Claudin 4-targeted nanographene phototherapy using a Clostridium perfringens enterotoxin peptide-photosensitizer conjugate

Abstract

In this study we designed a claudin 4-directed dual photodynamic and photothermal system, in which a 30-amino acid claudin 4-binding peptide, Clostridium perfringens enterotoxin (CPE), was linked to a photodynamic agent chlorin e6 (Ce6) through a polyethylene glycol spacer (CPC) and anchored onto reduced graphene oxide (rGO) nanosheets to form CPC/rGO nanosheets. For comparison, a conjugate of polyethylene glycol and Ce6 (PC) was anchored onto the rGO nanosheets to generate PC/rGO. Both PC and CPC generated reactive oxygen species upon irradiation at 660 nm. Application of CPC/rGO to claudin 4-overexpressing U87 glioblastoma cells in vitro resulted in a significantly higher cellular uptake compared to application of PC/rGO. Upon irradiation at 660 and 808 nm, the CPC/rGO-treated U87 cells generated significantly higher reactive oxygen species and caused significantly higher temperature increase, and showed most potent anticancer effect compared to the other groups. Taken together, these results suggest that CPC/rGO is potentially useful as a tumor-specific combined phototherapy.

Figure 1

Dual-photoresponsive cancer therapy: CPC/rGO on claudin 4-overexpressing cancer cells. Schematic diagram showing the CPC/rGO (A) nanosheets. CPC/rGO was composed of the targeting ligand, the photothermal rGO, and the PS Ce6 as an anchoring moiety. CPC/rGO may enter the cells via claudin 4-mediated endocytosis due to the presence of the CPE30 ligand (B). Box (C) illustrates that CPE30 specifically binds to the secondary loop of the claudin 4 receptor, which extends into the extracellular matrix. Irradiation at 660 nm induces ROS generation by Ce6 to apply photodynamic anticancer activity. Irradiation at 808 nm enabled the rGO nanosheets to exert photothermal anticancer activity.

Figure 2

Singlet oxygen production of Ce6 during LED irradiation. The production of singlet oxygen from free Ce6 (A), PC (B), and CPC (C) was observed during LED irradiation.

Figure 3

Size and zeta potentials of the surface-modified rGO nanosheets. Transmission electron microscopy images of PC/rGO (A) and CPC/rGO (B) are presented (scale bars indicate 100 nm). AFM images of PC/rGO (C) and CPC/rGO (D) are presented (scale bars indicate 1 μm). (E) The lateral sizes of rGO, PC/rGO, and CPC/rGO were determined after incubation in serum. (F) Zeta potentials of rGO, PC/rGO, and CPC/rGO were determined after incubation in TDW or serum for 1 h.

Figure 4

Photothermal activity of the surface-modified rGO nanosheets. The real-time temperature increases of the rGO, PC/rGO, or CPC/rGO were recorded upon irradiation at 808 nm. Results are reported as the mean±SD of three independent experiments.

Figure 5

In vitro release of CPC from rGO nanosheets. The release of CPC from rGO was measured at acidic and neutral pH conditions. Results are presented as the mean±SD of three independent experiments.

Figure 6

Cellular uptake of CPC/rGO. U87 cells were treated with PC/rGO or CPC/rGO (with or without CPE30 pretreatment). After treatment for 1 h, the fluorescence of Ce6 was measured by confocal microscopy (scale bar indicates 20 μm). Representative flow cytometry data are presented (B), and the fluorescence-positive cell populations were quantified (C). Results are reported as the mean±SD of three independent experiments.

Figure 7

Dual photo-responsiveness of the CPC/rGO-treated cells. U87 cells were treated with rGO, PC/rGO, or CPC/rGO for 1 h. The intracellular ROS accumulation was measured after irradiation at 660 nm (A). The real-time temperature increases were recorded upon irradiation at 808 nm (B). The highest cell suspension temperature was determined using the FLIR QuickReport 1.2 software (C). Results are reported as the mean±SD of three independent experiments. ^***P<0.005 (ANOVA and Student-Newman-Keuls test, n=5).

Figure 8

Photodynamic and photothermal anticancer effect. U87 cells were treated with various concentrations of CPC on rGO and irradiated at two wavelengths (660 nm and 808 nm). After 24 h, live cells were stained with calcein and the cell viability was measured using fluorometry (A). U87 cells, either treated or untreated with rGO, PC/rGO, or CPC/rGO, were irradiated at 808 nm or two wavelengths. After 24 h, live cells were stained with calcein and the percentage of viable cells was measured using fluorometry (B) and observed by fluorescence microscopy (C). Results are reported as the mean±SD of three independent experiments. The scale bar indicates 100 μm. ^***P<0.005 (ANOVA and Student-Newman-Keuls test, n=3).