Mitochondria-targeting for improved photodynamic therapy

Photodynamic therapy (PDT) is an emerging cancer therapeutic modality, with great potential to selectively treat surface cancers, thus minimizing systemic side effects. PDT employs a combination of tumor localizing photosensitizing agents, low energy light (600 - 850 nm), and tissue oxygen to bring about cytotoxic effects in cancerous cells. Since singlet oxygen, the predominant cytotoxic agent in PDT, has a very short lifetime in biological systems(< 40 ns) and consequently a very short radius of action (< 20 nm), the loci of singlet oxygen generation is often the loci of maximum photodamge. Thus the photosensitizer's subcellular localization site at the time of irradiation plays an important role in determining both the therapeutic efficiency and mechanism of cell death. Although, several clinically approved photosensitizers have been shown to localize in critical cell organelles such as mitochondria, this has been in many cases by serendipity. Thus, there is a need to rationally design photosensitizers which can target critical cell organelles such as the mitochondria. In this dissertation, two approaches to deliver photosensitizers to the mitochondria were investigated: 1) Reducing photosensitizer sizes to improve endocytosis and lysosomal localization. Upon irradiation the photosensitizers would then produce singlet oxygen which could rupture the lysosomal membrane releasing the lysosomally trapped photosensitizers to the cytosol, from where they could relocalize to mitochondria by passive diffusion (photochemical internalization). 2) Using delocalized lipophilic cationic dyes (DLCs) to exploit membrane potential differences between the cytoplasm and mitochondria in delivering photosensitizers to mitochondria. A potential difference of ~ -180 mV exists across the mitochondrial membrane of mammalian cells and according to Nernst equation every -60 mV difference could lead to a 10-fold accumulation of delocalized monocations, sufficiently lipophilic to traverse the mitochondrial lipid bilayer. We also investigated the ability of rhodamine B, a delocalized lipophilic cationic dye (DLC) with a relatively large two-photon cross-section (200 GM at 800 nm), to deliver photosensitzers to the mitochondria while enhancing their two-photon cross-sections for improved NIR absorption. To investigate the effects of steric hindrance on mitochondrial localization and photodynamic response, a series of eight thiaporphyrins, previously synthesized at the University of Sunny at Buffalo were studied. Two new thiaporphyrin analogues 6 and 8 with reduced steric hindrance at the 10- and 15- meso positions were studied in comparison to 5,20-diphenyl-10, 15-bis[4 (carboxymethyleneoxy)-phenyl]-21,23- dithiaporphyrin 1, previously validated as a potential second generation photosensitizer. To evaluate the effects of the structural changes on the photophysical properties: the UV/Visible absorption spectra, molar extinction coefficients, rates of singlet oxygen generation, n-octanol/pH74 buffer partition coefficients, and aggregation tendencies were studied. To evaluate the effects of the structural modifications on photodynamic activity: the dark toxicities, phototoxicities, cellular uptake, and sub-cellular localization patterns were evaluated in vitro using rat mammary adenocarcinoma cells (R3230AC). Although 6 showed an extraordinarily high uptake (7.6 times higher than 1), it was less potent than 1 (IC50 = 0.18 μM versus 0.13 μM) even though they both showed similar sub-cellular localization patterns. This low potency was attributed to its high aggregation tendency in aqueous media (4 times higher than 1), which might have affected its ability to generate singlet oxygen in vitro. 8 on the other hand showed an even lower potency than 6 (2.28 vs 0.18 μM). However this was attributed to its low cellular uptake (20 times less than 6) and inefficient generation of singlet oxygen. Overall, although the structural modifications did improve the cellular uptake of 6, 6 was still less potent than the lead photosensitizers 1. Thus, other strategies to target mitochondria for improved photodynamic activity were investigated In a continuing project, we evaluated the ability of delocalized lipophilic cationic dyes to deliver photosensitizers to mitochondria by exploiting the membrane potential difference between the cytoplasm and mitochondria. Two conjugates: a porphyrinrhodamine B conjugate (TPP-Rh) and a porphyrin-acridine orange conjugate (TPP-AO), each possessing a single delocalized lipophilic cation, were designed and synthesized. The conjugates were synthesized by conjugating a monohydroxy porphyrin (TPP-OH) to rhodamine B (Rh B) and acridine orange base (AO), respectively, via saturated hydrocarbon linkers. To evaluate the efficiency of the conjugates as photosensitizers, their photophysical properties and in vitro photodynamic activities were studied in comparison to those of TPP-OH, the parent porphyrin photosensitizer. Although fluorescence energy transfer (FRET) was observed in the conjugates, they were capable of generating singlet oxygen at rates comparable to TPP-OH. Biologically, TPP-Rh showed very promising results, with a much higher phototoxicity [IC50 , 3.95 μM: upon irradiation with 400 - 850 nm light (3 mWcm-2) for 1 h], than either TPP-OH or Rh B (IC50, >20 μM). Furthermore, TPP-Rh showed no significant dark toxicity at concentrations up to 20 μM. This improved photodynamic activity was presumably due to a greater cellular uptake and preferential localization in the mitochondria. The cellular uptake of TPP-Rh was 8 and 14 times greater than TPP-OH and Rh B, respectively. In addition, fluorescence imaging studies suggest that TPP-Rh localized more in mitochondria than TPP-OH. TPP-AO, on the other hand showed dark toxicity at