The in vivo efficacy of phthalocyanine–nanoparticle conjugates for the photodynamic therapy of amelanotic melanoma
Introduction
Photodynamic therapy (PDT) is a well-established modality for the treatment of localised tumours which is used in clinical practice as an alternative or adjuvant to conventional therapies, such as radiotherapy, surgery or chemotherapy. PDT selectively destroys neoplastic lesions by the combined action of a light-activated drug, termed a photosensitiser, and visible light. The wavelengths of the light that are typically used for PDT are in the red or near infrared spectral range as these wavelengths exhibit a greater penetration depth into most human tissues and are not absorbed by normal tissue constituents. Upon irradiation, the photosensitiser is promoted to the long-lived lowest excited triplet state. The energy of the excited state photosensitiser molecule is transferred to the ground state of oxygen to produce the singlet state oxygen species. Singlet oxygen is cytotoxic and it is the production of this species which results in the destruction of cancerous tissue.1 Alternatively, the triplet photosensitiser can promote electron transfer processes with nearby substrates with the generation of radical species (including the superoxide anion and the hydroxyl radical), which can in turn induce different types of damage in the microenvironment of the photosensitiser binding site.1
Approval of PDT for oncological indications was first granted in 1993. Currently four drugs have received regulatory approval in North America and/or the European Union2: Photofrin (a complex mixture of haematoporphyrin derivatives, Axcan Pharma Inc., Mont-Saint-Hilaire, Canada), for advanced and early lung cancer, superficial gastric cancer, oesophageal adenocarcinoma, cervical cancer and bladder cancer; Foscan (meta-tetrahydroxyphenyl chlorin, Biolitec Pharma Ltd., Dublin, Ireland), for palliative head and neck cancer; the other two drugs, Levulan (5-aminolevulinic acid; ALA, Dusa Pharmaceuticals Inc., Wilmington, MA, USA) and Metvix (methyl 5-aminolevulinate, Photocure ASA, Oslo, Norway), are not themselves photoactive but when applied to basal cell carcinoma, for example, are metabolically converted to protoporphyrin IX (or a methyl ester derivative for Metvix) via the haem biosynthetic pathway. Protoporphyrin IX is a photosensitiser and consequently produces singlet oxygen when stimulated by light. Photodynamic therapy with these four photosensitiser drugs has been used to treat several thousand patients worldwide.3, 4 While excellent clinical results are achieved, the full exploitation of the potential of PDT is impeded by factors such as the somewhat limited selectivity of tumour targeting; the prolonged persistence of the available PDT agents in the skin, causing a generalised cutaneous photosensitivity; and the chemical heterogeneity and inefficient red light absorption typical of Photofrin and ALA-derived protoporphyrin.1, 3 The clinical status of photodynamic therapy for cancer treatment has been recently reviewed.2, 3, 4
Novel approaches are being investigated in order to overcome the present limitations of PDT, including the development of second-generation photosensitisers with improved photochemical and tumour-localising properties. Many of the compounds developed as second-generation photosensitisers have limited water solubility as hydrophobic photosensitisers typically exhibit a better tumour targeting and substantially greater PDT efficacy.1, 5 The obvious limitation of such water-insoluble photosensitisers is that they require a vehicle to deliver the drug to the tumour tissue.
While numerous drug delivery vehicles exist, particularly encouraging results have been obtained recently which suggest that nanoparticle-carried PDT agents are accumulated in significant amounts by a variety of tumour cells.6, 7, 8, 9 Upon accumulation in the tumour and subsequent activation by suitable visible-light wavelengths, these photosensitiser–nanoparticle conjugates induce an efficient damage of the malignant tissue.9, 10, 11 Photophysical investigations have demonstrated that the association of a photosensitiser with nanoparticles does not necessarily alter its excited state properties nor the efficiency of singlet oxygen generation.10, 11, 12 Thus, PDT could take advantage of the increasing evidence supporting the concept that nanoparticles have a large potential to act as a viable enhancer of tumour targeting by anti-neoplastic drugs.13, 14
Many different materials have been used to formulate nanoparticles for drug delivery. Various photosensitisers, many of which are hydrophobic, have been encapsulated within water-soluble polymers such as polylactic-co-glycolic acid and polylactic acid, to overcome the problems associated with solubilisation. For example, meso-tetra(4-hydroxyphenyl)porphyrin,15, 16 bacteriochlorophyll-a,17 verteporfin,18 methylene blue,19 hypericin20 and various phthalocyanines21, 22, 23 have all been encapsulated in such water-soluble polymers. Silica nanoparticles have been used successfully for the delivery of the readily available PDT photosensitisers, such as pheophorbides and chlorins,7, 24 or with unusual sensitisers, such as fullerene.25 Cyclodextrin-based nanoparticles also have been used to entrap photosensitisers and shown to exhibit good PDT efficiency.26, 27
We28, 29 and others30 have developed gold nanoparticles as carriers of hydrophobic phthalocyanines, examples of second-generation photosensitisers. In this instance, rather than the photosensitiser being encapsulated within the nanoparticle carrier, the photosensitiser is bound to the surface of the gold nanoparticles via a thiol tether specifically designed so that the molecule will form a self-assembled monolayer on the gold nanoparticle surface.28 The formation of the phthalocyanine on the gold nanoparticle surface has the potential advantage that the hydrophobic character of the photosensitiser is maintained so that the conjugates retain the enhanced tumour targeting and greater PDT efficacy. It has also been suggested that the binding of the photosensitiser to the surface of the nanoparticle may represent an advantage as singlet oxygen does not need to diffuse out of the polymeric/silica nanoparticle structure as per encapsulated photosensitisers.11
While the in vitro results are extremely promising, to date there are only a limited number of studies in which the in vivo efficacy of photosensitiser–nanoparticle conjugates has been investigated.18, 30, 31, 32, 33, 34, 35 In this paper, we describe the pharmacokinetic behaviour and PDT efficacy of phthalocyanine–gold nanoparticle conjugates for the treatment of a sub-cutaneously implanted amelanotic melanoma. This tumour was selected as a model for the present investigations since it is a common skin tumour that favourably responds to PDT treatment36 and it has been repeatedly adopted for assessing the efficacy of the experimental PDT with a number of photosensitising agents.37, 38
Section snippets
Photosensitising agents
The following photosensitising agents were used in the pharmacokinetic and experimental PDT studies: 1,1′,4,4′,8,8′,15,15′,18,18′,22,22′-tetradecakisdecyl-25,25′-(11,11′dithiodiundecyl)diphthalocyanine (C11Pc), (the free, non-bound, phthalocyanine photosensitiser) and the nanoconstruct made by conjugating the C11Pc photosensitiser with gold nanoparticles (C11Pc-Np). The chemical structure of the C11Pc is shown in Fig. 1. The synthesis of C11Pc and the formulation of the C11Pc-Np nanoparticle
Pharmacokinetic studies with C11Pc and the C11Pc-Np conjugate
The time-dependent distribution of the i.v.-injected C11Pc in C57 mice bearing a sub-cutaneously transplanted amelanotic melanoma (Fig. 2A) is similar to that previously determined for structurally analogous octa-decyl or octa-pentyl phthalocyanine derivatives albeit in a different animal model.40, 41 Thus, the largest accumulation of the photosensitising agent was found to occur in the constituents of the reticulo-endothelial system, such as liver and spleen. This observation is in agreement
Discussion
The utilisation of delivery vehicles for the administration of photodynamic agents by systemic routes has been the subject of active debate and several experimental studies among both basic investigators and clinical users in the field of PDT.43 In practice, the photosensitisers which usually exhibit the largest affinity for tumour tissues are characterised by a relatively high degree of hydrophobicity, hence their direct intravenous injection into the bloodstream is greatly facilitated by
Conclusions
The pharmacokinetic data reveal that by conjugating the C11Pc photosensitiser to gold nanoparticles an enhanced accumulation within a sub-cutaneously implanted amelanotic melanoma is achieved as compared with the free phthalocyanine. Photodynamic therapy studies confirm that tumour growth is optimally slowed following light treatment 3 h post-i.v. injection. Electron microscopy studies have confirmed that the mechanism of photodamage of the tumour is via destruction of the vasculature. The data
Conflict of interest statement
None declared.
Acknowledgement
Funding from Cancer Research UK (Grant C22031/A7097) to DAR and GJ which supported this work is gratefully acknowledged.
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