Coumarin-containing photo-responsive nanocomposites for NIR light-triggered controlled drug release via a two-photon process

Article abstract of DOI:10.1039/c3tb21206h

A new multifunctional nanovehicle for tumor therapy and cell imaging was fabricated by coating NIR light-responsive polymers (HAMAFA-b-DDACMM) onto the surface of octadecyltrimethoxysilane (C18)-modified hollow mesoporous silica nanoparticles (HMS@C18) via self-assembly. First, the targeting NIR light-responsive block copolymer was synthesized by the RAFT living polymerization of [7-(didodecylamino) coumarin-4-yl] methyl methacrylate with hydroxyethylacrylate and N-(3-aminopropyl) methacrylamide hydrochloride and then grafted with folic acid (FA). The copolymers could be disrupted by excitation by a femtosecond NIR light laser (800 nm) via a two-photon absorption process due to the high two-photon absorption cross-section of the coumarin moiety. In order to enhance the drug loading capacity and biological stability of the nanovehicle, HMS nanoparticles modified by hydrophobic octadecyl chains were selected as the "core", which had a considerable drug loading efficiency of more than 70%. Then the core-shell nanocomposites (HMS@C18@HAMAFA-b-DDACMM) were obtained by coating the amphiphilic copolymers onto the core via self-assembly. Under excitation by NIR light at 800 nm, the pre-loaded drugs could be released from the nanocomposites due to the degradation of the light-responsive copolymers and the release efficiency was correlated with the irradiation time and light power. The in vitro experiments indicated that the nanocomposites were easily targeted into the tumor cells that over-expressed folic acid receptor (FR(+)) such as KB cells by endocytosis. Furthermore, the copolymer itself had strong fluorescence, which could be used to track the process of drug delivery.

Full text of DOI:10.1039/c3tb21206h

Journal of
Materials Chemistry B
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Coumarin-containing photo-responsive
nanocomposites for NIR light-triggered controlled
Cite this: J. Mater. Chem. B, 2013, 1,
5942
drug release via a two-photon process†
a
ac
Dongyun Chen,a Xiuxiu Qi,a Wenwei Sha,a Yang Jiao,b
*
Weidong Ji, Najun Li,
Qingfeng Xuac and Jianmei Lu
ac
*
A new multifunctional nanovehicle for tumor therapy and cell imaging was fabricated by coating NIR light-
responsive polymers (HAMAFA-b-DDACMM) onto the surface of octadecyltrimethoxysilane (C18)-modified
hollow mesoporous silica nanoparticles (HMS@C18) via self-assembly. First, the targeting NIR light-
responsive block copolymer was synthesized by the RAFT living polymerization of [7-(didodecylamino)
coumarin-4-yl] methyl methacrylate with hydroxyethylacrylate and N-(3-aminopropyl) methacrylamide
hydrochloride and then grafted with folic acid (FA). The copolymers could be disrupted by excitation by
a femtosecond NIR light laser (800 nm) via a two-photon absorption process due to the high two-
photon absorption cross-section of the coumarin moiety. In order to enhance the drug loading capacity
and biological stability of the nanovehicle, HMS nanoparticles modified by hydrophobic octadecyl chains
were selected as the “core”, which had a considerable drug loading efficiency of more than 70%. Then
the core–shell nanocomposites (HMS@C18@HAMAFA-b-DDACMM) were obtained by coating the
amphiphilic copolymers onto the core via self-assembly. Under excitation by NIR light at 800 nm, the
pre-loaded drugs could be released from the nanocomposites due to the degradation of the light-
responsive copolymers and the release efficiency was correlated with the irradiation time and light
power. The in vitro experiments indicated that the nanocomposites were easily targeted into the tumor
cells that over-expressed folic acid receptor (FR(+)) such as KB cells by endocytosis. Furthermore, the
copolymer itself had strong fluorescence, which could be used to track the process of drug delivery.
Received 30th August 2013
Accepted 9th September 2013
DOI: 10.1039/c3tb21206h
www.rsc.org/MaterialsB
controlled by spatial or temporal control to occur at a desired
time by illumination. However, most of light-responsive photo-
Introduction
Recently, much research has involved the study of stimuli-
responsive block copolymers (BCPs)1,2 based on the stimuli of
ultrasound,3–5 magnetic eld,6,7 enzymes,8 electrical eld,9
pH,10–13 temperature14 and light15–20 for the triggered release of
hydrophobic drugs in controlled drug delivery systems due to
several advantages, such as signicantly increasing drug water-
solubility, prolonging circulation time, decreasing side effects
and improving drug bioavailability. Compared with tempera-
ture or pH sensitive strategies,41,42 photo-cleavage has attracted
growing interest, because the use of light enables the remote
activation of the release process and the drug release can be
chromic moieties respond to UV or visible light, whose energy
can be easily weakened by tissues in vivo and even cause great
damage to the human body, which hampers their possible bio-
logical or biomedical applications.21,22 Near-infrared (NIR) light
with a longer wavelength in the range of about 700–1000 nm is
less detrimental to healthy cells and can have a deeper penetra-
tion through tissue due to its reduced absorption and scattering
by water and biological substances.23 So stimuli-responsive BCPs
triggered by NIR light are more suitable for drug delivery and
controlled release than those triggered by UV or visible light.24–28
[7-(Diethylamino) coumarin-4-yl] methyl (DEACM) is an
attractive photo-responsive group because of its UV-induced
cleavage via single-photon absorption.29,30 Additionally, DEACM
can undergo the same cleavage reaction via a two-photon
process under NIR laser light.31,32 Recently, Yue Zhao and
coworkers used NIR light to photo-activate a coumarin-based
micellar system for the controlled release of Rifampicin and
Paclitaxel, which indicated the potential application in NIR
light-controlled drug delivery systems.33,34 In addition,
coumarin itself has strong blue uorescence, which provides
can be used to track the process of drug delivery.
aJiangsu Key Laboratory of Advanced Functional Polymer Design and Application,
College of Chemistry, Chemical Engineering and Materials Science, Soochow
University, Suzhou, 215123, China. E-mail: linajun@suda.edu.cn; lujm@suda.edu.cn;
Fax: +86 (0) 512 6588 0367; Tel: +86 (0) 512 6588 0367
bSchool of Radiation Medicine and Protection, Medical College of Soochow University,
Suzhou, Jiangsu 215123, China
cState Key Laboratory of Treatments and Recycling for Organic Effluents by Adsorption
in Petroleum and Chemical Industry, Suzhou, 215123, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3tb21206h
5942 | J. Mater. Chem. B, 2013, 1, 5942–5949
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Because of the water-insolubility and toxicity of the common anticancer drug) was encapsulated in the above polymer-coated
anti-cancer drugs, micellar aggregates formed by amphiphilic HMS. The resulting nanocomposite was specialized due to the
BCPs are being actively investigated as nanovehicles for drug photo-sensitive layer, which could release pre-loaded DOX upon
delivery and controlled release, including the work by Yue Zhao photocleavage of the chromophore in the polymer layer. To
mentioned above. However, the low drug loading capacity and further assess the potential application of this NIR-responsive
instability of micelles shows that this drug delivery system is not biocompatible nanocomposite system for drug release, the NIR
perfect. On the other hand, compared to polymeric micelles, light-triggered release of DOX, cytotoxicity and cellular uptake
inorganic nanoparticles (e.g. SiO2, Fe3O4, Au, etc.) with well- of the nanocomposite were also investigated.
controlled morphologies have shown better tissue permeability
and greater cell penetrability. Therefore, hollow mesoporous
silica (HMS) nanoparticles which have hollow cores and meso-
Experimental
Materials
porous silica shells have been proved to have excellent
biocompatibility, good monodispersity and high drug
loading.35–37 A lot of research into HMS has also been reported
previously by our group.12,13,18,38,39 However, as a smart drug
nanovehicle, the HMS nanoparticles should be modied with
stimuli-responsive groups for controlled release and targeting
groups for cancer cells (Scheme 1).
Here we fabricated a novel nanovehicle for controlled drug
release and cell imaging by coating a photo-responsive amphi-
philic polymer onto modied HMS. The multifunctional
amphiphilic polymer was synthesized by RAFT polymerization
of [7-(didodecylamino) coumarin-4-yl] methyl methacrylate
(DDACMM) and hydroxyethylacrylate (HEA) and successfully
conjugated with folic acid for selective cancer targeting. Aer
modication with hydrophobic octadecyl chains (C18), the HMS
nanoparticles could be easily coated with hydrophobic portions
of amphipathic copolymers through the strength of van der
Waals forces via a simple self-assembly strategy. The as-
synthesized nanocomposite had excellent biocompatibility and
Azobisisobutyronitrile (AIBN) and 4,40-azobis(4-cyanovaleric
acid) (V-501) were purchased from Aldrich. N-Hydroxy-
succinimide (NHS), N-(3-aminopropyl) methacrylamide hydro-
chloride (APMA), dicyclohexacarbodiimide (DCC) and 4-
dimethylaminopyridine (DMAP) were purchased from Alfa
Aesar. Methacryloyl chloride, methoxycarbonyl chloride, aceto-
acetic ester and SeO2 were purchased from TCI. Hydroxy-
ethylacrylate (HEA) was distilled under vacuum and stored at
15 ꢀC under an inert gas atmosphere. Other reagents were
commercially available and used as received.
Synthesis of hollow mesoporous spherical particles (HMS)
and modication of the external surface of HMS with
octadecyltrimethoxysilane (C18) (HMS@C18)
Hollow mesoporous spherical particles (HMS) were synthesized
by the polystyrene (PS) latex template method according to the
literature12,13 with some modications, and the detailed steps
are shown in the ESI.† The PS latex templates (ꢁ130 nm) were
prepared by emulsion polymerization and initiated by 2,20-
azobis(2-methylpropionamide) dihydrochloride (V-50). Then,
the PS nanospheres were coated with mesoporous silica, and
the silica thickness could be controlled by adjusting the amount
of tetraethoxysilane (TEOS). Finally, the material was calcined
NIR light-responsivity. Doxorubicin (DOX,
a widely used
ꢀ
in air at 600 C to remove the PS templates.
Synthesis of NIR light-responsive coumarin-based monomer
[7-(didodecylamino) coumarin-4-yl] methyl methacrylate
(DDACMM)
The 7-amino-4-methylcoumarin (AMC) compound was synthe-
sized according to the literature40 and this is shown in the ESI.†
A mixture of AMC (2.66 g, 15.2 mmol) and bromododecane
(11.1 mL, 45.6 mmol) was dissolved in 250 mL dime-
thylformamide. Then K2CO3 (12.6 g, 91.2 mmol) was added to
ꢀ
the solution and the obtained mixture was stirred at 80 C for
12 h. Aer ltration, the solution was evaporated to remove
DMF, then a sample of water and ethyl acetate was added. The
organic layer was washed with water and saturated NaCl solu-
tion, dried with MgSO4 and evaporated. The residue was puri-
ed by column chromatography (ethyl acetate : petroleum
ether ¼ 1 : 4) to give 2.5 g (4.89 g, 32% yield) DDAMC. 1H NMR
(CDCl3, 400 MHz): d 7.36 (d, J ¼ 8.0 Hz, 1H, Ar-H), 6.55 (d, J ¼ 8.0
Hz, 1H, Ar-H), 6.49 (s, 1H, Ar-H), 5.99 (s, 1H, Ar-H), 3.64 (t, J ¼
12.0 Hz, 1H, N–CH2CH2), 3.16 (t, J ¼ 12.0 HZ, 3H, N–CH2CH2),
Scheme 1 Schematic depiction of the fabrication of HMS@C18@HAMAFA-b-
DDACMM for drug delivery and controlled release by degradation upon NIR light
exposure.
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2.34 (s, 3H, CH3), 1.72–1.47 (m, 4H, N–CH2), 1.45–1.15 (m, 36H, 1.22 g of HEA (10.5 mmol) and 268 mg of APMA (1.5 mmol).
Then, the tube was sealed and cycled between vacuum and
CH2), 0.88 (t, J ¼ 16 Hz, 6H, CH3) (Scheme 2).
DDAMC (2.0 g, 3.91 mmol) and SeO2 (0.87 g, 7.83 mmol) were nitrogen three times. Aer reaction for 5 h in an oil bath at
ꢀ
suspended in 100 mL xylene, and the reaction mixture was 70 C, the mixture was concentrated and washed with a large
reuxed with vigorous stirring for 16–24 h. Then the mixture amount of acetone. The obtained copolymer was dried under
was ltered through a pad of Celite and concentrated under vacuum and stored in a desiccator for further polymerization.
pressure. The dark brown residual oil was dissolved in meth- 1H NMR (d6-DMSO, 400 MHz), d (ppm): 4.32–3.56 (COOCH2CH2
anol (70 mL), then sodium borohydride (0.3 g, 7.83 mmol) was and CH2OH), 2.88 (NHCH2 and CH2NH2), 2.19–2.03 (CH2).
added to the ask, and the mixture was stirred for 4 h. Aer the
The amphiphilic diblock polymer HAMA-b-DDACMM was
solution was neutralized with 1 M HCl, the solution was synthesized using a HAMA macromolecular transfer agent. In a
extracted three times with CHCl3, dried over magnesium sulfate typical polymerization procedure, 200 mg of HAMA was added
and evaporated under vacuum. The residue was puried by to a DMF solution containing 3 mg AIBN and 50 mg (0.08 mmol)
ꢀ
ash chromatography (dichloromethane : acetone
¼
5 : 1) DDACMM and then placed in an oil bath at 70 C for 5 h. The
affording 800 mg (1.52 mmol, 39% yield) of DDAHC as an mixture was concentrated and washed with a large amount of
orange solid. 1H NMR (DMSO, 400 MHz): d 10.14 (s, 1H, Ar-H), ethyl ether. Aer washing, the obtained diblock polymer was
1
7.61 (t, J ¼ 19.6 Hz, 2H, Ar-H), 7.39 (d, J ¼ 10.4 Hz, 1H, Ar-H), dried under vacuum overnight and stored in a desiccator. H
6.33 (s, 1H, N–CH2CH2), 5.64 (t, J ¼ 11.2 Hz, 1H, OH), 4.73 (d, J ¼ NMR (d6-DMSO, 400 MHz), d (ppm): 4.32–3.56 (COOCH2CH2
4.8 Hz, 3H, N–CH2CH2), 4.13 (t, J ¼ 13.2, 2H, OCH2), 1.63–1.66 and CH2OH), 2.88 (NHCH2 and CH2NH2), 2.39–2.01 (CH2 in
(m, 4H, N–CH2), 1.25 (s, 36H, CH2), 0.86 (t, J ¼ 13.2, CH3).
DDAHC (527 mg, 1 mmol) was dissolved in dry THF along
with triethylamine (557 mL, 4 mmol), then a solution of meth-
acꢀrylyl chloride (304 mL, 3 mmol) in THF was added dropwise at
0 C. Aer stirring for 6 h at room temperature, the resulting
insoluble amine hydrochloride salt was removed by ltration,
and the THF was removed by rotary evaporation. The residue
was dissolved with dichloromethane and washed with K2CO3
solution and saturated NaCl solution, dried with MgSO4, and
evaporated. Aer recrystallization in hexane and vacuum
drying, the coumarin based monomer was obtained to give 548
mg (0.92 mmol, 92% yield) DDACMM. 1H NMR (CDCl3, 400
MHz): d 7.29 (s, 1H, Ar-H), 6.52 (t, J ¼ 18.8 Hz, 3H, Ar-H), 6.25 (s,
1H, CCH2), 6.19 (s, 1H, CCH2), 5.69 (s, 1H, N–CH2CH2), 5.28 (d,
J ¼ 32.4, 3H, N–CH2CH2), 3.17 (t, J ¼ 14.4, 2H, CH2), 2.02 (s, 3H,
CH3), 1.67–1.63 (m, 4H, N–CH2), 1.47–1.26 (m, 36H, CH2), 0.88
(t, J ¼ 12.8, 6H, CH3).
HEA), 1.92–0.62 (CH2 and CH3 in DACMM).
Synthesis of folate-conjugated diblock polymer HAMAFA-b-
DDACMM
The conjugation procedure was achieved according to the
literature as reported elsewhere.11,13 First, the thiocarbonylthio
end group of the HAMA-b-DDACMM was removed according to
the procedure in the literature with some modications. 500 mg
of HAMA-b-DDACMM was dissolved in 10 mL DMF containing
30 mg of AIBN. The DMF solution was sealed and cycled
between vacuum and nitrogen three times. Aer stirring at
70 ꢀC for 5 h, the mixture was precipitated in anhydrous ether.
The obtained diblock polymer was dried under vacuum and
stored in a desiccator for further use. Second, folate NHS ester
was prepared by the following procedure: 0.62 g of DCC and
0.51 g NHS were added to dry dimethylformamide (50 mL)
solution containing 2.0 g of folic acid for 12 h at room
temperature in the dark. The solution was ltered off and
precipitated in diethyl ether. The obtained yellow powder was
washed several times with anhydrous ether and used immedi-
ately for the next step.
Synthesis of HAMA-b-DDACMM
HAMA was prepared by RAFT polymerization of 2-hydroxy-
ethylacrylate (HEA) and N-(3-aminopropyl) methacrylamide
hydrochloride (APMA) using 4-cyanopentanoic acid dithio-
benzoate (CAD, shown in ESI†) as a transfer agent. Typically,
5.3 mg (0.02 mmol) CAD was added to the aqueous solution
containing 2.5 mg of 4,40-azobis(4-cyanovaleric acid) (V-501),
Then, FA–NHS (30 mg) was added to the dry pyridine con-
taining 100 mg of the above polymer. The solution was shaken
for 12 h at room temperature and the mixture was precipitated
in 40% acetone in diethyl ether. The ltrate was concentrated
1
ꢀ
and stored in the dark at 4 C. H NMR (d6-DMSO, 400 MHz),
d
(ppm): 8.63–6.63 (folic acid and CH2NH2), 4.32–3.56
(COOCH2CH2 and CH2OH), 2.88 (NHCH2 and CH2NH2), 2.0–0.7
(CH2 and CH3).
Self-assembly of HAMAFA-b-DDACMM with HMS@C18
(HMS@C18@HAMAFA-b-DDACMM)
HMS@C18 (2 mg) and HAMAFA-b-DDACMM (10 mg) were dis-
solved in tetrahydrofuran (1 mL). Then, 5 mL of distilled water
was added to this solution with vigorous shaking. The mixture
was stirred for 24 h at room temperature to evaporate tetrahy-
drofuran. Then, the product was separated by centrifugation
and washed with distilled water several times to remove the
Scheme 2 Synthetic route to DDACMM.
5944 | J. Mater. Chem. B, 2013, 1, 5942–5949
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unbound copolymer. The obtained product was denoted as
HMS@C18@HAMAFA-b-DDACMM.
In vitro cytotoxicity. In vitro cytotoxicity was assessed by the
standard MTT assay. A p-value of less than 0.05 was considered
statistically signicant. Each data point is represented as mean
ꢄ standard deviation (SD) of eight independent experiments
(n ¼ 8, n indicates the number of wells in a plate for each
experimental condition). The dose dependence of the cytotox-
icity was investigated at different particle concentrations.
To test the cytotoxicity of various materials with and without
NIR irradiation, KB cells were seeded in a 96-well plate at a
density of 105 cells per well and cultured in 5% CO2 at 37 ꢀC for
24 h. Then, different NPs were added to the medium. For
materials without NIR irradiation, the cells were incubated in
5% CO2 at 37 ꢀC for various time durations. For materials with
NIR irradiation, the NPs were removed aer 3 h of incubation,
followed by exposure to different amounts of light. Then, the
cells were further incubated for 24 h in the dark. At the end of
the incubation, the medium was removed, and 100 mL of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
solution (diluted in a culture medium with a nal concentration
of 0.5 mg mLꢂ1) was added and incubated for another 4 h. The
medium was then replaced with 100 mL dimethyl sulfoxide
(DMSO) per well, and the absorbance was monitored using a
microplate reader (Bio-TekELx800) at a wavelength of 490 nm.
The cytotoxicity was expressed as the percentage of cell viability
compared to the untreated control cells.
Drug loading and release
To evaluate the drug loading and release efficiency, doxorubicin
(DOX) was used as a model anticancer agent. Free water-insol-
uble DOX was extracted from doxorubicin hydrochloride
(DOX$HCl) according to the procedure reported previously.
DOX$HCl (4 mg) was dissolved in 0.75 mL DMSO, and aer the
solution turned to reddish orange, 1.92 mL triethylamine was
added, and a dark red solution was obtained. With stirring
overnight in darkness at room temperature, we removed the
upper colorless liquid and obtained a concentration of 5
mg$mLꢂ1. The DOX solution (40 mL) was diluted with water (1
mL), then HMS@C18 (4.0 mg, 2.0 mg, 0.4 mg) was added to this
solution. Aer stirring for 24 h in the darkness at room
temperature, the HMS@C18 with adsorbed DOX was separated
by centrifugation. The concentration of the remaining DOX
solution was determined using a UV-Vis spectrophotometer at
l ¼ 480 nm. To conrm the amount of drug stored by the
nanoparticles, a standard plot was prepared under identical
conditions.
Drug loading content (wt%) ¼ (weight of loaded drug/
weight of nanocomposites) ꢃ 100%
Cellular uptake of the HMS@C18@HAMAFA-b-DDACMM
Drug loading efficiency (%) ¼ (weight of loaded drug/
weight of drug in feed) ꢃ 100%
KB cells and A549 cells were seeded in 96-well plates (1.3 ꢃ 104
per well) and incubated overnight at 37 ꢀC in a humidied
incubator. The dispersion of the HMS@C18@HAMAFA-b-
DDACMM sample was prepared in RPMI-1640 medium. Cells
were cultured with the HMS@C18@HAMAFA-b-DDACMM
solution for a certain time and observed using confocal laser
uorescence microscopy (Olympus, FV 1000) aer washing
three times with PBS.
The drug release test was performed by suspending the DOX-
loaded nanoparticles in distilled water under light exposure. To
determine the release amount at any given time, 100 mL of the
solution was withdrawn aer centrifugation, and the same
volume of water was added to keep the volume constant. The
drug concentration of the withdrawn solution was analyzed by
measuring the UV absorbance at l ¼ 480 nm. The experiments
were conducted in triplicate and the results are shown as
averages with standard deviations.
Characterization
FT-IR measurements were performed using KBr pellets on a
Nicolet 4700 spectrometer (Thermo Fisher Scientic) in the
range of 400–4000 cmꢂ1. Gel permeation chromatography
(GPC) analysis was carried out on a Waters 1515 pump and a
In vitro experiments
In vitro drug-release study. The prepared DOX-loaded differential refractometer, and DMF was used as a mobile phase
HMS@C18@HAMAFA-b-DDACMM sample was suspended in at a ow rate of 1.0 mL minꢂ1. 1H NMR spectra were measured
phosphate buffer solution (PBS, pH 7.4) with shaking in a water by an INOVA 400 MHz NMR instrument. TEM images were
ꢀ
bath maintaining the temperature at 37 C under continuous obtained using a TecnaiG220 electron microscope at an accel-
light (UV or NIR) exposure. To determine the release amount at eration voltage of 200 kV. Brunauer–Emmett–Teller (BET) and
any given time, 0.1 mL of the solution was withdraw aer Barrett–Joyner–Halenda (BJH) analyses were used to determine
centrifugation and analyzed by UV-Vis spectroscopy. The the surface area, pore size and pore volume and measurements
experiments were conducted in triplicate and the results are were obtained with a Quantachrome Autosorb 1C apparatus at
averages with standard deviations.
ꢂ196 ꢀC under continuous adsorption conditions. Confocal
Cell culture and preparation. A549 FR(ꢂ) and human KB laser scanning microscopy (CLSM) images were observed using
FR(+) cell lines (purchased from Shanghai cell Institute Country a confocal laser scanning microscope (Olympus, FV 1000). The
Cell Bank, China) were cultured as a monolayer in RPMI-1640 UV-Vis absorption spectra were measured on a TU-1901 spec-
medium supplemented with 10% heat-inactivated fetal bovine trophotometer. Emission and excitation spectra were obtained
ꢀ
serum at 37 C in a humidied incubator (5% CO2 in air, v/v). using an Edinburgh-920 uorescence spectra photometer.
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distribution of HMS, HMS@C18 and DOX-loaded HMS@C18
are shown in Fig. S2.†
Light exposure
The NIR photons were generated from a Coherent Vitesse
Ti:Sapphire laser at 800 nm with a repetition rate of 1 kHz. The
output power of the laser was controlled at 2.3–2.7 W. An NIR
beam whose spot size was approximately 2 mm in diameter was
focused onto a 1 cm-thick cuvette. For UV (365 nm) irradiation,
a 125 W high-pressure mercury lamp from Philips was used.
The FT-IR spectra of HMS, HMS@C18, and DOX-loaded
HMS@C18 are shown in Fig. 1. As we can see, the Si–OH bond
(950 cmꢂ1) in the spectrum of HMS indicates that a great
number of silanol groups exist on the surface of the HMS for
coupling alkyl chains. In the spectrum of HMS@C18, strong
C–Hx bands (2850 cmꢂ1 and 2920 cmꢂ1) suggested the
successful conjunction of alkyl chains on the silica. For the
DOX-loaded HMS@C18, a new band (1740 cmꢂ1) appeared
according to the spectrum of HMS@C18 and DOX, implying
that the DOX was loaded into the nanoparticles successfully.
Results and discussion
Synthesis of HAMAFA-b-DDACMM and HMS@C18
Scheme 3 shows the synthetic route of the amphiphilic photo-
responsive block copolymer, denoted as HAMAFA-b-DDACMM.
The 1H NMR spectrum of HAMAFA-b-DDACMM (not shown
here, but the characteristic data are listed above in the experi-
ment section) showed the successful conjugation of FA. The
number-average molecular weight (Mn), weight-average molec-
ular weight (Mw) and polydispersity index (PDI) of the polymers
are shown in Table S2.† From the increase of the molecular
weight, we conrmed the successful FA conjugation (ꢁ2.3%).
Based on an evaluation of the characterization data of the
copolymers, HAMA-b-DDACMM with a monomer feed ratio of
4 : 1 was chosen for further investigation (Table S1†). A typical
1H NMR spectrum of the copolymer HAMA-b-DDACMM in
DMSO is shown in Fig. S1a.† Degradation was observed by
comparing the spectra of the copolymer before and aer light
exposure. Aer light exposure for several hours, the specic
chemical shi appeared at d ¼ 12.29 and d ¼ 8.0–8.7, which
correspond to the carboxylic acid group and coumarin hydroxyl
group. This clearly indicates the obvious photo-degradation
process of the polymer chain under light exposure (Fig. S1b†).
C18 was used here to simply modify the surface of the HMS.
As can be seen, there was almost no change in the diameter of
the particles between HMS and HMS@C18. N2 adsorption–
desorption isotherms and the corresponding pore size
Fabrication of HMS@C18@HAMAFA-b-DDACMM via self-
assembly
The surface of the HMS shell was rst modied with C18 to
change the wettability of the particle, and TEM images of HMS
(Fig. 2a) and HMS@C18 (Fig. 2b) did not show any difference.
Then the shell was coated with polymer through a self-assembly
process. HMS@C18 was dispersed in tetrahydrofuran by soni-
cation to get solution A, then HAMAFA-b-DDACMM was
immersed in this solution. Then 5 mL of distilled water was
added to the above solution, and THF was evaporated under
continued shaking, and the amphiphilic polymer would form
micelles to encapsulate the hydrophobic polymer guest mole-
cules. The obtained HAMAFA-b-DDACMM-coated HMS@C18
remained hydrophilic because of the hydrophilic segment of
HAMAFA-b-DDACMM. The detailed morphological and struc-
tural features of the prepared nanoparticles were examined by
TEM. In the TEM image of HMS@C18@HAMAFA-b-DDACMM
(Fig. 2c), we can clearly see that the polymer has completely
coated the surface of the HMS@C18.
The prepared nanoparticles had an average diameter of
about 250 nm and maintained good monodispersity and DLS
(dynamic light scattering) experiments showed nearly the same
result (Fig. 3). When the nanoparticles were under light expo-
sure for a number of hours, the photosensitive polymer layer
was disrupted and disappeared (Fig. 2d), conrming the orig-
inal idea. Hence, nanoparticles composed of HMS@C18 and
HAMAFA-b-DDACMM were successfully prepared.
Scheme 3 Schematic of the synthetic route of HAMAFA-b-DDACMM.
5946 | J. Mater. Chem. B, 2013, 1, 5942–5949
Fig. 1 FT-IR spectra of HMS, HMS@C18 and DOX-loaded HMS@C18.
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Fig.
2
TEM images of HMS (a), HMS@C18 (b), HMS@C18@HAMAFA-b-
DDACMM (c) and HMS@C18@HAMAFA-b-DDACMM after light irradiation.
Fig. 4 The degradation degree of HAMAFA-b-DDACMM under different light
exposures (a) and the release of DOX in vitro from DOX-loaded
HMS@C18@HAMAFA-b-DDACMM (b).
increased continuously over time, indicating signicant release
from the nanocomposites.
Fig. 3 DLS of HMS@C18 and HMS@C18@HAMAFA-b-DDACMM.
Evaluation of cytotoxicity and drug uptake
Evaluation of drug loading and release
We used the MTT assay to assess the cytotoxicity of the
To evaluate the drug loading and release efficiency of the HAMAFA-b-DDACMM-coated HMS@C18 at various concentra-
HAMAFA-b-DDACMM-coated HMS@C18, doxorubicin (DOX) tions. We incubated KB cells in the culture medium with
was used as a model anticancer agent. The drug loading content NIR excitation (800 nm,
1 kHz, 2.4 W) for 60 min,
was determined by measuring the drug concentration before HMS@C18@HAMAFA-b-DDACMM (nanocarrier), nanocarrier
and aer loading with HMS@C18, using a UV-Vis spectropho- under NIR excitation, and DOX-loaded nanocarrier as controls,
tometer at l ¼ 480 nm. A standard curve was produced under and with DOX-loaded nanocarrier under NIR excitation. In this
identical conditions in advance to measure the DOX concen- assay, aer irradiation by NIR for 60 min the cells showed little
tration. The theoretical drug loading contents were set at 5%, indication of toxicity due to NIR. Aer 3 h of incubation, the NPs
10% and 50%. The DOX loading efficiencies were measured as were removed and the cells were further incubated for 24 h in
77.52%, 75.35% and 71.56% (Table S3†).
the dark. Both before and aer NIR excitation, the viability of
To evaluate the drug release behavior, we rst analyzed the the cells incubated in the nanocarrier was maintained at a high
hydrolysis proles of the HAMAFA-b-DDACMM under different level, conrming the safety and biocompatibility of the nano-
conditions (Fig. 4a), which were determined by UV-Vis spec- particle itself and the residue aer loaded drug release. Treat-
troscopy. We found that with UV light (365 nm, 125 nm) and a ment with nanocarrier loaded with and without DOX did not
femtosecond pulse NIR laser (800 nm, 1 kHz) the polymer could result in a signicant decrease in the cell viability, indicating
be easily hydrolyzed within 200 min, while a smaller change was the negligible delivery of DOX into the KB cells. In contrast,
observed under the two other light conditions. Due to the better upon irradiation at 800 nm, the nanoparticles showed a
biocompatibility of the NIR light, a femtosecond pulse NIR laser signicantly enhanced cytotoxicity to the cells, which could be
was required in this experiment. The in vitro cumulative DOX explained by the controlled drug release and signicant accu-
release proles of the nanocomposite were investigated at 37 ꢀC mulation of DOX inside the cells (Fig. 5).
and under NIR light exposure as shown in Fig. 4b. It was seen
Since the polymer contains uorescent coumarin moieties,
that without NIR irradiation, little absorption of DOX could be the uorescence intensity could be used to indicate the intra-
detected, while aer NIR irradiation, the absorption of DOX cellular activity of the nanoparticles in FR(+) (KB cells) and the
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(HMS@C18@HAMAFA-b-DDACMM) (Fig. 6c and d) and without
(HMS@C18@HAMA-b-DDACMM) FA groups (Fig. 6a and b) for
0.5 and 2 h were studied by uorescence microscopy. Moreover,
by way of contrast, uorescence images of A549 cells incubated
with HMS@C18@HAMAFA-b-DDACMM are shown in Fig. 6e
and f.
From the photos using a confocal laser scanning microscope
(CLSM), a brighter uorescence intensity, not merely aer 0.5 h
but also 2 h, can be seen in the KB cells incubated with FA-
conjugated nanocomposites compared to the two other
samples. This result demonstrates that the targeting moiety
offered by folic acid is efficient at enhancing the tumor cell
targeting in vitro because the FA-conjugated nanocomposite was
taken up by KB cells through folate receptor-mediated
endocytosis.
Fig. 5 Inhibition of KB cell growth with excitation by a laser (980 nm, 60 min),
and with HMS@C18@HAMAFA-b-DDACMM (nanocarrier) and DOX-loaded
nanocarrier without or with NIR light exposure for various durations/dosages
after 24 h of incubation (the concentration of the materials was 1 mg mLꢂ1).
Conclusions
In summary, we have designed an NIR-triggered nanocomposite
for anti-cancer drug delivery via a simple self-assembly strategy.
As a result of the large cross section of the coumarin moiety for
800 nm NIR light excitation via a two-photon absorption, NIR-
induced removal of coumarin groups from the BCPs shis the
hydrophilic–hydrophobic balance toward the destabilization of
the polymer layer in aqueous solution. A signicant number of
drug molecules (>50%) could be released from the nanocarriers
upon NIR light irradiation. The introduction of HMS highly
enhanced the drug loading capacity and biological stability. The
whole process of drug delivery and release based on the nano-
vehicles in cells could be tracked by the uorescence intensity of
the coumarin moiety. Since the BCPs were conjugated with folic
acid (FA) groups, the prepared nanomaterials could easily target
the FR(+) KB cells and showed good selectivity compared to
FR(ꢂ) A549 cells. We are convinced that this smart drug
nanovehicle is potentially useful for cancer chemotherapy.
FR(ꢂ) (A549) cancer cell line. Here, in vitro experiments
based on these two cells were used to prove the targeting
properties of the nanocomposites. The KB cell uptake and
intracellular distribution of the nanocomposites with
Acknowledgements
We gratefully acknowledge the nancial support provided by
National Natural Science Foundation of China (21076134,
21176164), Natural Science Foundation of Jiangsu Province
(BK2012625), Natural Science Foundation of the Jiangsu Higher
Education Institutions of China (13KJB430022), a project
funded by the Priority Academic Program Development of
Jiangsu Higher Education Institutions (PAPD) and Joint
Research Projects of SUN-WIN Joint Research Institute for
Nanotechnology.
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