New approach to hydrophobic cyanine-type photosensitizer delivery using polymeric oil-cored nanocarriers: Hemolytic activity, in vitro cytotoxicity and localization in cancer cells

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Abstract

We report on encapsulation of cyanine IR-768 in oil-in-water (o/w) microemulsion, i.e. fabrication of templated polymeric nanocapsules as effective nanocarriers for a new generation of photodynamic agents suitable for photodynamic therapy (PDT). Discussed here are nanocapsule imaging, their in vitro biological evaluation, cyanine encapsulation potential, and the cellular localization of cyanine IR-768 delivered in the nanocapsules to MCF-7 cancer cells. Oil-cored poly(n-butyl cyanoacrylate) (PBCA) nanocapsules were prepared by interfacial polymerization in o/w microemulsions formed by the nonionics Tween 80 (polysorbate 80, polyoxyethylene 20 sorbitan monooleate), and Brij 96 (polyoxyethylene 10 oleyl ether). Iso-propyl myristate (IPM), ethyl oleate (EOl), iso-octane (IO), and oleic acid (OA) were used as the oil phases and iso-propanol (IP) and propylene glycol (PG) as the cosurfactants. Such o/w droplets, also containing hydrophobic IR-768 in the oil phase, were applied in the interfacial polymerization of n-butyl cyanoacrylate at 10 °C at pH 5.0. The isolated cyanine-loaded nanoparticles were visualized by atomic force microscopy (AFM) and scanning electron microscopy (SEM), which proved their unimodal size distribution and spherical shape, with diameters dependent upon the monomer content and the template type. The entrapment efficiency of cyanine increased with increasing n-butyl cyanoacrylate concentration and varied from 65.7% to 91.7%. The results of in vitro erythrocyte hemolysis and the cell viability of breast cancer MCF-7 cells showed that the PBCA nanocapsules are quite safe carriers of IR-768 in the circulation, having a very low hemolytic potential and being non-toxic to the studied cells. Fluorescence microscopy visualized the cyanine intracellular distribution and, furthermore, demonstrated that PBCA-nanocarriers effectively delivered the IR-768 molecules to the MCF-7 doxorubicin-sensitive and -resistant cell lines. Photoirradiation of the cancer cells with entrapped photosensitizer decreased cell viability, demonstrating that this effect may be utilized in PDT.

Introduction

Photodynamic therapy (PDT) uses a combination of light and non-toxic dyes or photosensitizers to induce cancer cellular and tissue damage and is considered a minimally invasive therapeutic modality in oncology (Castano et al., 2005, Huang et al., 2008). In clinical PDT, however side effects were observed as a result of dark toxicity of the photosensitizers (Castano et al., 2004). Increased photosensitizer affinity to tumor cells which reduces side effects on normal cells may be obtained by various delivery strategies, which has led to the generation of new photosensitizers (Josefsen and Boyle, 2008). Among the recently described compounds with photodependent biological activity some cyanine dyes showed very promising properties (Delaey et al., 2000, Kassab, 2002). However, in vitro and in vivo instability, thermal- and photodegradation, as well as high protein binding impede the utility of free cyanines in PDT (Saxena et al., 2004, Saxena et al., 2006). Their drawback is the hydrophobicity leading to their insolubility, impeding the use of physiologically acceptable aqueous vehicles for intravenous administration. The delivery of greater amounts of such molecules to cancer cells is, however, possible through incorporation of them into biocompatible nanoparticles (mainly nanocapsules) (Bechet et al., 2008). Recently, indocyanine green (ICG) was entrapped in poly(dl-lactic-co-glycolic acid) nanoparticles, which permitted an increase in ICG circulation time in plasma and also elevated ICG uptake and accumulation in tissues (Saxena et al., 2004, Saxena et al., 2006).

An ideal drug delivery system should be biodegradable, biocompatible, and unassociated with incidental adverse effects. Furthermore, it is very important to achieve the lowest possible cytotoxicity of nanocarriers not influencing the therapeutic effect of the loaded bioactive substances. The most restrictive property of drug carriers in parenteral formulations is their interaction with components of the erythrocyte membrane, a commonly used model for biological membranes, denoted as hemolytic action. Particular efforts have to be undertaken to find effective delivery systems suitable for anticancer activity whose components should be selectively retained by the malignant tissue.

In the literature there are many examples of polymer-based colloidal drug-delivery systems designed for a variety of carriers (Breunig et al., 2008, Cho et al., 2008, Torchilin, 2007), including those which comprise nanocapsules consisting of a liquid core (mainly water) surrounded by a thin polymer envelope (Guterres et al., 2007, Johnston et al., 2006). Some of these structures can be obtained by interfacial polymerization, i.e. reactions carried out at the interfaces of micellar aggregates, mainly emulsions and liposomes (Gasco and Trotta, 1986, Lherm et al., 1992, Allemann et al., 1993, Watnasirichaikul et al., 2000a, Watnasirichaikul et al., 2000b; Vauthier et al., 2003; Graf et al., 2008a, Graf et al., 2008b). There have been attempts to apply a very unique type of aggregate as the template, i.e. transparent, isotropic, thermodynamically stable microemulsions that may spontaneously form as dispersed systems of water, oil, and a surfactant mixture in the presence of a cosurfactant (a pseudoternary system) or its absence (a ternary system), whose droplet diameters remain within the range of 10–140 nm (Moulik and Paul, 1998). The application of microemulsions as templates in interfacial polymerization has mainly been limited to water-in-oil, bicontinuous, and solution-type microemulsions (Graf et al., 2008a, Krauel et al., 2005) as well as water-free microemulsions (Krauel et al., 2006). Oil-containing nanocapsules were obtained mainly by the polymerization of alkyl cyanoacrylates at the oil/water interface of very fine o/w emulsion (Al Khoury-Fallouh et al., 1986). In the literature profound representatives of poly(alkyl cyanoacrylate) nanocapsules are presented which are suitable for the delivery of a water-soluble bioactive compound, for example as a potential system for delivering oligonucleotides (Lambert et al., 2000), proteins (Li et al., 2001), and peptide drugs (Graf et al., 2008b, Graf et al., 2009, Watnasirichaikul et al., 2000a).

As a continuation of our search for new carriers (Wilk et al., 2009a, Zielińska et al., 2008a) and new templates (Wilk et al., 2009b, Zielińska et al., 2008b), we focused our systematic studies on cytotoxicity and hemolysis of oil-cored poly(n-butyl cyanoacrylate) (PBCA)-type nanoparticles, mainly including photosensitizer-loaded PBCA nanocarriers obtained by the template-directed polymerization of n-butyl cyanoacrylate solubilized in the o/w droplet interfacial area. The microemulsion templates comprised o/w microemulsion systems formed by commercially available components, i.e. the nonionic surfactants Tween 80 (polysorbate 80, polyoxyethylene 20 sorbitan monooleate) and Brij 96 (polyoxyethylene 10 oleyl ether), the oil phases iso-propyl myristate (IPM), ethyl oleate (EOl), iso-octane (IO), and oleic acid (OA), and the cosurfactants iso-propanol (IP) and propylene glycol (PG). The biological impact of the obtained nanocapsules, both without and those loaded with hydrophobic cyanine IR-768 (for its structure see Fig. 1), was investigated. The hemolytic action was determined on human red blood cells (RBC). Moreover, the influence of the nanocapsules on cancer cells in vitro was assessed in terms of cell viability using MCF-7 human breast cancer cells. The MCF-7 cell line was established from the pleural effusion of a patient with a breast carcinoma (Soule et al., 1973). Recalling literature data the MCF-7/DX cells – subline of the MCF-7 – are resistant to various treatments, including these with cisplatin, doxorubicin (Chorna et al., 2004, Chorna et al., 2005), and some types of photodynamic therapeutic agents (Doyle et al., 1998, Crescenzi et al., 2004). Taking into account the MCF-7/DX multidrug resistant system it is not possible to treat the cells with free anticancer agents, above mentioned, and nanoencapsulation approaches by means of e.g. polymeric micelles (Mohajer et al., 2007) or nanocapsules described in the present contribution are necessary to be designed and investigated. In recent studies, however, the usefulness of polymeric micelles for the chemotherapeutic agent delivery of doxorubicin to both wild-type and doxorubicin-resistant MCF-7 cells has been demonstrated (Mohajer et al., 2007).

We report here on a new generation of oil-cored PBCA nanocapsules fabricated by means of the o/w microemulsion templates formed by polyoxyethyleneted nonionics as promising nanocarriers for the delivery of hydrophobic cyanine-type photosensitizers.

Section snippets

Materials

All reagents were of analytical grade and used as provided. Most of the chemicals, i.e. the polyoxyethyleneted nonionics Tween 80 (polysorbate 80, polyoxyethylene 20 sorbitan monooleate) and Brij 96 (polyoxyethylene 10 oleyl ether), chloroform, ethanol, dimethylsulfoxide (DMSO), Dulbecco's modified Eagle medium (DMEM), and cyanine IR-768 perchlorate, were purchased from Sigma–Aldrich. Iso-propyl myristate, ethyl oleate (both from Fluka) and oleic acid (Sigma–Aldrich) were used as the oil phase.

Nanocapsule preparation and imaging

The main approach to obtaining the oil-cored nanocapsules is the preparation and characterization of o/w microemulsions stabilized by the selected nonionics polyoxyethylene 20 sorbitan monooleate (Tween 80/polysorbate 80) and polyoxyethylene 10 oleyl ether (Brij 96), the most commonly used surfactants in pharmaceutical applications due to their high biocompatibility and low cytotoxicity and irritancy (Thadros, 2005, von Rybinski and Hill, 2003, Lawrence and Rees, 2000, Söderlind et al., 2003).

Conclusions

Poly(n-butyl cyanoacrylate) nanocapsules loaded with IR-768 prepared by interfacial polymerization in Tween 80 and Brij 96 stabilized o/w microemulsion templates are relatively innocuous for both human erythrocytes and the MCF-7/WT and MCF-7/DX cell lines. Some of the microemulsions had the capacity to cause defects in human erythrocyte membrane. However, the PBCA nanocapsules appeared nontoxic to erythrocytes and MCF-7 cells and were able to deliver cyanine IR-768 to the breast cancer cell

Acknowledgments

This work was financially supported by the Polish Ministry of Science and Higher Education (grant no. N205013854) and Wroclaw Medical University (grant no. 1603).

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