Photodynamic therapy (PDT), which canablate cancer cells or diseased tissue by the generated reactive oxygen species (ROS) once the photosensitizers (PSs) are excited by light with specific wavelength, has attracted various attention in the last decades due to its unique advantages, including non-invasiveness, few side-effects, etc. The advancement of PDT has been significantly restricted by the penetration depth of the excitation light and sub-cellular organelles targeting capability. Here, an effective carbon dots (C-dots) photosensitizer with intrinsic nucleolus-targeting capability is synthesized, characterized, and employed for in vitro photodynamic anticancer therapy with enhanced treatment performance at a low dose of PS and light irradiation.
The optical system, which included a microscope and femtosecond laser, was designed for two-photon phototherapy. The nucleolus-targeted C-dots were synthesized using microwave heating. The characteristics of synthesized C-dots including particles size, absorption and emission, two-photon fluorescence, photobleaching, biocompatibility, etc., were measured by DLS measurement, UV-Visible spectrophotometer, fluorescence spectrometer, femtosecond laser, CCK-8 kit, respectively. The nucleolus-targeting capability of C-dots was investigated using fluorescence imaging. The HeLa cells were incubated with C-dots and irradiated with a femtosecond laser before cell viability was examined using Calcein-AM/PI staining and fluorescence imaging.
The microwave heating method selects citric acid and ethylenediamine as raw materials to synthesize the C-dots. The synthesized C-dots were studied using dynamic light scattering measurement, and the average size of the C-dots was approximately 1 nm [Fig. 2(a)]. The C-dots absorbed light in various wavelengths from 300 to 700 nm, with the main absorption peak at 360 nm and a shoulder peak at 450 nm [Fig. 2(b)]. The C-dots exhibited excitation-dependent emission [Fig. 2(c)] and significant two-photon fluorescence when exposed to femtosecond laser irradiation [Fig. 2(d)]. The ROS-generation capability of the C-dots in aqueous solutions was investigated using ABDA as the ROS indicator under white light irradiation (400-700 nm, 100 mW/cm2). The ABDA was almost decomposed after 10 min illumination [Fig. 2(e)], indicating the ROS-generation capability of the C-dots. The long-term stability and photostability of the C-dots were characterized by measuring the absorption spectra at different time points [Fig. 2(f)] and after continuous irradiation [Fig. 2(g)], respectively. The experimental results showed that the C-dots had good long-term stability and photostability. CCK-8 kits were used to evaluate the biocompatibility of the C-dots before undergoing in vitro photodynamic therapy. For 24 and 48 h, no significant difference existed between control cells and cells treated with the C-dots in the mass concentration range of 250-750 g/mL, indicating the excellent biocompatibility of the C-dots. The C-dots were treated with HeLa cells to investigate their intracellular position, followed by fluorescence imaging. The fluorescence signal of the C-dots was observed in some round areas, i.e., nucleoli. HeLa cells were co-stained with the C-dots and one commercial nucleolus imaging probe, SYTO RNASelect, to demonstrate the C-dots’ nucleolus-targeting capacity. Figure 3 showed the fluorescence of the C-dots completely overlapped with that of SYTO RNASelect, confirming that the C-dots could specifically stain the nucleolus. HeLa cells were cultured with/without the C-dots (500 g/mL) for 3 h before being exposed to femtosecond laser irradiation to study the two-photon PDT efficiency of the C-dots (740 nm, 28 mW). The treated cells were incubated for 4 h following the irradiation and stained with Calcein-AM and PI. Figure 4 showed that more cells were PI-positive with an increment of irradiation time. When the irradiation time reached 45 s, almost all cells were necrotic, suggesting the excellent cancer cell ablation potential of nucleolus-targeted two-photon photodynamic treatment. The identical irradiation did not result in necrosis in the absence of the C-dots, indicating that laser irradiation had no effect.
We designed and synthesized novel C-dots with intrinsic nucleolus-targeting and ROS-generation capabilities. The nucleolus-targeted two-photon PDT exhibits outstanding cancer cell ablation efficiency at a low dose of the C-dots and light irradiation because the C-dots generated ROS is positioned within the nucleolus, which is an efficient cancer therapy site. Additionally, the developed C-dots possess some unique advantages, including ultrasmall size, long-term stability, and excellent biocompatibility, making them promising for practical two-photon PDT applications.
