Gabriela Di Venosa

Mechanisms of 5‐aminolevulic acid ester uptake in mammalian cells

The porphyrin precursor 5‐aminolevulinic acid (ALA) is being widely used in photodynamic therapy of cancer. Improvement in ALA delivery has been sought through the use of ALA derivatives, in particular the esterification of ALA with aliphatic alcohols, which in certain cases can improve cellular penetration and selectivity. ALA uptake systems appear to be distinctive for each cell type. The LM3 mammary adenocarcinoma cell line takes ALA up by BETA transporters. In this work, we investigated ALA derivative transport systems through the inhibition of radiolabelled ALA uptake in the LM3 cells. We also performed inhibition studies of γ‐aminobutyric acid (GABA) uptake. The more lipohilic ALA derivatives hexyl‐ALA and undecanoyl‐ALA inhibit ALA uptake, whereas methyl‐ALA, R, S‐ALA‐2‐(hydroxymethyl)tetrahydropyranyl ester and the dendron aminomethane tris methyl 5‐ALA does not inhibit ALA uptake. A similar pattern was found for GABA, except that the dendron inhibited GABA uptake. However, hexyl‐ALA and undecanoyl‐ALA are not taken up by BETA transporters, but by simple diffusion, although they still inhibit ALA uptake by binding to the cell membrane. These results show that different modifications to the ALA molecule lead to different uptake mechanisms. Whereas ALA is taken up by BETA transporters, none of the ALA derivatives shares the same mechanism. Knowledge of the mechanisms of ALA derivatives entry into the cells is essential to understand and improve ALA‐mediated PDT and to the design of new ALA derivatives that may be taken up at a higher rate than ALA.

Sustained and efficient porphyrin generation in vivo using dendrimer conjugates of 5-ALA for photodynamic therapy

The use of endogenous protoporphyrin IX (PpIX) after administration of 5-aminolaevulinic acid (ALA) has led to many applications in photodynamic therapy (PDT). However the efficacy of ALA-PDT is sub-optimal for thicker tumours and improved ALA delivery and therapeutic response are required. We have investigated the conjugation of ALA to a second-generation dxcendrimer for enhancing porphyrin synthesis in vitro and in vivo in a murine tumour model using systemic i.p. administration. In vitro, the dendrimer was more efficient than ALA for porphyrin synthesis at low concentrations in good correlation with higher cellular ALA dendrimer accumulation. In vivo, the porphyrin kinetics from ALA exhibited an early peak between 3 and 4 h in most tissues, whereas the dendrimer induced sustained porphyrin production for over 24 h and basal values were not reached until 48 h after administration. Integrated porphyrin accumulation from the dendrimer and ALA, at equivalent molar ratios, was comparable showing that the majority of ALA residues were liberated from the dendrimer. The porphyrin kinetics appear to be governed by the rate of enzymatic cleavage of ALA from the dendrimer, which is consistent with in vitro results. ALA dendrimers may be useful for metronomic PDT, and multiple low-dose ALA-PDT treatments.

Characterisation of liposomes containing aminolevulinic acid and derived esters

Liposomes of different compositions have been designed to improve delivery of aminolevulinic acid (ALA) and its esterified derivatives ALA-Hexyl ester (He-ALA) and ALA-Undecanoyl ester (Und-ALA) for its use in photodynamic therapy (PDT). Egg yolk phosphatidyl choline (PC), phosphatidic acid (PA) and phosphatidyl glycerol (PG) were employed in the preparation of the liposomes. Sonicated vesicles composed of PC, PC-PG (80:20) or PC-PA (80:20) containing ALA or derivatives were obtained and purified by a minicolumn centrifugation method. PC liposomes presented encapsulation percentages around 6% for 2 mM ALA, 13% for 2 mM He-ALA and 51% for 2 mM Und-ALA. The addition of PG or PA to the formulation, resulted in an increased entrapment: 19% for 2 mM ALA, 69% for 2 mM He-ALA and 87% for 2 mM Und-ALA in PC-PG liposomes and 21% for 2 mM ALA, 60% for 2 mM He-ALA and 87% for 2 mM Und-ALA in PC-PA liposomes. Higher concentrations of ALA or derivatives resulted in lower percentages of entrapment. The three formulations containing ALA or derivatives were stable up to 1 week upon storage at 4 degrees C. However, upon dilution with medium, ALA leaked from the liposomes, while on the contrary, He-ALA was highly retained, being therefore a good choice for its use in PDT. The stability of Und-ALA upon dilution could not be tested, but Und-ALA proved to have the highest entrapment efficacy.

Disorganisation of cytoskeleton in cells resistant to photodynamic treatment with decreased metastatic phenotype

The appearance of cells resistant to photodynamic therapy (PDT) is crucial for the outcome of this antitumoral treatment. We had previously isolated two sublines resistant to PDT derived from the mammary adenocarcinoma LM3 [A. Casas, C. Perotti, B. Ortel, G. Di Venosa, M. Saccoliti, A. Batlle, T. Hasan, Induction of murine tumour cell lines resistant to ALA-mediated Photodynamic Therapy, Int. J. Oncol. 29 (2006) 397-405.]. These clones have severely impaired its metastatic potential in vivo together with decreased general anchorage-dependent adhesion and invasion. In the present work we analyzed the differential expression and distribution of cytoskeleton and adhesion proteins in these cell lines. In both resistant clones, loss of actin stress fibers and disorganization of the actin cortical rim was observed. E-cadherin and beta-catenin and vinculin distribution was also disorganized. However, Western blot assays did not show differential expression of actin, E-cadherin, vinculin or beta-catenin. It was demonstrated that PDT strongly affects cell-cell and cell-substrate adhesion through the reorganization of some cytoskeletal and adhesion proteins. Changes in the metastasis phenotypes previously found are likely to be ascribed to these differences.

Decreased metastatic phenotype in cells resistant to aminolevulinic acid-photodynamic therapy

Photodynamic therapy (PDT) is a novel cancer treatment utilising a photosensitiser, visible light and oxygen. PDT often leaves a significant number of surviving tumour cells. In a previous work, we isolated and studied two PDT resistant clones derived from the mammary adenocarcinoma LM3 line (Int. J. Oncol. 29 (2006) 397-405). The isolated Clon 4 and Clon 8 exhibited a more fibroblastic, dendritic pattern and were larger than the parentals. In the present work we studied the metastatic potential of the two clones in comparison with LM3. We found that 100% of LM3 invaded Matrigel, whereas only 19+/-6% and 24+/-7% of Clon 4 and Clon 8 cells invaded. In addition, 100% of LM3 cells migrated towards a chemotactic stimulus whereas 38+/-8% and 73+/-10% of Clones 4 and 8, respectively, were able to migrate. In vivo, 100% of the LM3 injected mice developed spontaneous lung metastasis, whereas none of the Clon 8 did, and only one of the mice injected with Clon 4 did. No differences were found in the proteolytic enzyme profiles among the cells. Anchorage-dependent adhesion was also impaired in vivo in the resistant clones, evidenced by the lower tumour take, latency time and growth rates, although both clones showed in vitro higher binding to collagen I without overexpression of beta1 integrin. This is the first work where the metastatic potential of cells surviving to PDT has been studied. PDT strongly affects the invasive phenotype of these cells, probably related to a higher binding to collagen. These findings may be crucial for the outcome of ALA-PDT of metastatic tumours, although further studies are needed to extrapolate the results to the clinic employing another photosensitisers and cell types.

Porphyrin synthesis from aminolevulinic acid esters in endothelial cells and its role in photodynamic therapy

Photodynamic therapy (PDT) may cause tumour cell destruction by direct toxicity or by inducing microcirculatory shutdown. Protoporphyrin IX generated from 5-aminolevulinic acid (ALA) has been widely used as an endogenous photosensitiser in PDT. However, the hydrophilic nature of the ALA molecule limits its penetration through the stratum corneum of the skin and cell membranes and thus, ALA alkyl-esters have been developed to improve ALA permeation. The aim of this work was to study Protoporphyrin IX synthesis from ALA and its derivatives ALA methyl ester (Me-ALA) and ALA hexyl ester (He-ALA) in the microvascular endothelial cell line HMEC-1 derived from normal skin, and to evaluate their response to PDT. We found that lower light doses are required to photosensitise HMEC-1 endothelial cells than to photosensitise PAM212 transformed keratinocytes, showing some possible selectivity of ALA-PDT for vascularisation in skin. Employed at concentrations leading to equal Protoporphyrin IX synthesis, ALA, He-ALA and Me-ALA presented the same efficacy of HMEC-1 photosensitisation. However, He-ALA was a promising compound for the use in the enhancement of Protoporphyrin IX in HMEC-1 cells employed at low concentrations at both short and long time exposures whereas Me-ALA should be employed at high concentrations and longer time periods in order to surpass the Protoporphyrin IX levels obtained with ALA. The advantage of Me-ALA over ALA was based on its lower dark toxicity. This is the first work to report vascular cell photosensitisation employing alkyl-esters of ALA, and we demonstrated that these derivatives could exert the same effect as ALA and under certain conditions enhance photosensitisation of vasculature.

Comparation of liposomal formulations of ALA Undecanoyl ester for its use in photodynamic therapy

ALA administration has been used to induce the endogenous photosensitiser Protoporphyrin IX for photodynamic therapy (PDT) of tumours. However, the hydrophilic nature of ALA limits its ability to penetrate through skin restricting the use of ALA-PDT to superficial diseases. Lipophilic derivatives of ALA such as ALA Undecanoyl ester (Und-ALA) were designed to have better diffusing properties. However, Und-ALA, applied topically on the skin over the tumour, induced low porphyrin content. To improve Und-ALA efficacy we tested the efficacy of Und-ALA as porphyrin inducer, delivered in phosphatidylcholine and phosphatidylglycerol (PC-PG) or phosphatidylcholine and phosphatidic acid (PC-PA) liposomal formulations. Entrapment of Und-ALA into PC-PA or PC-PG liposomes resulted in a dramatic impairment of toxicity in the mammary tumour LM3 cells. However, liposomal Und-ALA induced lower intracellular porphyrin content compared to free ALA, although total porphyrins content (intracellular+media) from free Und-ALA resulted equal compared to liposomal Und-ALA, due to induction of porphyrins release induced by the latter. Topical administration of Und-ALA in PC-PG or PC-PA liposomes over the skin of LM3 subcutaneously injected mice, induced equal amount of tumour porphyrins as compared to free Und-ALA. The kinetics of porphyrins synthesis from Und-ALA is similar for free and liposomal formulations both in vivo and in vitro, showing that release of Und-ALA from liposomes is not gradual and suggesting that liposome membranes either fuses or binds to the cell membranes. To sum up, the incorporation of Und-ALA into liposomes of PC-PA or PC-PG composition does not improve the rate of porphyrin synthesis either in vitro or in vivo, due to a massive release of extracellular porphyrins and a poor cytoplasmatic release of the liposome content. The design of new liposome compositions either favouring endocytosis or coated with natural polymers to prevent Und-ALA interaction with cellular membrane are desired to overcome intracellular porphyrin release after long-chained ALA esters treatment.

Distribution of 5-aminolevulinic acid derivatives and induced porphyrin kinetics in mice tissues

Purpose: Porphyrins synthesised from 5-aminolevulinic acid (ALA) have been successfully used for the photodiagnosis and photodynamic treatment of cancer. To find a more efficient pro-photosensitiser, we synthesised two ALA esters: R,S-ALA-2-(hydroxymethyl)tetrahydropyranyl ester (THP-ALA) and ALA-Undecanoyl ester (Und-ALA). Methods: In mice bearing a subcutaneous mammary adenocarcinoma, we studied the distribution of the porphyrins formed from these esters in tissues after systemic administration, to establish if these esters are retained in any specific tissue, which could potentially be targeted for photodynamic treatment with ALA derivatives. We also investigated the topical use of these esters. Results: After systemic administration, tumour and skin overlying tumour porphyrin levels were lower from the ALA esters than from ALA. Other tissues such as liver, colon, kidney, skin and spleen also accumulated less porphyrins from the esters, showing that there is no specific retention of the esters in these tissues. However, the brain was the only organ that synthesised more porphyrins from THP-ALA than from ALA. The kinetics of porphyrin synthesis from ALA esters is comparable to those from ALA in almost all tissues, showing that esterases activities are not limiting the availability of the hydrolysed ALA. Both THP-ALA and Und-ALA, applied topically on the skin over the tumour, exhibited higher selectivity than ALA for the site of application, whereas the amount of tumour porphyrin was the same from ALA and THP-ALA but lower from Und-ALA. Conclusions: THP-ALA may be useful for the treatment of brain tumours after systemic administration, whereas THP-ALA and Und-ALA may be used more suitable for the treatment of superficial tumours due to their higher selectivity.

The use of dipeptide derivatives of 5-aminolaevulinic acid promotes their entry to tumor cells and improves tumor selectivity of photodynamic therapy.

The use of endogenous protoporphyrin IX generated after administration of 5-aminolaevulinic acid (ALA) has led to many applications in photodynamic therapy (PDT). However, the bioavailability of ALA is limited by its hydrophilic properties and limited cell uptake. A promising approach to optimize the efficacy of ALA-PDT is to deliver ALA in the form of prodrugs to mask its hydrophilic nature. The aim of this work was to evaluate the potential of two ALA dipeptide derivatives, N-acetyl terminated leucinyl-ALA methyl ester (Ac-Leu-ALA-Me) and phenylalanyl-ALA methyl ester (Ac-Phe-ALA-Me), for their use in PDT of cancer, by investigating the generation of protoporphyrin IX in an oncogenic cell line (PAM212-Ras), and in a subcutaneous tumor model. In our in vitro studies, both derivatives were more effective than ALA in PDT treatment, at inducing the same protoporphyrin IX levels but at 50- to 100-fold lower concentrations, with the phenylalanyl derivative being the most effective. The efficient release of ALA from Ac-Phe-ALA-Me appears to be consistent with the reported substrate and inhibitor preferences of acylpeptide hydrolase. In vivo studies revealed that topical application of the peptide prodrug Ac-Phe-ALA-Me gave greater selectivity than with ALA itself, and induced tumor photodamage, whereas systemic administration improved ALA-induced porphyrin generation in terms of equivalent doses administered, without induction of toxic effects. Our data support the possibility of using particularly Ac-Phe-ALA-Me both for topical treatment of basal cell carcinomas and for systemic administration. Further chemical fine-tuning of this prodrug template should yield additional compounds for enhanced ALA-PDT with potential for translation to the clinic. Mol Cancer Ther; 14(2); 440–51. ©2014 AACR.

Reversal of the Migratory and Invasive Phenotype of Ras-Transfected Mammary Cells by Photodynamic Therapy Treatment

Photodynamic therapy (PDT) is a non‐thermal technique for inducing tumor damage following administration of a light‐activated photosensitizing drug (PS). In a previous work we found that PDT induces cytoskeleton changes in HB4a‐Ras cells (human mammary breast carcinoma HB4a cells transfected with the RAS oncogene). In the present work we have studied the migratory and invasive features and the expression of proteins related to these processes on HB4a‐Ras cells after three successive cycles of PDT using different PSs: 5‐aminolevulinic acid (ALA), Verteporfin (Verte), m‐tetrahydroxyphenylchlorin (m‐THPC), and Merocyanine 540 (MC). A slight (1.25‐ to 2‐fold) degree of resistance was acquired in cell populations subjected to the three successive PDT treatments. However, complete cell killing was achieved after a light dose increase. Regardless of the PS employed, all the PDT‐treated populations had shorter stress fibres than the untreated control HB4a‐Ras cells, and the number of dorsal stress fibres was decreased in the PDT‐treated populations. E‐Cadherin distribution, which was already aberrant in HB4a‐Ras cells, became even more diffuse in the PDT‐treated populations, though its expression was increased in some of them. The strong migratory and invasive ability of HB4a‐Ras cells in vitro was impaired in all the PDT‐treated populations, with a behavior that was similar to the parental non‐tumoral HB4a cells. MMP‐2 and ‐9 metalloproteinase activities were also impaired in the PDT‐treated populations. The evidence presented herein suggests that the cells surviving PDT would be less metastatic than the initial population. These findings encourage the use of PDT in combination with other treatments such as intraoperative or post‐surgery therapeutic procedures. J. Cell. Biochem. 118: 464–477, 2017.