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P4 Intracellular uptake of liposomal chloroaluminum phthalocyanine and this antitumor activity on 3D melanoma cell culture

Barbugli, P.A.a; Alves, C.P.b; Espreafico, E.M.b; Tedesco, A.C.c

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doi: 10.1097/01.cmr.0000382836.66984.da
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Introduction Skin cancer, such as melanoma, is one of the most aggressive diseases; it normally presents a higher therapeutical resistance to chemotherapy and radiotherapy. Based on this behavior, the development of new therapeutic strategies for the treatment of melanoma skin cancer becomes necessary [1]. Liposomes vesicles encapsulated with AlClPc phtosensitizer was development, reaching 90% of melanoma cell death after PDT also suggesting apoptose (Barbugli et al., 2010). As a second part of this work, the liposomal AlClPc intracellular uptake was evaluated as well as this antitumor activity on 3D melanoma cell cultures.

Methods Melanoma cells in different phases of the tumor progression were used: WM1552C (radial growth phase), WM278 (vertical growth fase) and WM1617 (metastatic). The cells were given by Dr. Herlyn from Winstar Institute (Philadelphia, USA). The intracellular uptake was measured by fluorescence spectroscopy (quantitave assay) and by confocal microscopy (qualitative assay). The 3D cell cultures were developed as multicellular spheroids, prepared by growing the metastatic cells WM1617 in Petri dishes on a 1.5% agar; and as 3D cultures on collagen matrix. In all experiments, the cells were treated with liposomal AlClPc 0.29 μg ml−1 and irradiated with a laser visible light at 675 nm at 70, 140 and 700 mJ cm−2.

Results In the quantitative assay, the photosensitizer intracellular uptake was almost the same for all melanoma cells in a monolayer model, about 30 ng ml−1 of the photosensitizer after 3 h of incubation with liposomal AlClPc. The confocal fluorescence microscopy indicated that AlClPc was distributed throughout the cytoplasm of the cells, with no detectable penetration into the nucleus, reinforce the idea that liposomal AlClPc have no mutagenic effect as already proved by other studies using this dye. The multicellular spheroids size decreased according to the PDT treatment, reaching the lower size of them after 700 mJ cm−2 of laser irradiation in the presence of 0.29 μg ml−1 of the photosensitizer. In the collagen 3D dermal matrix, the metastatic cell colonies development was also decreased after PDT treatment, with no finding of metastatic cell colonies during two weeks after the laser irradiation (700 mJ cm−2).

Conclusion The liposomal AlClPc have a great antitumor activity against melanoma cells in 3D metastatic cultures, with no mutagenic effect and this is an open field to treat melanoma, which has been excluded from clinical treatment by PDT until now.

Acknowledgement

FAPESP Grants ♯ 06/61414-3,08/53719-4 & 07/55319-0 CAPES, CNPQ Grants ♯ 306351/2006-4.

References

1. Villanueva J, Herlyn M. Melanoma and the tumor microenvironment. Curr Oncol Rep 2008; 10:439–446.
© 2010 Lippincott Williams & Wilkins, Inc.