Sonodynamic and photodynamic mechanisms of action of the novel hypocrellin sonosensitizer, SL017: mitochondrial cell death is attenuated by 11, 12-epoxyeicosatrienoic acid.

Development of sonosensitizers for sonodynamic therapy (SDT) which selectively target abnormal cells can limit undesired side effects in chemotherapeutic applications. Hypocrellin-B (HB) derivatives are low molecular weight compounds which belong to the perylenequinone family of photosensitizing and sonosensitizing compounds. In this study, we investigate the cytotoxic mechanisms of a novel HB-derived photo- and sonosensitizer, SL017. Human fibroblast WI-38 cells were treated with SL017 (0 µM, 0.1 µM or 10 µM) and subjected to photodynamic therapy (PDT) or SDT. Studies demonstrate that maximal uptake of SL017 occurs within 30 min, with a mitochondrial subcellular localization. Activation of SL017 by either visible light or ultrasound resulted in significant increases in reactive oxygen species (ROS) production as measured by CM-H2-DCFDA (5-(and-6)-chloromethyl-2'7'-dichlorodihydrofluorescein diacetate acetyl ester). Co-administration of the antioxidant, ascorbic acid, attenuated ROS production. Low concentrations of SL017 (100 nM) induced a rapid (<90 s) loss of mitochondrial membrane potential (ΔΨm). Epoxyeicosatrienoic acids (EETs), cytochrome P450-derived metabolites of arachidonic acid (AA) involved in maintaining homeostasis and protection against cell injury, were able to attenuate loss of ΔΨm, however ascorbic acid was not. SL017 treatment resulted in increased mitochondrial fragmentation which followed loss of ΔΨm. Our studies demonstrate that SL017 targets mitochondria, triggering collapse of mitochondrial membrane potential, generates ROS and subsequently results in mitochondrial fragmentation.

Target specificity of 188Re-labeled B27.1 monoclonal antibodies to ovarian cancer cells in vivo.

BACKGROUND: Currently we are exploring a new multistep pretargeting approach involving administration of a bispecific antibody (B27.1 x P54) which has an anti-CA-125 (B27.1) and antibiotin (P54) paratope. It is followed by the administration of radiolabeled biotinylated liposomes to target the 188Re to the ovarian cancer cells. As a preliminary step to realize this goal, we determined the target specificity of the monoclonal antibodies (B27.1) to the ovarian cancer cells in vivo. MATERIALS AND METHODS: B27.1 monoclonal antibodies were photoreduced using UV light and incubated with reduced 188Re for 30 min at 25degreesC. 188Re-labeled B27.1 antibodies were purified using size exclusion chromatography. A comparative biodistribution of Re-B27.1 and 188Re was performed in nude mice xenografted with NIH:OVCAR-3 cells. RESULTS: While free rhenium distributed preferentially into thyroid and stomach with insignificant accumulation in the cancer cells, about 20% of the injected dose of 188Re-B27.1 was recovered in ascites cells with insignificant localization in other organs four hours after administration. CONCLUSION: The study validates the affinity of the B27.1 antibodies to the ovarian cancer cells in vivo.

Optimization of a two-step desolvation method for preparing gelatin nanoparticles and cell uptake studies in 143B osteosarcoma cancer cells.

PURPOSE: To establish a matrix of parameters to synthesize nanoparticles of different sizes and to investigate the cellular uptake of these nanoparticles by osteosarcoma cancer cells in order to investigate their potential as therapeutic drugdelivery carriers. METHODS: Gelatin A and B were used to synthesize nanoparticles by a two-step desolvation process. Different parameters were investigated, including temperature, pH, concentration of glutaraldehyde, type of desolvating agent and nature of gelatin. For cell uptake studies, Texas Red labeled nanoparticles were incubated with 143B osteosarcoma cells and then evaluated using confocal laser scanning microscopy (CLSM). RESULTS: The systematic investigation of the synthesis parameters showed that it is possible to prepare gelatin-based nanoparticles with different particle sizes and a narrow size distribution. Temperature and nature of the gelatin were the most important synthesis factors. Bioimaging using CLSM showed uptake of the nanoparticles by 143B osteosarcoma cancer cells. CONCLUSIONS: Osteosarcoma cancer cells take up gelatin nanoparticles. This might improve the clinical effectiveness of anti-cancer treatments if nanoparticles are used as a drug delivery system and has important implications for future cancer treatment strategies.

Distribution of photosensitizers in bladder cancer spheroids: implications for intravesical instillation of photosensitizers for photodynamic therapy of bladder cancer.

PURPOSE: Uniform intratumor distribution of sufficient photosensitizer is one of the important aspects of photodynamic therapy for solid tumors. METHODS: Multicellular spheroids derived from a human transitional cell carcinoma cell line (MGHU3) were used as a surrogate system of tiny solid tumors to study intratumor distribution of photosensitizers. Photosensitizers included Photofrin, hypocrellins (HBEA-R1/R2, HBBA-R2), aluminum phthalocyanine chloride (AlPC), benzoporphyrin derivative monoacid ring A (BPD-MA), protoporphyrin-IX (PpIX), and liposomal formulations of HBBA-R2 and BPD-MA. Spheroids were incubated with various doses of the above drugs for 1-4 hours, and were examined by confocal microscopy. RESULTS: Histology showed all cells were healthy in spheroids less than 400 microm in diameter. Scanning electron microscopy showed tight cell-to-cell interdigitation in spheroids. HBEA-R1/R2 distributed more uniformly in spheroids than other drugs. Free hypocrellins and BPD-MA penetrated spheroids centripetally deeper than AlPC, Photofrin, and PpIX. Liposomal HBBA-R2 and BPD-MA penetrated less than their free formulations. CONCLUSIONS: The spheroids mimic solid tumors prior to neovascularization. Based on drug distribution in spheroids, hypocrellins and BPD-MA appear superior to Photofrin, AlPC and PpIX for intravesical administration for bladder cancer phototherapy.