Chitosan-based nanocarriers with ph and light dual response for anticancer drug delivery

Article abstract of DOI:10.1021/bm400451v

Currently, the major challenge for cancer treatment is to develop new types of smart nanocarriers that can efficiently retain the encapsulated drug during blood circulation and quickly release the drug in tumor cells under stimulation. In this study, the

Full text of DOI:10.1021/bm400451v

Article
pubs.acs.org/Biomac
Chitosan-Based Nanocarriers with pH and Light Dual Response for
Anticancer Drug Delivery
Lili Meng,† Wei Huang,*,† Dali Wang,† Xiaohua Huang,‡ Xinyuan Zhu,† and Deyue Yan*,†
†School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University,
800 Dongchuan Road, Shanghai 200240, PR China
‡Key Laboratory of New Processing Technology for Nonferrous Metal & Materials Ministry of Education and School of Material
Science and Engineering, Guilin University of Technology, Guilin 541004, PR China
S
* Supporting Information
ABSTRACT: Currently, the major challenge for cancer
treatment is to develop new types of smart nanocarriers that
can efficiently retain the encapsulated drug during blood
circulation and quickly release the drug in tumor cells under
stimulation. In this study, the dual pH-/light-responsive cross-
linked polymeric micelles (CPM) were successfully prepared
by the self-assembly of amphiphilic glycol chitosan−o-
nitrobenzyl succinate conjugates (GC-NBSCs) and then
cross-linking with glutaraldehyde (GA), which was synthesized
by grafting hydrophobic light-sensitive o-nitrobenzyl succinate
(NBS) onto the main chain of hydrophilic glycol chitosan
(GC). The GC-NBSC CPMs exhibited good biocompatibility
according to the MTT assay against NIH/3T3 cells. The cell
viability was maintained higher than 75% after 24 h incubation with CPMs even at a high concentration of 1.0 mg mL−1. The
hydrophobic anticancer drug camptothecin (CPT) was selected as a model drug and loaded into GC-NBSC CPMs. The results
of in vitro evaluation showed that the encapsulated CPT could be quickly released at low pH with the light irradiation.
Meanwhile, the CPT-loaded CPMs displayed a better cytotoxicity against MCF-7 cancer cells under UV irradiation, and IC50 of
the loaded CPT was as low as 2.3 μg mL−1, which was close to that of the free CPT (1.5 μg mL−1). Furthermore, the flow
cytometry and confocal laser scanning microscopy (CLSM) measurements confirmed that the CPT-loaded CPMs could be
internalized by MCF-7 cells efficiently and release CPT inside the tumor cells to enhance the inhibition of cell proliferation.
Thereby, such excellent GC-NBSC CPMs provide a favorable platform to construct smart drug delivery systems (DDS) for
cancer therapy.
glycol)−poly(L-aspartic acid)−poly(L-phenylalanine) which
showed a rapid Dox release triggered by endosomal pH.19
Wang and co-workers displayed another kind of boronate
cross-linked micelles made from telodendrimer pair (PEG5K-
(nitroboronic acid/catechol)4-CA8), and the stability of such
CPMs was strongly interrupted by lowering the pH value from
7.4 to 5.0, leading to release of the encapsulated paclitaxel.22
Besides these single pH-responsive CPMs, more and more
CPMs with dual stimuli-responsive properties have been
fabricated recently to meet the conflicting requirements, i.e.,
the extracellular stability and intracellular release of drug.23−27
McCormick et al. manufactured imine shell cross-linked
micelles starting from a temperature-responsive triblock
copolymer for the first time and investigated the pH-triggered
release of loaded hydrophobic drug.25 Liu and co-workers also
developed novel thiol and pH dual-responsive CPMs based on
INTRODUCTION
■
In the past few decades, nanosized polymeric micelles have
emerged as promising carriers for anticancer drug delivery due
to their outstanding characteristics, such as high drug loading
capacity, long circulation in the bloodstream, and passive
targeting capability based on the enhanced permeability and
retention (EPR) effect.1−4 However, polymeric micelles formed
by supramolecular self-assembly of amphiphilic copolymers are
always too fragile to protect the encapsulated drugs while
circulating in blood due to the dynamic nature and also
inefficient in achieving controlled release.5 Therefore, there is
growing interest in designing intelligent micelles that are stable
under physiological conditions but can fall apart in a controlled
fashion upon stimulation to release the payloads. Up to now,
various stimuli-responsive CPMs have been developed for this
purpose.6−17 Among them, the CPMs with acid-cleavable cross-
linkers have been intensively investigated owing to the low pH
(about 5.0−6.5) in tumor tissues.18−22 For example, Jeong et al.
prepared a kind of shell cross-linked micelles with a pH-labile
ketal cross-linker based on the self-assembly of poly(ethylene
Received: March 30, 2013
Revised: July 1, 2013
© XXXX American Chemical Society
A
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and deuterated trifluoroacetic acid (CF3COOD) as solvents. Fourier-
transform infrared (FT-IR) spectra were recorded on a PerkinElmer
Paragon 1000 spectrometer by the KBr sample holder method. Spectra
were obtained by collecting and averaging 16 scans at frequencies
ranging from 500 to 4000 cm−1 at room temperature. The
thermogravimetric analysis (TGA) was measured on a PE TGA-7
thermogravimetric analyzer from 100 to 800 °C at a rate of 20 °C/min
under a nitrogen atmosphere. Dynamic light scattering (DLS)
measurements were performed with a Malvern Zetasizer Nano S
apparatus equipped with a 4.0 mW laser operating at wavelength of
633 nm. All samples were measured at a scattering angle of 173°. Every
data point was detected in triplicate and averaged. Transmission
electron microscopy (TEM) measurements were carried out on a
JEOL TEM-100CX-II instrument operating at a voltage of 200 kV.
Samples were prepared by dropping the aqueous solutions of micelles
or CPMs onto carbon-coated copper grids and then air-drying at room
temperature before measurements. Fluorescent spectra were recorded
using a PerkinElmer LS 50B luminescence spectrometer at room
temperature. The fluorescence measurements were taken at an
excitation wavelength of 550 nm, and the emission was monitored
from 570 to 750 nm. For the UV light irradiation, the CPMs solution
was placed vertically under a high-pressure mercury lamp (365 nm,
150 W) at a distance of 10 cm. In the cell experiments, an ultraviolet
lamp (365 nm, 25 W) was used.
an amphiphilic diblock copolymer (PCL-b-P(OEGMA-co-
MAEBA)), which could largely minimize the premature leakage
of drug under physiological conditions and exhibit accelerated
drug release under mildly acidic pH and/or reductive
microenvironment.26
Despite their potential for drug delivery, smart CPMs still
face some inevitable problems, such as achieving time and site-
controlled drug delivery only by virtue of the internal biological
stimuli related to the targeted diseased sites. Nowadays,
multiple external stimuli, such as light,28−31 heat,32,33 electric/
magnetic fields,34,35 and ultrasound,36,37 are being explored to
fabricate DDS which may show unprecedented control over
drug delivery.38,39 Light as a trigger for drug release has drawn a
great deal of attention since it provides a possibility for realizing
remote and spatiotemporal drug release by tuning the
wavelength, energy, and site of irradiation.31,40 Among the
numerous chromophores applied in light-responsive materials,
o-nitrobenzyl is of particular importance since it can undergo a
photolysis reaction under either UV light or near-infrared light,
disrupting the hydrophilic−hydrophobic balance of the o-
nitrobenzyl-containing polymer and thus the integrity of
assemblies.31,41 Until now, only a few CPMs are reported
with both internal and external stimuli response.42 On the other
hand, as an important hydrophilic derivative of chitosan, GC
has been widely used in biomedical fields due to its favorable
advantages, including abundant reactive groups for facile
modification, good biocompatibility and biodegradability, and
enhanced cell permeability with less toxicity.43−45
In this work, we attempted to synthesize amphiphilic GC-
NBSCs by covalently conjugating hydrophobic light-sensitive
NBS onto the main chains of GC. Then, the nanosized micelles
were prepared by the self-assembly of GC-NBSCs in water and
further stabilized by cross-linking their GC shells with GA to
form GC-NBSC CPMs. Finally, the hydrophobic anticancer
drug CPT was encapsulated into the CPMs, and their
performance as smart DDS was also investigated in terms of
the stabilization under physiological conditions, in vitro drug
release under pH-/light stimuli, anticancer activity in vitro, and
cellular internalization behavior.
Synthesis of o-Nitrobenzyl Succinate. o-Nitrobenzyl succinate
(NBS) was synthesized according to the previous report.46 Typically,
o-nitrobenzyl alcohol (4.0 g, 26.12 mmol), succinic anhydride (5.23 g,
52.24 mmol), and DMAP (1.60 g, 13.06 mmol) were dissolved
completely in dried chloroform (86 mL) and refluxed under a nitrogen
atmosphere for 24 h. After removing partial chloroform under reduced
pressure, the mixture was washed three times with 10% HCl and then
extracted with saturated NaHCO3 solution. The basic aqueous phase
was washed with ether and acidified to pH 5.0 with 10% HCl. The
white solid precipitate was collected and dried in vacuo at 40 °C
1
overnight to give NBS (6.08 g, 92%). H NMR (CDCl3): δ (ppm)
8.10−8.12 (d, 1H, ArH), 7.47−7.67 (m, 3H, ArH), 5.56 (s, 2H,
ArCH2), 2.75 (s, 4H, CH2CH2).
Synthesis of Glycol Chitosan−o-Nitrobenzyl Succinate
Conjugates. GC-NBSCs with different degree of substitution (DS)
were synthesized by grafting different amount of NBS onto the main
chain of GC according to the method in previous literature.42
Typically, GC (250.0 mg, 0.98 mmol glucosamine residues) was
dissolved completely in the mixed solvent of methanol and deionized
water (1:1 v/v, 60 mL). After that, NBS (56.7 mg, 0.22 mmol), EDC
(63.3 mg, 0.33 mmol), and NHS (38.0 mg, 0.33 mmol) were added
into the above mixture in sequence, and the reaction mixture was
stirred at ambient temperature for 24 h. The resulting mixture was
dialyzed in the mixed solvent of methanol/deionized water (4:1 v/v)
for 2 days and deionized water for another 2 days (MWCO 14 kDa)
and followed by lyophilization to produce pure GC-NBSC with the DS
of 19.3 (GC-NBSC19.3). Other samples with the DS of 10.0 and 4.8
were synthesized by the same procedure. The DS was determined by
measuring the concentration of unreacted NBS in the reaction mixture
through UV absorbance at 260 nm and calculated according to the
equation
EXPERIMENTAL SECTION
■
Materials. Glycol chitosan (degree of polymerization = 2800,
degree of deacetylation = 82.7%) was purchased from Sigma-Aldrich
(St. Louis, MO), which was purified by filtration and dialysis in
deionized water before use. Succinic anhydride, o-nitrobenzyl alcohol,
4-(dimethylamino)pyridine (DMAP), N-hydroxysuccinimide (NHS),
glutaraldehyde (GA) (25% in water), and Nile red were purchased
from Acros Organics and used without further purification. 1-Ethyl-3-
(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) was
obtained from Titan Chemical Corp. Propidium iodide (PI) and 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenytetrazolium bromide (MTT)
were from Sigma-Aldrich. Camptothecin was purchased from Shaanxi
Sciphar. Biotechnology Co. Chloroform, methanol, and dimethyl
sulfoxide (DMSO) were supplied by Sinopharm Chemical Reagent
Co. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine
serum (FBS), and phosphate buffered solution (PBS) were purchased
from PAA Laboratories GmbH. Chloroform was dried over calcium
hydride (CaH2) and distilled just before use. Dialysis tube (MWCO,
14 and 3.5 kDa) was obtained from Shanghai Lvniao Technology
Corp. All the other chemicals were purchased from domestic suppliers
and used as received. Clear polystyrene tissue culture treated 6-well
and 96-well plates were obtained from Corning Costar.
Mtotal − Munreacted
DS =
× 100
Mglucosamine
(1)
where Mtotal, Munreacted, and Mglucosamine are the amount (in moles) of
NBS in feed, NBS unreacted in reaction mixture, and glucosamine unit
in GC.
Critical Micelle Concentration (CMC) Measurement. The
CMC value of GC-NBSCs was determined by the fluorescence probe
technique. Here, Nile red was used as the fluorescence probe.
Typically, the solid GC-NBSC19.3 was dissolved in deionized water to
prepare the aqueous solutions with various concentration of GC-
NBSC19.3 (from 0.023 to 0.248 mg mL−1). Then, 25 μL of Nile red
acetone solution (1.6 × 10−4 M) was added into 4.0 mL of GC-
NBSC19.3 micelles solution and evaporated overnight to remove the
Methods. 1H NMR spectroscopy was performed on a Varian
Mercury-400 spectrometer with deuterated chloroform (CDCl3),
deuterated oxide (D2O), deuterated dimethyl sulfoxide (DMSO-d6),
B
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Scheme 1. Synthesis of NBS (A) and GC-NBSC (B)
acetone. The final concentration of Nile red in the solution was 1.0 ×
10−6 M. Finally, the fluorescence emission spectra of the solutions
were recorded in the range from 570 to 750 nm on a fluorescence
spectrometer with the exciting wavelength of 550 nm.
immersed in 50 mL of PBS solution (pH 7.4) or acetate buffer
solution (pH 5.0) in a shaking water bath at 37 °C to acquire sink
conditions. Meanwhile, the samples without UV light irradiation were
also prepared as a control. At the predetermined time interval, the
external buffer solution (3 mL) was taken out and replenished with an
equal volume of fresh media. The amount of released CPT was
determined by fluorescence measurement. All the drug release
experiments were performed in triplicate, and the results were the
average data.
Cell Culture. NIH/3T3 normal cells (a mouse embryonic
fibroblast cell line) and MCF-7 cancer cells (a human breast cancer
cell line) were cultivated in DMEM supplied with 10% FBS (fetal
bovine serum) and antibiotics (50 U mL−1 penicillin and 50 U mL−1
streptomycin) at 37 °C in a humidified atmosphere containing 5%
CO2.
Preparation of GC-NBSC CPMs. GC-NBSC19.3 (50.0 mg) was
dissolved in 20 mL of phosphate buffer saline (PBS, pH 7.4) under
mild stirring until the micelles formed. Then, 1 mL of GA aqueous
solution (1.22 × 10−2 mmol mL−1) was added dropwise into the above
micelle solution over 4 h under vigorous stirring at room temperature.
After that, the resulting mixture was kept stirring for another 4 h and
further purified by dialysis (MWCO 3500 Da) in PBS solution for 24
h, during which the PBS solution was replaced by fresh one every 4 h.
Finally, the mixture was filtrated through 0.45 μm Millipore filter, and
the pure GC-NBSC19.3 CPMs were obtained after lyophilizing the
filtrate with a yield of 80%.
Dual Stimuli Response of GC-NBSC CPMs. Typically, 3 mL of
aqueous solution of GC-NBSC19.3 CPMs purified by dialysis was
incubated in 0.1 M PBS solution (pH 7.4) or acetic buffer solution
(pH 5.0) at 37 °C for 5 h and then irradiated with UV light (365 nm,
150 W) for 10 min. As a control, the samples without UV light
irradiation were also prepared. After that, the size and morphology of
samples were determined by DLS and TEM measurement.
In Vitro Cytotoxicity Measurements. The relative cytotoxicity
of GC-NBSC19.3 CPMs was estimated by a MTT viability assay against
NIH/3T3 normal cells. In the MTT assay, NIH/3T3 cells were seeded
into 96-well plates with a density of 8 × 103 cell per well in 200 μL of
medium. After 24 h incubation, the culture medium was carefully
removed and replaced with 200 μL of fresh medium containing serial
dilutions of GC-NBSC19.3 CPMs. The cells were grown for another 24
h. Then, 25 μL of MTT assay stock solution (5 mg mL−1) in PBS was
added into each well. After incubation for an additional 4 h, the
medium containing unreacted dye was removed carefully. The
obtained blue formazan crystals were dissolved in 200 μL of DMSO
per well, and the absorbance were measured in a BioTek SynergyH4 at
a wavelength of 490 nm.
Activity Analysis. The cytotoxicity of CPT-loaded CPMs of GC-
NBSC19.3 against MCF-7 cancer cells was examined in vitro by the
MTT assay. MCF-7 cells were seeded into 96-well plates at an initial
density of 8 × 103 cells per well in 200 μL of medium. After incubation
for 24 h, the culture medium was replaced with fresh one, and 50 μL of
PBS solution containing serial dilutions of CPT-loaded CPMs was
added into each well. The cells were irradiated under UV light at 365
nm (25 W) for 30 min after incubation with CPT-loaded CPMs for 3
h and cultured for another 24 h. Meanwhile, cells without UV light
irradiation were also set as a control. The cytotoxicity of blank GC-
GC-NBSC19.3 CPMs with or without UV light irradiation was also
investigated as controls. Then, 25 μL of 5 mg mL−1 MTT assays stock
solution in PBS was added into each well, and the cells were further
grown for 4 h, followed by removing the medium carefully. The
obtained blue formazan crystals were dissolved in 200 μL of DMSO
per well, and the absorbance was measured in a BioTek SynergyH4 at
a wavelength of 490 nm.
Preparation of CPT-Loaded GC-NBSC CPMs. In brief, GC-
NBSC19.3 (50.0 mg) and CPT (3.0 mg) were simultaneously added
into DMSO (20 mL) under the vigorous stirring until both of them
dissolved completely. Then, the mixture was transferred into a dialysis
bag (MWCO 3500 Da) and dialyzed in PBS solution for 2 days,
during which the PBS solution was renewed every 4 h. During this
process, CPT-loaded micelles were formed. After that, 1 mL of GA
aqueous solution (1.22 × 10−2 mmol mL−1) was added dropwise into
the above micelle solution over 4 h under vigorous stirring at room
temperature. The resulting mixture was kept stirring for another 4 h
and further purified by dialysis (MWCO 3500 Da) in PBS solution for
24 h, during which the PBS solution was replaced by fresh one every 4
h. Finally, the mixture was filtrated through 0.45 μm Millipore filter to
remove the free CPT, and the CPT-loaded CPMs were obtained by
lyophilizing the filtrate. To determine the drug loading content (DLC)
and drug loading efficacy (DLE), the CPT-loaded CPMs were
incubated in PBS solution (pH = 5.0) for 24 h, then lyophilized, and
dissolved in DMSO again. The drug concentration was determined by
measuring the fluorescence intensity of CPT (excitated at 340 nm).
DLC and DLE were calculated according to the following equations:
DLE (%) = Wloaded/W × 100%
(2)
(3)
total
DLC (%) = Wloaded/(W
+ Wloaded) × 100%
polymer
Cellular Internalization of CPT-Loaded GC-NBSC CPMs. The
cellular internalization experiments of CPT-loaded CPMs of GC-
NBSC19.3 were performed by flow cytometry and CLSM measure-
ments.
Flow Cytometry. MCF-7 cells were seeded into six-well plates at 5
× 105 cells per well in 1 mL complete DMEM and cultured for 24 h.
Then, the medium containing CPT-loaded CPMs at a final CPT
concentration of 3.3 μg/mL was added into different wells, and the
where Wtotal, Wloaded, and Wpolymer are the weight of total CPT used, the
loaded CPT, and GC-NBSC19.3 CPMs.
In Vitro Drug Release. For in vitro drug release study, the
lyophilized CPT-loaded CPMs of GC-NBSC19.3 (10 mg) were
dispersed in 4 mL of PBS solution (pH 7.4) and then transferred
into a dialysis bag (MWCO 3500 Da). After being irradiated under UV
light at 365 nm (150 W) for 10 min, the mixture in dialysis bag was
C
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cells were incubated at 37 °C for 5, 60, and 180 min. Thereafter, the
samples were prepared for flow cytometry analysis by removing the
cell culture medium, rinsing with cold PBS, and treating with trypsin.
Data for 1.0 × 104 gated events were collected and analyzed by means
of a BDFACS Calibur flow cytometer and CELL Quest software.
CLSM Study. For the CLSM study, MCF-7 cells were seeded into
six-well plates at 2 × 105 cell per well in 1 mL of complete DMEM and
cultured for 24 h, followed by removing the culture medium and
adding 1 mL of medium containing CPT-loaded CPMs at a final CPT
concentration of 3.3 μg/mL into different wells. The cells were
incubated at 37 °C for predetermined intervals. Then, the culture
medium was removed, and cells were washed with PBS three times.
Subsequently, the cells were fixed with 4% paraformaldehyde for 30
min at room temperature, and the slides were rinsed with cold PBS
three times. Finally, the slides were mounted and observed with a
LSM510 META.
1
Figure 2. H NMR spectra of GC in D2O (a), GC-NBSC19.3 in D2O
(b), and GC-NBSC19.3 in the mixed solvent of DMSO-d6/CF3COOD
(1:1, v/v) (c).
RESULTS AND DISCUSSION
■
Synthesis and Characterization of GC-NBSC. Scheme 1
gives the synthesis route of GC-NBSC. First, NBS was
process of coupling reaction. The carboxyl acid group of NBS
was first activated by NHS and then coupled with the primary
amino groups in GC to form an amide linkage. The DS is a key
parameter to determine the hydrophobic/hydrophilic property
of GC-NBSC and finally affects the size of self-assembled
aggregates.43 Here the DS is defined as the number of NBS
groups per 100 glucosamine units of GC and can be easily
controlled by adjusting the feed ratio of NBS to GC. Therefore,
three samples of GC-NBSC with different DS were prepared at
various feed molar ratio of NBS to glucosamine units of GC
(0.230, 0.115, and 0.058) and fully characterized by FTIR and
TGA measurements. The FTIR spectra of GC-NBSC and GC
are shown in Figure 1. Compared with GC (a), a new shoulder
peak assigned to the carbonyl of ester group is observed at 1726
cm−1 in the spectra of GC-NBSC (b−d) and is gradually
intensified while increasing the feed ratio. Besides, the
characteristic peaks at 1654 and 1564 cm−1 observably increase
after conjugation, which are attributed to the carbonyl of amide
I band and N −H bending of amide II band, respectively. All
the above results confirm the formation of amide linkage
between the carboxyl group of NBS and the amino groups of
GC.
Figure 1. FTIR spectra of GC (a), GC-NBSC4.8 (b), GC-NBSC10.0
(c), and GC-NBSC19.3 (d).
Table 1. General Properties of GC-NBSC with Different DS
a
b
feed ratio
DS
CMC (mg mL−1
)
size (nm)
The DS value of GC-NBSC was determined by the UV
absorbance-based quantification according to eq 1, and the
results are shown in Table 1.44 Apparently, it increases with
increasing the feed ratio of NBS and GC. This result is also
confirmed by the TGA measurement. The TGA curves (Figure
S2 ) show an increased weight loss percentage of GC-NBSC in
the first stage with the increase of feed ratio.
GC-NBSC4.8
GC-NBSC10.0
GC-NBSC19.3
0.06
0.12
0.23
4.8
10.0
19.3
0.29
0.18
513.0
378.3
58.8
0.08
a
b
Molar ratio of NBS to glucosamine units of GC. DS measured by
the UV absorbance-based quantification.
Self-Assembly Behavior of GC-NBSC. Similar to other
polymeric amphiphiles, GC-NBSC might also be able to self-
assemble into nanosized micelles because of its amphiphilic
structure comprised of hydrophobic NBS groups and hydro-
philic GC backbone. The self-assembly behavior of GC-NBSC
was well investigated by means of 1H NMR, fluorescence
spectroscopy, dynamic light scattering (DLS), and transmission
electron microscopy (TEM).
synthesized from o-nitrobenzyl alcohol by the carboxylation
of succinic anhydride under the catalysis of DMAP in CHCl3 at
60 °C (Scheme 1A). The results show that the hydroxyl groups
of o-nitrobenzyl alcohol are able to react with succinic
anhydrides quantitatively to produce NBS with a high yield
1
of 92%. The chemical structure of NBS is confirmed by H
NMR measurement (Supporting Information Figure S1),
which clearly shows the signals of aromatic protons at 7.47−
8.12 ppm (4H, ArH), the benzyl methylene protons at 5.56
ppm (2H, CH2), and the succinic methylene protons at 2.75
ppm (4H, CH2CH2). Then, NBS was further coupled with the
primary amine groups in GC to form GC-NBSC in the
presence of EDC and NHS (Scheme 1B). Because of the
dramatically different water solubility of NBS and GC, a mixed
solvent of CH3OH/deionized water (1:1, v/v) was used in the
First, taking GC-NBSC19.3 as an example, 1H NMR was used
to study the self-assembly behavior, and the results are shown
in Figure 2. Compared with the spectrum of GC in D2O (a),
only some weak signals ascribed to the protons of appended
NBS groups are observed at 2.46, 5.4, and 7.4−8.0 ppm in the
spectrum of GC-NBSC19.3 in D2O (b). Especially those signals
at 7.4−8.0 ppm assignable to the aromatic protons are barely
visible, indicating that GC-NBSC19.3 are self-assembled into
D
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Figure 3. (A) Emission spectra of Nile red (λex = 550 nm) at various concentration of GC-NBSC19.3. (B) Emission intensity of Nile red versus the
concentration of GC-NBSC19.3
.
Scheme 2. Preparation of CPT-Loaded GC-NBSC CPMs and Intracellular Drug Release Triggered by pH and UV Light
Furthermore, the micellar self-assembly of GC-NBSC was
monitored by the fluorescence spectroscopy using Nile red as a
probe. As shown in Figure 3A, the fluorescence emission
intensity of Nile red increases slowly as the concentration of
GC-NBSC19.3 is low. However, it increases dramatically once
the concentration exceeds a certain value, implying the
formation of micelles and encapsulation of Nile red into the
hydrophobic cores. This concentration can be defined as the
critical micelle concentration (CMC). It is suggested in Figure
3B that the CMC of GC-NBSC19.3 in water is about 0.08 mg
mL−1. The CMC of other two conjugates with different DS
were also determined by the same method and listed in Table
1. Clearly, it decreases from 0.29 to 0.08 mg mL−1 with
increasing the DS, due to the enhanced hydrophobicity of the
conjugates. After that, the size of GC-NBSC micelles was
evaluated by DLS. Not surprisingly, it exhibits the same change
1
Figure 4. H NMR spectra of the cross-linking reaction between GA
with the DS, since the enhanced hydrophobicity induces the
and GC-NBSC19.3 micelles in D2O at pH 7.4.
formation of more dense hydrophobic cores.43 It is well-known
that the CMC of amphiphilic polymers and the size of their
self-assemblies are two important parameters for DDS. Low
CMC and small size makes micelles very stable in vivo and
core−shell structures in aqueous environment, and the
circulate longer in the blood. Therefore, GC-NBSC19.3 was
selected as a drug carrier and used to prepare the cross-linked
micelles as a potential DDS.
Preparation of Cross-Linked Micelles and Their Dual
Stimuli-Responsive Property. Generally, upon intravenous
injection, micelles can quickly be diluted to a concentration
hydrophobic NBS groups are confined within the core domain
shielded by hydrophilic GC corona, thus leading to weak
signals of NBS protons. However, when GC-NBSC19.3 is
dissolved in the mixed solvent of DMSO-d6/CF3COOD (1:1,
v/v) (c), the self-assemblies are disrupted, causing the increased
signals for NBS at 2.48, 5.48, and 7.35−7.90 ppm.
E
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Figure 5. TEM photographs (A) and DLS results (B) of GC-NBSC19.3 micelles (a), GC-NBSC19.3 CPMs at pH 7.4 (b), and GC-NBSC19.3 at pH 5.0
after UV irradiation (c).
shell of GC-NBSC19.3 micelles in PBS solution (pH 7.4)
because GA can easily react with amino groups of GC-NBSC19.3
to form acid-labile imine bonds which induces the formation of
Table 2. Dual Stimuli-Responsive Properties of GC-
NBSC19.3 CPMs
condition
size (nm)
pH 7.4
pH 5.0
pH 7.4 with
UV
pH 5.0 with
UV
1
dual stimuli-responsive GC-NBSC19.3 CPMs (Scheme 2). H
NMR spectroscopy was adopted to track the cross-linking
reaction, and the results are shown in Figure 4. The signal of
aldehyde protons in GA at 9.60 ppm weakens gradually with
increasing the reaction time and almost disappears after 4 h.
Unfortunately, the signal ascribed to the protons of imine
bonds is not found clearly because it is overlapped with the
signals assigned to the aromatic protons of NBS at 7.54−8.10
ppm. On the other hand, the signals of GC backbones (3.64
ppm) and methylene protons from GA (1.22−1.68 ppm) also
decrease with the reaction time, due to the increased viscosity
of the reaction mixture induced by the interparticle cross-
linking which is difficult to avoid when the reaction was
conducted in a magnetic tube without stirring. Besides, the
ATR-FTIR spectra of GC-NBSC19.3 and its cross-linked
product was measured and shown in Figure S3. The
characteristic peak of imine bonds cannot be observed due to
overlapping with the absorbance of amide I band. However, the
peak of N−H bending at 1525 cm−1 becomes very weak in the
spectrum of GC-NBSC19.3 CPMs, which indicates the
successful cross-linking reaction. TEM and DLS measurements
were also taken to evaluate the size and morphology change of
micelles after cross-linking. As shown in Figure 5A-b, the
morphology of CPMs is similar to that of non-cross-linked
micelles, while the diameter decreases to 30.0 nm after cross-
linking, indicating the formation of more compact structures.
The DLS result reveals a decreased average diameter of 37.8
nm for CPMs (Figure 5B-b), which is basically consistent with
that determined by TEM.
37.8 3.0
71.2 9.9 52.4 11.5
14.6 3.0
Figure 6. In vitro drug release behavior of CPT-loaded GC-NBSC19.3
CPMs under different stimulus conditions.
Thereafter, dual pH-/light-responsive properties of GC-
NBSC19.3 CPMs were studied through DLS and TEM. As
mentioned above, the average diameter of CPMs is about 37.8
nm by DLS at pH 7.4 (Table 2). When the pH was adjusted to
5.0 and kept for 5 h, the size of CPMs increases to 71.2 nm,
attributing to the cleavage of cross-linked imine bonds in the
shell. Besides, amino groups in GC are protonated at such a low
pH which greatly enhances the hydrophilicity of CPMs, leading
to the expanded volume. When this acid CPMs solution is
further irradiated under UV light for 10 min, the size measured
Figure 7. In vitro cytotoxicity of GC-NBSC19.3 CPMs against NIH/
3T3 cells after 24 h incubation.
below CMC and disaggregate, resulting to the drug leakage
before reaching the diseased site. Therefore, cross-linking of the
core or shell of micelles is a feasible and effective method to
improve the stability. Here we selected GA to cross-link the
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Figure 8. (A) Cytotoxicity of free CPT and CPT-loaded GC-NBSC19.3 CPMs with or without UV irradiation against MCF-7 cells after 24 h
incubation. (B) Cytotoxicity of blank GC-NBSC19.3 CPMs with or without UV irradiation against MCF-7 cells after 24 h incubation.
irradiation time increasing and levels off after 10 min, indicating
that the photocleavable reaction proceed completely in a short
time.
In Vitro Drug Release Study. In order to evaluate the
potential of this dual pH-/light-responsive GC-NBSC19.3 CPMs
as a smart DDS, the anticancer drug CPT was selected and
capsulated into the CPMs. We first evaluated the photostability
of CPT by using the fluorescence spectroscopy. The
fluorescence excitation and emission spectra of CPT in Figure
S8 are nearly the same before and after the UV irradiation,
confirming the high photostability of CPT for UV light
irradiation. Then the dual-triggered CPT release behavior was
investigated in vitro by the dialysis method. The DLE and DLC
Figure 9. Cellular uptake of CPT-loaded GC-NBSC19.3 CPMs by
MCF-7 cells versus the incubation time by flow cytometry analysis.
Inset is the representative flow cytometry histogram profiles of MCF-7
cells incubated with CPT-loaded CPMs for 3 h.
of the CPT-loaded CPMs are found to be 5.0% and 5.93%,
respectively, on the basis of the standard curve of CPT.
Different pH- and UV light-triggered drug release curves of
CPT-loaded CPMs are presented in Figure 6. At pH 7.4
without UV irradiation, only 18.35% of the loaded CPT is
released from CPMs within 12 h. However, at pH 7.4 with UV
irradiation, approximately 31.96% of the loaded CPT is released
within the same period. It indicates that the photolysis of NBS
side groups occurs in the core of CPMs, but the core−shell
structure of CPMs is still preserved due to the cross-linking
effect. Similarly, at pH 5.0 without UV irradiation, the cross-
linked imine bonds in the CPMs shell are destroyed, but the
core−shell structure is still integrated because of the
amphiphilic nature of GC-NBSC19.3. Thus, only about 49.08%
of the loaded CPT is released out within 12 h under this
condition. However, when the low pH and UV irradiation are
simultaneously exerted on the CPMs, a burst release of CPT
(45.30%) is observed during the initial first hour because of the
quick disaggregation of CPMs, and the cumulative amount of
released drug is up to 81.68% after 12 h. This is consistent with
the results of acid-stimuli hydrolysis of imine linkage and
photolysis of NBS. Overall, these results show that this smart
GC-NBSC19.3 CPMs are able to realize a quick release of the
encapsulated drugs only when it is synchronously triggered by
pH and UV light. On the contrary, the encapsulated drug
release would be dramatically suppressed with a sole
stimulation.
by DLS sharply decreases to about 14.6 nm (Figure 5B-c). At
the same time, no regular spherical micelles are observed except
some irregular tiny fragments in the TEM photograph (Figure
5A-c). This implies that light-sensitive side NBS groups are
cleaved from GC chains under UV irradiation, causing the
complete disaggregation of CPMs into the analogue of glycol
chitosan (Scheme S1). In order to confirm the above
speculation, the size and morphology of GC were also
investigated by DLS and TEM. As shown in Figure S4, some
similar small irregular particles with a diameter of less than 20
nm are found and verify the complete disaggregation of CPMs
into the analogue of glycol chitosan. On the other hand, if the
CPMs solution at pH 7.4 is irradiated by UV light for 10 min,
only small size change of CPMs is observed, increasing from
37.8 to 52.4 nm, suggesting that the core−shell structures are
still maintained even if the photolysis of NBS core occurs. The
acid-cleavable process of imine linkage was also investigated by
1H NMR spectroscopy, and the results are shown in Figure S5.
When GC-NBSC19.3 CPMs were incubated in D2O at pH = 5.0
and 37 °C for different times, the signal of aldehyde was
monitored and quantified by comparing with the signal of
CHCl3 as an external standard. Then the hydrolysis ratio of
GC-NBSC19.3 CPMs was calculated and displayed in Figure S6.
Obviously, half of the cross-linkage is broken in about 2 h, and
a complete hydrolysis of the cross-linkage is achieved within 5
h. Besides, the photolysis of NBS in GC-NBSC was also
evaluated by UV−vis spectroscopy by using the aqueous
solution of GC-NBSC19.3 micelles as a model (Figure S7). The
characteristic peak of NBS at 260 nm decreases with the
In Vitro Cytotoxicity of GC-NBSC CPMs and Anti-
cancer Activity of CPT-Loaded CPMs. As drug delivery
system materials, the biocompatibility or cytotoxicity of the
carrier is a key index for its biomedical application. Thus, the
cytotoxicity of GC-NBSC19.3 CPMs in vitro was evaluated by
the MTT method against a mouse embryonic fibroblast NIH/
3T3 cell line. Figure 7 shows the cell viability after 24 h
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Figure 10. CLSM images of MCF-7 cells incubated with the CPT-loaded GC-NBSC19.3 CPMs for 5 min (A), 30 min (B), and 1 h (C). Cell nuclei
were stained with PI.
incubation with CPMs at the concentration varying from 8 to
1000 μg mL−1. Obviously, the cell viability remains above
75.0% even when the concentration of CPMs increases to 1000
μg mL−1. Consequently, GC-NBSC19.3 CPMs have good
biocompatibility and are a suitable material for drug delivery
system.
same dose, probably due to the time-consuming drug release
from the CPMs and thus delayed nuclear uptake.48 Addition-
ally, in order to eliminate the possibility that the increased
anticancer efficacy of CPT-loaded CPMs after light irradiation
comes from the cell death under UV light or the cytotoxicity of
irradiated products, the in vitro cytotoxicity of blank CPMs
against MCF-7 cells with or without light irradiation was also
conducted by the MTT method. It is exhibited in Figure 8B
that when the CPMs concentration is up to 1.0 mg mL−1, the
cell viability without light irradiation still retains about 75%, and
only a slight decrease, 3%, is found after irradiation.
Cell Internalization Studies. It is an important factor for
therapeutic efficacy of anticancer drugs that whether they can
be quickly transported into cancer cells. Therefore, the flow
cytometry analysis was performed to measure the cellular
uptake of CPT-loaded GC-NBSC19.3 CPMs by the MCF-7 cell
line. The CPT-loaded CPMs solution with a CPT concen-
tration of 3.3 μg mL−1 was added into the culture medium, and
the cells were incubated in this culture medium at 37 °C for the
predetermined time intervals. As a control, the MCF-7 cells
were also incubated in the culture medium without the CPT-
loaded CPMs solution. As shown in Figure 9, the fluorescence
intensity of MCF-7 cells is found within 5 min, and its intensity
increases greatly with prolonging the incubation time. After 1 h
incubation, the relative geometrical mean fluorescence intensity
Besides, the anticancer activity of CPT-loaded GC-NBSC19.3
CPMs with or without UV irradiation was also investigated by
MTT assay against MCF-7 cancer cells, and the free CPT was
used as a control. Figure 8A shows that the dose of the
capsulated CPT required for 50% cellular growth inhibition
(IC50) with light irradiation is about 2.3 μg mL−1, while the one
without irradiation is about 5.0 μg mL−1. It is well reported that
tumor extracellular environment is acidic (pH 6.5), and the pH
values in endosome and lysosome are even lower (pH 5.0−
5.5).47 Therefore, after the CPMs are internalized by cancer
cells through an endocytosis process, the imine linkages are
quickly cleaved in the acidic intracellular compartments and the
amino groups of GC are protonated to facilitate the endosome/
lysosome escape. Combining the light stimulation, those CPMs
disassemble completely and release the drug intracellularly to
achieve high anticancer activities. As a comparison, the IC50 of
free CPT is 1.4 μg mL−1, suggesting a slightly better anticancer
effect than loaded CPT. The free drug often shows higher
activity than the loaded drug in polymeric nanoparticles at the
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of the CPT-loaded CPMs pretreated cells is about 1.2-fold of
that of the nonpretreated cells and further increases rapidly to
4-fold in a period of 3 h. The rapid enhancement of
fluorescence intensity indicates that the CPT-loaded CPMs
can be effectively internalized by MCF-7 cells.
AUTHOR INFORMATION
Corresponding Author
*E-mail: hw66@sjtu.edu.cn (W.H.); dyyan@sjtu.edu.cn (D.Y.).
Notes
■
The authors declare no competing financial interest.
Moreover, the cellular uptake behavior was further evaluated
by CLSM. By taking advantage of the red fluorescence from
propidium iodide (PI) and blue fluorescence from CPT, the
intracellular location of drug was studied. After CPT-loaded
GC-NBSC19.3 CPMs with a CPT concentration of 3.3 μg mL−1
were added into the culture medium, MCF-7 cells were
incubated at 37 °C for 5 min, 30 min, and 1 h. Thereafter, the
cell nuclei were stained with propidium iodide (PI), and the
treated samples were observed directly using CLSM. As shown
in Figure 10A, the cells pretreated with CPT-loaded CPMs for
5 min display weak blue fluorescence in cytoplasm. When the
incubation time was extended to 30 min or 1 h, the strong
fluorescence is observed mainly in cytoplasm while a small
amount in nuclei due to a small drug release from the CPT-
loaded CPMs (Figure 10B,C). Therefore, these results also
confirm the effective internalization of CPT-loaded CPMs by
cancer cells.
ACKNOWLEDGMENTS
■
This work was supported by the National Basic Research
Program (No. 2013CB834506, 2012CB821500,
2009CB930400) and the National Natural Science Foundation
of China (No. 21204048, 91127047, 21174086, 21074069).
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