Nanoparticulate carriers for photodynamic therapy of cholangiocarcinoma: In vitro comparison of various polymer-based nanoparticles

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Abstract

The photodynamic therapy with porphyrin derivatives is an established approach to targeted tumor therapy, but is still afflicted with disadvantages of the physicochemical characteristics of the photosensitizer. To overcome drug-related restrictions in photodynamic therapy, three 5,10,15,20-tetrakis(m-hydroxyphenyl) porphyrin (mTHPP)-loaded nanoparticulate formulations based on poly(dl-lactide-co-glycolide) (PLGA), poly(d,l-lactide) (PLA), and Eudragit® E were prepared in a consistent diameter range and compared with free mTHPP in vitro. Formulation behavior was investigated in two different cholangiocellular cell lines, EGI-1 and TFK-1.

High cytotoxicity was shown for all photosensitizer loaded nanoparticle (NP) formulations and free mTHPP, with EC50 values ranging from 0.2 to 1.3 μM. PLA based NP were not as effective in all performed tests as other formulations. Nanoparticulate embedded mTHPP remained photodynamically active and resulted in caspase-3 activation even at low concentrations of 250 nM. PLGA based NP exhibited highest caspase-3 activation.

For all formulations an effective intracellular accumulation of mTHPP was observed, whereby for mTHPP-Eudragit® E-NP a 200-fold drug accumulation was shown.

Polymer based nanoparticles were shown to be an effective and highly active transport vehicle for the photosensitizer mTHPP in vitro. Problems like low solubility of free drug can be circumvented by successful embedding into nanoparticulate carrier systems, maintaining therapeutic effects of the photosensitizer.

Graphical abstract

The study on cholangiocellular cell lines was conducted with mTHPP-loaded nanoparticles with different polymers as matrices. Nanoparticles were characterized and the effect of the carrier system was examined in vitro. Comparison of free substance, embedded substance and different materials revealed severe differences for cellular uptake and in vitro activity after activation by illumination.

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Introduction

In the last decade photodynamic therapy (PDT) has become more and more attractive for the treatment of cancer in comparison to conventional chemo- and radiation therapy (Paszko et al., 2011). Especially, in terms of adverse effects and selectivity, PDT offers advantages. After intravenous application of the photosensitizing drug and a period of latency for accumulation into the malignant tissue, the target region is illuminated with light of a suitable wavelength. Due to the activation of the used photosensitizer (PS), reactive oxygen species (ROS) are generated, which finally result in a destruction of malignant tumor tissue by mechanisms of apoptosis and necrosis. Although most PS are applied systemically, the therapeutic effect is rather local and very selective (Castano et al., 2005). Only the target tissue is illuminated and surrounding healthy regions can be spared of treatment. Nevertheless some disadvantages are still present. Most of the PS belong to class IV of the biopharmaceutical classification system (BCS) and therefore are characterized by a low solubility and low permeability and need to be administered intravenously (Chatterjee et al., 2008). Due to low solubility, they tend to aggregate in aqueous medium, leading to undesirable side effects (Chatterjee et al., 2008, Westerman et al., 1998). To overcome these obstacles, drug delivery systems are an important tool to increase applicable PS concentration and reduce the adverse effects such as skin or eye photosensitivity and pain during application. Several drug carrier systems for PS have been investigated in recent years (Chatterjee et al., 2008). A trend-setting drug carrier system can be seen in polymeric nanoparticles (NP), since they enable a high loading efficiency of the PS and a controlled release in vivo without influencing the PS activity (Paszko et al., 2011). As prominent examples, poly(dl-lactide-co-glycolide) (PLGA), poly(d,l-lactide) (PLA), and poly(butyl methacrylate-co-(2-dimethylaminoethyl) methacrylate-co-methyl methacrylate) 1:2:1 (Eudragit® E) have been used for drug delivery applications by other groups before (Khachane et al., 2011, Konan et al., 2003, Löw et al., 2011). PLGA is one of the most commonly used polymers for drug delivery. PLA has more lipophilic properties than PLGA, therefore the half-life in vivo is higher. Some of the big advantages of PLGA and PLA are the biodegradability and the biocompatibility (Wischke and Schwendeman, 2008). These properties are the most important factors to ensure safe drug release and to minimize cytotoxicity. Eudragit® E is an often used excipient for a protective coating of tablets and capsules or for sustained release formulations (Nollenberger and Albers, 2013). In this study it is investigated as an alternative material to PLGA and PLA. All the polymers used are approved by the Food and Drug Administration (FDA) for pharmaceutical use.

One emerging application of PDT is the palliative treatment of non-resectable cholangiocellular carcinomas (CCC) (Kiesslich et al., 2009). It is applied in patients with non-resectable carcinomas of the bile duct to regain biliary drainage and additionally reduce tumor size. Photosensitizers like porfimer sodium (Photofrin®) and 5,10,15,20-tetrakis(m-hydroxyphenyl) chlorin (mTHPC, Foscan®) can be used for this application and have been tested in several studies (Kniebühler et al., 2013, Lee et al., 2013). A study with low doses of the second-generation photosensitizer mTHPC demonstrated an improved standard PDT of CCC with a decrease of side effects and a good outcome (Kniebühler et al., 2013). In order to maximize the therapeutic effect, it is desirable to accumulate more photosensitizer in tumor tissue than in healthy tissue (Kiesslich et al., 2010). For this intention, nanoparticulate formulations can be used to passively target the tumor via the enhanced permeability and retention (EPR) effect, which is caused by the leaky vascularization and the lack of lymphatic drainage in tumor tissue (Brannon-Peppas and Blanchette, 2004).

In the present study the in vitro behavior of photosensitizer-loaded NP based on the aforementioned polymers in comparison to the free drug was evaluated in human cholangiocarcinoma cell lines using the photosensitizer 5,10,15,20-tetrakis(m-hydroxyphenyl) porphyrin (mTHPP) as a porphyrin equivalent for the approved drug mTHPC. During drug carrier development no loss of mTHPP activity was favored while the substance was embedded into different nanoparticulate carriers.

Section snippets

Materials

Poly(dl-lactide-co-glycolide) (PLGA) characterized by a copolymer ratio of 50:50 (Resomer® RG 502H), poly(d,l-lactide) (PLA, Resomer® R 203H) and poly(butyl methacrylate-co-(2-dimethylaminoethyl) methacrylate-co-methyl methacrylate) 1:2:1 (Eudragit® E) were purchased from Evonik Industries (Darmstadt, Germany). The photosensitizer mTHPP was kindly provided by biolitec research GmbH (Jena, Germany). Poly(vinyl alcohol) (PVA, average molecular weight 30–70 kDa) and trifluoroacetic acid were

Synthesis and characterization of mTHPP-loaded nanoparticles

In the present study different polymers were used to encapsulate the photosensitizer mTHPP. Using the emulsion diffusion technique for nanoparticle preparation, spherical nanoparticles could be achieved. The average particle diameter ranged from 200 to 300 nm depending on the polymer used for particle formation. The PDI showed a nearly monodisperse size distribution in a range of about 0.1 for all formulations (Table 1). A well-defined spherical particle morphology could be revealed by SEM

Discussion

Palliative photodynamic therapy of cholangiocarcinoma has been used since the late nineties with remarkable success in better bile drainage and improvement of life quality (Ortner et al., 1998). Used photosensitizers are porfimer sodium (Photofrin©, approved by the FDA as orphan drug for cholangiocarcinoma) and mTHPC (Foscan®, not FDA-approved) in vivo and 5-aminolevulinic acid in vitro (Kim et al., 2011, Kniebühler et al., 2013Lee et al., 2013). Yet, not all expectations in therapy have been

Conclusion

In summary, it can be stated that three different photosensitizer loaded polymer-based nanoparticle systems could be produced with reproducible and well-defined physico-chemical properties. The NPs were tested in two bile duct cancer cell lines in comparison to free drug in order to assess the in vitro activity. Polymer dependent differences in cell accumulation and cytotoxic effects were observed. A certain advantage was detected for mTHPP-PLGA-NP with regard to cytotoxicity and for m

Acknowledgements

The authors acknowledge the German Bundesministerium für Bildung für Forschung (BMBF) for funding (FKZ 13N11390). We also thank biolitec research AG (Jena, Germany) for the kindly providing of the photosensitizer mTHPP. Furthermore the authors like to thank Karin Possemeyer from the University of Muenster (Germany) for support with scanning electron microscopy.

References (35)

  • A. Preuß et al.

    Photosensitizer loaded HSA nanoparticles II: in vitro investigations

    Int. J. Pharm.

    (2011)
  • S.K. Sahoo et al.

    Residual polyvinyl alcohol associated with poly (d,l-lactide-co-glycolide) nanoparticles affects their physical properties and cellular uptake

    J. Controlled Release

    (2002)
  • S. Spek et al.

    Characterization of PEGylated PLGA nanoparticles comparing the bulk to the particle surface using UV/vis spectroscopy, SEC, 1H-NMR spectroscopy, and X-ray photoelectron spectroscopy

    Appl. Surf. Sci.

    (2015)
  • D. Tang et al.

    Caspase-8 activation and bid cleavage contribute to MCF7 cellular execution in a caspase-3-dependent manner during staurosporine-mediated apoptosis

    J. Biol. Chem.

    (2000)
  • T. Vanden Berghe et al.

    Determination of apoptotic and necrotic cell death in vitro and in vivo

    Methods

    (2013)
  • C. Wischke et al.

    Principles of encapsulating hydrophobic drugs in PLA/PLGA microparticles

    Int. J. Pharm.

    (2008)
  • C.E. Astete et al.

    Synthesis and characterization of PLGA nanoparticles

    J. Biomater. Sci. Polym. Ed.

    (2006)

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