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.