Near-infrared Photosensitizer Based on a Cycloimide Derivative of Chlorin p6: 13,15-N-(3′-Hydroxypropyl)Cycloimide Chlorin p6¶

Abstract

The 13,15‐N‐(3′‐hydroxypropylcycloimide) chlorin p6 (CIC), which absorbs at 711 nm, possesses considerable photoinduced cell‐killing activity. It is 43‐, 61‐ and 110‐fold more active than chlorin p6, 3‐formyl‐3‐devinyl chlorin p6 and Photogem, respectively, and has no cytotoxicity without irradiation as estimated on A549 human adenocarcinoma cells. To attain the highest intracellular penetration and activity the monomeric form of CIC should be stabilized. This stabilization in an aqueous environment can be achieved using 0.002–0.005% of Cremophor EL emulsion (polyoxyethylene derivative of hydrogenated castor oil). The intracellular accumulation of CIC occurs in cytoplasm in a monomeric form bound to cellular membranes. This form of the dye is characterized by a high quantum yield of singlet oxygen generation (0.66 ± 0.02). Besides diffuse staining of intracellular membranous structures, CIC accumulates 3‐ to 4‐fold more intensely in mitochondria and Golgi apparatus, thus indicating these organelles to be the initial targets of its photodynamic action. The incubation time providing 50% accumulation level of CIC in cells is 30 ± 5 min. The time for 50% release of CIC from the cells is 60 ± 10 min. A 10‐fold decrease in CIC intracellular penetration at 22°C proves that temperature‐sensitive mechanisms of transport, rather than diffusion, are responsible for the dye uptake. The average cytoplasmic concentration of CIC was seven times the extracellular concentration in the 0.2–1.6 μM range, used for the photodynamic activity measurements. The concentration of CIC and the light dose that correspond to ca 50% level of phototoxicity induce predominantly an apoptotic‐type of cell death, whereas the conditions providing 100% level of phototoxicity induced necrosis. The results obtained indicate that cycloimide derivatives of chlorin p6 may serve as a base for the development of an efficient near‐IR photosensitizer.

Dynamics of ultrastructural changes in the isolated crayfish mechanoreceptor neuron under photodynamic impact

“…If mitochondria have been traditionally considered as the most possible PDT target, our data have shown that the Golgi apparatus might also be a target for photodynamic treatment. This is in agreement with the accumulating data indicating that different hydrophobic photosensitizers such as ZnPC (Fabris et al, 2001), pheophorbide‐a methylester (Matroule et al, 2001), chlorine p6 derivative (Feofanov et al, 2002), Photofrin (Hsieh et al, 2003), Foscan (Teiten et al, 2003), or 2,4,5,7‐tetrabromrodamine 123 bromide (Ogata et al, 2003) can target Golgi apparatus. PDT‐induced Golgi dysfunction may lead to apoptosis (Matroule et al, 2001; Ogata et al, 2003).…”

Comparative Study of Photodynamic Properties of 13, 15‐N‐cycloimide Derivatives of chlorin p6¶

“…Air‐equilibrated samples of methylene blue (11.7 μ M in buffer), 2 (8.3 μ M in 0.1 or 1% CrEL), 3 (8.9 μ M in 0.1 or 1% CrEL), 4 , (5.6 μ M in 0.1 or 1% CrEL) and 5 (6.9 μ M in 0.3 or 1% CrEL) were supplemented with RNO (30 μ M ) and histidine (10 m M ) in 50 m M Tris‐HCI (pH 7.4) and irradiated with the He‐Ne laser (543.5 nm, 0.6 mW/cm 2 , 2 h). Other details of the measurements and calculations have been described elsewhere (1). The RNO‐based assay in the absence of histidine was used under the same experimental conditions for the detection of *OH radicals.…”

“…To probe the state of lysosomes and mitochondria after irradiation, the cells were loaded with 4 or 5 , irradiated and stained with a mixture of ethidium bromide (EtBr; 5 ng/mL) and Rh123 (50 ng/mL) or AO (0.5 μg/mL). To clarify the type of cell death, the procedure of the PE‐annexin‐EtBr staining of the cells was performed as described previously (1). The functioning of lipid transport was checked with the following procedure.…”

“…The quantitative two‐dimensional (2D) maps of CIC distribution were reconstructed for each measured cell, and the cytoplasmic concentration of CIC was averaged over 30–50 cells. Data on the uptake of the dyes were analyzed with the equation 1 where C cyr (t) is the average cytoplasmic concentration (ACC) of the dye as a function of incubation time t, C ex the extracellular concentration of the dye, K k the maximum C cyt ‐C ex ratio at the saturation of uptake and T up the incubation time to achieve a 50% level of K k .…”

“…The photodynamic properties of 13,15‐[ N ‐(3‐hydroxypropyl)]cycloimide chlorin p6 ( 1 , Fig. 1) were recently investigated in vitro (1). The insertion of an additional imide ring in the chlorin p6 molecule shifts its long‐wavelength absorption band (λ Q ) from 665 to 711 nm and, by doing so, provides a suitable basis for the light excitation of chromophore situated deep within living tissue.…”