Leandro Mamone

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.

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.

The natural flavonoid silybin improves the response to Photodynamic Therapy of bladder cancer cells.

Photodynamic Therapy (PDT) is an anticancer treatment based on photosensitisation of malignant cells. The precursor of the photosensitiser Protoporphyrin IX, 5-aminolevulinic acid (ALA), has been used for PDT of bladder cancer. Silybin is a flavonoid extracted from Silybum marianum, and it has been reported to increase the efficacy of several anticancer treatments. In the present work, we evaluated the cytotoxicity of the combination of ALA-PDT and silybin in the T24 and MB49 bladder cancer cell lines. MB49 cells were more sensitive to PDT damage, which was correlated with a higher Protoporphyrin IX production from ALA. Employing lethal light doses 50% (LD50) and 75% (LD75) and additional silybin treatment, there was a further increase of toxicity driven by PDT in both cell lines. Using the Chou-Talalay model for drug combination derived from the mass-action law principle, it was possible to identify the effect of the combination as synergic when using LD75, whilst the use of LD50 led to an additive effect on MB49 cells. On the other hand, the drug combination turned out to be nearly additive on T24 cells. Apoptotic cell death is involved both in silybin and PDT cytotoxicity in the MB49 line but there is no apparent correlation with the additive or synergic effect observed on cell viability. On the other hand, we found an enhancement of the PDT-driven impairment of cell migration on both cell lines as a consequence of silybin treatment. Overall, our results suggest that the combination of silybin and ALA-PDT would increase PDT outcome, leading to additive or synergistic effects and possibly impairing the occurrence of metastases.

Photodynamic inactivation of Gram-positive bacteria employing natural resources.

The aim of this paper was to investigate a collection of plant extracts from Argentina as a source of new natural photosensitizers (PS) to be used in Photodynamic Inactivation (PDI) of bacteria. A collection of plants were screened for phototoxicity upon the Gram-positive species Staphylococcus epidermidis. Three extracts turned out to be photoactive: Solanum verbascifolium flower, Tecoma stans flower and Cissus verticillata root. Upon exposure to a light dose of 55J/cm(2), they induced 4, 2 and 3logs decrease in bacterial survival, respectively. Photochemical characterisation of S. verbascifolium extract was carried out. PDI reaction was dependent mainly on singlet oxygen and to a lesser extent, on hydroxyl radicals, through type II and I reactions. Photodegradation experiments revealed that the active principle of the extract was not particularly photolabile. It is noticeable that S. verbascifolium -PDI was more efficient under sunlight as compared to artificial light (total eradication vs. 4 logs decrease upon 120min of sunlight). The balance between oxidant and antioxidant compounds is likely to be masking or unmasking potential PS of plant extracts, but employing the crude extract, the level of photoactivity of S. verbascifolium is similar to some artificial PS upon exposure to sunlight, demonstrating that natural resources can be employed in PDI of bacteria.

Cytotoxic effects of Argentinean plant extracts on tumour and normal cell lines.

In the search for possible new anti-cancer agents, we investigated the effects of 75 aqueous and methanol extracts from 41 Argentinean plant species. The effect in cell growth was evaluated in the LM2 mammary adenocarcinoma cells. In a second stage, the highly active selected extracts were assayed in 3 other tumour cell lines: melanoma B16, bladder MB49 and lung A549; and 3 normal cell lines: mammary Hb4a and keratinocytes PAM212 and HaCat. Eight methanol extracts were found to be highly cytotoxic: Collaea argentina leaf, Iochroma australe leaf, Ipomoea bonariensis flower, Jacaranda mimosifolia flower, Solanum amygdalifolium flower, Solanum chacoense leaf, Solanum sisymbriifolium flower and Solanum verbascifolium flower. However, extract inhibition on cell growth was highly dependent on cell type. In general, except for the highly resistant cell lines, the inhibitory concentrations 50% were in the range of 10-150 μg/ml The eight extracts highly inhibited cell growth in a concentration-dependent manner, and in general the methanol extracts were always more active than the aqueous. Murine cells appear to be more sensitive than human cells to the cytotoxic action of the plant extracts. The human melanoma B16 line was the most resistant to four of the extracts. In terms of selectivity, S. verbascifolium was the species which showed most selectivity for tumour cells. Overall, this is one of the first studies focusing on southern South American native plants and their biological effects. Since some species of 5 genera analyzed have been reported to possess different degrees of alkaloid content, we examined microtubule structures after extract treatments. The eight extracts induced destabilization, condensation and aggregation of microtubules in LM2 cells, although no depolarization, typical of Vinca alkaloids damage was observed. In a near future, antitumour activity of purified fractions of the extracts administered at non-toxic doses will be assayed in transplantable murine tumour models.

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.

Changes in actin and E-cadherin expression induced by 5-aminolevulinic acid photodynamic therapy in normal and Ras-transfected human mammary cell lines.

Photodynamic therapy (PDT) is an anticancer treatment based on light-induced destruction of photosensitised malignant cells. It has been reported that PDT strongly affects cell-cell and cell-substrate adhesion through the reorganization of some cytoskeletal and adhesion proteins. The aim of the present work was to study the changes induced by PDT employing aminolevulinic acid (ALA), on the cytoskeleton actin network and E-cadherin expression. We employed the normal mammary HB4a cell line and its tumor counterpart transfected with the oncogene H-Ras, which has been shown to be resistant to PDT. Ras insertion induces per se disorganization of both F-actin and E-cadherin distribution. ALA-PDT induces on HB4a cells a dramatic disorganization of actin stress fibers, resembling normal Ras-transfected cells. After 48h some features of disorganization remain present. In HB4a-Ras cells, F-actin exhibits signals of photodamage, but distribution is recovered 24h after treatment. On the other hand, PDT did not impact on E-cadherin distribution, other than a transient disorganization, which was recovered at 24h. Moreover, E-cadherin disorganization did not favoured cell-cell detachment after PDT of HB4a-Ras cells. Actin but not E-cadherin constitutes in this model an important target of PDT. The fact that some features of microfilament disorganization remain present in HB4a surviving cells but not in Ras-transfected cells, suggests that cytoskeletal structures such as F-actin may be involved in the mechanisms of resistance to PDT.

Sae regulator factor impairs the response to photodynamic inactivation mediated by Toluidine blue in Staphylococcus aureus

Photodynamic inactivation (PDI) involves the combined use of light and a photosensitizer, which, in the presence of oxygen, originates cytotoxic species capable of inactivating bacteria. Since the emergence of multi-resistant bacterial strains is becoming an increasing public health concern, PDI becomes an attractive choice. The aim of this work was to study the differential susceptibility to Toluidine blue (TB) mediated PDI (TB-PDI) of S. aureus mutants (RN6390 and Newman backgrounds) for different key regulators of virulence factors related to some extent to oxidative stress. Complete bacteria eradication of planktonic cultures of RN6390 S. aureus photosensitized with 13μM TB was obtained upon illumination with a low light dose of 4.2J/cm2 from a non-coherent light source. Similarly, complete cell death was achieved applying 1.3μM TB and 19J/cm2 light dose, showing that higher light doses can lead to equal cell death employing low photosensitizer concentrations. Interestingly, RN6390 in planktonic culture responded significantly better to TB-PDI than the Newman strain. We showed that deficiencies in rsbU, mgrA (transcription factors related to stress response) or agr (quorum sensing system involved in copper resistance to oxidative stress) did not modify the response of planktonic S. aureus to PDI. On the other hand, the two component system sae impaired the response to TB-PDI through a mechanism not related to the Eap adhesin. More severe conditions were needed to inactivate S. aureus biofilms (0.5mM TB, 157J/cm2 laser light). In mutant sae biofilms, strain dependant differential susceptibilities are not noticed.