New Highly Fluorescent Water Soluble Imidazolium-Perylenediimides: Synthesis and Cellular Response

Abstract

The synthesis and characterization of two new water soluble 2,6-bis(imidazolylmethyl)-4-methylphenoxy-containing perylenediimides, PDI-1 and PDI-2, are described. These compounds demonstrate a high fluorescence quantum yield in water and were investigated as potential photosensitizers for generating reactive oxygen species with applications in anticancer activities. The HeLa cell line (VPH18) was used to evaluate their efficacy. Fluorescence microscopy was employed to confirm the successful internalization of PDI-1 and PDI-2, while confocal microscopy revealed the specific locations of both PDIs within the lysosomes and mitochondria. In vitro studies were conducted to evaluate the anticancer activity of PDI-1 and PDI-2. Remarkably, these photosensitizers demonstrated a significant ability to selectively eliminate cancer cells when exposed to a specific light wavelength. The water solubility, high fluorescence quantum yield, and selective cytotoxicity of these PDIs toward cancer cells highlight their potential as effective agents for targeted photodynamic therapy. In conclusion, the findings presented here provide a strong foundation for the future exploration and optimization of PDI-1 and PDI-2 as effective photosensitizers in photodynamic therapy, potentially leading to improved treatment strategies for cancer patients.

Keywords: perylenediimide; photodynamic therapy; photosensitizer; reactive oxygen species.

Scheme 1

Synthetic route to obtain (A) PDI-1 and (B) PDI-2.

Figure 1

Part of COSY spectrum of PDI-3.

Figure 1

Part of COSY spectrum of PDI-3.

Figure 2

Part of HSQC spectrum of PDI-3.

Figure 3

Part of HSQC spectrum of PDI-3.

Figure 4

Absorption (blue line) and normalized emission spectra (red line) of: (A) PDI-3, (B) PDI-4, (C) PDI-1, and (D) PDI-2.

Figure 5

Cellular uptake and cytotoxicity of PDIs in HeLa cell line. (A) Confocal microscopy images of HeLa cells treated with 10 μM PDI-1 and (B) PDI-2, respectively. Hoechst 33342 was used to counterstain nuclei. Viability of HeLa cells treated with different concentrations of PDI-1 (C) and PDI-2 (D) for 24 h. Data are represented as mean  ±  SD of independent experiments (n  =  4). Scale bar: 10 microns.

Figure 6

Phototoxicity induced by PDIs in HeLa cell line. Effect of increasing time exposure using 547 nm light at 35 mW/cm² irradiance from 30 s to 3 min in cells treated with 10 μM of PDI-1 (A–E) or PDI-2 (F–J). (K,L) Viability percentage of cells treated during 24 h with PDIs and exposure to light irradiation (547 nm) for 30 s, 1 min, 2 min, and 3 min, respectively. Untreated cells (control, 100% of viability) were neither exposed to PDIs nor irradiated. Cells “PDI no light” were incubated with PDIs without specific irradiation. Data are represented as mean  ±  SD of independent experiments (n  =  4). Scale bar: 1 mm.

Figure 7

Phototoxicity of PDIs after 2 min light treatment at 35 mW/cm² light dose induced cell death. Representative confocal images of HeLa cells treated with 10 μM of PDI-1 (A) or PDI-2 (B) and irradiated with 547 nm light. Phototoxicity is located mainly in the spotlight area surrounded by a dashed line (1 spot). The white arrow points to a spotlight area and a detailed view of the area where cell death due to oxidative stress has become visible, while non-irradiated area (1′) shows no loss of viability and unaltered cell morphology. Scale bars: 1 mm and 20 microns.

Figure 8

Effect of irradiation on treated and control cells. Fluorescence images of the live/dead assay were acquired after 3 min of irradiation (35 mW/cm² light dose) with 10 μM of PDI-1 (A); PDI-2 (B); irradiated control cells without PDIs (C); non-irradiated control cells (D). Green fluorescence represents live cells, while red fluorescence indicates dead cells. (E) Quantitative analysis of live cells in different conditions: control cells, irradiated control cells, cells treated with PDI-1, and cells treated with PDI-2. Data are represented as mean  ±  SD of independent experiments (n  =  4). Scale bar: 100 microns.

Figure 9

Subcellular localization of PDIs in vitro. (A,B) Fluorescence images of HeLa cells treated with LysoTracker and 10 μM of PDI-1 or PDI-2 show partial colocalization (white arrows). (C,D) Similarly, colocalization of PDIs with Mitotracker was observed in HeLa cells (white arrows). Nuclei were stained with Hoechst 33342. Scale bar: 20 microns.