Visible-light degradable polymer coated hollow mesoporous silica nanoparticles for controlled drug release and cell imaging

Article abstract of DOI:10.1039/c3tb20922a

A core-shell nanocomposite based on photo-degradable polymer coated hollow mesoporous silica nanoparticles (HMS) was successfully prepared for targeted drug delivery and visible-light triggered release, as well as fluorescence cell imaging. The HMS nanoparticles were first modified by the long-chain hydrocarbon octadecyltrimethoxysilane (C18) and fluorescent agent Rhodamine B isothiocyanate (RITC), and then encapsulated by a photodegradable amphiphilic copolymer via a self-assembly process. The obtained nanocarrier showed a high drug loading content due to the hollow core and mesopores of the HMS and could target folic acid receptor over-expressed tumor cells efficiently for conjugating folic acid (FA) in the amphiphilic polymer. The drug release could be triggered by the irradiation of green light (500-540 nm) due to the photodegradation of amphiphilic copolymer coated on the HMS. Furthermore, the targeted drug delivery and controlled release processes could be tracked by fluorescence imaging for the doping of RITC on the HMS. The In vitro results suggested that a smart visible light responsive drug delivery system was successfully prepared for the potential applications of cancer diagnosis and therapy.

Full text of DOI:10.1039/c3tb20922a

Journal of
Materials Chemistry B
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PAPER
Visible-light degradable polymer coated hollow
mesoporous silica nanoparticles for controlled drug
release and cell imaging†
Cite this: J. Mater. Chem. B, 2013, 1,
628
4
a
ab
a
a
c
c
Shun Yang, Najun Li,* Dongyun Chen, Xiuxiu Qi, Yujie Xu, Ying Xu,
ab
ab
ab
Qingfeng Xu, Hua Li and Jianmei Lu*
A core–shell nanocomposite based on photo-degradable polymer coated hollow mesoporous silica
nanoparticles (HMS) was successfully prepared for targeted drug delivery and visible-light triggered
release, as well as fluorescence cell imaging. The HMS nanoparticles were first modified by the long-
chain hydrocarbon octadecyltrimethoxysilane (C18) and fluorescent agent Rhodamine B isothiocyanate
(
RITC), and then encapsulated by a photodegradable amphiphilic copolymer via a self-assembly process.
The obtained nanocarrier showed a high drug loading content due to the hollow core and mesopores
of the HMS and could target folic acid receptor over-expressed tumor cells efficiently for conjugating
folic acid (FA) in the amphiphilic polymer. The drug release could be triggered by the irradiation of
green light (500–540 nm) due to the photodegradation of amphiphilic copolymer coated on the HMS.
Furthermore, the targeted drug delivery and controlled release processes could be tracked by
fluorescence imaging for the doping of RITC on the HMS. The In vitro results suggested that a smart
visible light responsive drug delivery system was successfully prepared for the potential applications of
cancer diagnosis and therapy.
Received 1st July 2013
Accepted 8th July 2013
DOI: 10.1039/c3tb20922a
www.rsc.org/MaterialsB
1–9
temperature, pH, redox and light. Compared to other stimuli-
responsive polymers, light-triggered amphiphilic polymers have
attracted much attention because they do not rely on changes in
the specic chemical properties of the physiological environ-
Introduction
Nanostructured materials have been developed for anti-cancer
drug delivery due to their improved biodistribution and facile
controllable morphology. In order to conquer the insufficient ment in cancer cells, and the possibilities of remote activation
intracellular drug release and side effects of loaded anti-cancer as well as light-enabled spatial and temporal control are
drugs, an ideal nanocarrier should provide an excellent drug attractive features.
loading content, efficient tumor targeting and controlled drug
release. Modifying these nanostructured materials with func- azobenzene,10,11 o-nitrobenzyl and spiropyran,
tional groups via special chemical or physical processes is
continually being under way as one of the dominant develop-
ment strategies.
The known majority of photo-responsive species, including
12
13,14
were proved
to be responsive to ultraviolet light (UV, 250–380 nm). However,
15
UV radiation has a relatively high energy (ꢀ6 eV), which is
harmful to humans, and this always hampers the biological
Recently, many efforts have been made to develop multi- applications of photo-responsive polymers containing these
functional stimuli-responsive polymers for drug delivery, and kinds of species. Hence, developing a new kind of polymer
most of these nanocarriers are purposely designed to exhibit containing photochromic moieties with a low energy excitation
tunable degradation properties induced by an external stim- wavelength, such as visible light (400–700 nm, ꢀ2 eV), is quite
ulus, which is usually of a chemical or physical nature such as necessary. In earlier work, the photochromic moiety containing
the 9,10-dialkoxyanthracene (DN) group showed favorable
a
visible light responsivity with synergy with the green light
Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application,
College of Chemistry, Chemical Engineering and Materials Science, Soochow absorbing sensitizer (PS), eosin. Eosin could transform triplet
16
3
1
University, Suzhou, 215123 China. E-mail: linajun@suda.edu.cn; lujm@suda.edu. oxygen ( O ) into singlet oxygen ( O ) in solution when exposed
2
2
cn; Fax: +86 (0)512-6588 0367; Tel: +86 (0)512-6588 0367
b
to green light (500–540 nm), which can change 9,10-dialkoxy-
anthracene (DN) into 9,10-anthraquinone (AQ), and the eosin
molecules have strong tendency to be bound with the DN
State Key Laboratory of Treatments and Recycling for Organic Effluents by Adsorption
in Petroleum and Chemical Industry, Suzhou, 215123 China
c
Medical College of Radiation Medicine and Protection, Soochow University, Suzhou,
17
species by p–p stacking. Therefore, it is a appropriate photo-
degradable moiety with which to construct a light responsive
amphiphilic polymer.
2
15123, China
†
Electronic supplementary information (ESI) available: Experimental section. See
DOI: 10.1039/c3tb20922a
4
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Besides, inorganic nanoparticles, such as gold nano- addition, the drug delivery process can be tracked by uores-
1
8
particles, mesoporous silica nanoparticles and Fe O nano- cence imaging for modication of the HMS with RITC.
3
4
19
particles, have attracted intense interest when acting as drug
carriers, due to their excellent unique biocompatibility and
chemical stability. Among these inorganic nanocarriers, hollow Experimental section
mesoporous silica nanoparticles (HMS) with well dened
morphologies have attracted considerable interest. The hollow
Materials
(2-(Acryloyloxy)ethyl)trimethylammonium chloride (AETAC, 80
core of the HMS can act as storage reservoir to enhance the drug
loading capacity, whereas the mesopores provide controlled
and slow release pathways for the encapsulated drug molecules,
and the surface of the HMS is full with Si–OH which can be
easily modied with functional groups for imaging, targeting
0
wt% in water), Rhodamine B isothiocyanate, 2,2 -azobis-
2-methylpropionamidine) dihydrochloride (V-50, >97.0%),
(
hexa-decyltrimethylammonium bromide (CTAB, >99.0%), octa-
decyltrimethoxysilane (C18) and (3-aminopropyl)triethoxysilane
(APTES) were purchased from Aldrich. Tetraethoxysilane (TEOS)
was purchased from Alfa without any purication. Styrene (St,
20–22
and hydrophobic properties.
However, pristine HMS cannot
achieve stimulus-triggered drug delivery and thus limits their
application as a smart drug carrier. Modifying the outer surface
of HMS with a stimulus-responsive amphiphilic copolymer
looks rather promising.
In this paper, we prepared a visible light-triggered drug
delivery system for controlled drug release and cell imaging.
Aer modication by the long-chain hydrocarbon octadecyl-
trimethoxysilane (C18) and the uorescent agent Rhodamine B
isothiocyanate (RITC) successively, HMS loaded the drug
molecules and then was encapsulated by a photo-degradable
amphiphilic copolymer through a simple self-assembly process
>99.0%) was washed through an inhibitor remover column for
the removal of tert-butyl catechol and then distilled under
reduced pressure prior to use. The analytical regents were used
without further purication. 4,4-Azobis(4-cyanovaleric acid)
(V-501) and azobisisobutyronitrile (AIBN) were purchased from
Aldrich. N-Hydroxysuccinimide (NHS, 99%), N-(3-aminopropyl)-
methacrylamide hydrochloride (APMA), dicyclohexacarbo-
diimide (DCC, 99%) and 4-dimethylaminopyridine (DMAP,
99%) were purchased from Alfa Aesar. Methacryloyl chloride
and stearyl chloride were purchased from the Haimen Best Fine
Chemical Industry Co. Ltd. and used aer distillation.
Hydroxyethylacrylate (HEA) was distilled under vacuum and
stored at 15 C under an inert gas atmosphere. Other reagents
were commercially available and used as received.
(Scheme 1). The DN-containing copolymer was successfully
synthesized via a reversible addition-fragmentation chain
transfer (RAFT) polymerization and then conjugated with the
folic acid (FA) moiety. When recognized by the FA receptor over-
ꢁ
expressed tumor cells, the nanocarriers could be internalized
efficiently and followed by a controlled drug release process
under the irradiation of green light at 540 nm aer degradation
Modication of HMS with C18 (CHMS)
22
of the DN groups. A high drug loading content could be ach- The HMS were synthesized according to our previous works.
ieved due to the hollow core and mesopores of the HMS, and a To prepare the PS latex templates, 0.5 g of AETAC (80 wt% in
long-term drug release could be achieved due to the hydro- H O) was dissolved in 180 g of water in a 500 mL round-bottom
2
phobic interaction between the loaded drug and C18. In ask. Then, 20.0 g of styrene was added slowly and kept stirring
for 30 min. The mixture was purged with nitrogen for 20 min
ꢁ
and then heated to 90 C. Aerwards, 5 mL of an aqueous
solution containing 0.5 g V-50 was added. The emulsion was
ꢁ
kept at 90 C for 24 h under nitrogen to complete the poly-
merization. The polystyrene latex was collected by centrifuga-
tion and washed with ethanol several times. To prepare the
HMS, 0.8 g of CTAB was dissolved in a mixture of 80.0 g water,
6
0
0.0 g of ethanol and 1.5 mL of ammonium hydroxide solution.
.93 g of PS powders was dispersed in 10.0 g water by sonication
and then added dropwise to the above CTAB solution at room
temperature under vigorous stirring, followed by sonication for
10 min. The derived milky mixture was then stirred for 30 min
before adding 4.0 g of TEOS. The mixture was kept stirring at
room temperature for 48 h. The precipitate was harvested by
centrifugation and washed with ethanol, then dried at room
ꢁ
temperature. Finally, the material was calcined in air at 600 C
for 8 h to remove the organic matter.
100 mg of HMS was rst dispersed in 20 mL anhydrous
acetonitrile, then 5 mL of C18 was added. The obtained
suspension was stirred for 24 h and collected by centrifugation,
washed with acetonitrile and ethanol several times and dried
under vacuum to give CHMS.
Scheme 1 Schematic depiction of the structure of DOX@CRHMS@HFMP and
controlled release by degradation of HFMP under visible light.
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Modication of CHMS with uorescent dye RITC (CRHMS)
temperature for 12 hours. The product was puried by column
chromatography with a mixture of ethyl acetate–CH Cl (1/20,
2
2
Modifying the external surface of CHMS with uorescent dye
v/v) and dried in a vacuum to give 3-((10-(3-(methacryloyloxy)
propoxy)anthracen-9-yl)oxy)propyl stearate (MAPS). Yield:
Rhodamine B isothiocyanate (RITC) was done according to the
24
literature with little modication. The RITC (2.14 mg) was
dissolved in 2 mL of ethanol, and APTES (1 mg) was then added.
The mixture was agitated for 12 h in the dark, then 100 mg
CHMS was added and then stirred 24 h. The product (CRHMS)
was collected by centrifugation, washed with acetonitrile and
ethanol several times, and dried under vacuum.
1
yellow solid, (0.145 g, 0.22 mmol, 52%). HNMR (400 MHz,
DMSO-d
anthracene), 6.18 (s, 1H, CCH
.0, 2H, CH
OCO), 4.53 (t, J ¼ 6.4, 2H, CH
CH CH O), 2.45–2.35 (m, 6H, CH CH OCO, COCH
s, 3H, CH ), 1.69–1,65 (m, 2H, CH CH CH ), 1.29–1.25 (m, 30H,
6
) d (ppm): 8.24 (d, 4H, anthracene), 7.48 (d, 4H,
), 5.60 (s, 1H, CCH
), 4.64 (t, J ¼
OCO), 4.29 (m, 4H,
CH ), 2.01
2
2
6
2
2
2
2
2
2
2
2
(
3
2
2
2
CH ),0.88 (t, J ¼ 13.2, 3H, CH CH ).
2
2
3
Synthesis of hydrophobic monomer 3-((10-(3-
(
(
methacryloyloxy)propoxy) anthracen-9-yl)oxy)propyl stearate
MAPS)
Synthesis of RAFT agent 4-cyanopentanoic acid
dithiobenzoate (CAD)
3
-Iodo-1-propanol was synthesized according to the literature
CAD was synthesized according to the literature with some
25
with some modications. 3-Chloro-1-propanol (10 g, 0.1 mol)
27
modications. Briey, 12.8 g of benzyl chloride was added
dropwise to a sodium methoxide methanol solution (172 g, 12.6
wt%) containing 12.8 g of elemental sulfur. Subsequently, the
obtained mixture was reuxed for 10 h under an inert
atmosphere. The crude sodium dithiobenzoate solution was
extracted by diethyl ether, 1.0 M hydrochloric acid and sodium
hydroxide aqueous solutions, respectively and nally yielded a
solution of sodium dithiobenzoate. Potassium ferricyanide
solution (500 mL, 6.5 wt%) was added dropwise to the sodium
dithiobenzoate over a period of 1 h with vigorous stirring. The
obtained red precipitate was ltered, washed with deionized
water, and dried in vacuum at room temperature overnight.
Dithiobenzoyl disulde (8.50 g, 0.28 mol) was added slowly to
the distilled ethyl acetate (150.0 mL) solution containing 11.68 g
of V-501 (0.42 mol). Aer reuxing for 18 h, the reaction solu-
tion was concentrated in vacuum and puried by column
and sodium iodide (60 g, 0.4 mol) were added to dry acetone
ꢁ
(100 mL) and stirred at 60 C under N
2
for 24 h. The mixture was

ltered and the solvent evaporated. Then, a 1 : 1 mixture of
diethyl ether–hexane was added and stirred for 10 min and then
ltered. The solution was washed with a dilute sodium thio-

sulfate solution followed by water and then brine. The organic
layer was dried with MgSO , ltered and the solvent evaporated
4
1
in vacuum giving a pale yellow liquid (12.4 g, 67%). HNMR (400
MHz, CDCl
.09 (m, 2H, CH
, 10-Bis(3-hydroxypropyloxy)anthracene: nitrogen-saturated
water (150 mL) and CH Cl (150 mL) was added to a mixture of
,10-anthraquinone (2.57 g, 12.36 mmol), Na (4.3 g, 24.7
3
), d (ppm): 3.74 (t, 2H, CH
2 2
OH), 3.29 (t, 2H, CH I),
2
2
CH CH ).
2
2
9
2
2
9
2 2 4
S O
mmol) and Adogen 464 (4.64 g, 10 mmol). The mixture was
stirred for 5 min and then NaOH (4.94 g, 123.5 mmol) was added.
Stirring was continued for 10 min and 3-iodo-1-propanol was
chromatography (ethyl acetate–hexane, 2/3). Yield 8.27 g,
ꢁ
added dropwise. The mixture was stirred overnight at 25 C.
1
3
2
2
3
5.5%. HNMR (400 MHz, CDCl ), d (ppm): 7.91 (d, J ¼ 7.59 Hz,
3
Then the phases were separated and the water phase was washed
H, C
H, C
H, CH
6
H
4
), 7.58 (t, J ¼ 7.47 Hz, 1H, C
), 2.75 (m, 2H, CCH ), 2.45 (m, 2H, CH
).
6
H
4
), 7.41 (t, J ¼ 7.79 Hz,
2 2
with CH Cl . The combined organic phases were washed with
6
H
4
2
2
COOH), 1.95 (s,
water and dried over MgSO
4
. The solution volume was reduced to
ꢁ
3
4
0 mL and the product was precipitated overnight at ꢂ20 C. The
solid was puried by column chromatography with a mixture of
1
Synthesis of HAMA-b-MAPS
ethyl acetate–CH Cl (1/4, v/v). Yield 1.13 g, 28%. HNMR (400
2
2
MHz, DMSO-d
6
), d (ppm): 8.24 (dd, J ¼ 6.8, 3.2 Hz, 4H, anthra- The RAFT polymerization was done according to the literature
28,29
cene), 7.52 (dd, J ¼ 6.8, 3.2 Hz, 4H, anthracene), 4.63 (t, J ¼ 5.2 Hz, with some modications.
HAMA (Poly(HEA-b-APMA)) was
2
H, CH(O)), 4.16 (t, J ¼ 6.5 Hz, 4H, OCH
2
CH
2
), 3.76 (m, prepared by the RAFT polymerization of HEA and APMA using
CAD as a transfer agent according to the literature with some
CH CH OH, 4H), 2.1 (m, CH CH CH , 4H).
2
2
2
2
2
23
MAPS was synthesized according to the literature with little modication. Typically, 5.3 mg (0.02 mmol) CAD was added to
26
modication. 9,10-Bis(3-hydroxypropyloxy)anthracene (0.4 g, the aqueous solution containing 2.5 mg of V-501, 1.22 g of HEA
.22 mmol) and pyridine (0.1 g, 1.22 mmol) were dissolved in (10.5 mmol) and 268 mg of APMA (1.5 mmol). Then, the tube
dry CH Cl (100 mL), then stearyl chloride (0.185 g, 0.61 mmol) was sealed aer three cycles between vacuum and nitrogen.
1
2
2
ꢁ
was added dropwise to the solution under a nitrogen atmo- Aer 5 h reaction in oil bath at 70 C, the mixture was
sphere. The mixture was stirred at room temperature for 2 concentrated and washed with a large amount of acetone. The
hours. The product was puried by column chromatography obtained copolymer was dried under vacuum and stored in
with a mixture of ethyl acetate–CH
vacuum to give 3-((10-(3-hydroxypropoxy) anthracen-9-yl)oxy)-
2
Cl
2
(1/20, v/v) and dried in a desiccators for further polymerization.
The amphiphilic diblock polymer HAMA-b-MAPS was
propyl stearate. Yield: 0.25 g (0.42 mmol, 36%). Then the synthesized using the HAMA macro chain transfer agent. In a
product (0.25 g, 0.42 mol) and triethylamine (0.101 g, 1 mmol) typical polymerization procedure, 1.2 g of HAMA was added to a
were dissolved in dry CH Cl (50 mL), and methacryloyl chloride dimethylsulfoxide solution containing 3 mg AIBN and 200 mg
2
2
ꢁ
(
0.104 g, 1 mmol) was added dropwise to the solution under a (0.31 mmol) MAPS and then placed in an oil bath at 70 C for
nitrogen atmosphere. The mixture was stirred at room 5 h. The mixture was concentrated and washed with a large
4
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amount of ethyl ether. Aer washing, the obtained diblock In vitro experiments
polymer was dried under vacuum overnight and stored in
desiccators.
In vitro drug-release. To evaluate the drug loading capacity
and release properties, doxorubicin (DOX) was used as a model
21,33
drug according to the literature with some modications.
DOX was extracted from doxorubicin hydrochloride (DOX$HCl)
Synthesis of folate-conjugated diblock polymer HAMAFA-b-
MAPS
34
according to the procedure reported previously. The CRHMS
ꢂ1
(

4, 2 and 0.4 mg ml ) were dispersed in a 1 mL simulated body
A conjugation procedure was carried out according to the
ꢂ1
uid (SBF) buffer solution, then the DOX solution (5 mg mL
,
30–32
literature reported elsewhere.
Firstly, the thiocarbonylthio
4
0 mL) was added. Aer dispersion and stirring under light-
end group of the HAMA-b-MAPS was removed according to the
literature with some modications. Briey, 500 mg of HAMA-b-
DBAM was dissolved in 10 mL N,N-dimethylformamide (DMF)
containing 30 mg of AIBN. The DMF solution was sealed aer
cycling between vacuum and nitrogen three times. Aer stirring
sealed conditions for 24 h, the DOX@CRHMS were obtained by
centrifugation and washed with 20 mL of PBS (pH 7.4), then
ꢁ
dried at 60 C in vacuum.
The DOX@CRHMS@HFMP and DOX@CRHMS@HFMP
without eosin were prepared via self-assembly, as discussed
ꢁ
at 70 C for 5 h, the mixture was precipitated in anhydrous
before. A 5 mg sample was immersed in simulated body uid
ether. The obtained diblock polymer was dried under vacuum
and stored in desiccators for further use. Secondly, folate NHS
ester was prepared by the following procedure: 0.62 g of DCC
and 0.51 g NHS were added to a 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. Then, FA–NHS (30 mg) was added to dry
pyridine containing 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
ꢁ
(
SBF) at 37 C to maintain a constant temperature, and then
different stimuli were introduced to determine the release of
the loaded DOX.
Here, the DOX concentration was determined using a uo-
rescence spectrophotometer at lex ¼ 480 nm and lem ¼ 627 nm.
A standard plot was prepared under identical conditions to
conrm the amount of drug loaded by CRHMS. The drug
loading content and drug loading efficiency can be calculated as
follows:
Drug loading content (wt%) ¼ (weight of loaded drug/weight of
nanocomposites) ꢃ 100%;
ꢁ
1
concentrated and stored in the dark at 4 C. HNMR (400 MHz,
DMSO-d ), d (ppm): 8.63–6.62 (folic acid, CH NH and anthra-
cene), 4.21–3.76 (COOCH CH and CH OH), 2.78 (NHCH and
2 2 2 2
CH NH ), 1.21 (CH ).
2 2 3
Drug loading efficiency (%) ¼ (weight of loaded drug/weight of
drug in feed) ꢃ 100%.
6
2
2
Cell culture and preparation
A549 human alveolar adenocarcinoma (FRꢂ) and human KB
Preparation of CRHMS@HFMP
(
FR+) cell lines (purchased from Shanghai Cell Institute Country
HAMAFA-b-MAPS (20 mg) were dissolved in 1 mL tetrahydro-
furan, and then 0.6 mg eosin in 100 mL DMSO was added. Aer
a continuous stirring for 1 h at RT, eosin and HAMAFA-b-MAPS
would form into HFMP through a p–p stacking process, and
then CRHMS (10 mg) was dispersed in this mixture by soni-
cation for 5 min. Then, 5 mL of distilled water was added to
Cell Bank, China) were cultured as monolayers in a RPMI-1640
medium supplemented with 10% heat-inactivated fetal bovine
ꢁ
serum at 37 C in a humidied incubator (5% CO in air, v/v).
2
In vitro cytotoxicity
the above mixture with vigorous shaking and the resulting The aminoxanthene dye, sulforhodamine B (SRB), was used in
colloid was stirred continually vigorously for 24 h at room an assay for assessing the effects of drug carriers in various
35
temperature to evaporate the tetrahydrofuran. The prepared concentrations. In brief, well-growing KB cells were placed in
4
nanoparticles were isolated by centrifugation, washed with 96-well plates (1.3 ꢃ 10 cells per well) and four duplicate wells
water, dried under vacuum overnight and stored in a desic- were set up in the sample. The culture medium was replaced
cator for further use.
with the medium containing different concentrations of drug
carriers and cultured at 37 C in a humidied incubator (5%
ꢁ
CO
2
in air, v/v) with the cells anchored to the plates. Aer being
Photocleavage of HAMAFA-b-MAPS
cultured for 24 h, the medium was poured away and 10% (w/v)
To study the photocleavage of HAMAFA-b-MAPS, 1% eosin was trichloroacetic acid in Hank's balanced salt solution (100 mL)
ꢁ
added to DMSO-d
6
containing a certain amount of HAMAFA-b- was added and stored at 4 C for 1 h. Then, the stationary liquid
1
MAPS, the HNMR spectrum was evaluated aer the irradiation was discarded, the cells were washed with deionized water ve
of visible light for 2 h.
times before air drying and stained with 0.4% (w/v) SRB solution
To further conrm the photocleavage of the HAMAFA- (100 mL per well) for 30 min at room temperature. Aer
b-MAPS, gel permeation chromatography (GPC) of HAMAFA- removing the SRB, the cells were washed with 0.1% acetic acid
b-MAPS containing 3% eosin in DMF was determined with solution ve times. The bound SRB dye was solubilized with
ꢂ1
irradiation of visible light at certain times.
10 mmol L of a Tris-base solution (150 mL, pH ¼ 10.5). The
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optical density (OD) value of each individual well was calculated
using a spectrophotometer at 531 nm absorbance.
Cellular uptake of DOX@CRHMS@HFMP
4
KB cells were seeded in 96-well plates at 1.3 ꢃ 10 cells per well
ꢁ
and incubated overnight at 3 C in a humidied incubator.
The sample of the DOX@CRHMS@HFMP was dispersed in a
RPMI-1640 medium. The cells were cultured with the
DOX@CRHMS@HFMP solution for a certain time and observed
using confocal laser uorescence microscopy (Olympus, FV
1000) aer washing three times with PBS.
The controlled therapeutic experiment was based on KB
cells. The KB cells were seeded into 96-well cell culture plates
4
at 1 ꢃ 10 per well until adherent and then incubated with the
ꢂ1
DOX@CRHMS@HFMP at a concentration of 100 ug mL for
certain times. Aer removal of the nanoparticles, the cells were
transferred into fresh media and irradiated at 540 nm. Each
irradiation time lasted for 10 min at 30 min intervals, and the
ꢁ
cells were then incubated at 37 C for an additional 48 h before
the standard methyl thiazolyl tetrazolium (MTT, Sigma Aldrich)
assay was used to determine their viabilities, relative to the
control cells.
Characterization
The FT-IR measurements were performed as KBr pellets on a
Nicolet 4700 spectrometer (Thermo Fisher Scientic) in the
ꢂ1
1
range 400–4000 cm . The HNMR spectra were measured by an Scheme 2 Schematic illustration of the synthesis of HAMAFA-b-MAPS.
INOVA 400 MHz NMR instrument. The TEM images were
obtained using a Tecnai G220 electron microscope at an accel-
eration voltage of 200 kV. The number average molecular weight to the solution of the HAMA-b-MAPS. When we applied a green
(
M
n
), weight-average molecular weight (M
index (PDI) were measured by gel permeation chromatography light lter at 500–550 nm) to this solution for 1 h, two new
GPC) utilizing a Waters 1515 pump. The Brunauer–Emmett– aromatic signals at d ¼ 9.69 and 8.08 ppm appeared in the
w
) and polydispersity visible light (mercury lamp of 120 W combined with a green
(
1
Teller (BET) and Barrett–Joyner–Halenda (BJH) analyses were
used to determine the surface area, pore size and pore volume anthraquinone.
HNMR spectrum (inset Fig. S2†) and were proved to be from
and were obtained with a Quantachrome Autosorb 1C appa-
To further conrm the photocleavage of the HAMAFA-b-
ꢁ
ratus at ꢂ196 C under continuous adsorption conditions. The MAPS, GPC measurement was done before and aer the visible
emissions (lex ¼ 540 nm) were obtained using an Edinburgh- light irradiation. The decrease of M
with the irradiation time is
n
9
20 uorescence spectrophotometer. The UV-vis absorption shown in Fig. S3.† Without visible light, the M
n
of the HAMAFA-
spectra were recorded by a Perkin-Elmer Lambda-17 Spectro- b-MAPS showed almost no change, but aer the visible light
1
photometer. Confocal laser scanning microscopy (CLSM) irradiation, a sharp decrease in M
n 2
indicated the O -mediated
images were observed by a confocal laser scanning microscope reaction had occurred and the polymer was cleaved. This result
(
Olympus, FV 1000).
suggested the conversion of HAMA-b-MAPS hydrophobic
segments to hydrophilic ones, with the appearance of eosin
when exposed to the green light.
Results and discussion
Synthesis of HAMAFA-b-MAPS and CRHMS
36,37
The surface of the HMS was enriched with Si–OH,
could be simply modied with C18 and RITC. The FT-IR spectra
which
The amphiphilic copolymer HAMAFA-b-MAPS used here was of the HMS, CHMS and CRHMS are shown in Fig. 1. The Si–OH
ꢂ1
synthesized following the procedure shown in Scheme 2. The bond (950 cm ) in the spectrum of the HMS suggested that a
1
successful conjugation of the FA was conrmed via the HNMR large number of silanol groups existed on the surface of the
1
spectrum of HAMAFA-b-MAPS (Fig. S1†). H NMR spectroscopy HMS for coupling alkyl chains and functional molecules.
was next performed to prove the photocleavage of the DN As shown in the spectrum of CHMS, the strong bond of C–H
x
1
ꢂ1
groups. The H NMR spectrum of the HAMA-b-MAPS in (2890 cm ) revealed the successful growth of alkyl chains on
ꢂ1
different cases is shown in Fig. S2,† the proton signals at d ¼ the silica. Similarly, the absorption peaks around 1700 cm in
8.25 and 7.52 ppm could be ascribed to the DN species. Then, the spectra correspond to the –COOH groups of RITC. The
the eosin sensitizer (3% of copolymer concentration) was added uorescence emission spectrum (lex ¼ 540 nm) of CRHMS is
4
632 | J. Mater. Chem. B, 2013, 1, 4628–4636
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appeared in the diameter and structure between the HMS and
CRHMS. Both the HMS and CRHMS maintained a good mon-
odispersity with an average diameter of 200 nm and a very clear
prole. In contrast, the prole of the CRHMS@HFMP became
blurred and a thin layer of polymer shell with a thickness
around 10 nm was clearly observed aer the encapsulation of
the HFMP (Fig. 2c). Thus, the controlled release pathways for
the encapsulated drug molecules could be well blocked to
prevent premature drug release from the hollow core of the
HMS in normal physical environments, which is very important
to clinical cancer therapy. A boundary line between the polymer

lm and the HMS shell could be clearly observed in the enlarged
Fig. 1 FT-IR spectra of HMS, CHMS and CRHMS.
TEM images of the nanocomposites (Fig. S5†), which further
conrmed the encapsulation of the polymer lm. Furthermore,
the prepared CRHMS@HFMP still retained a good mono-
dispersity with an average diameter of about 220 nm, which was
in accordance with the ndings from the dynamic light scat-
tering (DLS) measurements (Fig. 2d).
shown in Fig. S4,† which further conrms the successful
conjugation between RITC and surface of HMS.
Assembly of CRHMS@HFMP
To ensure the appearance of eosin in the nanocomposites,
In our approach, CRHMS@HFMP was synthesized via a self- UV-vis was used. The UV-vis spectra of the nanocomposites with
assembly process. HAMAFA-b-MAPS (20 mg) were dissolved in and without eosin is shown in Fig. S6;† the enhanced absor-
1
mL tetrahydrofuran, and then 0.6 mg eosin in 100 uL DMSO bance at 525 nm strongly demonstrates the appearance of this
was added. Aer continuous stirring for 1 h, eosin had strong complex.
tendency to bind with the DN species to give HFMP because of
p–p stacking reactions. Then CRHMS (10 mg) was dispersed in
Drug loading and release
this solution followed by the addition of 5 mL of distilled water.
Aer evaporation of the THF at room temperature, the prepared DOX was used here, as an model anti-cancer drug, to examine
amphiphilic HFMP would form micelles in water and hydro- the drug loading and release behaviour of the CRHMS@HFMP.
phobic CRHMS would be encapsulated to form core–shell DOX was rst loaded into the hollow core of the CRHMS, and
nanocomposites, and long alkyl chains of hydrophobic groups the N adsorption–desorption isotherms (Fig. S7†) of the HMS,
2
of polymer would strongly associate with the hydrophobic C18 CRHMS and DOX@CRHMS revealed that the modication of
alkyl chains on the HMS surface through hydrophobic van der C18 and RITC and loading of the DOX molecules have not
38,39
Waals interactions.
altered the pore structure of the HMS, which was necessary for
The detailed morphological features of the prepared nano- the drug loading and release. The drug loading content was
particles were examined by TEM. As shown in Fig. 2, aer being examined by measuring the DOX concentration before and aer
graed with C18 and RITC (Fig. 2b), no signicant difference being loaded by the CRHMS. The theoretical loading content
was set at 5%, 10% and 50% and the DOX loading efficiency was
measured at 78, 75 and 70%, respectively (Table 1). As can be
seen, the drug loading content and efficiency showed that the
HMS was much more suitable for drug delivery than common
mesoporous silica, owing to its hollow core. Furthermore, the
HMS modied with the hydrophobic long alkyl chains surface
was more suitable than the pristine HMS for the water-insoluble
drug molecule loading.
Then, DOX@CRHMS@HFMP was prepared through a self-
40
assembly process at a low drug loading content (ca. 5 wt%),
and by evaluating the DOX concentration aer the centrifuga-
tion of the nanocomposites, 5.6% of the DOX loaded in the
HMS was lost in this process. Then, different stimuli were
introduced to determine the release of the loaded DOX. The
concentration of DOX was evaluated at certain times aer the
centrifugation of the nanocomposites. The release quantity
versus time in different cases is shown in Fig. 3. Without any
stimuli, only less than 5% of DOX was released from HMS aer
100 h. When we exerted green light and PS, a burst release
(>50% in 10 h) was observed due to the uncovered pore caused
Fig. 2 TEM images of HMS (a), CRHMS (b), CRHMS@HFMP (c), and DLS of HMS
and CRHMS@HFMP (d).
by the degradation of HAMAFA-b-MAPS and the different
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Paper
Table 1 Drug loading content and drug loading efficiency of CRHMS
Theoretical drug loading
content (wt%)
Drug loading efficiency
(%)
a
Drug loading content (wt%)
5
1
5
4.1
8.1
38.5
78
75
70
0
0
a
Drug loading content for DOX was determined by uorescence measurement.
To quantify the cellular uptake of the nanocarriers for the
two different cell lines, the mean uorescence intensity of the
FR+ and FRꢂ cells with CRHMS@HFMP and FA-free nano-
composites (CRHMS@HMP) in solution at different incubation
times are shown in Fig. 6. No signicant differences could be
observed in the uorescence intensity between the two nano-
composites when incubated with A549 and KB cells for 0.5 h.
But aer 2 h, a signicant increase in the uorescence intensity
could be seen in the KB cells incubated with the FA conjugated
nanocomposites when compared to other incubations. This
result demonstrated that both of the two nanocomposites were
internalized in a short time by the KB and A549 cells through
the same endocytosis process. 2 h later, the remarkable changes
in the uorescence intensity demonstrated that the targeting
moiety offered by folic acid is efficient at enhancing the tumor
cell targeting in vitro, because the FA-conjugated nano-
composites was taken up by the KB cells through a folate
receptor-mediated endocytosis.
For a further application of the nanocarriers, the DOX was
loaded (drug loading content set as 38.5%) to investigate
whether the therapeutic could be controlled by using the
current nanocarrier. As a control, the photothermal effects and
apoptosis induced by irradiation were rst investigated. The
results shown in Fig. 7 indicated that no obvious decrease in the
cell viability was observed aer irradiation with visible light,
which suggested that the visible light exerted little damage on
the living cells, as expected. Aer incubation of the nano-
carriers, almost no decrease in the living cells was observed
without visible light irradiation, which further conrmed that
the nanocarriers showed almost no cytotoxicity in living cells,
and could prevent the DOX leakage well.
Fig. 3 Release of DOX in vitro from the drug-loaded DOX@CRHMS@HFMP with
ꢁ
different stimuli at 37 C.
concentration of DOX inside and outside of CRHMS. Aer 20 h,
a lower concentration difference and the hydrophobic interac-
tion between the drug and the long alkyl chains could lead to a
long-term drug release (70% aer 100 h), which is important for
41
further cancer treatment.
This phenomenon strongly
demonstrated that the amphiphilic copolymer provides a
favourable sensitivity to visible light and the CRHMS@HFMP is
capable of serving as a controlled drug release nanocarrier.
Evaluation of cytotoxicity and drug uptake
The sulforhodamine B (SRB) assay was used to assess the
cytotoxicity of the nanocomposite. Aer incubation in the
nanocomposite for 72 h, the cells displayed a high cell viability
(>80%) as shown in Fig. 4. Even at a high concentration, the
cytotoxicity still remained at a low level. The results above
proved the polymer shell to be biocompatible and non-toxic.
These characteristics are necessary for further biomedical
applications.
The RITC was graed onto the surface of the HMS, and then
the CRHMS was encapsulated by the HFMP. For the graed
RITC on the surface of the HMS, the distribution of the nano-
composites could be observed by uorescence imaging. Herein,
in vitro experiments based on folate receptor positive (KB) and
negative (A549) cancer cells were used to demonstrate the
selective release and targeting properties of the nano-
composites. The uptake of the KB and A549 cells and the
intracellular distribution of the nanocomposites with FA groups
were studied by CLSM. Aer being incubated with the nano-
composites for 0.5 h and 2 h, the nanocomposite distributions
are shown in Fig. 5.
Fig. 4 In vitro cell viability of the CRHMS@HFMP at different concentrations.
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Journal of Materials Chemistry B
cells survived aer 6 h irradiation, which conrmed that the
therapeutic could be controlled by using the current
nanocarrier.
Conclusion
In summary, a visible light triggered drug delivery system was
successfully prepared for controlled drug release and cell
imaging. HMS with a hollow core was used as the drug
container and a high drug loading content could be achieved.
Then, the HMS was easily encapsulated by a FA-conjugated
visible light-sensitive amphiphilic polymer via a self-assembly
process, due to modication of C18 on the HMS. This nano-
carrier allowed drug release from the HMS when exposed to
green light, because of the degradation of the amphiphilic
polymer. Since the HMS was modied with RITC, the cancer
targeting process could be easily tracked by uorescence
microscopy.
Fig. 5 CLSM of FRꢂ and FR+ incubated with CRHMS@HFMP and CRHMS@HMP
for 0.5 h and 2 h.
Acknowledgements
We gratefully acknowledge the nancial support provided by
the National Natural Science Foundation of China (21076134,
2
(
1176164), the Natural Science Foundation of Jiangsu Province
BK2012625), a Project Funded by the Priority Academic
Program Development of Jiangsu Higher Education Institutions
PAPD), the Joint Research Projects of the SUN-WIN Joint
(
Research Institute for Nanotechnology and the Natural Science
Foundation of the Jiangsu Higher Education Institutions of
China (13KJB430022).
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