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. 2020 Nov 17;12(11):1102.
doi: 10.3390/pharmaceutics12111102.

Highly Red Light-Emitting Erbium- and Lutetium-Doped Core-Shell Upconverting Nanoparticles Surface-Modified with PEG-Folic Acid/TCPP for Suppressing Cervical Cancer HeLa Cells

Kyungseop Lim  1 , Hwang Kyung Kim  1 , Xuan Thien Le  1 , Nguyen Thi Nguyen  1 , Eun Seong Lee  2 , Kyung Taek Oh  3 , Han-Gon Choi  4 , Yu Seok Youn  1
Affiliations

Highly Red Light-Emitting Erbium- and Lutetium-Doped Core-Shell Upconverting Nanoparticles Surface-Modified with PEG-Folic Acid/TCPP for Suppressing Cervical Cancer HeLa Cells

Kyungseop Lim et al. Pharmaceutics. .

Abstract

Photodynamic therapy (PDT) combined with upconverting nanoparticles (UCNPs) are viewed together as an effective method of ablating tumors. After absorbing highly tissue-penetrating near-infrared (NIR) light, UCNPs emit a shorter wavelength light (~660 nm) suitable for PDT. In this study, we designed and prepared highly red fluorescence-emitting silica-coated core-shell upconverting nanoparticles modified with polyethylene glycol (PEG5k)-folic acid and tetrakis(4-carboxyphenyl)porphyrin (TCPP) (UCNPs@SiO2-NH2@FA/PEG/TCPP) as an efficient photodynamic agent for killing tumor cells. The UCNPs consisted of two simple lanthanides, erbium and lutetium, as the core and shell, respectively. The unique core-shell combination enabled the UCNPs to emit red light without green light. TCPP, folic acid, and PEG were conjugated to the outer silica layer of UCNPs as a photosensitizing agent, a ligand for tumor attachment, and a dispersing stabilizer, respectively. The prepared UCNPs of ~50 nm diameter and -34.5 mV surface potential absorbed 808 nm light and emitted ~660 nm red light. Most notably, these UCNPs were physically well dispersed and stable in the aqueous phase due to PEG attachment and were able to generate singlet oxygen (1O2) with a high efficacy. The HeLa cells were treated with each UCNP sample (0, 1, 5, 10, 20, 30 μg/mL as a free TCPP). The results showed that the combination of UCNPs@SiO2-NH2@FA/PEG/TCPP and the 808 nm laser was significantly cytotoxic to HeLa cells, almost to the same degree as naïve TCPP plus the 660 nm laser based on MTT and Live/Dead assays. Furthermore, the UCNPs@SiO2-NH2@FA/PEG/TCPP was well internalized into HeLa cells and three-dimensional HeLa spheroids, presumably due to the surface folic acid and small size in conjunction with endocytosis and the nonspecific uptake. We believe that our UCNPs@SiO2-NH2@FA/PEG/TCPP will serve as a new platform for highly efficient and deep-penetrating photodynamic agents suitable for various tumor treatments.

Keywords: cancer; hypoxia; near infrared; photodynamic therapy; tetrakis(4-carboxy-phenyl)porphyrin; upconverting nanoparticles.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic illustration of the preparation of core-shell upconverting nanoparticles surface-modified with silica, PEG-folic acid (FA/PEG), and TCPP (UCNPs@SiO2@FA/PEG/TCPP) for photodynamic therapy based on singlet oxygen generation.
Figure 2
Figure 2
(A) TEM images of UCNPs depending upon the ratio between oleic acid (OA) and 1-octadecene (ODE) (3:7 for left, 6:15 for middle, and 4:15 for right). (B) TEM images of core@shell UCNPs depending upon the molar ratio between the core (erbium) and shell (lutetium) components (1:0.5 for left, 1:1 for middle, and 1:1.5 for right).
Figure 3
Figure 3
HR-TEM images and histograms of the particle size of (A) core-only UCNPs, (B) core@shell UCNPs, and (C) core@shell UCNPs@SiO2. Data are presented as means ± SDs (n = 3).
Figure 4
Figure 4
(A) Photographs of core or core@shell upconverting nanoparticles (UCNPs) emitting green fluorescence. (B) Photographs of core, core@shell, or core@shell@ SiO2 UCNPs emitting red fluorescence. (C) Photo images of silica-coated upconverting nanoparticles (UCNPs@SiO2) (10 mg) modified with (a) mPEG (0.1 mg) and FA/PEG (0.1 mg); (b) mPEG (0.2 mg) and FA/PEG (0.1 mg); (c) mPEG (0.5 mg) and FA/PEG (0.1 mg); (d) mPEG (1.0 mg) and FA/PEG (0.1 mg); (e) mPEG (2.0 mg) and FA/PEG (0.1 mg); and (f) mPEG (2.0 mg), FA/PEG (0.1 mg), and TCPP (1.0 mg).
Figure 5
Figure 5
(A) UV-Vis-NIR absorption spectra of core and core@shell UCNPs (400–850 nm). (B) Zeta potentials of UCNPs@SiO2, UCNPs@SiO2, UCNPs@SiO2@FA/PEG, and UCNPs@SiO2@FA/PEG/TCPP (n = 3). * p < 0.001 over UCNPs@SiO2@FA/PEG; ** p < 0.005 over UCNPs@SiO2@FA/PEG/TCPP; *** p < 0.005 over UCNPs@SiO2@FA/PEG. (C) UV-Vis-NIR absorption spectra (450–750 nm) and (D) singlet oxygen generation assay of DW, free TCPP, UCNPs@SiO2@ FA/PEG, and UCNPs@SiO2@FA/PEG/TCPP (DW: 808 nm, 1.5 W/cm2; free TCPP: 0.5 µg/mL, 660 nm, 10 mW/cm2; UCNPs@SiO2@FA/PEG: 808 nm, 1.5 W/cm2; UCNPs@ SiO2@FA/PEG/TCPP: 0.35 µg/mL, 808 nm, 1.5 W/cm2) (n =3). * p < 0.001 over Dw and UCNPs@SiO2@FA/PEG.
Figure 6
Figure 6
Cytotoxicity evaluation of free TCPP, UCNP@ SiO2@FA/PEG, and UCNP@ SiO2@FA/PEG/TCPP under normoxic or hypoxic conditions with or without laser irradiation (660 nm, 30 min, 10 mW/cm2 for free TCPP; 808 nm, 30 min, 1.5 W/cm2 for others). Data are presented as means ± SDs (n = 7). * p < 0.003 over Laser(−) and ** p < 0.001 over Laser(−).
Figure 7
Figure 7
Live/Dead assay of PBS, free TCPP, UCNP@SiO2@FA/PEG, and UCNP@SiO2@FA/PEG/TCPP under normoxic or hypoxic conditions with or without laser irradiation (660 nm, 30 min, 10 mW/cm2 for free TCPP; 808 nm, 30 min, 1.5 W/cm2 for others).
Figure 8
Figure 8
(A) Images for the Live/Dead assay in 3D multicellular HeLa cell spheroids under the predetermined conditions (808 nm, 30 min, 1.5 W/cm2). (B) Z-stack images of six 10–20-μm slices in the HeLa cell spheroids.
Figure 9
Figure 9
(A) Two-dimensional monolayer images of HeLa cells treated with core-shell upconverting nanoparticles surface-modified with silica, PEG-folic acid (FA/PEG), and TCPP (UCNP@SiO2@FA/PEG/TCPP) at predetermined time points (blue: nuclei, green: lysosomes, red: TCPP). (B) Bio-TEM images of HeLa cells treated with UCNP@SiO2@FA/PEG/TCPP. (C) In vitro spheroid model images (Z-stack of ten 20-μm slices) for predicting the cellular uptake of UCNP conjugates into three-dimensional tumor tissues.
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