Novel Photosensitizers Trigger Rapid Death of Malignant Human Cells and Rodent Tumor Transplants via Lipid Photodamage and Membrane Permeabilization

BACKGROUND: Apoptotic cascades may frequently be impaired in tumor cells; therefore, the approaches to circumvent these obstacles emerge as important therapeutic modalities. METHODOLOGY/PRINCIPAL FINDINGS: Our novel derivatives of chlorin e(6), that is, its amide (compound 2) and boronated amide (compound 5) evoked no dark toxicity and demonstrated a significantly higher photosensitizing efficacy than chlorin e(6) against transplanted aggressive tumors such as B16 melanoma and M-1 sarcoma. Compound 5 showed superior therapeutic potency. Illumination with red light of mammalian tumor cells loaded with 0.1 µM of 5 caused rapid (within the initial minutes) necrosis as determined by propidium iodide staining. The laser confocal microscopy-assisted analysis of cell death revealed the following order of events: prior to illumination, 5 accumulated in Golgi cysternae, endoplasmic reticulum and in some (but not all) lysosomes. In response to light, the reactive oxygen species burst was concomitant with the drop of mitochondrial transmembrane electric potential, the dramatic changes of mitochondrial shape and the loss of integrity of mitochondria and lysosomes. Within 3-4 min post illumination, the plasma membrane became permeable for propidium iodide. Compounds 2 and 5 were one order of magnitude more potent than chlorin e(6) in photodamage of artificial liposomes monitored in a dye release assay. The latter effect depended on the content of non-saturated lipids; in liposomes consisting of saturated lipids no photodamage was detectable. The increased therapeutic efficacy of 5 compared with 2 was attributed to a striking difference in the ability of these photosensitizers to permeate through hydrophobic membrane interior as evidenced by measurements of voltage jump-induced relaxation of transmembrane current on planar lipid bilayers. CONCLUSIONS/SIGNIFICANCE: The multimembrane photodestruction and cell necrosis induced by photoactivation of 2 and 5 are directly associated with membrane permeabilization caused by lipid photodamage

Effect of gramicidin A on the dipole potential of phospholipid membranes.

The effect of channel-forming peptide gramicidin A on the dipole potential of phospholipid monolayers and bilayers has been studied. Surface pressure and surface potential isotherms of monolayers have been measured with a Langmuir trough equipped with a Wilhelmy balance and a surface potential meter (Kelvin probe). Gramicidin has been shown to shift pressure-area isotherms of phospholipids and to reduce their monolayer surface potentials. Both effects increase with the increase in gramicidin concentration and depend on the kind of phosphatidylcholine used. Application of the dual-wavelength ratiometric fluorescence method using the potential-sensitive dye RH421 has revealed that the addition of gramicidin A to dipalmitoylphosphatidylcholine liposomes leads to a decrease in the fluorescence ratio of RH421. This is similar to the effect of phloretin, which is known to decrease the dipole potential. The comparison of the concentration dependences of the fluorescence ratio for gramicidin and phloretin shows that gramicidin is as potent as phloretin in modifying the membrane dipole potential

bcm_1038.qxd

Abstract-Using dialkylphospholipid (diphytanyl phosphatidylcholine) instead of the conventional diacylphospholipid (diphytanoyl phosphatidylcholine) in planar lipid bilayer membranes (BLM) led to an increase in the diffusion potential of the penetrating cation plastoquinonyl-decyl-triphenylphosphonium (SkQ1), making it close to the Nernst value, and accelerated translocation of SkQ1 across the BLM as monitored by the kinetics of a decrease in the transmembrane electric current after applying a voltage (current relaxation). The consequences of changing from an ester to an ether linkage between the head groups and the hydrocarbon chains are associated with a substantial reduction in the membrane dipole potential known to originate from dipoles of tightly bound water molecules and carbonyl groups in ester bonds. The difference in the dipole potential between BLM formed of the ester phospholipid and that of the ether phospholipid was estimated to be 100 mV. In the latter case, suppression of SkQ1-mediated proton conductivity of the BLM was also observed

Photosensitizer binding to lipid bilayers as a precondition for the photoinactivation of membrane channels.

The photodynamic activity of sulfonated aluminum phthalocyanines (AlPcS(n), 1 </= n </= 4) was found to correlate with their affinity for membrane lipids. Adsorbing to the surface of large unilamellar vesicles (LUVs), aluminum phthalocyanine disulfonate induced the highest changes in their electrophoretic mobility. AlPcS(2) was also most efficient in mediating photoinactivation of gramicidin channels, as revealed by measurements of the electric current across planar lipid bilayers. The increase in the degree of sulfonation of phthalocyanine progressively reduced its affinity for the lipid bilayer as well as its potency of sensitizing gramicidin channel photoinactivation. The portion of photoinactivated gramicidin channels, alpha, increased with rising photosensitizer concentration up to some optimum. The concentration at which alpha was at half-maximum amounted to 80 nM, 30 nM, 200 nM, and 2 microM for AlPcS(1), AlPcS(2), AlPcS(3), and AlPcS(4), respectively. At high concentrations alpha was found to decrease, which was attributed to quenching of reactive oxygen species and self-quenching of the photosensitizer triplet state by its ground state. Fluoride anions were observed to inhibit both AlPcS(n) (2 </= n </= 4) binding to LUVs and sensitized photoinactivation of gramicidin channels. It is concluded that photosensitizer binding to membrane lipids is a prerequisite for the photodynamic inactivation of gramicidin channels