Laser NIR Irradiation Enhances Antimicrobial Photodynamic Inactivation of Biofilms of Staphylococcus aureus.

OBJECTIVES: Photodynamic inactivation (PDI) is a powerful technique for eradicating microorganisms, and our group previously demonstrated its effectiveness against planktonic cultures of Staphylococcus aureus bacteria using 5,10,15,20-tetrakis[4-(3-N,N-dimethylaminopropoxy)phenyl]porphyrin (TAPP) and visible light irradiation. However, biofilms exhibit a lower sensitivity to PDI, mainly due to limited penetration of the photosensitizer (PS). In the context of emerging antibacterial strategies, near-infrared treatments (NIRTs) have shown promise, especially for combating resistant strains. NIRT can act either through photon absorption by water, causing a thermal effect on bacteria, or by specific chromophores without a significant temperature increase. Our objective was to enhance biofilm sensitivity to TAPP-PDI by pretreatment with NIRT. This combined approach aims to disrupt biofilms and increase the efficacy of TAPP-PDI against bacterial biofilms. MATERIALS AND METHODS: In vitro biofilm models of S. aureus RN6390 were utilized. NIRTs involved a 980 nm laser (continuous mode, 7.5 W/cm2, 30 s, totaling 225 J/cm2) post-TAPP exposure to enhance photosensitizer accumulation. Subsequent visible light irradiation at 180 J/cm2 was employed to perform PDI. Colony-forming unit counts evaluated the synergistic effect on bacterial viability. Scanning electron microscopy visualized the architectural changes in the biofilm structure. TAPP was extracted from bacteria to estimate the impact of NIRT on biofilm penetration. RESULTS: Using in vitro biofilm models, NIRT application following biofilm exposure to TAPP increased PS accumulation per bacteria. Under these conditions, NIRT induced a transient increase in the temperature of PBS to 46.0 ± 2.6°C (ΔT = 21.5°C). Following exposure to visible light, a synergistic effect emerged, yielding a substantial 4.4 ± 0.1-log CFU reduction. In contrast, the PDI and NIRT treatments individually caused a decrease in viability of 0.9 ± 0.1 and 0.8 ± 0.2-log respectively. Interestingly, preheating TAPP-PBS to 46°C had no significant impact on TAPP-PDI efficacy, suggesting the involvement of thermal and nonthermal effects of NIR action. In addition to the enhanced TAPP penetration, NIRT dispersed the biofilms and induced clefts in the biofilm matrix. CONCLUSION: Our findings suggest that NIR irradiation serves as a complementary treatment to PDI. This combined strategy reduces bacterial numbers at lower PS concentrations than standalone PDI treatment, highlighting its potential as an effective and resource-efficient antibacterial approach.

Hydrogen sulfide decreases photodynamic therapy outcome through the modulation of the cellular redox state.

Photodynamic therapy (PDT) is a non-surgical treatment that has been approved for its human medical use in many cancers. PDT involves the interaction of a photosensitizer (PS) with light. The amino acid 5- aminolevulinic acid (ALA) can be used as a pro-PS, leading to the synthesis of Protoporphyrin IX. Hydrogen sulfide (H2S) is an endogenously produced gas that belongs to the gasotransmitter family, which can diffuse through biological membranes and have relevant physiological effects such as cardiovascular functions, vasodilatation, inflammation, cell cycle and neuro-modulation. It was also proposed to have cytoprotective effects. We aimed to study the modulatory effects of H2S on ALAPDT in the mammary adenocarcinoma cell line LM2. Exposure of the cells to NaHS (donor of H2S) in concentrations up to 10 mM impaired the response to ALA-PDT in a dose-dependent manner. The addition of 3 doses of NaHS showed the highest effect. This decreased response to the photodynamic treatment was correlated to an increase in the GSH levels, catalase activity, a dose dependent reduction of PpIX and increased intracellular ALA, decreased levels of oxidized proteins and a decrease of PDT-induced ROS. NaHS also reduced the levels of singlet oxygen in an in vitro assay. H2S also protected other cells of different origins against PDT mediated by ALA and other PSs. These results suggest that H2S has a role in the modulation of the redox state of the cells, and thus impairs the response to ALA-PDT through multifactor pathways. These findings could contribute to developing new strategies to improve the effectiveness of PDT particularly mediated by ALA or other ROS-related treatments.

Novel meso-substituted porphyrin derivatives and its potential use in photodynamic therapy of cancer.

BACKGROUND: Photodynamic therapy (PDT) is an anticancer treatment that utilizes the interaction of light and a photosensitiser (PS), promoting tumour cell death mediated by generation of reactive oxygen species. In this study, we evaluated the in vitro photoactivity of four meso-substituted porphyrins and a porphyrin coupled to a fullerene. METHODS: The cell line employed was the LM3 mammary adenocarcinoma, and the PS with the best photokilling activity was administered to mice bearing the LM3 subcutaneously implanted adenocarcinoma. The TEMCP4+ porphyrin and its analogue TEMCC4+ chlorine contain four identical carbazoyl substituents at the meso positions of the tetrapyrrolic macrocycle and have A4 symmetry. The TAPP derivative also has A4 symmetry, and it is substituted at the meso positions by aminopropoxy groups. The DAPP molecule has ABAB symmetry with aminopropoxy and the trifluoromethyl substituents in trans positions. The TCP-C604+ dyad is formed by a porphyrin unit covalently attached to the fullerene C60. RESULTS: The PSs are taken up by the cells with the following efficiency: TAPP> TEMCP4+ = TEMCC4+ > DAPP >TCP-C604+, and the amount of intracellular PS correlates fairly with the photodamage degree, but also the quantum yields of singlet oxygen influence the PDT outcome. TAPP, DAPP, TEMCC4+ and TEMCP4+ exhibit high photoactivity against LM3 mammary carcinoma cells, being TAPP the most active. After topical application of TAPP on the skin of mice bearing LM3 tumours, the molecule is localized mainly in the stratum corneum, and at a lower extent in hair follicles and sebaceous glands. Systemic administration of TAPP produces a tumour: normal skin ratio of 31.4, and high accumulation in intestine and lung. CONCLUSION: The results suggest a potential use of topical TAPP for the treatment of actinic keratosis and skin adnexal neoplasms. In addition, selectivity for tumour tissue after systemic administration highlights the selectivity of and potentiality of TAPP as a new PS.

Apoptotic cell death induced by dendritic derivatives of aminolevulinic acid in endothelial and foam cells co-cultures.

Photodynamic therapy (PDT) is an effective procedure for the treatment of lesions diseases based on the selectivity of a photosensitising compound with the ability to accumulate in the target cell. Atherosclerotic plaque is a suitable target for PDT because of the preferential accumulation of photosensitisers in atherosclerotic plaques. Dendrimers are hyperbranched polymers conjugated to drugs. The dendrimers of ALA hold ester bonds that inside the cells are cleaved and release ALA, yielding PpIX production. The dendrimer 6m-ALA was chosen to perform this study since in previous studies it induced the highest porphyrin macrophage: endothelial cell ratio (Rodriguez et al. in Photochem Photobiol Sci 14:1617-1627, 2015). We transformed Raw 264.7 macrophages to foam cells by exposure to oxidised LDLs, and we employed a co-culture model of HMEC-1 endothelial cells and foam cells to study the affinity of ALA dendrimers for the foam cells. In this work it was proposed an in vitro model of atheromatous plaque, the aim was to study the selectivity of an ALA dendrimer for the foam cells as compared to the endothelial cells in a co-culture system and the type of cell death triggered by the photodynamic treatment. The ALA dendrimer 6m-ALA showed selectivity PDT response for foam cells against endothelial cells. A light dose of 1 J/cm2 eliminate foam cells, whereas less than 50% of HMEC-1 is killed, and apoptosis cell death is involved in this process, and no necrosis is present. We propose the use of ALA dendrimers as pro-photosensitisers to be employed in photoangioplasty to aid in the treatment of obstructive cardiovascular diseases, and these molecules can also be employed as a theranostic agent.

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. © 2016 Wiley Periodicals, Inc.

Methods for the detection of reactive oxygen species employed in the identification of plant photosensitizers.

Over the past ten years, alternative methods for the rapid screening of PSs have been developed. In the present work, a study was undertaken to correlate the phototoxicity of plant extracts on either prokaryotic or eukaryotic cells, with the total oxidation status (TOS) as well as with their ability to produce 1O2. Results demonstrated that the extracts containing PSs that were active either on eukaryotic cells or bacteria increased their TOS after illumination, and that there was a certain degree of positive correlation between the extract phototoxic efficacy and TOS levels. The production of 1O2 by the illuminated extracts was indirectly measured by the use of the fluorescence of "singlet oxygen sensor green", which is a method that has proved highly sensitive for such measurement. 1O2 was detectable only upon illumination of the most active extracts. In addition, the oxidation of tryptophan and was employed as a method capable of measuring ROS generated by both type I and II ROS reactions. However, it turned out to be not sensitive enough to detect the species generated by plant extracts. Results demonstrated that the TOS method, initially developed to measure the oxidant status in plasma, can be readily applied to plant extracts. Unlike the method used to detect 1O2, the method employed for the detection of TOS proved to be accurate, since all the extracts that displayed a high phototoxic activity on either prokaryotic or eukaryotic cells, presented high TOS levels after illumination.

The role of cytoskeleton and adhesion proteins in the resistance to photodynamic therapy. Possible therapeutic interventions.

It is known that Photodynamic Therapy (PDT) induces changes in the cytoskeleton, the cell shape, and the adhesion properties of tumour cells. In addition, these targets have also been demonstrated to be involved in the development of PDT resistance. The reversal of PDT resistance by manipulating the cell adhesion process to substrata has been out of reach. Even though the existence of cell adhesion-mediated PDT resistance has not been reported so far, it cannot be ruled out. In addition to its impact on the apoptotic response to photodamage, the cytoskeleton alterations are thought to be associated with the processes of metastasis and invasion after PDT. In this review, we will address the impact of photodamage on the microfilament and microtubule cytoskeleton components and its regulators on PDT-treated cells as well as on cell adhesion. We will also summarise the impact of PDT on the surviving and resistant cells and their metastatic potential. Possible strategies aimed at taking advantage of the changes induced by PDT on actin, tubulin and cell adhesion proteins by targeting these molecules will also be discussed.

Light fractionated ALA-PDT enhances therapeutic efficacy in vitro; the influence of PpIX concentration and illumination parameters.

Light fractionation, with a long dark interval, significantly increases the response to ALA-PDT in pre-clinical models and in non-melanoma skin cancer. We investigated if this increase in efficacy can be replicated in PAM 212 cells in vitro. The results show a significant decrease in cell survival after light fractionation which is dependent on the PpIX concentration and light dose of the first light fraction. This study supports the hypothesis that an underlying cellular mechanism is involved in the response to light fractionation in which a first light fraction leads to sub-lethally damaged cells that are sensitised to a second light fraction 2 hours later. The current study reveals the in vitro circumstances under which we can investigate the cellular pathways involved.

Ras-transfected human mammary tumour cells are resistant to photodynamic therapy by mechanisms related to cell adhesion.

AIMS: Photodynamic therapy (PDT) is a treatment modality for several cancers involving the administration of a tumour-localising photosensitiser (PS) and its subsequent activation by light, resulting in tumour damage. Ras oncogenes have been strongly associated with chemo- and radio-resistance. Based on the described roles of adhesion and cell morphology on drug resistance, we studied if the differences in shape, cell-extracellular matrix and cell-cell adhesion induced by Ras transfection, play a role in the resistance to PDT. MATERIALS AND METHODS: We employed the human normal breast HB4a cells transfected with H-RAS and a panel of five PSs. KEY FINDINGS: We found that resistance to PDT of the HB4a-Ras cells employing all the PSs, increased between 1.3 and 2.5-fold as compared to the parental cells. There was no correlation between resistance and intracellular PS levels or PS intracellular localisation. Even when Ras-transfected cells present lower adherence to the ECM proteins, this does not make them more sensitive to PDT or chemotherapy. On the contrary, a marked gain of resistance to PDT was observed in floating cells as compared to adhesive cells, accounting for the higher ability conferred by Ras to survive in conditions of decreased cell-extracellular matrix interactions. HB4a-Ras cells displayed disorganisation of actin fibres, mislocalised E-cadherin and vinculin and lower expression of E-cadherin and ß1-integrin as compared to HB4a cells. SIGNIFICANCE: Knowledge of the mechanisms of resistance to photodamage in Ras-overexpressing cells may lead to the optimization of the combination of PDT with other treatments.

Synthesis and cytotoxicity evaluation of olivacine-indole hybrids tethered by alkyl linkers.

In this work, eleven new derivatives were prepared of the alkaloid olivacine (1), which was isolated from the bark of Aspidosperma australe. These compounds (7a-k) are hybrids of olivacine and indoles or carbazole, tethered by alkyl chains of variable lengths (C-4, C-5 or C-6). Compounds 7a-k showed increased cytotoxicity towards a panel of four cell lines. The subcellular localization of olivacine and of the synthetic derivatives was studied by fluorescence microscopy. The cycles of K562 cells exposed to olivacine or compounds 7a-k were analysed by flow cytometry, and showed, for some of the new derivatives, a different profile of cell distribution among the phases of the cycle when compared to olivacine, which is indicative of lysosomal apoptosis.

Antiparasitic Derivatives of the Furoquinoline Alkaloids Kokusaginine And Flindersiamine.

We report the synthesis of 16 new compounds obtained from kokusaginine and flindersiamine, the main alkaloids isolated from the bark of Balfourodendron riedelianum. The activity of the compounds against axenic cultures of Trypanosoma cruzi epimastigtotes and trypomastigotes, as well as intracellular amastigotes, is described, together with their cytotoxic activity against three different human cell lines. The synthetic strategy for the preparation of the new compounds was based on the reactivity at the C4 position of the furoquinoline core towards nucleophiles. The new derivatives were synthesized by a Buchwald-Hartwig reaction, in most cases under green, solvent-free conditions. Compounds 1 c and 1 e displayed better in-vitro activity against trypomastigotes than benznidazole and nifurtimox (positive controls) with IC50 <4 µM. In addition, both compounds were not cytotoxic against the three human cell lines K562 (erytroleukimia), LM2 (breast cancer), and HaCat (keratinocyte). Interestingly, when evaluated against intracellular amastigotes, compound 1 c was able to significantly reduce the number of this parasite form, compared to the negative control.

Photodynamic therapy of cutaneous T-cell lymphoma cell lines mediated by 5-aminolevulinic acid and derivatives.

The delta-amino acid 5-aminolevulinic acid (ALA), is the precursor of the endogenous photosensitiser Protoporphyrin IX (PpIX), and is currently approved for Photodynamic Therapy (PDT) of certain superficial cancers. However, ALA-PDT is not very effective in diseases in which T-cells play a significant role. Cutaneous T-cell lymphomas (CTCL) is a group of non-Hodgkin malignant diseases, which includes mycosis fungoides (MF) and Sézary syndrome (SS). In previous work, we have designed new ALA esters synthesised by three-component Passerini reactions, and some of them showed higher performance as compared to ALA. This work aimed to determine the efficacy as pro-photosensitisers of five new ALA esters of 2-hydroxy-N-arylacetamides (1f, 1 g, 1 h, 1i and 1 k) of higher lipophilicity than ALA in Myla cells of MF and HuT-78 cells of SS. We have also tested its effectiveness against ALA and the already marketed ALA methyl ester (Me-ALA) and ALA hexyl ester (He-ALA). Both cell Myla and SS cells were effectively and equally photoinactivated by ALA-PDT. Besides, the concentration of ALA required to induce half the maximal porphyrin synthesis was 209 µM for Myla and 169 µM for HuT-78 cells. As a criterion of efficacy, we calculated the concentration of the ALA derivatives necessary to induce half the plateau porphyrin values obtained from ALA. These values were achieved at concentrations 4 and 12 times lower compared to ALA, according to the derivative used. For He-ALA, concentrations were 24 to 25 times lower than required for ALA for inducing comparable porphyrin synthesis in both CTCL cells. The light doses for inducing 50% of cell death (LD50) for He-ALA, 1f, 1 g, 1 h and 1i were around 18 and 25 J/cm2 for Myla and HuT-78 cells respectively, after exposure to 0.05 mM concentrations of the compounds. On the other hand, the LD50s for the compound 1 k were 40 and 57 J/cm2 for Myla and HuT-78, respectively. In contrast, 0.05 mM of ALA and Me-ALA did not provoke photokilling since the concentration employed was far below the porphyrin saturation point for these compounds. Our results suggest the potential use of ALA derivatives for topical application in PDT treatment of MF and extracorporeal PDT for the depletion of activated T-cells in SS.

Photosensitization of a subcutaneous tumour by the natural anthraquinone parietin and blue light.

Photodynamic therapy (PDT) is an anticancer treatment involving administration of a tumour-localizing photosensitizer, followed by activation by light of a suitable wavelength. In previous work, we showed that the natural anthraquinone (AQ) Parietin (PTN), was a promising photosensitizer for photodynamic therapy of leukemic cells in vitro. The present work aimed to analyze the photosensitizing ability of PTN in the mammary carcinoma LM2 cells in vitro and in vivo in a model of subcutaneously implanted tumours. Photodynamic therapy mediated by parietin (PTN-PDT) (PTN 30 µM, 1 h and 1.78 J/cm2 of blue light) impaired cell growth and migration of LM2 cells in vitro. PTN per se induced a significant decrease in cell migration, and it was even more marked after illumination (migration index was 0.65 for PTN and 0.30 for PTN-PDT, *p < 0.0001, ANOVA test followed by Tukey's multiple comparisons test), suggesting that both PTN and PTN-PDT would be potential inhibitors of metastasis. Fluorescence microscopy observation indicated cytoplasmic localization of the AQ and no fluorescence at all was recorded in the nuclei. When PTN (1.96 mg) dissolved in dimethyl sulfoxide was topically applied on the skin of mice subcutaneously implanted with LM2 cells, PTN orange fluorescence was strongly noticed in the stratum corneum and also in the inner layers of the tumour up to approximately 5 mm. After illumination with 12.74 J/cm2 of blue light, one PDT dose at day 1, induced a significant tumour growth delay at day 3, which was not maintained in time. Therefore, we administered a second PTN-PDT boost on day 3. Under these conditions, the delay of tumour growth was 28% both on days 3 and 4 of the experiment (*p < 0.05 control vs. PTN-PDT, two-way ANOVA, followed by Sidak's multiple comparisons test). Histology of tumours revealed massive tumour necrosis up to 4 mm of depth. Intriguingly, a superficial area of viable tumour in the 1 mm superficial area, and a quite conserved intact skin was evidenced. We hypothesize that this may be due to PTN aggregation in contact with the skin and tumour milieu of the most superficial tumour layers, thus avoiding its photochemical properties. On the other hand, normal skin treated with PTN-PDT exhibited slight histological changes. These preliminary findings encourage further studies of natural AQs administered in different vehicles, for topical treatment of cutaneous malignancies.

Photodynamic therapy of tumour cells mediated by the natural anthraquinone parietin and blue light.

Photodynamic therapy (PDT) is a treatment for superficial tumours involving the administration of a photosensitiser followed by irradiation. The potential of the natural anthraquinone parietin (PTN) in PDT is still relatively unexploited. In the present work, PTN isolated from the lichen Teoloschistes nodulifer (Nyl.) Hillman (Telochistaceae) was evaluated as a potential photosensitiser on tumour cells employing UVA-Vis and blue light. Blue light of 2 J/cm2 induced 50% death of K562 leukaemic cells treated 1 h with 30 µM PTN (Protocol a). Higher light doses (8 J/cm2) were needed to achieve the same percentage of cell death employing lower PTN concentrations (3 µM) and higher exposure times (24 h) (Protocol b). Cell cycle analysis after both protocols of PTN-PDT revealed a high percentage of sub-G1 cells. PTN was found to be taken up by K562 cells mainly by passive diffusion. Other tumour cells such as ovary cancer IGROV-1 and LM2 mammary carcinoma, as well as the normal keratinocytes HaCaT, were also photosensitised with PTN-PDT. We conclude that PTN is a promising photosensitiser for PDT of superficial malignancies and purging of leukaemic cells, when illuminated with blue light. Thus, this light wavelength is proposed to replace the Vis-UVA lamps generally employed for the photosensitisation of anthraquinones.

Photodynamic inactivation mediated by 5-aminolevulinic acid of bacteria in planktonic and biofilm forms.

Bacterial photodynamic inactivation (PDI) employing endogenous production of porphyrins from 5-aminolevulinic acid (ALA) - named ALA-PDI-, is a new promising tool to achieve bacteria control in non-spread infections. The technique combines the action of the porphyrins acting as photosensitisers with light, to produce reactive oxygen species to target the pathogen. To date, some clinical applications of ALA-PDI have been reported although variable responses ranging from total eradication to absence of photokilling were found. ALA-PDI conducted at suboptimal conditions may lead to misleading results and the complexity of haem synthesis in bacteria hinders the optimization of the treatment. The present work aimed to gain insight on the variables affecting ALA-PDI in Gram-positives and Gram-negatives bacteria growing on planktonic and biofilm cultures and to correlate the degree of the response with the amount and type of porphyrin synthesised. Staphylococcus epidermidis and Escherichia coli clinical isolates and Pseudomonas aeruginosa ATCC27853 and Staphylococcus aureus ATCC25923 strains were utilised, and the optimal conditions of concentration and time exposure of ALA, and light dose were set. In both Gram-positive species analysed, a peak of porphyrin synthesis was observed at 1-2 mM ALA in biofilm and planktonic cultures, which fairly correlated with the decrease in the number of CFU after PDI (5 to 7 logs) and porphyrin content was in the same order of magnitude. In addition, ALA-PDI was similarly effective for planktonic and biofilm S. aureus cultures, and more effective in S. epidermidis planktonic cultures at low light doses. Beyond a certain light dose, it was not possible to achieve further photosensitization. Similarly, a plateau of cell death was attained at a certain ALA incubation time. Accumulation of hydrophilic porphyrins at longer incubation periods was observed. The proportion of porphyrins changed as a function of ALA concentration and incubation time in the Gram-positive bacteria, though we did not find a clear correlation between the porphyrin type and PDI response. As a salient feature was the presence of isococroporphyrin isoforms in both Gram-positive and Gram-negative bacteria. Gram-negative bacteria were quite refractory to the treatment: P. aeruginosa was slightly inactivated (4-logs reduction) at 40 mM ALA, whereas E. coli was not inactivated at all. These species accumulated high ALA quantities and the amount of porphyrins did not correlate with the degree of photoinactivation. Our microscopy studies show that porphyrins are not located in the envelopes of Gram-negative bacteria, reinforcing the hypothesis that endogenous porphyrins fail to attack these structures.

Disaccharides obtained from carrageenans as potential antitumor agents.

Carrageenans are sulfated galactans found in certain red seaweeds with proven biological activities. In this work, we have prepared purified native and degraded κ-, ι-; and λ-carrageenans, including the disaccharides (carrabioses) and disaccharide-alditols (carrabiitols) from seaweed extracts as potential antitumor compounds and identified the active principle of the cytotoxic and potential antitumor properties of these compounds. Both κ and ι-carrageenan, as well as carrageenan oligosaccharides showed cytotoxic effect over LM2 tumor cells. Characterized disaccharides (carrabioses) and the reduced product carrabiitols, were also tested. Only carrabioses were cytotoxic, and among them, κ-carrabiose was the most effective, showing high cytotoxic properties, killing the cells through an apoptotic pathway. In addition, the cells surviving treatment with κ-carrabiose, showed a decreased metastatic ability in vitro, together with a decreased cell-cell and cell-matrix interactions, thus suggesting possible antitumor potential. Overall, our results indicate that most cytotoxic compounds derived from carrageenans have lower molecular weights and sulfate content. Potential applications of the results emerging from the present work include the use of disaccharide units such as carrabioses coupled to antineoplasics in order to improve its cytotoxicity and antimetastatic properties, and the use of ι-carrageenan as adjuvant or carrier in anticancer 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.

Investigation of a novel dendritic derivative of 5-aminolaevulinic acid for photodynamic therapy.

Photodynamic therapy is a treatment for malignant and certain non-malignant lesions that involves administration of a photosensitising drug. The use of 5-aminolaevulinic acid-induced porphyrins has become one of the most active fields of photodynamic therapy research. Since the efficacy of the treatment is somewhat limited by the hydrophilic nature of 5-aminolaevulinic acid, chemical modifications such as esterification with aliphatic alcohols have been made to induce higher porphyrin production. In an attempt to improve delivery of 5-aminolaevulinic acid to tissue, we have investigated the use of dendritic derivatives capable of bearing several drug molecules. The aim of this work was to evaluate in vivo and in vitro the efficacy of the first generation dendron, aminomethane tris-methyl 5-aminolaevulinic acid (containing three 5-aminolaevulinic acid residues) in terms of porphyrin synthesis. In LM3 cells, the dendron induced similar porphyrin levels compared to equimolar concentrations of 5-aminolaevulinic acid. Although the dendron is taken up with comparable efficiency to 5-aminolaevulinic acid, we found that there is only partial intracellular liberation of 5-aminolaevulinic acid residues. Both systemic and topical administration of the dendron to tumour-bearing mice induced higher porphyrin levels than the widely investigated hexyl ester derivative in most tissues studied, although it was not possible to surpass the levels induced by 5-aminolaevulinic acid. In conclusion, aminomethane tris-methyl 5-aminolaevulinic acid is capable of being taken up by cells efficiently, and liberating the active residues, although in vivo it was not possible to improve upon the efficacy of 5-aminolevulinic acid. Studies of accessibility and regulation of the esterases are needed to improve the design of these dendritic derivatives.

Study of the mechanisms of uptake of 5-aminolevulinic acid derivatives by PEPT1 and PEPT2 transporters as a tool to improve photodynamic therapy of tumours.

Endogenous porphyrin accumulation after administration of 5-aminolevulinic acid is employed in photodynamic therapy of tumours. Due to its low membrane permeability, esterified 5-aminolevulinic acid derivatives less hydrophilic than the parental compound are under investigation. Knowledge of the mechanisms of 5-aminolevulinic acid derivatives uptake into target cells is essential to understand and improve photodynamic therapy and useful in the design of new derivatives with better affinity and with higher selectivity for tumour cells in specific tissues. The aim of this work was to assess the interaction of 5-aminolevulinic acid derivatives with the intestinal PEPT1 and renal transporter PEPT2 expressed in Pichia pastoris yeasts. We found that Undecanoyl, Hexyl, Methyl and 2-(hydroxymethyl)tetrahydropyranyl 5-aminolevulinic acid esters and the dendron 3m-ALA inhibited (14)C-5-aminolevulinic acid uptake by PEPT2. However, only the Undecanoyl ester inhibited 5-aminolevulinic acid uptake by PEPT1. We have also found through a new developed colorimetric method, that Hexyl and 2-(hydroxymethyl)tetrahydropyranyl 5-aminolevulinic acid esters display more affinity than 5-aminolevulinic acid for PEPT2 whereas none of the compounds surpass 5-aminolevulinic acid affinity for PEPT1. In addition, the Undecanoyl ester binds with high affinity to the membranes of PEPT2 and PEPT1-expressing yeasts and to the control yeasts. The main finding of this work was that some derivatives have the potential to improve 5-aminolevulinic acid-based photodynamic therapy by increased efficiency of transport into cells expressing PEPT2 such as kidney, mammary gland, brain or lung whereas in tissues expressing exclusively PEPT1 the parent 5-aminolevulinic acid remains the compound of choice.

Mechanisms of 5-aminolevulinic 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 gamma-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.