In the present work, we have found that TAPP, DAPP, TEMCC4+ and TEMCP4+ exhibit high photoactivity against LM3 mammary carcinoma cells. Among them, TAPP was the most active molecule, and its biodistribution was tested after topical application and systemic administration.
Previous experiments showed that TEMCC4+ was a highly effective agent to photo-induce the hemolysis of human red blood cells, whereas TEMCP4+ induced hemolysis at a lower extent due to haemoglobin absorption . Furthermore, TAPP demonstrated to be an efficient generator of singlet oxygen in the presence of oxidizable substrates, such as 9,10-dimetylanthracene and L-tryptophan in biomimetic media . On the other hand, porphyrin-fullerene C60 dyads with high ability to form photoinduced charge-separated state were evaluated as PS with potential application in the photoinactivation of S. aureus .
Cellular uptake of the PSs employed in the present work suggests that all the PSs assayed are incorporated linearly as a function of the added concentration, suggesting a major involvement of passive transport in the uptake of the PSs. The different slopes of the curves of incorporated PS as a function of the concentration suggest different incorporation rates.
Our results suggest that at 3 hours of exposure, the amount of intracellular PS correlates fairly with the photodamage degree. However, even when at 24 h the intracellular accumulated PS is higher than at 3 h, a direct correlation with the photodamage degree was not always observed. Whereas TEMCC4+, TEMCP4+ and DAPP were 10 to 20% more photoactive at 24 h as compared to 3 h, TAPP employed at 5 µM concentration, was less photoactive at longer incubation times and TCP-C604+ was the only PS that increased remarkably its efficiency of photosensitisation.
The different behaviour for each PS suggests that depending on the molecule and the intracellular concentration, the photoactivity is variable. The different substituents, as well as the degree of axial symmetry, have been suggested to contribute to the cellular uptake. The positive charges located in the molecule periphery, increased the amphipathic character of the PS, thus contributing to the interaction with cell membranes . In the present study, TAPP is taken up by the cells at a higher rate than the rest of the PSs.
TAPP bears four mobile dimetilaminopropyl groups as precursors of cationic centres. Considering the pKa values of these substituents , a high percentage of the amine groups can acquire positive charges by protonation in water, favouring a better interaction with cells. In the cases of TEMCP4+ and TEMCC4+, although these porphyrin derivatives have four carbazoyl substituents with intrinsic cationic groups, the cationic groups are in a rigid macrocycle structure and less exposed to interact with cells. On the other hand, DAPP has only two amine groups, while the dyad TCP-C604+ contains four rigid charges and a bulky and highly lipophilic C60 group that decreases an effective interaction with the cells.
Besides, TAPP, DAPP, and TEMCC4 + have quantum yields of singlet oxygen production in the range of 0.49 to 0.53, and TEMCP4 + has a slightly lower (0.40) quantum yield.
In terms of efficiency of photosensitization, it is worth to note that after 3 h of incubation, TAPP induced on LM3 cells an LD50s of 35.7 and 22.9 mJ/cm2 employing 2.5 µM and 5 µM concentrations, respectively. On the other hand, after 3 h exposure of the same cell line to the already marketed pro-photosensitizer 5-aminolevulinic acid (25 µM), it was previously reported by us that a light dose of 150 mJ/cm2 (same light array) was required to induce the same level of cell killing . This comparison suggests that the porphyrin TAPP is a promising PS.
The impaired action of 5 µM TAPP at longer incubation times may be ascribed to saturation of the intracellular targets and, at higher concentrations, TAPP molecules could be aggregated, thus inducing a diminished activity. On the other hand, the higher action of the porphyrin coupled to the fullerene at long incubation periods may suggest better solubilization as monomers since this structure is prone to form aggregates in aqueous media, increasing the photodynamic activity. Besides, the action of proteases may be needed to act at long times of exposure to release the porphyrin from the C60 structure.
TCP-C604+ is the least photoactive porphyrin, and this is correlated with a poor cellular uptake and, in addition to a low singlet oxygen quantum yield in polar media, which is 6-fold lower as compared to the other PSs. The poorer uptake of the molecule is probably due to the presence of a voluminous group and the scarce endocytic ability of the LM3 cells. In addition, as suggested previously , drugs coupled to fullerenes can significantly differ from the free drug in terms of cellular uptake and subcellular distribution.
A major contribution of passive transport in the cellular uptake of TAPP is line with its reported Log P of 1.67 . Besides, Golgi major localization, as well as moderate localization in lysosomes and mitochondria and diffuse cytoplasmatic pattern, is observed at 3 h of TAPP exposure. Lysosomes and the trans-Golgi network usually have an acidic lumen with pH of approximately 5 and 6.5, respectively, so that weak bases are likely to be charged at a higher extent , thus exhibiting more marked fluorescence. The subcellular location of a PS is known to have a strong influence on the cell death response to photoinactivation .
Among the 5 analysed molecules, TAPP was the most photoactive in the cell line employed, and thus, was the chosen PS to be employed in a tumour bearing mice model. After topical application, TAPP was almost completely retained in the stratum corneum of the skin, without diffusion to the deeper layers of the skin. However, some fluorescence can be distinguished in the hair follicles and sebaceous glands suggesting a contribution of the transfollicular route of drug delivery. Specific localization of TAPP in the stratum corneum suggests the use of TAPP-PDT for the treatment of actinic keratosis.
As well, recent reports demonstrated that the transepidermal route, hair follicles, and sebaceous glands significantly contribute to topical or transdermal delivery. Malignant cutaneous adnexal neoplasms form a group of rare, typically low-grade-malignancy carcinomas with follicular, sebaceous, apocrine, or eccrine differentiation or a combination of the first 3 subtypes, and PDT has already been performed in some of these diseases . The presence of TAPP in hair follicles and sebaceous glands, suggests the possibility to use this molecule in the photodynamic treatment of skin adnexal neoplasms.
Systemic administration of TAPP produce accumulation of high quantities of TAPP in tumour and SOT (tumour invaded skin), showing a high tumour: normal skin ratio of 31.4. It is also interesting to note the lack of accumulation of TAPP in brain tissue, evidencing that the molecule does not passage through the blood-brain barrier. Besides, high intestine and lung TAPP values suggest the possibility of employing TAPP-PDT for the treatment of colon and lung cancer, since both organs are easily accessible by optic fibres if further studies confirm the accumulation of TAPP in tumours of that origin.
The key conclusion of this study was that TAPP was the PS that showed the best performance in vitro. Upon topical application to tumour bearing mice, TAPP was retained in the stratum corneum and adnexal glands, having a potentiality in the treatment of superficial malignancies or pre-malignancies such as actinic keratosis and skin adnexal neoplasms after topical application. After systemic administration, it is highly selective for tumour tissue, thus having promising uses in PDT treatment of non-dermatologic conditions.