The aim of the study was to develop a liposomal carrier for novel 2-(morpholin-4-yl)ethoxy-substituted phthalocyanines (Pcs 3 – 5) and to determine at what extent liposomal incorporation of Pcs 3 - 5 can affect physicochemical properties of these compounds and in vitro antiproliferative activity against oral cancer cells.
Phthalocyanine derivatives were studied both as free compounds and after incorporation into liposomes, used as drug delivery vehicles. Pc 3,4 had eight bulky groups in the periphery, whereas Pc 5 possessed 2 morpholiethoxy groups.1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), L-a-phosphatidyl-DL-glycerol (chicken egg, PG), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP) and cholesterol (chol) were used as components of liposomal membranes. Liposomes were prepared by a thin-film hydration method and extruded through polycarbonate filters. Free compounds were dissolved in spectroscopic grade solvents (purified water, DMSO, DMF). Subsequently, steady-state absorption solutions of free Pcs 3 – 5 or incorporated in liposomes were collected. 1-Octanol/water partition coefficients, Po/w, for Pcs 3 – 5 were determined following the shake-flask method and the binding constant to liposomes, Kb, by a spectroscopic titration technique. The in vitro photodynamic activity of Pcs 3 – 5 was evaluated against two oral squamous cell carcinoma lines, HSC-3 and H413, derived from the tongue and buccal mucosa, respectively. The cellular uptake of compounds was determined and subsellular localization was evaluated by fluorescent microscopy. The dark- and light-cytotoxicity of 3 – 5 was measured at concentrations: 0.1, 1.0 and 10.0 μM and IC50 for Pc 5 was determined. To determine the photocytotoxicity of these compounds, the cells treated with Pcs were irradiated with a total light dose of 3.6 J cm-2 using a wavelength of either 690 or 730 nm, depending on the compound.
The increasing number of 2-(morpholin-4-yl)ethoxy groups led to compounds that strongly absorb red light, which is favorable for photodynamic therapy applications. Furthermore, Pcs possessing eight substituents were more hydrophilic (logP value, lower Kb value, no aggregation in aqueous solution) than the disubstituted molecules. However, the hydrophilicity was found to attenuate the photodynamic therapy against oral cancer cells, as Pcs with eight substituents did not show significant cytotoxicity. The more hydrophobic derivative with two 2-(morpholin-4-yl)ethoxy moieties inhibited very effectively the viability of SCC cells (IC50 = 22 nM against HSC-3 cells). The subcellular localization and cellular uptake study showed that Pc 5 interacts effectively with oral cancer cells and is able to trigger the photodynamic process, in contrast to 3 and 4. This suggests that the chemical structure of 5 facilitates the interaction of this Pc with cells, and furthermore that the presence of eight substituents in the Pc molecules 3 and 4 effectively hinders this interaction. Liposomal incorporation of the Pcs influenced much more the properties of the hydrophobic compound 5 than that of the octasubstituted 3 and 4. These changes are related to the increased amount of the monomeric form of 5 following liposome incorporation.
Incorporation of hydrophobic phthalocyanines into liposomal membranes increases the fraction of the monomeric form of the compound and provides a hydrophobic matrix for a water-insoluble compound, enabling its administration in an aqueous environment. Among the different types of liposomes, the POPC/PG formulation of 5 can be considered as a suitable carrier for Pc 5 as this provides a desired antiproliferative activity at very low concentrations.
Available at: http://works.bepress.com/nejat-duzgunes/170/