3.5 In Vitro phototoxicity test of UCNP-based nanocarriers
To determine the PDT efficacy of the UCNP-based Pha nanocarrier, thein vitro cytotoxicity of free Pha, FA-PEAH-UCNPs-Pha, and CH3-PEAH-UCNPs-Pha was measured. For the phototoxicity test, we investigated the phototoxicity against MCF7 cells using various concentrations of Pha (0, 5, 10, 20, and 30 µg/ml) and laser exposure times (0, 0.5, 1, and 5 min). After 980 nm laser radiation for 5 min at 0.1 mW/cm2, FA-PEAH-UCNPs-Pha and CH3-PEAH-UCNPs-Pha exhibited significantly enhanced phototoxicity compared to free Pha as shown in Figure 8A. As the Pha concentration increased, the cell viability gradually decreased. Notably, the viability of MCF7 cells treated with FA-PEAH-UCNPs-Pha sample decreased to nearly 25% after a 5 min treatment at a concentration of 30 µg/ml. In order to determine the effect of laser exposure time on the phototoxicity, we also determined the in vitro phototoxicity of free Pha, FA-PEAH-UCNPs-Pha, and CH3-PEAH-UCNPs-Pha after 980 nm laser radiation for 0, 0.5, 1 and 5 min at 0.2 mW/cm2 (10 µg/ml, Pha equiv.). Under the dark condition, free Pha, FA-PEAH-UCNPs-Pha, and CH3-PEAH-UCNPs-Pha exhibited more than 90% cell viability and no significant dark toxicity as shown in Figure 8B (0 min of laser exposure time, 10 µg/ml Pha concentration). However, the cell viability significantly decreased as the laser exposure time increased (Figure 8B).
The PDT efficiency of FA-PEAH-UCNPs-Pha was obviously higher than free Pha and CH3-PEAH-UCNPs-Pha. These results are probably due to the increased solubility of hydrophobic Pha molecules in aqueous environments by loading into the block copolymer chain-immobilized UCNP carriers, resulting enhanced 1O2quantum yield37 of Pha. In addition, FA-conjugated FA-PEAH-UCNPs-Pha could exhibit the higher phototoxicity than free Pha and CH3-PEAH-UCNPs-Pha since the cellular uptake was improved by the FA receptor-medicated endocytosis process.
4. CONCLUSIONS
In order to demonstrate the UCNP-based cancer therapies, particularly NIR-light induced photodynamic therapy, we have designed the NIR light-triggered theranostic system based on hexagonal-phase NaYF4:Yb/Er UCNPs for efficient PDT with enhanced deep tissue penetration ability and fluorescence imaging. Hexagonal-phase NaYF4:Yb/Er UCNPs were synthesized by a hydrothermal method and the nanoparticles were monodisperse with a uniform size of about 20 nm. The crystalline morphology of the synthesized NaYF4:Yb/Er UCNPs showed a thermodynamically stable hexagonal β-phase. Since the UCNPs have no intrinsic aqueous solubility and lack functional moieties for subsequent biological functionalization, these UCNPs were modified with FA-conjugated biocompatible block copolymers through a bidentate dihydrolipoic acid linker. The FA-PEAH copolymer-modified UCNPs (FA-PEAH-UCNPs) showed improved solubility and dispersibility in aqueous solution. Then, the hydrophobic PS, Pha, was conjugated to the stable vectors through a pH-sensitive linkage. These water dispersible UCNPs have a much stronger luminescence property compared with hydrophobic UCNPs. The upconversion fluorescence spectra of FA-PEAH-UCNPs excited with a 980 nm laser showed sharp green emissions between 510 and 530 nm and between 530 and 570 nm as well as a red emission between 645 and 680 nm. These FA-PEAH-UCNPs-Pha that produce high energy visible photons from low energy radiation in the NIR region could be very promising materials for bioimaging and PDT. The advantage of NIR radiation use is less harmful to cells, minimizes auto-fluorescence from biological tissues, and penetrates tissues to a greater extent. Due to the active tumor targeting FA ligand conjugation, the cellular uptake and phototoxicity against MCF7 cells of FA-PEAH-UCNPs-Pha were significantly enhanced compared with free Pha and FA-ligand unconjugated CH3-PEAH-UCNPs-Pha. These UCNP-based Pha nanocarriers containing tumor targeting FA ligands and pH-sensitive cleavage sites could improve the solubility of Pha and increase 1O2 quantum yield in the weakly acidic conditions of tumor tissue, as well as promote PDT treatment efficiency. In addition, this nanocarrier could be triggered by NIR which has deep tissue penetration. These results suggest that the FA-PEAH-UCNPs-Pha system has potential use as an effective PS delivery system for tumor PDT applications in deep tissue.