Pharmaceutical NanotechnologyImproved photodynamic activity of porphyrin loaded into nanoparticles: an in vivo evaluation using chick embryos
Introduction
Photodynamic therapy (PDT) is based on the administration of a photosensitizing agent (also known as a photosensitizer, PS), which is further activated by external irradiation with light. This therapy results in a sequence of photochemical and photobiological processes that trigger irreversible damage to the irradiated tissues. The main therapeutic applications of PDT are cancer therapy (Dougherty et al., 1998) and the treatment of neovascularization-related disorders such as choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD), one of the leading causes of blindness in elderly people in Western countries (Ferris et al., 1984, Klein et al., 1992). Since photosensitizers are inactive without light activation, this treatment can be considered to be selective to the illuminated area and a decrease in adverse effects is expected especially in cancer treatment. Most photosensitizers are porphyrin-like macrocycles and include porphyrins, chlorins and bacteriochlorins (Sternberg et al., 1998). Hydrophobic porphyrins, such as benzoporphyrin derivative monoacid ring A (BPD-MA), are potentially interesting molecules for PDT either in the treatment of solid cancers or ocular vascularization diseases (Sharman et al., 1999, Schmidt-Erfurth and Hasan, 2000, Renno and Miller, 2001). Indeed, BPD-MA encapsulated into liposomes (Visudyne®) is the first PS approved for clinical PDT of classic subfoveal choroidal neovascularization. The development of hydrophobic photosensitizers is, however, hampered by formulation problems due to their lipophilicity especially when intravenous (IV) administration is needed. Different approaches have been proposed such as the incorporation of PS into liposomes, micelles, polymeric particles, and low density lipoproteins, and the development of hydrophilic polymer-drug complexes, as recently reviewed by Konan et al. (2002). Micellar systems can be regarded as suitable vehicles for hydrophobic PS. However, emulsifying agents used for their preparation, such as Cremophor-EL, have been reported to elicit acute hypersensitivity and anaphylactic reactions in vivo (Gelderblom et al., 2001). Different liposomal strategies have been developed to target PS to tumour tissues, as reviewed by Derycke and de Witte (2004). Although liposomal formulations can substantially improve both PDT efficacy and PS safety, conventional liposomes have limitations such as short shelf life and chemical and physical instability in biological environments. However, improvement of liposomal formulations has been reported with the design of liposomes with specifically modified moieties (Woodle, 1998, Drummond et al., 1999, Derycke and de Witte, 2004). Polymeric nanoparticles offer numerous advantages over the conventional drug delivery systems including high drug loading, controlled release, and a large variety of carrier materials and manufacturing processes (Leroux et al., 1996, Konan et al., 2002). Recently, a hydrophobic photosensitizer, meso-tetra(p-hydroxyphenyl)porphyrin (Fig. 1a), was incorporated into polymeric biodegradable nanoparticles (Konan et al., 2003a). This system proved to be more effective than free porphyrin in inhibiting in vitro mammary tumour cell growth following PDT. The relatively low drug concentration and the short times of incubation of nanoparticles with cells required to induce satisfactory photodynamic damages demonstrated that p-THPP-loaded nanoparticles offer superior photoactivity compared to the free drug (Konan et al., 2003b). In order to further investigate these carriers, the efficacy of the encapsulated p-THPP was assessed in the chick chorioallantoic membrane (CAM) assay. The developing chicken embryo is surrounded by a chorioallantoic membrane, which becomes vascularized as the embryo develops. This in vivo model has been extensively used to study both angiogenesis and anti-angiogenesis (Ribatti et al., 2001). Moreover, the CAM model has been used to study the photothrombic effects of PDT after topical application of PS (Toledano et al., 1998, Hammer-Wilson et al., 1999), injection of PS into the yolk sac (Gottfried et al., 1995), intraperitoneal administration (Hornung et al., 1999), and intraamniotical injection (Peterka and Klepacek, 2001). Lange et al. (2001) demonstrated the feasibility of an IV administration of PS into CAM vessels. This approach allowed the CAM model to be used as a screening procedure for new PS intended for PDT of choroidal neovascularization (CNV). The distribution and the possible leakage from the vascular system of intravenously injected PS or fluorescent dyes in the CAM is followed by measuring the fluorescence of the vascularized and non-vascularized tissues; hence pharmacokinetic data can be obtained. Furthermore, the photodynamic activity of the PS can be assessed by evaluation of the vascular occlusion achieved after irradiation in a pre-defined area of the CAM. In this context, the CAM is a convenient model for monitoring the modifications of the vasculature. The transparency of its superficial layers allows an examination of structural changes of each blood vessel in real time. This in vivo model offers the advantage of being easily accessible, inexpensive and easy to handle (Lange et al., 2001). Furthermore, it is possible to use and compare different administration routes.
The objective of this work was to compare the vascular effects of p-THPP either as a free solution or encapsulated in polymeric nanoparticles on the CAM vessels. An aqueous solution was first developed and tested for its suitability as a reference. Then, the distribution and the photodynamic activity of the drug were compared when administered in both systems.
Section snippets
Chemicals
Poly(d,l-lactide-co-glycolide) (PLGA) with a copolymer ratio of 50:50 and molecular weight of 12 kDa (Resomer® RG502) was obtained from Boehringer Ingelheim (Ingelheim, Germany). Meso-tetra(p-hydroxyphenyl)porphyrin (p-THPP) was provided by Aldrich (Steinheim, Germany). Poly(vinyl alcohol) 87.7% hydrolyzed with a molecular weight of 26 kDa (Mowiol® 4-88), was obtained from Hoechst (Frankfurt/Main, Germany). d(+)-Trehalose dihydrate and phosphate buffered saline (PBS) were purchased from
Preparation and characterization of nanoparticles
p-THPP was encapsulated in PLGA 50:50 via the emulsification–diffusion technique as described by Konan et al. (2003a). Nanoparticles were purified by cross-flow filtration and freeze-dried in the presence of trehalose (trehalose: nanoparticle mass ratio of 2:1). The mean diameter of the freeze-dried nanoparticles, determined by photon correlation spectroscopy (Zetasizer 5000, Malvern, Worcesterhire, UK), was 117 ± 7 nm with a polydispersity index of 0.2 on a scale from 0 to 1. For the
Development of an injectable soluble formulation for p-THPP
In order to compare the nanoparticles to a reference, it was necessary to develop a suitable formulation for IV injection of p-THPP, a highly hydrophobic photosensitizer. Various solvent systems containing 20% v/v of an organic solvent (either ethanol, benzyl alcohol or DMSO), 30% v/v of PEG 400 and 50% v/v of water were tested. p-THPP was soluble in all systems at concentrations up to 2.5 mg/ml. The absorption spectra of p-THPP formulations show all the characteristic bands of porphyrins, and
Development of an injectable soluble formulation of p-THPP
Since p-THPP is a very hydrophobic molecule that cannot be intravenously administered as a simple aqueous solution, cosolvents are needed to inject this compound for photodynamic purposes. Three different formulations using water and organic cosolvents (50% v/v of water, 30% v/v of PEG 400, and 20% v/v of either ethanol, DMSO or benzyl alcohol) were tested in this study. Polyethylene glycols (PEG 200 to PEG 600) are low toxicity compounds used as solvents for IV formulations (Mottu et al., 2000
Conclusion
We have demonstrated that PDT-induced vascular occlusion of the photosensitizer p-THPP is enhanced when encapsulated into nanoparticle delivery systems. The superiority of nanoparticles over solubilized p-THPP might be related to the reduced diffusion of p-THPP nanoparticles out of the vessels. Free p-THPP appeared to leak out before generating an efficient vascular occlusion, whereas nanoparticles appear to confer a longer residence time inside the vasculature and may also interact differently
Acknowledgments
The authors thank Professor Dr. Michael Eid (Department of Psychology, University of Geneva) for performing the statistical analysis of the experimental data.
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