Efficient anti-tumor effect of photodynamic treatment with polymeric nanoparticles composed of polyethylene glycol and polylactic acid block copolymer encapsulating hydrophobic porphyrin derivative

Abstract

To develop potent and safer formulation of photosensitizer for cancer photodynamic therapy (PDT), we tried to formulate hydrophobic porphyrin derivative, photoprotoporphyrin IX dimethyl ester (PppIX-DME), into polymeric nanoparticles composed of polyethylene glycol and polylactic acid block copolymer (PN-Por). The mean particle size of PN-Por prepared was around 80 nm and the zeta potential was determined to be weakly negative. In vitro phototoxicity study for PN-Por clearly indicated the significant phototoxicity of PN-Por for three types of tumor cells tested (Colon-26 carcinoma (C26), B16BL6 melanoma and Lewis lung cancer cells) in the PppIX-DME concentration-dependent fashion. Furthermore, it was suggested that the release of PppIX-DME from PN-Por would gradually occur to provide the sustained release of PppIX-DME. In vivo pharmacokinetics of PN-Por after intravenous administration was evaluated in C26 tumor-bearing mice, and PN-Por exhibited low affinity to the liver and spleen and was therefore retained in the blood circulation for a long time, leading to the efficient tumor disposition of PN-Por. Furthermore, significant and highly effective anti-tumor effect was confirmed in C26 tumor-bearing mice with the local light irradiation onto C26 tumor tissues after PN-Por injection. These findings indicate the potency of PN-Por for the development of more efficient PDT-based cancer treatments.

Introduction

Photodynamic therapy (PDT) is a unique cancer treatment modality based on the dye-sensitized photo-oxidation of biological substances in the target tissue (Agostinis et al., 2011, Kübler, 2005). Basically, PDT-based cancer treatment consists of the following two major steps: (i) topical or systemic administration of photosensitizers (PS) and (ii) irradiation of non-thermal light in the visible range (635–760 nm) to tumor tissues. The light irradiation to the tumor leads to the excited singlet state of PS within tumor tissues from ground singlet state. A fraction of the excited singlet state PS is transformed via intersystem crossing to the relatively long-lived excited triplet state. The interaction of the excited triplet PS and molecular oxygen results in the formation of singlet oxygen (¹O₂), which is the primary phototoxic species generated upon the light irradiation. The damage induced by the singlet oxygen results first in injury to cellular function and structure, and ultimately in the death of cancer cells and regression of tumor growth (O'Connor et al., 2009). Since these reactive oxygen species are only generated when all of the three factors, PS, light with specific range of wavelength, and oxygen molecule, co-exist, the tissue selectivity of this treatment toward tumor tissues can be expected (Buytaert et al., 2007, Robertson et al., 2009). In addition, unlike other conventional cancer chemotherapy, drug resistance is expected to be hardly acquired and therefore repeated treatments would be possible (Dolmans et al., 2003).

However, PDT has several disadvantages that have to be improved. Firstly, most of PS are lipophilic and essentially poorly water-soluble under physiological conditions and are easily aggregated after administration, which not only complicates the formulation of PS, but also dramatically reduces the photodynamic activity of PS against tumor (de Visscher et al., 2011). Secondly, even if PS is solubilized in the aqueous vehicle and is given as solution, it is known that PS nonspecifically distributes throughout the body and induces various side effects in the skin and eyes that are exposed to daylight. Skin photosensitivity reactions are characterized by erythema, edema, blistering, hyperpigmentation and sunburn (Wolf et al., 1993), and these phototoxicities to skin significantly reduce the quality of life of patients who receive PDT. To overcome these problems that associate with poor solubility and nonspecific in vivo disposition characteristics of PS, a lot of attention has been paid to the development of a safer dosage form for PS with higher solubilizing capability and better tumor targeting properties while reducing non-specific disposition to other tissues/organs. To date, although various nanoparticulate PS formulations such as liposome (Bovis et al., 2012, García-Díaz et al., 2012), emulsion (Garbo et al., 1998) or others (Konan et al., 2003, Sibata et al., 2004) have been examined, and all of these formulations achieved certain improvements in PS solubility, the outcome especially from in vivo studies is still limited and unsatisfactory. Among nanoparticulate drug carriers available, polymeric nanoparticles composed of an amphiphilic diblock copolymer are one of promising drug carriers (Ayen et al., 2011, Yang et al., 2011). In terms of structural and functional properties, polymeric nanoparticles have a hydrophobic core where hydrophobic drugs can be easily incorporated, and their hydrophilic outer shell is associated with an aqueous layer, which protects from the interaction with plasma proteins such as opsonins enhancing phagocytosis by macrophages in the liver and spleen, and which therefore provides a prolonged blood circulation time. It is well known that nanoparticles with certain physicochemical properties preferentially accumulate in many types of solid tumors due to the unique pathophysiological characteristics of tumors, so-called “enhanced permeability and retention effect (EPR effect)” (Maeda et al., 2000, Matsumura and Maeda, 1986). On the other hand, since nanoparticles usually cannot permeate the small vessels, the tissue distribution should be highly limited. Thus, polymeric nanoparticles could be a suitable vehicle for hydrophobic compounds including PS to achieve their efficient tumor delivery with lower side effects.

In the present study, therefore, we used a hydrophobic porphyrin derivative, photoprotoporphyrin IX dimethyl ester (PppIX-DME), as PS, and incorporated it into polymeric nanoparticles (PN) composed of poly(ethylene glycol)-block-polylactic acid (PEG–PLA) (PN-Por). The stable incorporation of hydrophobic drugs within PN is dependent on the physicochemical properties of the copolymer used. In general, block copolymers with a similar length of hydrophobic and hydrophilic segments with molecular weights ranging from 5000 to 15,000 are used for preparing polymeric micelles (Lee et al., 2007, Liu et al., 2007). However, due to the unstable incorporation of drugs in these conventional polymeric micelles in the systemic circulation, the chemical conjugation of doxorubicin to a diblock copolymer (Nakanishi et al., 2001) or chemical introduction of a hydrophobic group to paclitaxel (Negishi et al., 2006) was necessary to more stably incorporate these drugs into, and to reduce their excess release from, polymeric micelles in the systemic circulation. In the present study, to achieve stable incorporation of PppIX-DME in the blood circulation, a PEG–PLA polymer (Mw: 42,000) composed of long PLA chain (37,000) was applied (Araki et al., 2012). First, the in vitro phototoxicity of PN-Por was assessed for various types of cancer cells and the release property of PppIX-DME from PN-Por prepared was also evaluated. Moreover, in vivo disposition characteristics of PN-Por labeled with ³H-cholesteryl hexadecylether (³H-CHE) and in vivo anti-tumor effect of PN-Por with the local light irradiation to tumor tissue was assessed after its intravenous injection into Colon-26 carcinoma (C26) solid tumor-bearing mice.