Characterization of a Novel Amphiphilic Cationic Chlorin Photosensitizer for Photodynamic Applications

Abstract

A novel amphiphilic cationic chlorin e₆ derivative was investigated as a promising photosensitizer for photodynamic therapy. Two cationic -N(CH₃)₃⁺ groups on the periphery of the macrocycle provide additional hydrophilization of the molecule and ensure its electrostatic binding to the mitochondrial membranes and bacterial cell walls. The presence of a hydrophobic phytol residue in the same molecule results in its increased affinity towards the phospholipid membranes while decreasing its stability towards aggregation in aqueous media. In organic media, this chlorin e₆ derivative is characterized by a singlet oxygen quantum yield of 55%. Solubilization studies in different polymer- and surfactant-based supramolecular systems revealed the effective stabilization of this compound in a photoactive monomolecular form in micellar nonionic surfactant solutions, including Tween-80 and Cremophor EL. A novel cationic chlorin e₆ derivative also demonstrates effective binding towards serum albumin, which enhances its bioavailability and promotes effective accumulation within the target tissues. Laser confocal scanning microscopy demonstrates the rapid intracellular accumulation and distribution of this compound throughout the cells. Together with low dark toxicity and a rather good photostability, this compound demonstrates significant phototoxicity against HeLa cells causing cellular damage most likely through reactive oxygen species generation. These results demonstrate a high potential of this derivative for application in photodynamic therapy.

Keywords: chlorin photosensitizers; intracellular distribution; photodynamic therapy; photostability; phototoxicity; protein binding; singlet oxygen generation; solubilization studies.

Figure 1

UV–Vis absorption and fluorescence (insert) spectra of 1 in DMSO, H₂O, and micellar surfactant solutions.

Figure 2

Photostability of 1 in micellar nonionic surfactant solutions.

Figure 3

Singlet oxygen generation efficiency for 1 in DMSO and micellar nonionic surfactant solutions.

Figure 4

Triplet–triplet absorption spectrum of Compound 1 (5 × 10⁻⁷ M) in DMSO (240 μs after flash). Insert: Compound 1 triplet decay kinetics at 460 nm.

Figure 5

UV–Vis absorption and fluorescence (insert) spectra of 1 in polymer aqueous solutions.

Figure 6

Comparison of fluorescence and absorption (insert) spectra (a) and particle size distribution (b) for 1 in pure PBS and in the presence of BSA.

Figure 7

BSA fluorescence quenching upon titration with 1 (λ_ex = 290 nm, C_BSA = 5 μM) and the corresponding Stern–Volmer plot (insert).

Figure 8

Fluorescence spectra of 1 in PBS, BSA, PDDA, and BSA-PDDA PEC (a) and optical micrographs of coacervate microdroplets of BSA-PDDA PEC (b–e).

Figure 9

Intracellular distribution of 1 (red) in HeLa cells after incubation for 2 h (right). Untreated control (left). Blue–nuclei stained with Hoechst 33342. The sections were created at the middle height of the cells.

Figure 10

Light-induced and dark cytotoxicity of 1 analyzed by MTT assay. The percentage of cell viability was determined relative to the viable control cells. Survival of HeLa cells was measured at 72 h after treatment.

Figure 11

Structure of Compound 1.