Photodiagnosis and Photodynamic Therapy 16 (2016) 100–105 Contents lists available at ScienceDirect Photodiagnosis and Photodynamic Therapy jou rn al hom epage: www.elsevier.com/locate/pdpdt In vitro effects of photodynamic therapy induced by chloroaluminum phthalocyanine nanoemulsion Leonardo Pereira Franchi a , Camila F. Amantino a , Maryanne T. Melo a , Ana Paula de Lima Montaldi b , Fernando L. Primo c , Antonio Claudio Tedesco a,∗ a Department of Chemistry, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, USP, Ribeirão Preto, SP, Brazil b Department of Biology, Faculty of Philosophy, Sciences and Letters of RibeirãoPreto, USP, Ribeirão Preto, SP, Brazil c Department of Bioprocess and Biotechnology, School of Pharmaceutical Sciences of Araraquara, UNESP, Araraquara, SP, Brazil a r t i c l e i n f o Article history: Received 30 July 2016 Received in revised form 30 August 2016 Accepted 4 September 2016 Available online 7 September 2016 Keywords: Cytotoxicity Flow cytometry Chloroaluminum phthalocyanine DNA repair Cancer cell lines a b s t r a c t Background: The photodynamic therapy (PDT) has been used to treat cancer mainly by inducing oxidative stress. Our aim was to evaluate the effect of PDT and its combination with methoxyamine (MX), a blocker of base excision repair (BER), in cells expressing high levels of the APE1 protein, which is involved in cell oxidative damage response. Methods: The HeLa and A549 cells were treated for 3 h with chloroaluminum phthalocyanine incorpo- rated into a well-designed nanoemulsion (ClAlPc/NE); and then irradiated by visible light (@670 nm) with doses of 0.1, 0.5 and 1.0 J/cm 2 . A simultaneous combination of MX + ClAlPc/NE was performed and then irradiated with the selected dose of 0.5 J/cm 2 . The treatments were evaluated in terms of viability, clonogenicity, DNA fragmentation, and cell death mechanism by apoptosis and/or necrosis. Results: The APE1 protein expression observed was higher in HeLa than in A549. Both cell lines exhibited substantial differences in cell cytotoxicity. The PDT decreased the clonogenicity of HeLa by inducing apoptosis (sub-G1 and annexin detection). Additionaly, the MX potentiates the PDT-effects in HeLa. Otherwise, low cytotoxicity was observed in A549 cells. Conclusion: The PDT induced apoptosis in high APE1 expressive HeLa cells, and the blockage of BER by MX increased its effects. © 2016 Elsevier B.V. All rights reserved. 1. Background The photodynamic therapy (PDT) has become an emerging promise to anticancer therapy [1,2]. The chloroaluminum phthalo- cyanine (ClAlPc) are molecules excited by visible light (@ 670 nm) which generate reactive oxygen species (ROS) causing oxidative stress [3]. It may be incorporated into a nanoemulsion as a drug delivery system (ClAlPc/NE) and used as photosensitizer in PDT [4]. The oxidative DNA damage is repaired by Base Excision Repair (BER), being its main component, APE1 (apurinic/apyrimidinic endonuclease 1), overexpressed in numerous solid cancers [5]. Methoxyamine (MX) is an organic amine that interrupts BER by ∗ Corresponding author at: Center of Nanotechnology and Tissue Engineer, Pho- tobiology and Photomedicine Research Group–Department of Chemistry, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, University of São Paulo Av Bandeirantes, 3900, Monte Alegre, 14049 900, Ribeirão Preto, SP, Brazil. E-mail address: atedesco@usp.br (A.C. Tedesco). tightly interacting with the DNA apurinic/apyrimidinic sites [6], which are processed by APE1. Thus, this study aimed to compare the responses of cell lines with high APE1 protein basal levels (A549 and HeLa) to PDT expo- sure, as well as, evaluate the influence of BER blockage (PDT plus MX), in order to potentiate the cytotoxicity of this therapy. 2. Materials and methods The GM07492 (human fibroblast cells, Coriell Institute for Med- ical Research); the A549 (lung adenocarcinoma) and HeLa (cervical adenocarcinoma) cells (both from American Type Culture Collec- tion) were cultured in Ham-F12 + DMEM 1:1 medium, 10% fetal bovine serum (Cultilab, Brazil), antibiotics (1%) at 37 ◦ C in a humid- ified 5% CO 2 -incubator. The nanoemulsion of ClAlPc is type oil-in-water (o/a) obtained by spontaneous emulsification process and quantified as described by Siqueira-Moura et al. [7]. Briefly, the organic phase with ace- tone was prepared containing natural soy phospholipids and ClAlPc at 55 ◦ C. Subsequently, this organic solution was added into the http://dx.doi.org/10.1016/j.pdpdt.2016.09.003 1572-1000/© 2016 Elsevier B.V. All rights reserved.