS assay method in HeLa cells
were decreased by DOX-loaded polymeric micelles P3, and the
cytotoxicity of DOX-HCl and free DOX was examined by the
same method, as shown in Fig. S13.† All of the concentration of
the free DOX exhibited low cytotoxicity towards HeLa cells,
while the cell viability decreased sharply by 95% for DOX-HCl,
while for DOX-loaded polymeric micelles P3, the cell viability
decreased sharply by 70% aer 48 h of incubation; these results
provide further proof that DOX-loaded polymeric micelles P3
have sustained release effect on DOX.
In vitro cellular uptake of DOX
The intracellular uptake of DOX-HCl, free DOX and DOX-loaded
P3 was investigated using confocal lasing scanning microscopy
(CLSM) (Fig. 3). When the cells were incubated with DOX-HCl,
the cellular uptake of DOX-HCl was the same, regardless of
the different time duration of 2, 4, 8, 12, 24 and 48 h. DOX-HCl
was distributed in the nuclear region within 2 h of incubation,
and strong uorescence was observed in the nucleus, as re-
ported previously.21 From column DOX-HCl (P)-DOX-HCl (R),
aer 48 h incubation, it was clearly observed that the HeLa cell
morphology was completely destroyed. However, when the cells
were incubated with free DOX, the cellular uptake of free DOX
was also the same, regardless of different time durations of 2 h,
4 h, 8 h, 12 h, 24 h and 48 h, and free DOX was distributed in the
cytoplasm region during long-time incubation, with the aggre-
gation of free DOX with increasing time. Moreover, it was
difficult to for the free DOX to transfer into the nucleus even
aer 48 h. When the cells were incubated with DOX-loaded
polymeric micelle P3 in two hours, considerable uorescence
intensity was detected mainly in the cytoplasm, suggesting
novel cellular uptake ability of DOX-loaded polymeric micelle
P3, and with the prolongation of the incubation time, the
cellular uorescence was much higher. We found that aer 4
hours, the DOX molecules were localized in the cell nuclei, and
as expected from the drug release in vitro experiment, DOX
localized in the cell nuclei is likely to be intercalated into DNA
strands, thereby showing its toxicity against tumour cells. This
dual cellular uptake prole of DOX molecules is likely due to the
rapid demicellization of the DOX-loaded polymeric micelles
under cellular conditions, leading to the rapid release of DOX
from polymeric micelles and its subsequent internalization into
cell nuclei. As shown in Fig. S12,† the average uorescence
intensity in the nucleus is a measure of the amount of DOX that
enters the nucleus. When the cells were incubated with DOX-
HCl, the intensity mean value was approximately 80% aer
24 h incubation, and with the incubation time extended to 48 h,
the average uorescence intensity of HeLa nuclei was higher
than 200%. However, even aer 48 hours, the average uores-
cence intensity of the cells incubated with free DOX remained at
20%. The value obtained for DOX-loaded P3 was between these
values for free DOX and DOX-HCl and increased with incuba-
tion time, so that aer 48 h, the uorescence intensity increased
signicantly to 80%. These ndings suggested that DOX-loaded
polymeric micelles may be suitable for stimulus-responsive
nanocarriers.
Conclusions
In summary, we have successfully constructed a novel high-
drug-loading, dual stimulus-responsive drug-release system
based on a rearrangement of borate ester bond and a hydrolysis
of an ester bond. Compared to the common drug control release
systems, P3 exhibits high entrapment efficiency and drug
loading capacity, and the synergetic characteristics of two
stimuli. Such a dramatic loading capacity and synergetic char-
acteristics is anticipated to achieve unprecedented advances in
the development of stimuli-sensitive drug-delivery systems that
are deemed to be a promising vehicle for drug-delivery
nanocarriers.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
This work was nancially supported by the National Natural
Science Foundation of China (No. 21572147 and 21877082).
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This journal is © The Royal Society of Chemistry 2019