The synthesis of a series of new zinc phthalocyanine-peptide conjugates targeting the gastrin-releasing peptide (GRP) and integrin receptors is reported. Two alternative synthetic methods based on Sonogashira cross-coupling of an iodinated zinc phthalocyanine with acetylenic bombesin or arginine-glycine-aspartic acid (RGD) derivatives, either in solution or on solid phase, are presented. The water-soluble conjugates were screened for their photodynamic efficacy against several cancer cell lines expressing different levels of GRP and integrin receptors, and their intracellular localization was evaluated via confocal fluorescence microscopy. Variations in photocytotoxicity between the conjugates correlate to differences in hydrophobicity as well as receptor-mediated cell uptake. In the case of the phthalocyanine-bombesin conjugate, competition experiments confirm the involvement of the GRP receptor in both the phototherapeutic activity as well as intracellular localization. These findings warrant further in vivo studies to evaluate the potential of this conjugate as photosensitizer for photodynamic therapy (PDT) of cancers overexpressing the GRP receptor.

To identify optimal features of metalated sulfophthalocyanine dyes for their use as photosensitizers in the photodynamic therapy of cancer, we synthesized a series of alkynyl-substituted trisulfonated phthalocyanines and compared their amphiphilic properties to a number of parameters related to their photodynamic potency. Varying the length of the substituted alkynyl side-chain modulates the hydrophobic/hydrophilic properties of the dyes providing a linear relationship between their n-octanol/water partition coefficients and retention times on reversed-phase HPLC. Aggregate formation of the dyes in aqueous solution increased with increasing hydrophobicity while monomer formation was favored by the addition of serum proteins or organic solvent. Trisulfonated zinc phthalocyanines bearing hexynyl and nonynyl substituents exhibited high cellular uptake with strong localization at the mitochondrial membranes, which coincided with effective photocytotoxicity toward EMT-6 murine mammary tumor cells. Further increase in the length of the alkynyl chains (dodecynyl, hexadecynyl) did not improve their phototoxicity, likely resulting from extensive aggregation of the dyes in aqueous medium and reduced cell uptake. Aggregation was evident from shifts in the electronic spectra and reduced capacity to generate singlet oxygen. When monomerized through the addition of Cremophor EL all sulfonated zinc phthalocyanines gave similar singlet oxygen yields. Accordingly, differences in the tendency of the dyes to aggregate do not appear to be a determining factor in their photodynamic potency. Our results confirm that the latter in particular relates to their amphiphilic properties, which facilitate cell uptake and intracellular localization at photosensitive sites such as the mitochondria. Combined, these factors play a significant role in the overall photodynamic potency of the dyes.

This study demonstrates the efficacy of a three-step (64)Cu pretargeting procedure for PET imaging of apoptosis. Our data also confirm the usefulness of small animal PET to evaluate cancer treatment protocols.

Two cationic porphyrins bearing an isothiocyanate group for conjugation to monocolonal antibodies have been synthesized. The two porphyrins conjugated efficiently to three monoclonal antibodies (anti-CD104, anti-CD146 and anti-CD326), which recognize antigens commonly over-expressed on a range of tumour cells. In vitro, all conjugates retained the phototoxicity of the porphyrin and the immunoreactivity of the antibody. Mechanistic studies showed that conjugates formed from the mono- and tri-cationic porphyrin and anti-CD104 antibody mediated apoptosis following irradiation with non-thermal red light of 630 ± 15 nm wavelength. In vivo antibody conjugates caused suppression of human LoVo tumour growth in immunodeficient NIH III mice, similar to the commercial photodynamic therapy (PDT) agent Photofrin®, but at administered photosensitizer doses that were more than two orders of magnitude lower. Positron emission tomography (PET) following PDT showed a large, early increase in uptake of 18fluorodeoxyglucose (FDG) by tumours treated with the anti-CD104 conjugates. This effect was not observed with Photofrin® or with conjugates formed from the same photosensitizers conjugated to an irrelevant antibody.

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