Reduction/temperature/pH multi-stimuli responsive core cross-linked polypeptide hybrid micelles for triggered and intracellular drug release

Article abstract of DOI:10.1016/j.colsurfb.2018.06.015

The high toxicity, poor stability, premature drug release, and lack of intracellular stimuli responsibility of current polymeric micelles still hinder them for potential clinical applications. To address these challenges, a novel type of multi-stimuli responsive, core cross-linked polypeptide hybrid micelles (CCMs) was developed for triggered anticancer drug delivery in tumor microenvironment. The CCMs was prepared via free radical copolymerization by using N,N’-methylene-bis-acylamide (BACy) as the cross-linking agent, 2,2-azobisisobutyronitrile (AIBN) as the initiator, where poly (γ-benzyl-L-glutamate) (PBLG) and N-isopropylacrylamide (NIPPAM) as comonomers. The doxorubicin (DOX) was then introduced into the CCMs by hydrazone bond to prepare the drug-incorporated core cross-linked micelles (CCMs-DOX). By the experimental results, the CCMs showed reduction responsibility due to the degradable disulfide bond in the polymer network. The hydrazone bond can be broken under acidic condition causing a controllable drug release for CCMs-DOX. Compared to only 7.7% DOX release under pH 7.4 at 37°C, a much higher DOX release rate up to 85.3% was observed under 10 mM GSH (pH 5.0, 42°C). In vitro cell assays showed that the blank CCMs showed almost no toxicity against HUVEC cells while the CCMS-DOX exhibited significant cancer cell killing effect. These experimental results suggested that the prepared multi-stimuli responsive polymeric micelles could serve as a smart and promising drug delivery candidate for anti-cancer therapy.

Full text of DOI:10.1016/j.colsurfb.2018.06.015

Colloids and Surfaces B: Biointerfaces 170 (2018) 373–381
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces
journal homepage: www.elsevier.com/locate/colsurfb
Reduction/temperature/pH multi-stimuli responsive core cross-linked
polypeptide hybrid micelles for triggered and intracellular drug release
a
a
a
a
a,⁎
b,⁎
Jing Qu , Qiu-yue Wang , Kang-long Chen , Jian-bin Luo , Qing-han Zhou , Juan Lin
a
College of Chemical and Environment Protection, Southwest Minzu University, First Ring Road, 4th Section No.16, Chengdu, Sichuan 610041, China
b
School of Biomedical Sciences and Technology, Chengdu Medical College, 783 Xindu Road, Chengdu, Sichuan 610500, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Core cross-linked micelles
Polypeptide
Multiple-stimuli responsive
Drug delivery
The high toxicity, poor stability, premature drug release, and lack of intracellular stimuli responsibility of
current polymeric micelles still hinder them for potential clinical applications. To address these challenges, a
novel type of multi-stimuli responsive, core cross-linked polypeptide hybrid micelles (CCMs) was developed for
triggered anticancer drug delivery in tumor microenvironment. The CCMs was prepared via free radical copo-
lymerization by using N,N’-methylene-bis-acylamide (BACy) as the cross-linking agent, 2,2-azobisisobutyroni-
trile (AIBN) as the initiator, where poly (γ-benzyl-L-glutamate) (PBLG) and N-isopropylacrylamide (NIPPAM) as
comonomers. The doxorubicin (DOX) was then introduced into the CCMs by hydrazone bond to prepare the
drug-incorporated core cross-linked micelles (CCMs-DOX). By the experimental results, the CCMs showed re-
duction responsibility due to the degradable disulfide bond in the polymer network. The hydrazone bond can be
broken under acidic condition causing a controllable drug release for CCMs-DOX. Compared to only 7.7% DOX
release under pH 7.4 at 37°C, a much higher DOX release rate up to 85.3% was observed under 10 mM GSH (pH
5.0, 42°C). In vitro cell assays showed that the blank CCMs showed almost no toxicity against HUVEC cells while
the CCMS-DOX exhibited significant cancer cell killing effect. These experimental results suggested that the
prepared multi-stimuli responsive polymeric micelles could serve as a smart and promising drug delivery can-
didate for anti-cancer therapy.
1. Introduction
proteins, high electrolyte concentration, variation of pH and tempera-
ture, massive dilution, and mechanical shear forces in the blood cir-
Recently, stimuli-responsive polymeric micelles serving as func-
culation still remains challenges for DDS. The premature drug release
caused by micellar instability may cause serious toxicity problems and
hinder the effect of drug delivery at aimed sites, which would extremely
limit their application if used in vivo. Therefore, it is of great im-
portance for drug delivery vectors to endure the complex biological
environment. Covalent cross-links in micelles can remarkably enhance
the structural stability rather than the weak noncovalent intermolecular
interactions in linear polymer micelles. Therefore, the high stability of
polymer micelles with cross-linked structure have promising applica-
tions in DDS [15,16]. However, the permanently cross-linked micelles
cannot be disintegrated and may accumulate in the host cells or tissues
causing a long-term toxicity. In order to address the dilemma, in the last
decade various strategies have been developed for the fabrication of
degradable crosslinked micelles, which can maintain their nano-struc-
tures in the complicated extracellular environment, but undergo the
biodegradation process in response to intracellular microenvironment
[17].
tional drug vehicles have emerged as the most promising technology
platform for various drug delivery system (DDS) due to their small size,
core-shell structure, drug-loading capacity, and triggered drug release
in response to biological stimuli such as differences in redox potential,
pH, and temperature between normal cells and tumor cells [1–5]. The
stimuli-responsive polymeric micelles consist of a hydrophobic core and
a hydrophilic shell, and possess the functional group for controlled drug
release [6]. Therefore, various anti-cancer drugs can be incorporated in
the micellar core such as paclitaxel or doxorubicin (DOX), and the
hydrophilic shell can protect them from degradation by enzymes, pre-
vent micelle aggregation, improve the bioavailability of drugs in water,
and decrease side effects on healthy cells [7–10]. Therefore, the stimuli-
responsibility can realize the triggered release of anti-cancer drugs in
cancer tissue for targeted delivery [11–14].
However, for the conventional polymeric micelles from linear am-
phiphilic block copolymers, the micellar instability caused by plasma
⁎
Corresponding authors.
E-mail addresses: zhqinghan@swun.edu.cn (Q.-h. Zhou), linjuan@cmc.edu.cn (J. Lin).
https://doi.org/10.1016/j.colsurfb.2018.06.015
Received 18 April 2018; Received in revised form 8 June 2018; Accepted 11 June 2018
Available online 18 June 2018
0927-7765/ © 2018 Published by Elsevier B.V.

J. Qu et al.
Colloids and Surfaces B: Biointerfaces 170 (2018) 373–381
On the other hand, although synthesis of tailored biopolymers with
temperature for another 12 h. The organic phase was extracted with
controllable architectures and compositions has been facilitated by new
polymerization process, comparing to the proteins or peptides, syn-
thetic biopolymers have not yet match the natural structures and bio-
functional diversity so far. Due to the good biodegradability, bio-
compatibility, biofunctionality, and structural analogous to natural
biomolecules, synthetic polypeptides have gained great interest for DDS
dichloromethane, and then dried over anhydrous MgSO
4
. The BACy
was finally purified by recrystallization from ethyl acetate/hexane
1
mixture (1:1, v/v). The H spectrum of BACy in CDCl
Fig. 1a. 1.60 g (61 wt%). 1H-NMR (400 MHz): δ (ppm) = 2.89 (t, 4H,
–CONHCH CH S–), 3.68 (t, 4H, –CONHCH CH S–), 5.66–6.31 (m, 6H,
CH =CHCO–), and 6.61 (brs, 2H, –CONH–).
3
was shown in
2
2
2
2
2
[
18]. Recently, various kinds of polypeptides with stimuli responsibility
have been developed, such as reduction, temperature, pH, and enzyme
as well [19–25]. Owing to good colloidal stability, the polypeptide
based cross-linked polymeric micelle has been intensively studied for
drug, protein, and gene delivery. For example, by using a ring-opening
polymerization, Zhang et al. reported a reduction-sensitive shell-
crosslinked polyglutamate-b-polysaccharide micelles for DOX in-
tracellular delivery [26]. Ren et al. reported the preparation of a pH/
sugar-sensitive, core-cross-linked, polyion complex micelles based on
poly glutamic acid via a phenylboronic acid–catechol interaction for
protein intracellular delivery [27]. Gao et al. developed a reduction-
and temperature-sensitive core-cross-linked polyglutamate hybrid mi-
celle with pendant diethylene glycol with lower critical aggregation
concentration for DOX delivery against HeLa cells [28]. Via a reduc-
tion-responsive disulfide cross-linked stearyl-peptide-based micelle
system, Yao et al. achieved co-delivery of DOX and microRNA-34a for
prostate cancer therapy [29]. Recently, our group reported a reduction-
responsive core cross-linked polyethylene glycol-polypeptide hybrid
micelle with high loading capacity and efficient intracellular drug re-
lease [30]. However, due to the difficulty in fabrication schemes that
involve complex and multi-step synthesis procedures, it is still chal-
lenging to integrate reduction, temperature, and pH stimuli-responsi-
bility into one cross-linked micelle based on polypeptide for in-
tracellular and efficient drug delivery [31].
2.1.2. Synthesis of vinyl poly (γ-benzyl-L-glutamate) (PBLG)
γ-Benzyl-L-glutamate N-carboxyanhydride (BLG-NCA) was prepared
according to a reported literature procedure [32]. Allylamine (5 μL,
66.2 μmol), BLG-NCA (0.70 g, 2.7 mmol), and 3 mL of DMF were added
to a Schlenk flask. After degassed by three freeze-thaw cycles, the
mixture solution was reacted for 24 h at room temperature by gently
stirring under N
2
atmosphere. The product was precipitated in ice-
1
cooled ether and dried under vacuum. The H spectrum of PBLG in
1
CDCl
δ (ppm) = 1.80–2.16 (–COOCH
2.54 (CH =CHCH –), 4.48–4.67 (–CONHCH–), 4.99–5.19 (C
=CHCH –), 7.21–7.39 (C
3
was show in Fig. 1b. Yield: 0.90 g (79 wt%). H-NMR (400 MHz):
2
CH
2
–), 2.31–2.52 (–COOCH
2
CH
CH
2
–),
–),
2
2
6
H
5
2
5.46∼6.67 (CH
(–CONHCH–).
2
2
6
H
5
–), and 7.72–7.92
2.1.3. Preparation of CCMs
In a typical experiment, NIPPAM (0.61 g, 5.3 mmol), PBLG (0.20 g,
0.1 mmol), BACy (0.04 g, 0.2 mmol), AIBN (0.04 g, 0.2 mmol), and
150 mL of toluene were added into a three-necked flask, and the mix-
ture solution was stirred for 12 h at 85℃ under N atmosphere. The
2
product was precipitated in a mixture of ice-cooled ether/ tetra-
hydrofuran solution. The product was then filtrated and dried under
1
vacuum. The H spectrum of the cross-linked copolymer was shown in
1
Fig. 1c. Yield: 1.50 g (70 wt%).
(ppm) = 0.99∼1.30 (–CH in
(–COOCH CH –, in PBLG unit), 2.32–2.53 (–COOCH
unit), 3.66–3.78 (–CONHCH CH
NIPPAM unit), 4.52–4.67 (–CH–, in PBLG polymer backbone),
4.96–5.20 (C CH –, in PBLG unit), 7.18∼7.37 (C –, in PBLG unit),
H-NMR (400 MHz):
δ
Herein, we developed a simple approach to fabricate the core cross-
linked polypeptide hybrid micelles (CCMs) via free radical copoly-
merization, and the anticancer drug, DOX, was then introduced to the
CCMs through an acid-sensitive hydrazone bond to prepare a novel type
of reduction, temperature, and pH multi-stimuli responsive core cross-
linked micelles (CCMs-DOX). Their chemical structure, size, and mor-
phology were fully characterized, and the release experiments demon-
strated that the CCMs-DOX exhibited multi-stimuli responsive drug
release. The blank CCMs showed almost no toxicity against HUVEC cells
3
,
NIPPAM
unit),
CH
1.77–2.15
–, in PBLG
2
2
2
2
2
2
S–, in BACy unit), 3.89–4.02 (–CH–, in
6
H
5
2
6 5
H
7.70∼7.98 (–CONHCH–, in PBLG unit and NIPPAM unit).
The CCMs were prepared by a dialysis method. 10 mg of cross-
linked copolymer was dissolved in 4 mL of DMF. The solution was drop-
wisely added to 5 mL of deionized water and stirred for 1 h. After that,
dialysis method was used to remove the unbonded molecules and or-
ganic solvents with a dialysis tube with molecular weight cut-off
(MWCO) of 12,000 for 3 days. Finally, the CCMs were freeze-dried into
white powder.
(
normal cell) while the CCMs-DOX showed great promise of anti-tumor
efficacy and intracellular drug delivery against HeLa cells (cancer cell)
by the CCK8 assay and CLSM analysis. Scheme 1 illustrates the pre-
paration of the CCMs, CCMs-DOX, and the overall mechanism of the
controlled drug release in response to the simulated tumor micro-
environment.
2.1.4. Preparation of CCMs-DOX
Briefly, 0.50 g of CCMs was dissolved in 50 mL of DMF, and then
10 mL of anhydrous hydrazine and excessive DOX (0.10 g, 0.2 mmol)
were added into the mixture solution under stirring for 24 h at room
2. Experimental
2.1. Materials
2
temperature with the exclusion of light and protection of N atmo-
sphere. The product solution was dialyzed against PBS buffer (pH 7.4,
10 mM) with a dialysis tube (MWCO 12,000), and the PBS buffer was
refreshed every 5 h for 3 days to remove the excess DOX. Finally, the
product solution was freeze-dried into a red powder.
The CCMs-DOX were prepared by a dialysis method. 10 mg of as
prepared product was dissolved in 4 mL of DMF. The solution was drop-
wisely added to 5 mL of deionized water and stirred for 1 h. Then the
solution was transferred into a dialysis tube (MWCO 12,000) and dia-
lyzed against PBS buffer (pH 7.4, 10 mM) for 3 days to prepare the
CCMs-DOX. Finally, the product was freeze-dried into a brown powder.
N-isopropylacrylamide (NIPPAM) (98%) and Acryl amide (99%)
were obtained from Best Reagent Ltd. 2,2-azobisisobutyronitrile (AIBN)
99%) purchased from Kemio Chemical Reagent Ltd. Hydrazino (80%)
were obtained from Zhiyuan Chemical Reagent Ltd. Glutathione (GSH)
98%) and DOX (98%) purchased from Hua Feng Chemical Materials
Ltd. N,N-dimethylformamide (DMF), toluene, dichloromethane, tetra-
hydrofuran (THF), and ethyl acetate were used after distilled.
(
(
2
.1.1. Synthesis of N,N’-methylene-bis-acylamide (BACy)
Cysteamine hydrochloride (2.30 g, 10.2 mmol) was dissolved in
8 mL water and cooled in ice-water bath for 30 min. Acryloyl chloride
1
2.2. Characterization
(
1.8 mL, 21.5 mmol) was dissolved in 3 mL of THF. Both above solu-
tions were added slowly into three-necked flask within 5 min; at the
same time, NaOH solution (1.60 g, 40 mM) was also drop-wisely added.
The mixture was stirred for 3 h in ice-water bath, and then at room
1H-NMR spectra of BACy, PBLG, and CCMs were obtained using a
Bruker 400-MHz spectrometer with deuterated chloroform (CDCl
3
) as
solvent using tetramethylsilane (TMS) as the internal standard. FTIR
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J. Qu et al.
Colloids and Surfaces B: Biointerfaces 170 (2018) 373–381
Scheme 1. (a) Synthetic route to the CCMs. (b) Schematic illustration of the multi-stimuli responsiveness of CCMs and triggered drug release process of the CCMs-
DOX.
spectra of BACy, PBLG, and CCMs were gathered in solid state on a PE
Spectrum One FTIR spectrophotometer under room temperature at 4
cm resolution from 400 to 4000 cm . The micelle diameter of CCMs
were determined by Dynamic light scattering (DLS, Malvern Zetasizer
Nano-ZS90). Each sample of 1 mg/mL was added into a 2.0 mL quartz
cuvette and measured under ambient in three times. The turbidity
measurement was carried out to monitor the degradation behavior of
the micelles by UV‐vis spectroscopy, and the relative turbidity of
sample solution was determined by calculating the ratio of the absor-
bance of the micelles in presence of GSH to that of the initial non-
degraded micelles. GPC measurements were performed to obtain the
−
1
−1
n
number average molecular weight (M ) on a Waters 1515 solvent pump
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J. Qu et al.
Colloids and Surfaces B: Biointerfaces 170 (2018) 373–381
Fig. 2. Properties of CCMs-DOX. (a) Comparison chart of PBLG, NIPPAM,
BACy, and DOX feeding and loading ratios in copolymer. (b) Absorbance and
fluorescence spectra of DOX in CCMs-DOX.
373 and 384 nm (I373/I384) were calculated and plotted against the
logarithm of the micelle concentrations. Finally, the CMC value was
obtained from the intersection of the tangent to the curve at the in-
flection with the horizontal tangent at low concentration.
Fig. 1. 1H NMR spectra of BACy (a), PBLG (b), and CCMs (c) in CDCl
.
3
2
.2.2. LCST measurement and temperature-responsibility of CCMs
The lower critical solutions temperature (LCST) of the CCMs was
with a Waters 2410 refractive index detector. HPLC grade DMF was
chosen as the eluent at a flow rate of 1.0 mL/min. The linear poly-
styrene was chosen as standards to obtain the calibration curve.
Morphologies of micelles were observed by transmission electron mi-
croscopy (Hitachi H-600, Japan). Each sample was prepared by drop-
ping the solution of micelles onto carbon-coated copper grids and ne-
gatively stained with 1% phosphotungstic acid for TEM observation.
Raman spectroscopy was used to confirm the disulfide bond in polymer
network on a LabRam confocal Raman microscope (Renishaw CR-IM16-
obtained by measuring the optical absorbance of the samples in aqu-
eous solutions at various temperatures at 500 nm with a UV–vis spec-
trometer. Prior to measurements the sample solution was thermo-stated
at different temperatures, and the LCST is defined as the temperature
exhibiting a 50% decrease of the total decrease in transmission rate.
2.2.3. In vitro drug release
139, USA). UV–vis spectrometer (Mapada TU1950, China) was used to
According to a standard curve obtained from DOX/DMF solutions,
the drug loading and encapsulation efficiency was measured on a
UV–vis spectrophotometer at the absorbance of 480 nm. The drug
loading was defined as the weight ratio of loaded drug to the drug-
entrapped polymer micelles. The encapsulation efficiency of DOX was
obtained from the weight ratio of the drug loaded in micelles to that
used in the preparation. The in vitro DOX drug release study was per-
formed at 37℃ and 42℃. 2 mL of CCMs-DOX at a concentration of
1 mg/mL in a dialysis tube (MWCO 12,000) were incubated in different
media: 1) PBS (pH 7.4, 37 °C) without GSH, 2) PBS (pH 7.4, 37 °C) with
10 mM GSH, 3) PBS (pH 5.0, 37 °C) without GSH, 4) PBS (pH 5.0, 37 °C)
with 10 mM GSH, and 5) PBS (pH 5.0, 42 °C) with 10 mM GSH under
gentle stirring, respectively. At each time interval, 1.5 mL of medium
investigate the pH-responsibility of the micelles by tracing the absor-
bance of sample solution in different pH from 0.5 to 7.4 at a con-
centration of 1.0 mg/mL under room temperature.
2.2.1. Determination of critical micelle concentration (CMC)
The CMC value of the CCMs were determined using fluorescence
spectroscopy (RF-5301PC, Shimadzu, Japan) using pyrene as fluor-
escent probe. Briefly, a large number of scans for different concentra-
−
4
tions of the CCMs (5 × 10
to 1 mg/mL) were gathered from fluor-
escence spectroscopy, in which the final concentration of pyrene in
−
6
each sample was set to 6.0 × 10
mol/L. According to the excitation
spectra of pyrene, the ratios of pyrene probe fluorescence intensity at
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J. Qu et al.
Colloids and Surfaces B: Biointerfaces 170 (2018) 373–381
Fig. 3. (a) Size distribution of copolymer micelles by DLS measurement, and their TEM photo (b). (c) Hydrodynamic size of CCMs in cell culture media (DMEM with
0% FBS) as a function of time. (d) Fluorescence emission spectrum of pyrene at 335 nm, and (e) plots of the intensity ratio vs log C.
1
was used for measuring and replaced with 1.5 mL of fresh medium.
3. Results and discussion
3.1. Preparation and structural characterization of the CCMs and CCMs-
2.2.4. In vitro cell assay
DOX
Cancer cell viability of the CCMs and CCMs-DOX were determined
by Cell Counting Kit-8 (CCK-8) assay. The HeLa cells (human cervical
carcinoma cells, cancer cells) and HUVEC cells (human umbilical vein
endothelial cells, normal cells) were cultured in DMEM (dulbecco's
modified eagle medium) at 37 °C in a humidified atmosphere with 5%
In this work, to prepare the CCMs-DOX, CCMs was firstly synthe-
sized via free radical copolymerization by using BACy as crosslinker
and AIBN as radical initiator, where PBLG and NIPPAM as comonomers.
The preparation of CCMs was a simple single-step procedure where all
the comonomers were directly mixed by using toluene as the solvent.
Since PBLG, NIPPAM, and BACy contain vinyl groups, they can be di-
rectly incorporated into the backbone of CCMs (Scheme 1a). Thus,
comparing to the conventional method where each component is in-
dividually attached to polymer network, this single-step method elim-
inates the need of multiple conjugation steps, and the amount of each
component can be easily controlled.
2
CO and supplemented with 10% heat-inactivated fetal bovine serum.
The logarithmic growth phase cells were washed with PBS, digested
with trypsin, and centrifugalized to remove liquid supernatant. Cells
5
were seeded into 96-well plate at a density of 5 × 10 cells/well, and
incubated for 24 h to allow cell attachment. After that, the medium was
removed and replaced with 100 μL of different concentrations of free
DOX, blank CCMs, or CCMs-DOX. The cells were incubated with dif-
ferent samples for several hours. At predetermined intervals, 10 mL of
CCK-8 solution were added into each well, and the cells were incubated
for 3 h. After incubation, the absorbance was measured at 450 nm to
evaluate the cell viability by a micro-plate reader (Thermo Fisher
Multlskan FC, USA).
The DOX was then introduced into the CCMs by hydrazone bond to
prepare the multi-responsive core cross-linked micelles, CCMs-DOX, by
the dialysis method. The CCMs-DOX could keep stable in aqueous so-
lution due to the formation of hydrogen bonds between hydrophilic
PNIPPAM outer shell and water molecules, which acted as a hydration
barrier. In brief, the CCMs-DOX contains three basic units: BACy as a
disulfide bonded cross-linker to provide reduction-responsibility, the
biocompatible polypeptide was used to bond DOX to provide pH-re-
sponsivity, and NIPPAM as the hydrophilic unit to provide temperature-
responsibility. The synthetic route and stimuli-responsive drug release
process of the micelles were illustrated in Scheme 1.
2.2.5. Confocal laser scanning microscopy
Confocal Laser Scanning Microscope (CLSM) was used to investigate
the cellular uptake of DOX. The cells were incubated in an UV disin-
5
fected 6-well plate with cover glass at a density of 5 × 10 cells/well.
1
After 24 h incubation period, samples of the free DOX or CCMs-DOX
were added into each well. At 2 h and 24 h, the suspension was re-
moved, and the samples were washed with fresh DMEM and PBS before
fluorescence observation. The samples were then mixed with fluor-
escent mounting medium to be observed by CLSM (Motic AE31, Japan)
with an imaging software.
H NMR spectra were used to confirm the chemical structures of the
1
synthesized BACy, PBLG, and CCMs. The representative H NMR
spectrum of BACy (in CDCl
3
) was depicted in Fig. 1a. The resonance
signals of –CH – (e and d) neighboring to the disulfide bond of the
2
BACy molecule, vinyl group (a and b), and –NH– (c) was observed at
δ = 2.89 (e), 3.68 (d), 5.66–6.31 (a and b), and 6.61 (c), respectively,
as described in experimental section. As shown in Fig. 1b, the chemical
structure of PBLG was evidenced by 1H NMR in CDCl
with 15% tri-
fluoracetic acid. The resonance signals of protons of β- and γ-methylene
3
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Colloids and Surfaces B: Biointerfaces 170 (2018) 373–381
Fig. 4. Investigations on the redox-induced micelle degradation: (a) and (b)The size change of copolymer micelles in response to GSH by DLS measurement. (c) The
relative turbidity in the presence and absence of 10 mM GSH. (d) Investigation on the reduction-responsibility by GPC measurement of PBLG (up, before degradation)
and copolymer micelle (down, after degradation).
initiator residue at δ = 5.46–6.67 indicated the successfully synthesis of
PBLG. The 1H NMR spectrum of CCMs (in CDCl
) was depicted in
3
Fig. 1c. The resonance signals at δ = 0.99–1.30 (j), 3.66–3.78 (k), and
.89–4.02 (l) were belong to the methyl group in NIPPAM units (j),
eCH e group (k) of the BACy crosslinker, and methine group in
NIPPAM units (l). As shown in Fig. 1c, the appearance of the benzyl unit
in PBLG (δ = 7.18–7.37), eCH e group (k) signals in BACy, and me-
3
2
2
thine group (l) in NIPPAM unit, and disappearances of the vinyl group
in PBLG unit (δ = 5.46–6.67) indicated the successfully synthesis of the
CCMs. The FTIR and Raman spectra of crosslinked micelles were shown
in Figure S1.
The CCMs-DOX were prepared by introducing DOX molecule into
the CCMs via pH-responsive hydrazone bond by the dialysis method. As
shown in Fig. 2a, herein, the polymer component weight ratios were
referred to the feeding and loading ratios before and after poly-
merization, respectively. The small difference between the feeding and
loading of monomers indicated efficient conversion during the poly-
merization reaction. Therefore, the drug/polymer composition in the
end product can be precisely controlled by altering the feeding ratio of
monomers. CCMs-DOX were also characterized by UV–vis and fluor-
escence spectroscopy (Fig. 2b) to further confirm and quantify the DOX
loading. Free DOX emits red fluorescence at 590 nm and the spectrum
depicted in Fig. 2b confirms that CCMs-DOX conserved the fluorescent
property of DOX with the absorbance and emission maxima at 480 and
Fig. 5. In vitro DOX release profiles of CCMs-DOX under different simulated
physiological conditions.
groups (f and g), –CH – in initiator residue (c), α-methine group (e) in
2
polymer backbone, methylene group of benzyl (h), phenyl group (i),
and amide group (d) appeared at δ = 1.80–2.16 (f), 2.31–2.52 (g), 2.54
590 nm, respectively. By using the absorbance measured from CCMs-
(
c), 4.48–4.67 (e), 4.99–5.19 (h), 7.21–7.39 (i), and 7.72–7.92 (d), re-
DOX, the drug loading was quantified that the weight of DOX accounts
for 6.3% of the total drug entrapped polymer weight, and 56.0% for
spectively. The resonance signals of vinyl group (a and b) in the
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Colloids and Surfaces B: Biointerfaces 170 (2018) 373–381
Fig. 6. CLSM images of HeLa cells cultured with free DOX over incubation: (a–d) 2 h and (e–h) 24 h; Cells cultured with CCMs-DOX: (i–l) 2 h and (m–p) 24 h. I,
stained by DAPI; II, DOX fluorescence images; III, merged images; IV the bright field. The scale bar corresponds to 25 μm.
encapsulation efficiency.
by DLS measurements. In Fig. 4a and b, a rapid degradation was ob-
served for CCMs in the first 2 h, in which the average micelle size in-
creased from about 160 nm to around 350 nm, reaching over 450 nm
after 4 h. It was indicated that the reductive cleavage of the disulfide
bonds had resulted in the degradation of the cross-linked micelles and
formation of a swelling structure. When proceeding to over 8 h, a re-
markable size decrease of the nanoparticles was observed, which can be
explained by the disassembly of the micelles into linear polymer chains,
finally reaching 220 nm after 24 h degradation. Fig. 4c shows the de-
gradation extent of the CCMs by relative turbidity in PBS buffer 7.4 at
37 °C in the presence of 10 mM GSH. In the first 2 h after the addition of
GSH, the relative turbidity changed very quickly and then the relative
turbidity decreased gradually as shown due to the degradation of CCMs.
When proceeding to 24 h, the translucent emulsion of micelles changed
into a clear solution indicating that the CCMs were degraded into linear
polymeric chains. However, the CCMs kept stable and no significant
change in relative turbidity was observed in the solution without GSH.
In Fig. 4d, the GPC experiments indicated that the PBLG (before co-
polymerization) had a similar number-average molecular weight
The CCMs were prepared by dialysis method and formed a core-
shell structure, which consisted of a hydrophobic polymer network
inner core and a hydrophilic PNIPPAM outer shell as a hydration bar-
rier. DLS measurements showed that the cross-linked copolymer formed
micelles with average sizes of about 160 nm and a narrow unimodal
distribution with a PDI of 0.24 as shown in Fig. 3a. As shown in Fig. 3b,
TEM micrograph revealed that these micelles had an oval morphology
with an average size of about 100 nm. The smaller size (about 100 nm)
observed by TEM as compared to that determined by DLS is probably
due to shrinkage of the hydrophilic shell through overnight dehydration
before TEM observation. After 7 days, no significant changes in size
were observed in DMEM media, indicating that the micelles retained
good stability due to the presence of hydrophilic PNIPPAM outer shell
and the covalent cross-linked polymer network. Therefore, the CCMs
could enable long circulation time in blood and reduce liver and mac-
rophage uptake when used in vivo. As shown in Fig. 3d and e, the CMC
values of CCMs were determined to be 31.6 mg/L by the intersection of
the tangent to the curve of I373/I384 at the inflection with the horizontal
tangent. Because of the low CMC value, it was indicated that the CCMs
could provide excellent colloidal stability in very dilute aqueous solu-
tion such as bloodstream and body fluids.
(M
(M
n
= 1900) to that of the degraded linear polymer chains
= 2000). It was suggested that the cross-linked polymer network in
n
micelles were chemically cleaved through disulfide-thiol exchange re-
action, leading to the formation of the short linear polymers. Ad-
ditionally, the temperature- and pH-responsibility of the CCMs-DOX
was also investigated and shown in Figure S2 and S3).
3.2. Redox degradation of the CCMs
To monitor the redox-responsive behavior of the micelles, the de-
gradation experiments of the micelles with redox agent were carried out
by DLS, UV–vis, and GPC. GSH was chosen as the reducing agents to
study the degradation behavior of CCMs, and the size change of CCMs
in response to 10 mM GSH in PBS buffer (pH 7.4) at 37 °C was followed
3.3. In vitro drug release
To investigate the potential application of CCMs-DOX as multi-re-
sponsive drug delivery vehicle in chemical therapy, the DOX releasing
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Colloids and Surfaces B: Biointerfaces 170 (2018) 373–381
3.4. In vitro cell assays
To confirm our expectation and evaluate the therapeutic properties,
the in vitro cell assays were carried out to investigate the intra-cellular
drug release of CCMs-DOX by CLSM. Fig. 6 showed the CLSM images of
the cells incubated for 2 and 24 h with free DOX (Fig. 6a–h) and CCMs-
DOX (Fig. 6i–p) solution. As shown in Fig. 6b and j, comparing to the
free DOX that a weaker DOX fluorescence was observed in the HeLa
cells after 2 h incubation with CCMs-DOX. It was indicated that only
small amount of released DOX from CCMs-DOX entered cells via en-
docytosis, which could be explained by the slow release kinetics of
CCMs-DOX and the quicker diffuse rate of the free DOX. When HeLa
cells cultured with CCMs-DOX for 24 h, the gradually bright red fluor-
escence was observed within the cell nuclei in Fig. 6n indicating that
more CCMs-DOX entered cells via endocytosis and more DOX was re-
leased. Based on the experimental results it was suggested that it took
period of time for CCMs-DOX to be endocytosed in to cell and release
incorporated drugs into the cells comparing to the quicker diffuse of
free DOX. On the anther hand, the in vitro cell assays showed that
CCMs-DOX release DOX at a slow rate for the first 2 h, that is, the
reasonable stability of disulfide bonds would allow sufficient time for
CCMs-DOX to reach the tumor site and be internalized by tumor cells,
without premature release of DOX. After 24 h, the release rate of DOX
increases, suggesting that drug release would be greatly accelerated
after CCMs-DOX are endocytosed by cancer cells where a lower pH and
a high concentration of GSH environment in endosomes or lysosomes
enhances the cleavage of hydrazone bonds and disulfide bonds. In ad-
dition, for in vivo medical applications the nanoparticles should have a
desirable range of hydrodynamic size from 10 nm (avoiding elimination
by kidneys) to 200 nm (minimizing liver and spleen uptake) and keep
their stability in the presence of salts with high concentrations to pre-
vent phagocytosis. Herein, the hydrodynamic sizes of CCMs-DOX are
about 160 nm and this kind of nanoparticle shows excellent stability
and minor aggregation in culture media (Fig. 3c), which can be at-
tributed to the presence of PNIPPAM units in the nanoparticle surface
coating, providing excellent steric stabilization. In brief, the CLSM ex-
periment confirmed the successfully triggered release of DOX from the
CCMs-DOX and its efficient drug delivery to the cell nuclei, demon-
strating the expected results.
Fig. 7. Cell survival assay of cells. (a) Cytotoxicity of blank CCMs to HUVEC
cells following 24 h incubation. (b) HeLa cells cultured with free DOX, blank
CCMs, and CCMs-DOX for 24 h. The concentration of blank CCMs was shown on
the top x-axis.
The in vitro cell assays were also carried out for evaluation of the
cytotoxic activity for free DOX, blank CCMs, and CCMs-DOX within
HUVEC and HeLa cells. As shown in Fig. 7a, it should be noted that the
blank CCMs showed almost non-toxicity against HUVEC cells up to
sample concentration of 80 μg/mL following 24 h incubation. In Fig. 7b,
free DOX and CCMs-DOX effectively reduced viability of HeLa cells. It
was shown that the cell viability was dose-dependent, and CCMs-DOX
had a lower cytotoxicity to HeLa cells comparing with that of the free
DOX. The amount of DOX from CCMs required to achieve IC50 for HeLa
cells was 49.8 μg/mL at 24 h after incubation and was higher than that
of free DOX (19.6 μg/mL), which was likely due to the quicker diffuse
rate of the free DOX through cell membrane and slow release kinetics of
CCMs-DOX. However, the CCMs-DOX exhibited significant cancer cell
killing effect that 52.6% of the cells remained viable at a DOX dose of
capacity was tested. As shown in Fig. 5, the release behavior of the free
DOX in PBS (pH 7.4) and the incorporated DOX from CCMs-DOX was
monitored under different conditions as shown in the experimental
section. In contrast with the burst release of free DOX in the first 6 h,
the sustained drug release from CCMs-DOX was observed. For CCMs-
DOX at 37 °C, it was indicated that the DOX cumulative release was less
than 7.7% at pH 7.4 for 24 h, indicating that the micelles were re-
markably stable with minimum premature drug release under physio-
logical conditions. On the other hand, the release rate of DOX was much
faster and had a higher cumulative release in stimuli conditions. In the
presence of 10 mM GSH, the cumulative release of DOX increased to
4
5 μg/mL, while 22.1% viability of the cells remained for free DOX at
58% at pH 7.4, while reached 83.1% at pH 5.0 after 24 h. When the
the same concentration.
temperature was set to 42 °C, the cumulative release of CCMs-DOX
reached a maximum of 85.3% at pH 5.0 in the presence of 10 mM GSH
after 24 h. It could be explained by that the collapse of PNIPPAM units
and formation of defects in the micellar shell helped DOX to diffuse
from the inner micellar core to the outer medium, so the cumulative
drug release became higher above the LCST of the CCMs at 42 °C.
Overall, the release behavior of the CCMs-DOX suggested that the as
prepared polymeric micelles with multi-responsibility could be used as
a promising drug delivery candidate for cancer therapy and potentially
facilitated in the tumor microenvironment.
4
. Conclusions
In summary, we demonstrated a simple method to prepare a multi-
stimuli responsive micelle, CCMs-DOX, for triggered anticancer drug
delivery in tumor microenvironment. The CCMs-DOX exhibited a
minimal drug release in physiological environment without stimuli
comparing with an effective drug release in response to the simulated
tumor microenvironment. The cellular uptake and cytotoxicity of the
micelles demonstrated that the CCMs showed excellent biocompat-
ibility, and the CCMs-DOX could be internalized by HeLa cells and
380

J. Qu et al.
Colloids and Surfaces B: Biointerfaces 170 (2018) 373–381
demonstrated killing capacity toward cancer cells. Based on the above
discussions, we are convinced that this as prepared multi-functional
micelle with excellent stability, good biocompatibility, and multi-sti-
muli responsibility holds great promise for constructing a safe and ef-
fective triggered drug release system in cancer therapy.
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Acknowledgements
This work was financially supported by Sichuan Science and
Technology Program (18YYJC0265), and the Research Project of
Sichuan Provincial Department of Education (16ZA0284), and the
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