2. EXPERIMENTAL
2.1 Materials
Yttrium(Ш) chloride hexahydrate
(YCl3⋅6H2O), ytterbium(Ш) chloride
hexahydrate (YbCl3⋅6H2O), erbium(Ш)
chloride hexahydrate (ErCl3⋅6H2O),
ammonium fluoride (NH4F), oleic acid, triphosgene, and
4-(dimethylamino) pyridine (DMAP) were purchased from Aldrich Chemical
Co. (Milwaukee, WI, USA). Sodium borohydride, N-hydroxysuccinimide
(NHS), and N, N’-dicyclohexylarbodiimide (DCC) were purchased from Fluka
(Buchs, Switzerland). 4-Hydroxy-2-butanone was purchased from TCI
(Tokyo, Japan). α-Lipoic acid (LA), folic acid (FA), PEG-bis(amine)
(molecular weight: 3.350 kDa), β-benzyl-L-aspartate (BLA), triethylamine
(TEA), hydrazine monohydrate, sodium bicarbonate, and
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) were
purchased from Sigma Chemical Co. (St. Louis, MO, USA). Dimethyl
sulfoxide (DMSO), tetrahydrofuran (THF), n-hexane, benzene, N,
N-dimethylformamide (DMF), chloroform, diethyl ether, 1, 4-dioxane,
methanol, dichloromethane (DCM), and acetic acid were obtained from
Samchun Pure Chemical Co., Ltd. (Gyeonggi-do, Korea). Pheophorbidea (Pha) was purchased from Frontier Scientific, Inc. (Logan, UT,
USA). Spectra/Por membranes were purchased from Spectrum Laboratories,
Inc. (Rancho Dominguez, CA, USA). All other chemicals were analytical
grade.
2.2 Synthesis of tumor-targeted ligand and
photosensitizer-conjugated
UCNPs
2.2.1 Synthesis of hexagonal phase
NaYF4:Yb/Er
UCNPs
YCl3⋅6H2O (0.8 mmol),
YbCl3⋅6H2O (0.18 mmol), and
ErCl3⋅6H2O (0.02 mmol) were mixed with
25 ml oleic acid in a 250 ml flask. The solution was heated to 160 °C to
form a homogeneous solution, and then cooled to room temperature. A 10
ml methanol solution containing NaOH (2.5 mmol) and NH4F
(4 mmol) was slowly added into the flask and stirred for 30 min. The
solution was heated slowly to 100°C for 10 min to evaporate the
methanol, and then heated to 300°C and maintained for 1 h under an
N2 atmosphere. After the solution was cooled naturally
to room temperature, the nanocrystals were precipitated from the
solution using ethanol and then washed three times with an ethanol/water
(v/v=1:1) mixture (Z. Li & Zhang, 2008).
2.2.2 Surface modification of UCNPs
FA-conjugated block copolymer (FA-PEAH block copolymer), composed of
tumor targeting ligand FA, PEG, poly(aspartate) and a dihydrolipoic acid
end group, for surface modification of UCNPs was prepared as we reported
previously (Zhao, Kim, Ahn, Kim, & Kim, 2013). The surface modification
of UCNPs was performed by a ligand exchange method using the synthesized
FA-PEAH block copolymers. The UCNPs (80 mg) were dispersed in 10 ml
aqueous solution. The dispersion was stirred for 2 h while maintaining
the pH at 4 by adding 0.1 M HCl solution to remove the oleate ligands.
After the reaction, the aqueous solution was mixed with hexane to remove
the oleic acid by extraction with hexane, and repeated three times. The
combined hexane layers were re-extracted with water. In addition, the
water layers were combined and re-extracted with hexane. The ligand-free
UCNPs in the water dispersible fraction were collected by centrifugation
after precipitation with cold acetone. The product was re-dispersed in
acetone and the particles were collected by centrifugation. Finally, the
particles were dispersed in water (20 ml) for future use. FA-PEAH (160
mg) was dissolved in 10 ml water, and the solution was added into
ligand-free UCNP aqueous solution. The mixture was stirred at room
temperature for 24 h, and then purified by dialysis against deionized
water for 6 h. The resulting product was freeze-dried for further study
(Bogdan, Vetrone, Ozin, & Capobianco, 2011; Naccache, Vetrone,
Mahalingam, Cuccia, & Capobianco, 2009).
2.2.3 Preparation of Pha-conjugated UCNP nanocarriers
(FA-PEAH-UCNPs-Pha)
To introduce the ketone groups to Pha molecules, Pha (0.176 mol) was
dissolved completely in methanol (30 ml). EDC (0.53 mmol), DMAP (0.53
mmol), and 4-hydroxy-2-butanone (1.39 mmol) were added to the Pha
solution. The reaction mixture was stirred at 400 rpm for 24 h at room
temperature in the dark. Next, the solvent from the resulting mixture
was removed in a vacuum oven, and the residue was washed with deionized
water several times. The ketone group-modified Pha (Pha-HB) product was
collected by centrifugation, and then freeze-dried.
Next, FA-PEAH-modified UCNPs (120 mg) were dissolved in DMSO (10 ml).
The modified Pha (40 mg) dissolved in DCM (4 ml) was slowly added into
the FA-PEAH-UCNPs solution. Subsequently, four drops of acetic acid were
added into the mixture. The reaction mixture was stirred at 500 rpm for
24 h at room temperature in the dark. After the reaction, the DCM was
removed under vacuum, and then DMSO was added into the residue to adjust
the concentration of the mixture to about 3 mg/ml. After mixing, the
solution was dialyzed against NaHCO3 (pH 8.0) solution
for 1 day, and then against deionized water for another 12 h. The
resulting product was centrifuged to remove unreacted Pha, and then the
product was freeze-dried. In addition, an FA-unconjugated UCNP carrier
sample (CH3-PEAH-UCNPs-Pha) was synthesized using a
similar method as a control group.
2.3 Characterization
The modification of copolymers was confirmed by 600 MHz1H NMR (AVANCE Ⅲ 600, Bruker, Rheinstetten, Germany)
using D2O, and DMSO-d6 as the solvent.
Sizes and size distributions of UCNPs were determined by dynamic light
scattering (DLS) (ELS-Z, OTSUKA, Japan) at 25ºC using a He-Ne laser (633
nm) as a light source. The scattered light was measured at 90º and
collected by an autocorrelator. The surface modification of UCNPs was
determined by UV-visible spectrophotometry (UVmini-1240, Shimadzu,
Japan), and energy dispersive spectroscopy (EDS) (Tecnai
G2 F30 TEM system). The morphologies of the
nanoparticles were observed by field-emission transmission electron
microscopy (FE-TEM) (Tecnai G2 F30, FEI,
Amsterdam, the
Netherlands). The
crystalline morphology of nanoparticles was also investigated by
selected area electron diffraction (SAED) and X-ray Diffraction (XRD)
(D8 DISCOVER, Bruker, Rheinstetten, Germany).
2.4 Cellular uptake of
FA-PEAH-UCNPs-Pha
The MCF7 breast cancer cell is a FA receptor-overexpressing cell line
(K. Li et al., 2011; Meier et al., 2010), used here for cellular uptake
and phototoxicity tests. MCF7 cells (1×105 cells/well)
were seeded onto 6-well plates and cultured in RPMI 1640 supplemented
with 10% FBS and 1% penicillin-streptomycin at 37 ℃ in a humidified
5% CO2-95% air atmosphere. After 24 h, the medium was
replaced with 1.5 ml of fresh medium containing free Pha (10 µg/ml)
and
FA-PEAH-UCNPs-Pha (68 µg/ml, 10 µg/ml Pha equiv.), and then incubated
for 4 h. The cells were then washed with PBS and harvested using 0.05%
trypsin-EDTA. 4’, 6-Diamidine-2-phenylindol (DAPI) and Alexa Fluor 488
phalloidin solutions were added to stain the cell nucleus and F-actin,
respectively, at room temperature. All experiments were carried out in a
dark room to prevent photodegradation of the probes. The cells were
visualized using a confocal laser scanning
microscope
(LSM800, Carl-Zeiss, Germany).
2.5 In vitrophototoxi city
assay of FA-PEAH-UCNPs-Pha
MCF7 cells (1×104 cells/well) were seeded onto 96-well
plates in
200
μl RPMI 1640 and allowed to attach for 24 h. After cell attachment, the
medium was replaced with 100 μl of fresh medium containing free Pha and
FA/CH3-PEAH-UCNPs-Pha under a series of concentrations
(0, 5, 10, 20, and 30 µg/ml, Pha equiv.) with laser (980 nm) treatment
at 0.1 mW/cm2 for 5 min. Then, the irradiated cells
were incubated at 37 ºC for 24 h and cell viability was evaluated using
a cell viability assay kit (CCK-8, DoGenBio, Korea). Untreated cells
served as 100% viable cells. Data presented are averaged results of
triplicate experiments.
To
determine the effect of laser exposure time on the phototoxicity, we
also investigated the in vitro phototoxicity of free Pha,
FA-PEAH-UCNPs-Pha, and CH3-PEAH-UCNPs-Pha samples after
laser (980 nm) radiation for 0, 0.5, 1, and 5 min at 0.2
mW/cm2, the concentration of Pha was selected at 10
µg/ml (Pha equiv.). Dark-toxicity of the FA-PEAH-UCNPs-Pha was also
evaluated by incubating for 4 h under 10 µg/ml (Pha equiv.) without
laser irradiation.
3. RESULTS AND
DISCUSSION
3.1 Synthesis and characterization of the UCNP-based
nanocarrier
The synthetic scheme of the FA-PEAH-UCNPs-Pha nanocarrier is illustrated
in Figure 2. Characterization of the biocompatible block copolymer,
FA-PEAH, used for UCNPs modification was described in detail in our
previous report33. As shown in Figure 3A, the
synthesis of Pha-conjugated UCNP nanocarrier (FA-PEAH-UCNPs-Pha) were
confirmed by the presence of the characteristic peaks of Pha at 8.9 ppm,
9.4 ppm, and 9.8 ppm, as well as characteristic peaks of FA at 7.2 ppm,
PEG at 3.5 ppm, P(Asp) at 8.2 ppm, and DHLA at 1.2 ppm. In addition, the
conjugation content of Pha to FA-PEAH-UCNPs was evaluated by1H NMR using the relative intensity ratio of the
methylene protons of the PEG chain
(-OCH2CH2-, 3.5 ppm) to the methane
protons of Pha (8.9 ppm, 9.4 ppm, and 9.8 ppm). The conjugation content
of photosensitizer Pha in FA-PEAH-UCNPs-Pha carrier was determined as
about 14.7%.
Furthermore, the formation and surface modification of lanthanide-doped
NaYF4:Yb/Er UCNPs were characterized by UV-visible
spectroscopy and EDS measurements. Figure 3B showed the UV-visible
absorption spectra of FA-PEAH-UCNPs and FA-PEAH-UCNPs-Pha. As shown in
Figure 3B (b), the peak at about 279 nm was assigned to FA, while the
peaks at about 401 nm, and 690 nm were attributed to Pha. This
observation indicated that the Pha was introduced successfully to
FA-PEAH polymer chain.
EDS was also employed to investigate the elemental composition of UCNPs
before and after surface modification. As shown in Figure 3C, the
characteristic peaks of F, Na, Yb, Y, Er, and C were observed in the
free UCNPs sample. After FA-PEAH modification on the surface of UCNPs by
a ligand cap exchange method, new N and S characteristic peaks belonging
to the FA-PEAH copolymer appeared, and the relative intensity of the C
peak increased significantly (Figure 3D). These results indicated that
the NaYF4:Yb/Er UCNPs were formed and the FA-PEAH layer
was successfully immobilized onto the surface of UCNPs. After surface
modification, the solubility of UCNPs improved significantly at the
macroscopic level.
3.2 Morphology of UCNP-based
nanocarriers
Size, size distribution, morphology and crystalline morphology of free
UCNPs, FA-PEAH-UCNPs, and FA-PEAH-UCNPs-Pha were investigated by DLS,
FE-TEM, SAED and XRD.
The morphologies of UCNPs, FA-PEAH-UCNPs and FA-PEAH-UCNPs-Pha were
observed by FE-TEM. As shown in Figures 4A-C, These
NaYF4:Yb/Er nanocrystals were uniform submicron in size
and monodisperse size distribution. The particle size was about 20 nm in
dimeter with a hexagonal plate-like shape. The sizes of UCNPs were
almost the same before and after modification in TEM images as shown in
Figures 4A-C, while the sizes before and after modification were quite
different in the DLS data (Figure 4D). Since TEM measurement is
sensitive only to the electron dense metal particles, the size of all
samples in the TEM images were almost the same and the polymers used for
surface modification were not clearly observed. However, DLS measurement
is sensitive to the hydrodynamic diameter of the whole nanocomposite.
Thus, the DLS results of surface-modified UCNPs, FA-PEAH-UCNPs and
FA-PEAH-UCNPs-Pha samples, exhibit a larger size than the TEM results.
Typical average particle size distributions measured by DLS for free
UCNPs, FA-PEAH-UCNPs, and FA-PEAH-UCNPs-Pha are shown in Figure 4D as
996.0, 68.6, and 90.3 nm, respectively. Since free UCNPs were quite
hydrophobic due to the hydrophobic oleate capping ligand before surface
modification, the macroscopic aggregations were observed. Thus, DLS data
of free UCNPs exhibited a much larger size compared with the
surfaced-modified UCNP samples (FA-PEAH-UCNPs and FA-PEAH-UCNPs-Pha).
However, after surface modification of the hydrophilic FA-PEAH polymer
instead of the hydrophobic oleate ligand, the dispersity and solubility
in aqueous solution significantly improved. As shown in Figure 4D, the
size of the nanoparticles significantly decreased (about 68.6 nm for
FA-PEAH-UCNPs and 90.3 nm for FA-PEAH-UCNPs-Pha), and the size
distribution maintained a narrow monodisperse unimodal pattern.
In order to evaluate the deep-penetration PDT application,
hexagonal-phase UCNPs are the best choice, because hexagonal-phase
NaYF4:Yb/Er UCNPs usually produce a bright green
emission (around 550 nm) along with a weak dark red emission (around 660
nm) under 980 nm NIR irradiation(F. Wang & Liu, 2009). Also, it has
been reported that hexagonal-phase
NaYF4(βNaYF4) crystals are the most
efficient host materials for upconverting lanthanide ions due to the low
phonon energy of the crystal lattice. The crystalline morphology of the
synthesized NaYF4:Yb/Er UCNPs was investigated by the
SAED pattern, as shown in Figure 4E. The SAED pattern of the UCNPs was
shown as spotty polycrystalline diffraction rings, which can be indexed
to the (100), (110), (101), (110), (200), (111), (201), (210), (002),
(300), (211), and (321) planes of hexagonal NaYF4lattice. We also employed XRD to further confirm the crystalline
morphology of NaYF4:Yb/Er UCNPs. In Figure 5, the peak
positions and intensities of the free UCNPs agree well with the standard
pattern of hexagonal phase NaYF4 crystal (Figure 5A; ■,
JCPDS 16-0334). These results indicated that the synthesized
NaYF4:Yb/Er UCNPs have the hexagonal β -phase.
Additionally, in Figure 5B, the peak positions between 15 and 30 degrees
were well matched with the standard pattern of PEG (▼, JCPDS 49-2095).
It also could be evidence that the FA-PEAH layer was successfully
immobilized onto the surface of UCNPs.
3.3 Luminescence properties of
UCNPs
The upconversion fluorescence spectra of surface modified
NaYF4:Yb/Er UCNPs (FA-PEAH-UCNPs) in aqueous solution
excited with a 980 nm laser at room temperature are shown in Figure 6.
The FA-PEAH-UCNPs sample shows three district Er3+emission bands. The sharp green emissions between 510 and 530 nm and
between 530 and 570 nm were assigned to the2H11/2 →4I15/2 and4S3/2 →4I15/2 transitions, respectively. A
red emission was also observed between 645 and 680 nm corresponding to
the 4F9/2 →4I15/2 transition. The inset in Figure
6 exhibits photographs of free UCNPs and surface modified
NaYF4:Yb/Er UCNPs in aqueous solution under 980 nm laser
irradiation. The free UCNPs and FA-PEAH-UCNPs sample show a yellowish
green color upon excitation by a 980 nm laser. The red emission band of
UCNPs between 645 and 680 nm exhibits a good match for the main
absorption band of Pha between 645 and 735 nm. This result indicates
that the photosensitizer Pha molecules could be activated by
luminescence intensity of NaYF4:Yb/Er UCNPs upon 980 nm
laser irradiation.