Facile preparation of coating fluorescent hollow mesoporous silica nanoparticles with pH-sensitive amphiphilic diblock copolymer for controlled drug release and cell imaging

Article abstract of DOI:10.1039/c2sm07320j

A smart fluorescent drug carrier based on hollow mesoporous silica (HMS) nanoparticles was prepared step by step. First, HMS nanoparticles were doped with lanthanide rare-earth nanocrystals (YVO₄:Eu³⁺). Then the surface of HMS@YVO

Full text of DOI:10.1039/c2sm07320j

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PAPER
Facile preparation of coating fluorescent hollow mesoporous silica
nanoparticles with pH-sensitive amphiphilic diblock copolymer for controlled
drug release and cell imaging†
a
a
a
a
a
a
b
Xiao Mei, Dongyun Chen, Najun Li, Qingfeng Xu, Jianfeng Ge, Hua Li, Baixia Yang, Yujie Xu
b
*
*
a
*
and Jianmei Lu
Received 8th December 2011, Accepted 14th February 2012
DOI: 10.1039/c2sm07320j
A smart fluorescent drug carrier based on hollow mesoporous silica (HMS) nanoparticles was prepared
step by step. First, HMS nanoparticles were doped with lanthanide rare-earth nanocrystals
(YVO4:Eu3+). Then the surface of HMS@YVO4:Eu3+ was modified by octadecyltrimethoxysilane
(C18). Afterwards, it was coated by designed pH-sensitive amphiphilic diblock copolymer
(poly(MPEG-b-DBAM), PMD) through hydrophobic van der Waals interactions. The results of
characterization such as transmission electron microscopy (TEM) and Fourier transform infrared
spectroscopy (FT-IR) reveal that the material shows excellent monodisperse spherical morphology and
narrow size distribution (180 nm) with hollow-core@mesoporous-silica-shell@thin-polymer-film
structure. The multifunctional system HMS@YVO4:Eu3+@C18@PMD was utilized to deliver the
model drug ibuprofen (IBU), and the drug loading content of the system is as high as 834 mg/g (drug/
carrier). Due to the coated pH-sensitive polymer film, the loaded drug is selectively released in mildly
acidic environment. The time of release of about 80% drug was however prolonged from 50 to 150 h (at
pH ¼ 5.0) by the effect of modified C18, which has thus achieved longer-term release. Besides, the
prepared material is easily imported into human mouth epidermal carcinoma (KB) cells and showed
good and stable red fluorescence, which is suitable for cell imaging.
and HMS,21–26 are generally considered as ideal drug delivery
candidates due to their high level of biocompatibility and
adjustable morphologies, effective property to deliver anticancer
drug into tumors and excellent tumor suppressing effect,27 and
modifiable outer surface with abundant Si–OH.
Introduction
Today, thousands of anti-cancer drugs such as doxorubicin
(DOX), paclitaxel (PTX) and hydroxycamptothecine (HCPT)1–4
have been applied in clinical treatment. However, common anti-
cancer drugs are water-insoluble, and display toxicity and side
effects towards normal cells. Hence, many researchers pay great
attention to ‘‘drug delivery’’, and a variety of materials such as
inorganic nanoparticles,5–8 polymeric nanoparticles,9–11 lipo-
somes,12 dendrimers,13,14 and others15 have been designed as
smart materials which are able to deliver anti-cancer drugs to
targeted sites in a controlled manner. Among the drug delivery
materials mentioned above, the family of mesoporous silica
nanoparticles (MSNs), such as MCM-n,16,17 SBA-n,18,19 CMS20
To achieve controlled drug release, MSNs are often designed
to be coated by other functional materials, such as functional
nanoparticles,6,28–30 supramolecular nanovalves31,32 and bio-
macromolecular caps,33–35 especially coating of MSNs with
functional polymers.36–38 Due to its broad applicability in drug
delivery, gene delivery and other biomedical applications, poly-
mer-coated systems have been extensively studied. Many
methods including free radical polymerization, reversible addi-
tion–fragmentation chain transfer (RAFT), emulsion polymeri-
zation, and solid-oil-in water (S/O/W) emulsification have been
developed. For example, Gao and co-workers coated several
layers of poly-methylacrylic acid-co-vinyl triethoxysilane (PMV)
onto mesoporous silica by the free radical polymerisation
method, and this material showed pH responsive release of
ibuprofen.36 Lin and co-workers coated thermosensitive poly-N-
isoproplyacrylamide (PNIPAm) onto the outer surface of MSNs
via RAFT and this composite can be utilized to prepare nano-
valves based on thermally responsive MSN, where drugs can be
released at a certain temperature.39 However, in such systems, the
aLaboratory of Absorbent Materials and Techniques for Environment,
College of Chemistry, Chemical Engineering and Materials Science,
Soochow University, Suzhou, China 215123. E-mail: lujm@suda.edu.cn;
linajun@suda.edu.cn; Fax: +86 (0) 512-6588 0367; Tel: +86 (0)512-
6588 0368
bJiangsu Provincial Key Laboratory of Radiation Medicine and Protection,
Medical College of Radiation Medicine and Protection, Soochow
University, Suzhou, China, 215123. E-mail: dbnm@suda.edu.cn; Fax:
+86 (0) 512-65884830; Tel: +86 (0) 512-6588 006
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c2sm07320j
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Soft Matter, 2012, 8, 5309–5316 | 5309

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drug loading content is actually poor due to the blocking of the
polymer in pore channels of MSNs when the drug was loaded
after polymer was coated onto MSNs. Hence, it is crucial to
develop a strategy that can offer the dual advantage of high drug
loading content and effective functional polymer coating.40
In this paper, we designed an easy method to solve the problem
above (Scheme 1). We first chose HMS as support for a high drug
loading content owing to its hollow core,22,23 then the surface of
HMS was modified with long-chain hydrocarbon octadecyl-
trimethoxysilane (C18) moieties, and this strategy could have
several advantages. First, it can modify the chemical interaction
between the pore surface and the adsorbed drug, which can
decrease the wettability of the surface by aqueous solutions and
increase the amount of hydrophobic drug adsorption and further
delay the drug release.41 Secondly, the modified alkyl chains are
not able to close the pores but are suitable to interact with the pH
stimuli-responsive amphiphilic diblock copolymer we designed
through hydrophobic van der Waals interactions,42,43 so the
HMS would be easily coated by functional polymer. The drug
was loaded in HMS and then blocked by coated polymer. When
specifically recognized and internalized by tumor cells, the coated
pH-responsive polymer would hydrolyze into biocompatible
polymer due to cleavage of acetal moieties in the weakly acidic
endosomal/lysosomal compartments, resulting in encapsulated
drug release. Furthermore, the surface of HMS was modified
with fluorescent material of lanthanide-doped rare-earth nano-
crystals for cell imaging rather than organic dyes or quantum
dots (QDs), due to their good optical properties, high chemical
and photochemical stability and low toxicity.44–46 Hence, the
polymer-coated system, prepared by an easy strategy, has
potential applications of controlled drug release and cell
imaging.
Experimental
Materials
(2-(Acryloyloxy)ethyl)trimethylammonium chloride (AETAC,
80 wt% in water), 2,20-azobis(2-methylpropionamidine) dihy-
drochloride (V-50, >97.0%), hexadecyltrimethylammonium
bromide (CTAB, >99.0%) and octadecyltrimethoxysilane (C18)
were purchased from Aldrich. Methoxy poly(ethylene glycol)
(MPEG-OH, 5000 g molꢀ1, Fluka) were dehydrated by azeo-
tropic distillation of water in toluene, precipitated with cold ethyl
ether, filtered, washed with diethyl ether and dried in vacuum.
Tetraethoxysilane (TEOS), 2-bromoisobutyryl bromide, euro-
pium oxide (Eu2O3), yttrium(III) oxide (Y2O3) and ammonium
vanadium oxide (NH4VO3) were purchased from Alfa without
any purification. Styrene (St, >99.0%) was washed through an
inhibitor remover column for removal of tert-butyl catechol and
then distilled under reduced pressure prior to use. Tetrahydro-
furan (THF) and cyclohexanone were purified by reduced pres-
sure distillation. CuBr was purified until colorless by stirring in
glacial acetic acid, consecutive washing with absolute ethanol
and diethyl ether, and keeping under vacuum. Other reagents
were commercially available and used as received.
Synthesis of hollow mesoporous spherical (HMS) particles
The hollow mesoporous spherical particles were synthesized
according to the literature47 with some modifications. To prepare
the polystyrene latex templates, 1.0 g of AETAC (80 wt% in
H2O) was dissolved in 390.0 g water in a 500 mL round-bottom
flask. 40.0 g styrene was added slowly to the solution and kept
stirring at 800 rpm by mechanical raking for 30 min. The mixture
ꢁ
was purged with nitrogen for 20 min and then heated to 90 C
with an oil-bath. Afterwards, 10 mL of an aqueous solution
containing 1.0 g V-50 was added. The emulsion was kept at
90 ꢁC for 24 h under nitrogen. The polystyrene latex was
obtained by centrifugation at 18000 rpm for 15 min, and washed
with ethanol several times. To obtain the HMS, 0.8 g of CTAB
was dissolved in a mixture of 29.0 g of water, 12.0 g of ethanol
and 1.0 ml of aqueous ammonia solution. 930 mg 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 magnetically stirred for 30 min
before adding dropwise 4.0 g of TEOS. The resulting mixture
was kept stirring at room temperature for 48 h before the mes-
oporous silica coated latex was harvested by centrifugation at
7000 rpm for 40 min. The precipitate was washed with copious
amounts of ethanol and then dried at room temperature. Finally
the material was calcined in air at 600 ꢁC for 8 h using a heating
Scheme 1 Schematic depiction of the structure of IBU@HMS@Y-
VO4:Eu3+@PMD and controlled release by degradation under weakly
acidic conditions.
rate at 3 C minꢀ1
.
ꢁ
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Luminescence functionalization of HMS by YVO4:Eu3+
(HMS@YVO4:Eu3+)
J ¼ 12.92 Hz, 2H, CHCH2O), 3.95 (t, J ¼ 6.59 Hz, 2H,
C6H4OCH2), 2.01 (s, 3H, CH3), 1.81–1.72 (m, 2H, CH2CH2O),
1.49–1.38 (m, 2H, CH2CH2CH2O), 1.26 (m, 16H, CH2), 0.88 (t,
J ¼ 6.68 Hz, 3H, CH2CH3). 13C NMR (75 MHz, CDCl3),
d 167.45 (s, 1C, COO), 159.95 (s, 1C, C), 136.28 (s, 1C, CCH3),
132.25 (s, 1C, C), 127.57 (s, 2C, CH), 126.50 (s, 1C, CCH2),
114.52 (s, 2C, CH), 74.21 (s, 1C, CH2CO), 69.25 (s, 2C, CH2CO),
68.28 (s, 1C, CHCH2O), 32.16 (s, 1C, CH2CH2CH3), 29.28–
29.88, (m, 7C, CH2), 26.24 (s, 1C, CH2CH2CH2O), 22.94 (s, 1C,
CH2CH3), 18.52 (s, 1C, CCH3), 14.39 (s, 1C, CH3).
Y2O3 (0.429 g, 1.9 mmol), Eu2O3 (0.0352 g, 0.1 mmol) and
NH4VO3 (0.232 g, 2 mmol) were added into dilute HNO3
(15 mL, 3 mol Lꢀ1). After dissolving completely, the solution was
added to a water–ethanol solution (v/v ¼ 1/7), then citric acid
(0.84 g, 4 mmol) and poly(ethylene glycol) (PEG, 10000 g/mol)
was added. After stirring for 1 h, the solution turned into a blue
homogenous sol, followed by adding 200 mg HMS and stirring
for another 3 h. The product was separated by centrifugation,
dried at 100 ꢁC for 1 h, and calcined at 500 ꢁC for 2 h. The
obtained product was denoted as HMS@YVO4:Eu3+.
Synthesis of diblock copolymer poly(MPEG-b-DBAM) (PMD)
The diblock copolymer MPEG-b-DBMA was synthesized by
atom transfer radical polymerization (ATRP). First, the mac-
roinitiator MPEG-Br was synthesized by reacting MPEG-OH
with 2-bromoisobutyryl bromide. A solution of MPEG-OH
(5000, 10 g, 2 mmol) and triethylamine (400 mg, 4 mol) in
anhydrous THF (35 mL) was slightly cooled in an ice–water
bath. Then, a solution of 2-bromoisobutyryl bromide (1.72 mL,
14 mmol) in anhydrous THF (5 mL) was slowly added to the
reaction mixture. The solution was warmed to room temperature
and stirred for 48 h. The mixture was poured into water (100 mL)
and extracted with dichloromethane (5 ꢂ 20 mL). The organic
extracts were washed successively with HCl (1 M), NaOH (1 M)
and saturated NaCl solution, and dried by anhydrous MgSO4
overnight. Then the solvent was removed under reduced
pressure. The crude extract was dissolved in a minimum of
dichloromethane and then precipitated in diethyl ether. The
macroinitiator MPEG-Br was recovered by filtration.
Modification of the external surface of HMS@YVO4:Eu3+ with
octadecyltrimethoxysilane (C18) (HMS@YVO4:Eu3+@ C18)
100 mg HMS@YVO4:Eu3+ was first dispersed in 20 mL anhy-
drous acetonitrile, then 5 mL C18 was added, and the obtained
suspension was stirred for 24 h and collected by centrifugation,
washed with acetonitrile and ethanol several times, and dried
under vacuum. The obtained white solid was denoted as
HMS@YVO4:Eu3+@C18.
Synthesis of 4-n-dodecyloxybenzal acetal monomer (DBAM)
p-Hydroxybenzaldehyde (5 g, 40.9 mmol) was dissolved in
150 mL acetone solution containing anhydrous potassium
carbonate (16.8 g, 122 mmol), then 1-bromododecane (12.2 g,
49.2 mmol) was added slowly. After heating under reflux with
stirring for 24 h, the acetone was evaporated. Then 100 mL water
and 100 mL ethyl acetate were added. The organic layer was
collected and washed with saturated NaCl solution twice and
dried by anhydrous MgSO4 overnight. After evaporating the
ethyl acetate, the crude 4-n-dodecyloxybenzaldehyde (DBD)
product was purified by column chromatography (ethyl acetate–
petroleum ether, 1:5).
To synthesize PMD, MAPEG-Br (2 g, 0.4 mmol) was added to
a solution containing PMEDTA (76.25 mg, 0.44 mmol), CuBr
(45.76 mg, 0.44 mmol), and DBAM (4.32 g, 10 mmol) in cyclo-
hexanone (2 mL). The mixture was degassed with argon for 30 min
at room temperature and then heated overnight at 90 ꢁC. Then the
mixture was diluted by 10 mL THF and filtered on neutral
aluminum oxide gel, with THF as an eluent, to remove copper
bromide. Finally, the product was concentrated and then precip-
itated in diethyl ether. The PMD was recovered by filtration.
The obtained DBD (8.7 g, 30 mmol) was dissolved in 50 mL
toluene with p-toluenesulfonic acid (PTSA) (0.5 g) as catalyst.
Then glycerol (2.76 g, 30 mmol) was added and the mixture
refluxed with vigorous stirring for 14 h, and the water by-product
was removed by the oil–water separator. Then the mixture was
evaporated and washed with potassium carbonate solution (1%,
80 mL) to remove the acid catalyst and any remaining glycerol.
The crude product was purified by column chromatography
(ethyl acetate–petroleum ether, 1 : 2), and a white product 4-n-
dodecyloxybenzal acetal (DBA) was obtained.
Drug loading experiment (IBU@HMS@YVO4:Eu3+@C18)
Ibuprofen(IBU)wasusedasmodeldrugaccordingtotheliterature
with some modifications.22,23 Ibuprofen (300 mg) was dissolved in
hexane (5 mL), then, HMS@YVO4:Eu3+@C18 (100 mg) was
added to this solution. After stirring for 24 h at room temperature,
the HMS@YVO4:Eu3+@C18with adsorbed IBU was separated by
centrifugation and washed with a solution oꢁf pH 1.4 for several
times. Finally, the product was dried at 60 C in vacuum. This
product is denoted as IBU@HMS@YVO4:Eu3+@C18.
To synthesize the final product DBAM, DBA (4.0 g, 11 mmol)
and triethylamine (4.5 g, 44 mmol) were dissolved in anhydrous
ꢁ
tetrahydrofuran (20 mL) and the mixture was cooled to 0 C in
an ice-bath. Then anhydrous tetrahydrofuran (5 mL) with
methacryloyl chloride (2.3 g 22 mmol) was added slowly. After
stirring at room temperature overnight, the mixture was filtered
off and evaporated, and the crude product was purified by
column chromatography (ethyl acetate–petroleum ether, 1 : 8).
Anal. Calc. for C26H40O5: C, 72.19, H, 9.32, O, 18.49. Found: C,
72.30, H, 9.28, O, 18.42%. 1H NMR (400 MHz, CDCl3), d (ppm):
7.41 (d, J ¼ 8.39 Hz, 2H, C6H4), 6.89 (d, J ¼ 8.41 Hz, 2H, C6H4),
6.30 (s, 1H, CCH2), 5.65 (s, 1H, CCH2), 5.52 (s, 1H, C6H4CH),
4.75 (s, 1H, CHO), 4.31 (d, J ¼ 12.88 Hz, 2H, CHCH2O), 4.18 (d,
To evaluate the amount of drug loaded by HMS@Y-
VO4:Eu3+@C18, UV-Vis spectroscopy was used for analysis.
First, the calibration curve of ibuprofen was determined by
taking absorbance vs. ibuprofen concentration between 0 and
2 mg mlꢀ1 as parameters, and the calibration curve was fitted to
the Lambert–Beer law as follows:
A ¼ 0.02584 + 0.99556C
where A is the absorbance and C is the concentration (mg mlꢀ1).
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After adsorption, the IBU solution (0.1 mL) was extracted and
diluted to 10 mL, and then analyzed by UV-Vis spectroscopy at
a wavelength of 265 nm. The calculation of drug loading content
is discussed below.
prepared in RPMI-1640 medium. Cells were cultured with the
HMS@YVO4:Eu3+@C18@PMD solution for a certain time
and observed using confocal laser fluorescence microscopy
(Olympus, FV 1000) after washing three times with PBS.
Characterization. FT-IR measurement was performed as KBr
pellets on a Nicolet 4700 spectrometer (Thermo Fisher Scientific)
in the range of 400–4000 cmꢀ1. Gel permeation chromatography
(GPC) analysis was carried out on a Waters 1515 pump and
a differential refractometer, THF was used as a mobile phase at
Synthesis of PMD-coated IBU@HMS@YVO4:Eu3+@C18
(IBU@HMS@YVO4:Eu3+@C18@PMD)
IBU@HMS@YVO4:Eu3+@C18 (10 mg) and PMD (20 mg) were
dissolved in tetrahydrofuran (1 mL). Then, 5 mL of distilled
water was added to this solution with vigorous shaking. The
mixture was kept stirring for 24 h at room temperature to
evaporate tetrahydrofuran. After that, the product was sepa-
rated by centrifugation and washed with distilled water several
times to remove the unbound copolymer. The obtained product
was denoted as IBU@HMS@YVO4:Eu3+@C18@PMD.
a flow rate of 1.0 mL minꢀ1. H and 13C NMR spectra were
1
measured by an INOVA 400 MHz NMR instrument with CDCl3
as deuterated solvent. Transmission electron microscopy
(TEM) and high-resolution transmission electron microscopy
(HRTEM) images were obtained by a TecnaiG220 electron
microscope at an acceleration voltage of 200 kV. The sample for
TEM and HRTEM observations was prepared by placing a drop
of the dispersion of nanoparticles on copper grids. Energy
dispersive spectrometry (EDS) was taken on a Hitachi S-4700
equipped with an energy-dispersive X-ray spectrum. Room-
temperature emission and excitation spectra were carried out by
using an Edinburgh-920 fluorescence spectrophotometer. Bru-
nauer–Emmett–Teller (BET) and Barrett–Joyner–Halenda
(BJH) analysis used to determine the surface area, pore size and
pore volume were obtained with a Quantachrome Autosorb 1C
In vitro experiments
In vitro drug-release study. The prepared IBU@HMS@Y
VO4:Eu3+@C18@PMD sample (15 mg) was immersed in simu-
ꢁ
lated body fluid (SBF) at 37 C to maintain a constant temper-
ature. The obtained colloid was divided into three equivalent
parts and adjusted to different pH values by acetate buffer. The
drug concentration was analyzed by UV-Vis spectroscopy.
ꢁ
apparatus at ꢀ196 C under continuous adsorption conditions.
Cell culture and preparation. Human mouth epidermal carci-
noma (KB) cell line (purchased from Shanghai Cell Institute
Country Cell Bank, China) were cultured as a monolayer in
RPMI-1640 medium supplemented with 10% heat-inactivated
fetal bovine serum at 37 ꢁC in a humidified incubator (5% CO2 in
air, v/v).
The UV-Vis absorption spectra were measured on a TU-1901
spectrophotometer. Confocal laser scanning microscopy
(CLSM) images were observed by confocal laser scanning
microscope (Olympus, FV 1000).
Results and discussion
In vitro cytotoxicity. The aminoxanthene dye, sulforhodamine
B (SRB), was used as an assay for assessing the effects of drug
carriers in various concentrations.48 In brief, well-growing KB
cells were placed in 96-well plates 1.3 ꢂ 104 cells per well) and
four duplicate wells were set up in the sample. The culture
medium was replaced with the medium containing different
concentrations of drug carriers (0, 0.390625, 0.78125, 1.5625,
Synthesis and characterization of
IBU@HMS@YVO4:Eu3+@C18@PMD
Synthesis and characterization of diblock copolymer PMD. The
synthetic process of PMD is shown in Scheme 2. Three diblock
copolymers with different Mn and PDI were synthesized by
varying the mole ratio of the macroinitiator and the monomer.
Based on an evaluation of the characterization data of the
diblock copolymers, PMD polymerized with macroinitiator and
the monomer feed ratio of 1 : 25 was chosen for further investi-
3.125, 6.25, 12.5, 25, 50, 100 mg mLꢀ1) and cultured at 37 C in
ꢁ
a humidified incubator (5% CO2 in air, v/v) with the cells
anchored to the plates. After being cultured for 24 h, the medium
was poured away and 10% (w/v) trichloroacetic acid in Hank’s
balanced salt solution (100 mL) was added and stored at 4 ꢁC for
1 h. Then the stationary liquid was discarded, the cells were
washed with deionized water five times before air drying and
stained with 0.4% (w/v) SRB solution (100 mL per well) for 30
min at room temperature. After removing the SRB, the cells were
washed with 0.1% acetic acid solution five times. Bound SRB dye
was solubilized with 10 mmol Lꢀ1 tris-base solution (150 mL,
pH ¼ 10.5). The optical density (OD) value of each individual
well was calculated using a spectrophotometer at 531 nm
absorbance.
1
gation (Table S1, ESI†). In addition, H NMR of PMD clearly
showed peaks corresponding to DBMA besides signals assign-
able to MPEG (Fig. S1, ESI†). These results signified that the
structurally well-defined diblock copolymer PMD was success-
fully synthesized.
Synthesis
and
characterization
of
IBU@HMS@Y
VO4:Eu3+@C18. The surface of HMS shell was first functional-
ized by the deposition of YVO4:Eu3+ phosphor layer through
a sol–gel process. From Fig. 1a we can see the noticeable
difference between the hollow core and the mesoporous shell of
HMS@YVO4:Eu3+, which suggest that HMS@YVO4:Eu3+
maintain well-defined hollow, mesoporous structures and keep
the ability of loading drug. The clear lattice fringes deposited
on the SiO2 matrix in the HRTEM image (Fig. 1b) further
confirm the high crystallization of the product. The average
Cellular uptake of HMS@YVO4:Eu3+@C18@PMD. KB cells
were seeded in 96-well plates 1.3 ꢂ 104 cells per well) and incu-
ꢁ
bated overnight at 37 C in a humidified incubator. The disper-
sion of the HMS@YVO4:Eu3+@C18@PMD sample was
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of Si–OH (950 cmꢀ1) suggest a large number of silanol groups
still exist on the surface of the rare earth-modified HMS, which
play a key role for coupling alkyl chains. Besides, the band of OH
(3429 cmꢀ1), Si–O–Si (1092 cmꢀ1) and H2O (1635 cmꢀ1) are also
present. After coupling C18, the new band of C–Hx (2890 cmꢀ1
)
clearly confirms the successful modification of C18. For
IBU-loaded HMS@YVO4:Eu3+@C18, the obvious new band at
1720 cmꢀ1 which is assigned to –COOH confirms the successful
loading of IBU into HMS@YVO4:Eu3+@C18.23,46 After coating
the PMD, The absorption bands related with C]O (1740 cmꢀ1
)
and skeleton vibrations (1520 and 1620 cmꢀ1) of the benzene ring
were observed, confirming that the polymer coated the HMS
successfully.
Synthesis
and
characterization
of
IBU@HMS@Y
VO4:Eu3+@C18@PMD. In our strategy, the prepared PMD
would strongly associate with the hydrocarbon coupling agents
C18 on the surface of HMS through hydrophobic van der Waals
interactions owing to their large number of hydrophobic
alkyl chains. The macroscopic state and microstructure of
IBU@HMS@YVO4:Eu3+@C18@PMD were evaluated by
photographs and TEM, respectively. In visible light (Fig. 3a), the
pure HMS has excellent dispersibility in water owing to the
abundant silanol groups at the surface (Fig. 3a-1). After coupling
C18, the nature of HMS was transformed into hydrophobic,
which was easily dispersed in hexane and could no longer
disperse in water well (Fig. 3a-2). The C18 plays a key role in the
interaction between HMS and PMD, so IBU@HMS@Y-
VO4:Eu3+@C18 was easily coated by PMD via self-assembly.
Finally, the prepared IBU@HMS@YVO4:Eu3+@C18@PMD
would restore hydrophilicity because of the hydrophilic segment
in PMD (Fig. 3a-3). Under UV irradiation (Fig. 3b), no fluo-
rescence was observed in an aqueous solution of pure HMS
(Fig. 3b-1), while red fluorescence was evident for a hexane
solution of IBU@HMS@YVO4:Eu3+@C18 (Fig. 3b-2) and
aqueous solution of IBU@HMS@YVO4:Eu3+@C18@PMD
(Fig. 3b-3), respectively. These results indicated IBU@HM-
S@YVO4:Eu3+@C18@PMD with excellent water-solubility and
good fluorescence was prepared.
Scheme 2 Schematic illustration of the synthetic route of PMD.
To observe the microstructure, TEM images of IBU@HM
S@YVO4:Eu3+@C18@PMD (Fig. 3e–3f) were compared with
Fig. 1 TEM images (a), HRTEM (b) and EDX (c) (inset: acquire
HAADF images) of HMS@YVO4:Eu3+
.
distances between adjacent lattice fringes are 0.35 nm, which are
close to the d200 spacing (0.355 nm) of the tetragonal YVO4 phase
(JCPDS No. 17-0341). EDX (Fig. 1c) and EDS (Fig. S3, ESI†)
spectra further confirmed the presence of silicon (Si), oxygen (O),
yttrium (Y), and vanadium (V) in the HMS@ YVO4:Eu3+
sample. The signals of Cu and C in EDX are from the copper grid
and carbon membrane for measurement. All of the results above
reveal successful preparation of HMS@YVO4:Eu3+ with
a hollow core structure and mesoporous channels.
The FT-IR spectra of HMS@YVO4:Eu3+, HMS@Y
VO4:Eu3+@C18 and IBU@HMS@YVO4:Eu3+@C18 are shown
in Fig. 2. In the spectrum of HMS@YVO4:Eu3+, the strong bands
Fig. 2 FTIR spectra of HMS@YVO4:Eu3+, HMS@YVO4:Eu3+@C18
and IBU@HMS@YVO4:Eu3+@C18.
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reduction of UV absorbance between the IBU solution before
and after adsorption reflects the reduction of drug concentration,
which means drug was loaded into HMS@YVO4:Eu3+@C18; up
to 834 mg/g (drug/carrier) of the drug loading content was
calculated according to the Beer–Lambert law mentioned above.
Such a high drug loading content may be mainly attributed to the
hollow core which can store more drug molecules than normal
silica nanoparticles.23 Besides, the modification of the long alkyl
chains changed the surface of HMS into hydrophobic, increasing
the interaction between surface and drug molecules, making the
drug more easily loaded into HMS.41
Drug release behavior. To study the drug release behavior of
the prepared nanoparticles, we first compared the drug release
rate of pure HMS and HMS@YVO4:Eu3+@C18 in SBF at 37 ꢁC
(Fig. 5). It can be seen that within 10 h, about 70% of IBU can be
quickly released from pure HMS, but only about 20% of IBU
was released from HMS@YVO4:Eu3+@C18. Even 45 h later,
only 40% of IBU was released from HMS@YVO4:Eu3+@C18.
The relatively quick drug release of pure HMS may be because
that the release media would easily penetrate into the core of pure
HMS and the interaction between the Si–OH and carboxyl
groups of IBU is weak, so the drug would easily diffuse from the
system along release media filled pore channels. However, for the
sample modified by long alkyl chains, the wettability of system
would be reduced, and the penetration of release media would be
more difficult. Besides, the hydrophobic interaction between
drug molecules and long alkyl chains would further delay the
drug release. Therefore, a long-term drug release system can be
achieved by modifying alkyl chains on surface of HMS. Since the
anti-cancer drugs can not kill all of cancer cells in a short time
and any remaining cancer cells will regrow the tumor, this system
can improve the ratio of drug utilization and achieve sustained
treatment.
Fig. 3 Photographs of HMS (1), IBU@HMS@YVO4:Eu3+@C18 (2)
and IBU@HMS@YVO4:Eu3+@C18@PMD (3) under visible light
(a) and UV light (l ¼ 265 nm) (b), respectively. TEM images of
HMS (c), IBU@HMS@YVO4:Eu3+@C18 (d) and IBU@HMS@Y
VO4:Eu3+@C18@PMD (e, f).
To further investigate pH-stimulated drug release properties of
IBU@HMS@YVO4:Eu3+@C18@PMD, the prepared nano-
particles were immersed in SBF at two different pH values (pH
7.4 and 5.0) at 37 ꢁC (Fig. 6). With coating of the functional
polymer, the IBU would be blocked in the core of the nano-
particles, and less than 5% leaked out the carrier at pH 7.4 even
pure HMS (Fig. 3c) and IBU@HMS@YVO4:Eu3+@C18
(Fig. 3d). TEM images are utilized to observe the microstructure
of the particles. Almost no changes was observed between pure
HMS and IBU@HMS@YVO4:Eu3+@C18, and both of them
maintained good monodispersity with an average diameter of
180 nm and a very clear profile. In contrast, the profile of
IBU@HMS@YVO4:Eu3+@C18@PMD became blurred and
a thin layer of polymer film with thickness around 10 nm was
clearly observed. The coated polymer film would block the pores
of the sphere and hinder the penetration of drugs. Furthermore,
the prepared IBU@HMS@YVO4:Eu3+@C18@PMD still retains
good monodispersity and nanosize, which is very important for
application in drug delivery. DLS results (Fig. S2-d, ESI†) show
that the HMS@YVO4:Eu3+@C18@PMD have an average
diameter of 200 nm, with a narrow size distribution (PDI ¼ 0.03),
which are consistent with the TEM results.
Properties of IBU@HMS@YVO4:Eu3+@C18@PMD
Determination of drug loading content. Fig. 4 shows the UV
absorbance spectra in 60 mg mLꢀ1 IBU hexane solution before
and after being loaded by HMS@YVO4:Eu3+@C18. The visible
Fig. 4 UV-Vis absorption of ibuprofen solution before and after being
loaded by HMS@YVO4:Eu3+@C18.
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Fig. 5 Release of IBU in vitro from drug-loaded HMS and HMS@Y-
VO4:Eu3+@C18 at 37 ꢁC.
Fig. 7 In vitro cell viabilities, measured by SRB assay, after culture
of human mouth epidermal carcinoma (KB) cells with HMS@Y
VO4:Eu3+@C18@PMD as function of concentration and time: (a) exci-
tation (left) and emission (right) spectra of HMS@YVO4:Eu3+
@
after 150 h. When adjusting the pH value to 5.0, the acetal groups
of PMD would hydrolyse into hydroxyl groups and the hydro-
phobic segments would change into hydrophilic segments (1H
NMR of the hydrolyzed polymer and hydrolysis degree curve are
shown in Fig. S6 and Fig. S7, respectively, see ESI†), so the
polymer will no longer block the pore channels and the drug
would be released. However, given the hydrophobic interaction
between drug molecules and the long alkyl chains, the drug
release is slow and about 80% drug release occurs over 150 h.
Hence, a pH-stimulated response and long-term release drug
delivery system was prepared successfully.
C18@PMD (b); confocal fluorescence microscopy images of KB cells in
bright field (c), and exposed to HMS@YVO4:Eu3+@C18@PMD (50 mg
mLꢀ1) for 5 min (d), 60 min (e and f).
Fig. 7b, with excitation wavelength of 278 nm and emission
wavelength of 616 nm.46 Human mouth epidermal carcinoma
(KB) cells were exposed to HMS@YVO4:Eu3+@C18@PMD (50
mg mLꢀ1) for 5 and 60 min, respectively. After 5 min of exposure,
pale red signals were observed in the cytoplasm, revealed that the
samples were entering the KB cells. At an exposure time of up to
60 min (Fig. 7e and f), the fluorescence of cells became stronger,
and the profile of cells was clearly visible. Clearly HMS@Y-
VO4:Eu3+@C18@PMD with low cytotoxicity and red fluores-
cence can enter the cancer cells quickly, which is suitable for cell
imaging.
Cell imaging properties. The cytotoxicity of HMS@Y
VO4:Eu3+@C18@PMD was assessed at different concentrations
by SRB assay with human mouth epidermal carcinoma (KB)
cells used as model cells. As shown in Fig. 7a, no cytotoxicity was
evident in the preliminary observations of the cells that were
treated by HMS@YVO4:Eu3+@C18@PMD for 24 h, and even at
as high as 100 mg mLꢀ1 concentration of the carrier, the cell
viability was still maintained at more than 80%.
Conclusions
In summary, a facile method for coating HMS with functional
polymer through hydrophobic van der Waals interactions has
been established. The prepared material IBU@HMS@Y
VO4:Eu3+@C18@PMD via this method has a high drug loading
content and can achieve long-term drug release. Besides, this
multifunctional material was verified to be a pH stimuli-
responsive controlled-release system through designing the
structure of the coated polymer and a cell imaging system
through modification with a fluorescent material. Clearly,
different polymers with other characteristics such as tempera-
ture-, light- and enzyme-responsive properties could be easily
coated on the surface of silica nanoparticles to satisfy specific
requirements of drug delivery.
The PL properties of HMS@YVO4:Eu3+@C18@PMD were
characterized by excitation and emission spectra, as shown in
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (Grant Nos. 20876101, 20902065,
21071105 and 21076134), the Natural Science Foundation of the
Jiangsu Higher Education Institutions of China (Grant
No.09KJB430010) and Innovative Research Team of Advanced
Chemical and Biological Materials, Soochow University.
Fig.
6 Release of IBU in vitro from drug-loaded HMS@Y-
VO4:Eu3+@C18@PMD at different pH at 37 ꢁC.
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