RESUMO
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.
Assuntos
Ácido Aminolevulínico/farmacologia , Apoptose/efeitos dos fármacos , Células Espumosas/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Fármacos Fotossensibilizantes/farmacologia , Ácido Aminolevulínico/química , Animais , Linhagem Celular , Técnicas de Cocultura , Células Espumosas/fisiologia , Humanos , Macrófagos/fisiologia , Camundongos , Fármacos Fotossensibilizantes/químicaRESUMO
CONTEXT: 5-Aminolevulinic acid (5-ALA) is a prodrug used in photodynamic therapy (PDT) of tumors, including cancer of the oral mucosa. 5-ALA poorly penetrates oral tissues due to its high hydrophilicity, which impairs its local effects in PDT. OBJECTIVES: To examine whether α-bisabolol (α-Bis) influences the 5-ALA permeability in the porcine buccal mucosa, to an extent that improves its application in PDT (which requires low permeation and high retention in the buccal mucosa). METHODS: In vitro permeability studies with 5-ALA (1% and 10% w/w) associated with α-Bis (1% to 20% w/w) in propylene glycol were carried out at 4h and 24h using porcine buccal mucosa in a modified Franz cell system. The in vitro release profiles (0.5 to 48h) of the selected formulation and its respective control were determined using artificial membranes. Samples of buccal mucosa treated with the formulation were submitted to histopathological analysis, using a routine optical microscopy technique. RESULTS: The association of 1% 5-ALA and 5% α-Bis provided the best results; after 4h of treatment with this formulation, the 5-ALA permeation was low and its retention in the mucosa was six-fold higher than that promoted by the control formulation (5-ALA alone). Histological analysis of the porcine buccal mucosa evidenced that 5% α-Bis altered the tissue morphology, which probably promoted 5-ALA retention. We concluded that 5% α-Bis is a potential adjuvant in formulations containing 5-ALA that could improve its retention after topical oral administration for the PDT treatment of cancer.
Assuntos
Ácido Aminolevulínico/química , Ácido Aminolevulínico/metabolismo , Neoplasias Bucais/tratamento farmacológico , Fotoquimioterapia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/metabolismo , Sesquiterpenos/química , Ácido Aminolevulínico/uso terapêutico , Animais , Composição de Medicamentos , Luz , Sesquiterpenos Monocíclicos , Mucosa Bucal/metabolismo , Mucosa Bucal/efeitos da radiação , Permeabilidade/efeitos dos fármacos , Fármacos Fotossensibilizantes/uso terapêutico , Suínos , Fatores de TempoRESUMO
The use of endogenous protoporphyrin IX after administration of 5-aminolaevulinic acid (ALA) has led to many applications in photodynamic therapy (PDT). We have previously reported that the conjugation of ALA dendrimers enhances porphyrin synthesis. The first aim of this work was to evaluate the ability of ALA dendrimers carrying 6 and 9 ALA residues (6m-ALA and 9m-ALA) to photosensitise cancer cells. For this aim, we employed LM3 mammary carcinoma cells. In these tumour cells, at low concentrations porphyrin synthesis from dendrimers was higher compared to ALA, whereas at high concentrations, porphyrin synthesis was similar from both compounds. Topical application of ALA dendrimers on the skin overlying a subcutaneous LM3 implanted tumour showed no diffusion of the molecules either to distant skin sites or to the adjacent tumour, suggesting a promising use of the ALA macromolecules in superficial cancer models. As a second objective, we proposed the use of ALA-dendrimers in vascular PDT for the treatment of atherosclerosis. Thus, we focused our studies on ALA-dendrimer's selectivity towards macrophages in comparison with endothelial cells. For this aim we employed Raw 264.7 macrophages and HMEC-1 microvasculature cells. Porphyrin synthesis induced in macrophages by 6m-ALA and 9m-ALA (3 h, 0.025 mM) was 6 and 4.6 times higher respectively compared to the endothelial cell line, demonstrating the high affinity of ALA dendrimers for macrophages. On the other hand, ALA employed at low concentrations was slightly selective (1.7-fold) for macrophages. Inhibition studies suggested that ALA dendrimer uptake in macrophages is mainly mediated by caveloae-mediated endocytosis. Our main conclusion is that in addition to being promising molecules in PDT of superficial cancer, ALA dendrimers may also find applications in vascular PDT, since in vitro they showed selectivity to the macrophage component of the atheromatous plaque, as compared to the vascular endothelium.
Assuntos
Adenocarcinoma/tratamento farmacológico , Ácido Aminolevulínico/farmacologia , Dendrímeros/farmacologia , Neoplasias Mamárias Animais/tratamento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Ácido Aminolevulínico/química , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Dendrímeros/química , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Neoplasias Mamárias Animais/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Microvasos/efeitos dos fármacos , Microvasos/metabolismo , Estrutura Molecular , Transplante de Neoplasias , Fármacos Fotossensibilizantes/química , Porfirinas/metabolismo , Pele/efeitos dos fármacos , Pele/metabolismoRESUMO
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.
Assuntos
Ácido Aminolevulínico/uso terapêutico , Dipeptídeos/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Fotoquimioterapia , Ácido Aminolevulínico/administração & dosagem , Ácido Aminolevulínico/química , Ácido Aminolevulínico/farmacologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dipeptídeos/administração & dosagem , Dipeptídeos/química , Dipeptídeos/farmacologia , Humanos , Cinética , Masculino , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Neoplasias/patologia , Porfirinas/biossíntese , Proteínas ras/metabolismoRESUMO
Photodynamic therapy (PDT) is a relatively new method to treat various kinds of tumors, including those of the oral cavity. The topical 5-ALA-PDT treatment for tumors of the oral mucosa is preferred, since when administered systemically, there is a general photosensitization drawback in the patient. However, 5-ALA is a hydrophilic molecule and its penetration and retention is limited by topical route, including oral mucosa. We propose a topical delivery system of chitosan-based mucoadhesive film, aiming to promote greater retention of 5-ALA in tissue. The chitosan (CHT) films (4% w/w) were prepared using the solvent evaporation/casting technique. They were tested without 5-ALA resulting in permeability to water vapor (W.V.P=2.15-8.54 g mm/(h cm(2)Pa) swelling â¼300.0% (±10.5) at 4 h or 24 h and in vitro residence time >24 h for all tests. CHT films containing 10.0% (w/w) 5-ALA have resulted in average weight of 0.22 g and thickness of 0.608 mm as suitable characteristics for oral application. In the presence of CHT films both in vitro permeation and retention of 5-ALA (1.0% or 10.0%) were increased. However, 10.0% 5-ALA presented highest values of permeation and retention (â¼4 and 17 times respectively, compared to propylene glycol vehicle). On the other hand, in vitro mucoadhesion of CHT films was decreased (18.2-fold and 3.1-fold) by 5-ALA addition (1.0% or 10.0% respectively). However, CHT film containing 10.0% of 5-ALA can be a potential delivery system for topical use in the treatment of tumors of the oral cavity using PDT because it favored the retention of 5-ALA in this tissue and has shown convenient mucoadhesion.
Assuntos
Adesivos/química , Ácido Aminolevulínico/química , Quitosana/química , Neoplasias Bucais/tratamento farmacológico , Fármacos Fotossensibilizantes/química , Ácido Aminolevulínico/metabolismo , Ácido Aminolevulínico/farmacologia , Ácido Aminolevulínico/uso terapêutico , Animais , Liberação Controlada de Fármacos , Mucosa Bucal/metabolismo , Permeabilidade , Fotoquimioterapia , Fármacos Fotossensibilizantes/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/uso terapêutico , Solventes/química , SuínosRESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Fármacos Fotossensibilizantes/toxicidade , Silimarina/farmacologia , Ácido Aminolevulínico/química , Ácido Aminolevulínico/farmacologia , Ácido Aminolevulínico/toxicidade , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sinergismo Farmacológico , Quimioterapia Combinada , Flavonoides/química , Flavonoides/farmacologia , Flavonoides/uso terapêutico , Humanos , Luz , Silybum marianum/química , Silybum marianum/metabolismo , Fotoquimioterapia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/uso terapêutico , Silibina , Silimarina/química , Silimarina/uso terapêutico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologiaRESUMO
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.
Assuntos
Actinas/metabolismo , Ácido Aminolevulínico/farmacologia , Caderinas/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Proteínas ras/metabolismo , Ácido Aminolevulínico/química , Adesão Celular/efeitos dos fármacos , Adesão Celular/efeitos da radiação , Linhagem Celular , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/efeitos da radiação , Humanos , Luz , Glândulas Mamárias Humanas/efeitos dos fármacos , Glândulas Mamárias Humanas/efeitos da radiação , Fármacos Fotossensibilizantes/química , Transfecção , Proteínas ras/genéticaRESUMO
Syntheses of two water-soluble phthalocyanines (Pc) containing 5-aminolevulinic acid (ALA) linked to the core structure are described. These compounds were prepared by using original functionalizations, and they present remarkable structural and photophysical features, indicating that they could be applied to photodynamic therapy (PDT).
Assuntos
Ácido Aminolevulínico/síntese química , Indóis/síntese química , Água/química , Ácido Aminolevulínico/química , Indóis/química , Isoindóis , Estrutura Molecular , SolubilidadeRESUMO
The photodynamic activity of three photosensitizers (PS): AL-induced PPIX, the porphyrin derivative 5-(4-trimethylammoniumphenyl)-10, 5, 20-tris (2,4,6- trimethoxyphenyl) porphyrin (CP) and the molecular dyad porphyrin-C(60) (P-C(60)), the last two incorporated into liposomal vesicles, was evaluated on Hep-2 human larynx carcinoma cell line. ALA-induced accumulation of the endogenous PS PPIX, reached saturation values between 5 and 24 h incubation time; the maximal PPIX content was 5.7 nmol/106 cells. The same intracellular level was accumulated when the cationic porphyrin CP was used, while the amount of P-C(60) attained was 1.5 nmol/106 cells. Under violet-blue exciting light, the fluorescence of PPIX and P-C(60) was found in the cytoplasm showing a granular appearance indicating lysosomal localization. CP was mainly detected as a filamentous pattern characteristic of mitochondrial localization. No dark cytotoxicity was observed using 1mM ALA, 5 microM CP and 1 microM P-C(60) after 24 h incubation. Cell morphology was analyzed using Hoechst-33258, toluidine blue staining, TUNEL assay and DNA fragmentation, 24 h after irradiation with 54 J/cm2. When photosensitized with ALA and P-C(60), chromatine condensation characteristic of apoptotic cell death was found; instead, 58 % of necrotic cells were observed with CP. The results show that in the Hep-2 cells, of the three PS analyzed, the molecular dyad P-C(60) was more efficient than CP and PPIX, and confirm that PDT can induce different mechanisms of cell death depending on the PS and the irradiation dose.
Assuntos
Fármacos Fotossensibilizantes/metabolismo , Porfirinas/química , Ácido Aminolevulínico/química , Carcinoma/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fulerenos/química , Humanos , Neoplasias Laríngeas/tratamento farmacológico , Luz , Lipossomos/química , Lipossomos/metabolismo , Microscopia de Fluorescência , Fotoquimioterapia , Fármacos Fotossensibilizantes/uso terapêutico , Fármacos Fotossensibilizantes/toxicidade , Porfirinas/uso terapêutico , Porfirinas/toxicidade , Protoporfirinas/químicaRESUMO
Endogenous production of Protoporphyrin IX (PpIX) is successfully exploited for photodynamic therapy (PDT) on malignant cells, following 5-aminolevulinic acid (ALA) administration and light irradiation. This treatment kills cancer cells by damaging organelles and impairing metabolic pathways via cellular reactive oxygen species (ROS) generation. We studied the efficiency of PpIX synthetized from ALA on ROS generation, in the Vincristine resistant (LBR-V160), Doxorubicin resistant (LBR-D160) and sensitive (LBR-) murine leukemia cell lines. Cells were incubated 4 hr with 1 mM ALA and then irradiated during different times with fluorescent light. One hour later, production of ROS was analyzed by flow cytometry using different fluorescent probes: Hydroethidine (HE) for superoxide anion, 2',7' Dichlorodihydrofluorescein diacetate (DCFH-DA) for hydrogen peroxide; mitochondrial damage was examined with 3,3' Dihexyloxacarbocyanine iodide (DiOC6). We found that superoxide anion production in the three cell lines increased with irradiation time whereas no peroxide hydrogen was detected. Mitochondrial damage also increased in an irradiation time dependent manner, being higher in the Vincristine resistant line. Previous studies have demonstrated that apoptotic cell death increased with irradiation time, which is consistent with these results, indicating that ROS are critical in ALA-PDT efficiency to kill malignant cells.