Photochemical internalization: a novel technology for delivery of macromolecules into cytosol.

The therapeutic usefulness of macromolecules, such as in gene therapy, is often limited by an inefficient transfer of the macromolecule to the cytosol and a lack of tissue-specific targeting. The possibility of photochemically releasing macromolecules from endosomes and lysosomes into the cytosol was examined. Endocytosed macromolecules and photosensitizer were exposed to light and intracellular localization and the expression of macomolecules in the cytosol was analyzed. This novel technology, named photochemical internalization (PCI), was found to efficiently deliver type I ribosome-inactivating proteins, horseradish peroxidase, a p21ras-derived peptide, and a plasmid encoding green fluorescent protein into cytosol in a light-dependent manner. The results presented here show that PCI can induce efficient light-directed delivery of macromolecules into the cytosol, indicating that PCI may have a variety of useful applications for site-specific drug delivery, e.g., in gene therapy, vaccination, and cancer treatment.

Liposome-bound Zn (II)-phthalocyanine. Mechanisms for cellular uptake and photosensitization.

In the present study, cellular uptake of a liposomal formulation of ZnPc (CGP 55847) has been studied in human cervix carcinoma cells of the line NHIK 3025. The cellular uptake of ZnPc is found to be completed after 4-8 h of incubation. The maximum level of ZnPc in the cells after incubation with 1 microgram/ml ZnPc in E2a medium containing 3% serum is 60 ng/mg protein. The cellular uptake is attenuated by the presence of serum and at low temperature of the incubation medium, but the activation energy (30 kJ/mol) and fluorescence microscopic analysis of cells incubated with ZnPc at 0 degree C indicate that ZnPc is taken up into cells by a diffusion-mediated pathway. Measurements of subcellular marker enzymes have been performed immediately after light exposure of ZnPc-treated cells. The mitochondrial marker enzyme (cytochrome c oxidase) and the marker enzyme for the Golgi apparatus (UDP galactosyl transferase), but not those for lysosomes (beta-N-acetyl-D-glucosaminidase) and endoplasmic reticulum (NADPH cytochrome c reductase), are inactivated upon photodynamic treatment. These results indicate that ZnPc is mainly located in the Golgi apparatus and the mitochondria of NHIK 3025 cells. In contrast, photoactivated Photofrin is found to reduce the activity of UDP galactosyl transferase, but not that of NADPH cytochrome c reductase. The tetraphenylporphine TPPS2a and light reduce the activity of NADPH cytochrome c reductase, without influencing the activity of UDP galactosyl transferase. TPPS4 and light do not attenuate the activities of UDP galactosyl transferase and NADPH cytochrome c reductase.

Apoptosis induction by different pathways with methylene blue derivative and light from mitochondrial sites in V79 cells.

The importance of mitochondria for the induction of apoptosis by photodynamic therapy (PDT) was studied with a new photosensitizing dye, methylene blue derivative (MBD), and light. By using fluorescence microscopy and by measuring the MBD-PDT-induced inhibition of specifically subcellularly localized marker enzymes, we show that MBD is localized in mitochondria and not in lysosomes, endoplasmic reticulum or Golgi apparatus of V79 Chinese hamster fibroblasts. Cellular uptake kinetics and fluorescence properties of the dye in cells were characterized. Cell death was studied by a cell survival assay and by flow cytometry of cells stained using the terminal deoxynucleotidyl transferase (TdT) assay. MBD with light induced cell death by apoptosis via 2 different pathways, one rapid and one delayed, depending on the amount of dye in the cells. Cells treated with an MBD concentration higher than 0.05 microg/ml died by apoptosis within 3 hr after light exposure. At a concentration of 0.05 microg/ml MBD, cell death was induced slowly, and apoptotic cells appeared increasingly from the second day after PDT. Combination studies with 2-deoxyglucose (2-DOG) and carbonylcyanide-m-chlorophenylhydrazone (CCCP), inhibitors of glycolysis and oxidative phosphorylation, respectively, indicated that MBD and light inhibited mitochondrial oxidative phosphorylation. Abolishment of both energy sources led to cell death by necrosis within 6 hr. Inhibition of glycolysis alone induced apoptosis between 3 and 6 hr, while inhibition of mitochondrial oxidative phosphorylation alone led to delayed apoptosis within days.