Interaction dynamics of hypericin with low-density lipoproteins and U87-MG cells

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Abstract

The natural photosensitizer hypericin exhibits potent properties for tumor diagnosis and photodynamic therapy. Fluorescent properties of hypericin along with various technical approaches have been used for dynamic studies of its interaction with low-density lipoprotein and U87 glioma cells. Evidences for hypericin release from low-density lipoprotein towards cells plasmatic membrane are addressed. Subsequent subcellular bulk flow redistribution leading to non-specific staining of intracellular membranes compartment were observed within cells. It was shown, that monomers of hypericin are the only redistributive forms. Increasing concentration of hypericin leads to the formation of non-fluorescent aggregates within low-density lipoprotein as well as within the U87 cells, and can preclude its photosensitizing activities. However, the aggregation process can only account for a part of the observed emission decrease. As shown by the excited state lifetime measurements, this fluorescence quenching actually results from a combination of aggregation process and energy transfer from monomers to aggregates. In all experiments, hydrophobic character of hypericin appears as the driving force of its redistribution process.

Introduction

Hypericin (Hyp) is a natural photosensitizer characterized by high singlet oxygen production (Olivo et al., 2006, Cole et al., 2008, Higuchi et al., 2008, Kacerovská et al., 2008). Its specific accumulation in tumor cells can be used for photo-diagnosis of early epithelial cancer (Sim et al., 2005, Fu et al., 2007, Kah et al., 2008, Ritz et al., 2008a). These properties together with minimal dark toxicity, and high clearance rate from the host body make Hyp a promising agent for PDT of cancer (Falk, 1999, Agostinis et al., 2002, Miskovsky, 2002, Kiesslich et al., 2006).

As expected from its molecular structure (Fig. 1), Hyp is a highly hydrophobic compound that forms non-fluorescent aggregates in aqueous solution. However absorption and fluorescence emission spectra of Hyp monomer can be observed after binding with various biological macromolecules (Das et al., 1999, Falk, 1999, Petrich, 2000, Miskovsky et al., 2001, Agostinis et al., 2002, Miskovsky, 2002, Kiesslich et al., 2006). Formation of reactive oxygen species (ROS) can only be obtained from its monomeric form (Senthil et al., 1992, Guedes and Erikson, 2005, Gbur et al., 2008, Gbur et al., 2009). The very short lifetimes of ROS limit their diffusion through the cells. Consequently, the intracellular biological target and subsequent photo-induced cell death pathways (apoptotic or necrotic) are closely related to the subcellular localization of Hyp as well as to its monomers–aggregates balance. In this view, the intravascular transport of Hyp by LDLs and subsequent cellular uptake processes should be taken in consideration.

LDL is known as the main carrier of cholesterol in the human circulation system. It can be characterized as a spherical particle of 22 nm in diameter having three different regions. The outer surface layer, which consists of phospholipids molecules with a single apoB-100 protein; the core of LDL, which is enriched with triglycerides and cholesterol esters and an interfacial region between these two (Hevonoja et al., 2000). Due to the overexpression of specific receptors in many types of transformed cells (Brown and Goldstein, 1976, Vitols et al., 1992) LDL could play a key role in the targeted delivery of hydrophobic and/or amphiphilic photosensitizers to tumor cells for PDT (Jori, 1996, Reddi, 1997, Konan et al., 2002, Derycke and de Witte, 2004, Sherman et al., 2004). Previous investigations of Hyp interaction with LDL have shown non-specific binding of both its monomer and aggregate forms. Up to ∼30 monomer molecules of Hyp can enter one LDL (Kascakova et al., 2005). Hyp are most likely embedded in the phospholipids shell of LDL close to the apoB-100 protein (Kascakova et al., 2008, Lajos et al., 2008, Mukherjee et al., 2008) and cholesterol has recently been identified as the key determinant of the observed selectivity of Hyp in model membrane systems (Ho et al., 2009). Upon increasing the Hyp concentration, the fluorescence quantum yield is dramatically decreased, suggesting most likely self-quenching of the aggregated form. It was shown that mixing of photosensitizers with LDL before in vivo administration led to a better delivery to target tissues and to an improved photodynamic efficacy (Korbelik, 1992, Korbelik, 1993, Chowdhury et al., 2003). Consistently, we have shown that over expression of LDL receptors leads to an increased accumulation of Hyp within U87 glioma cells (Kascakova et al., 2008). Therefore, the question is; what is the importance of the in vivo plasmatic transport of Hyp by LDL on the cellular uptake process and on its subsequent subcellular distribution. Does it enter cells through the endocytosis pathway of LDL or might the release of Hyp from LDL towards cellular membrane – prior to LDL endocytosis – be the main process? In this view, steady-state and kinetic studies of LDL–Hyp interaction were performed in using the fluorescence properties of Hyp.

Section snippets

Chemicals

Hyp, LDL and LDL-bodipy (LDLb) were all purchased from Gibco-Invitrogen, France.

Cell culture

The U87-MG human glioma cells were grown in Dulbecco's modified Eagle's medium (D-MEM) containing l-glutamine (862 mg L−1), sodium pyruvate (110 mg L−1), glucose (4500 mg L−1), streptomycin (50 μg mL−1), penicillin (50 μg mL−1) and supplemented with 10% fetal bovine serum (FBS) or serum substitute 2% Ultroser G (Pall Corporation, France), in the presence of 5% CO2 humified atmosphere at 37 °C. For all experiment cells were

Dynamic studies of Hyp–LDL interaction

The basic fluorescence spectroscopic study of Hyp interaction with LDL has already been described in our previous studies (Kascakova et al., 2005, Mukherjee et al., 2008). The concentration dependence of Hyp steady-state fluorescence emission in the presence of LDLs was monitored (Kascakova et al., 2005) and result of a similar experiment is given here (Fig. 2). While increasing the number of Hyp molecules per LDL molecule, a saturation effect of the fluorescence intensity is first observed,

Conclusion

In this work, dynamic studies gives clear evidences that the monomer form of Hyp which was already considered as the only photoactive form should also be considered as the only redistributive form while aggregate form should be considered as a monomer delivery delay form. However, the transport of Hyp under aggregate form in overloaded LDL (Hyp/LDL = 200:1) not only allows precluding the toxic photo-oxidation process of LDL prior to cellular uptake but also leads to an higher intracellular

Acknowledgements

This work was supported by the Slovak Research and Development Agency contracts No. APVV-0449-07 and APVV-LPP-0337-06, the Scientific Grant Agency of the Ministry of Education of Slovak Republic (grant VEGA-0164-09). The authors thank the Synchrotron SOLEIL for providing the detection system of the DISCO beamline.

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