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 the2H11/24I15/2 and4S3/24I15/2 transitions, respectively. A red emission was also observed between 645 and 680 nm corresponding to the 4F9/24I15/2 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.