In vitro photodynamic therapy on human oral keratinocytes using chloroaluminum-phthalocyanine

In this study, oral carcinoma cells were used to evaluate chloroaluminum-phthalocyanine encapsulated in liposomes as the photosensitizer agent in support of photodynamic therapy (PDT). The genotoxicity and cytotoxicity behavior of the encapsulated photosensitizer in both dark and under irradiation using the 670-nm laser were investigated with the classical trypan blue cell viability test, the acridine orange/ethidium bromide staining organelles test, micronucleus formation frequency, DNA fragmentation, and cell morphology. The cell morphology investigation was carried out using light and electronic microscopes. Our findings after PDT include reduction in cell viability (95%) associated with morphologic alterations. The neoplastic cell destruction was predominantly started by a necrotic process, according to the assay with acridine orange and ethidium bromide, and this was confirmed by electronic microscopy analysis. Neither the PDT agent nor laser irradiation alone showed cytotoxicity, genotoxicity, or even morphologic alterations. Our results reinforce the efficiency of tight-irradiated chloroaluminum-phthalocyanine in inducing a positive effect of PDT. (C) 2008 Elsevier Ltd. All rights reserved.

Mitochondria, endoplasmic reticulum and actin filament behavior after PDT with chloroaluminum phthalocyanine liposomal in HeLa cells

Photodynamic therapy (PDT) for cancer is a therapeutic modality in the treatment of tumors in which visible light is used to activate a photosensitizer. Cell membranes have been identified as an important intracellular target for singlet oxygen produced during the photochemical pathway. This study analyzed the cytotoxicity in specific cellular targets of a photosensitizer used in PDT in vitro. The photosensitizing effects of chloroaluminum phthalocyanine liposomal were studied on the mitochondria, cytoskeleton and endoplasmic reticulum of HeLa cells. Cells were irradiated with a diode laser working at 670 nm, energy density of 4.5 J/cm(2) and power density of 45 mW/cm(2). Fluorescence microscopic analysis of the mitochondria showed changes in membrane potential. After PDT treatment, the cytoskeleton and endoplasmic reticulum presented basic alterations in distribution. The combined effect of AlPHCl liposomal and red light in the HeLa cell line induced photodamage to the mitochondria, endoplasmic reticulum and actin filaments in the cytoskeleton. (c) 2008 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

HPLC-FLD methods to quantify chloroaluminum phthalocyanine in nanoparticles, plasma and tissue: application in pharmacokinetic and biodistribution studies

Analytical and bioanalytical methods of high-performance liquid chromatography with fluorescence detection (HPLC-FLD) were developed and validated for the determination of chloroaluminum phthalocyanine in different formulations of polymeric nanocapsules, plasma and livers of mice. Plasma and homogenized liver samples were extracted with ethyl acetate, and zinc phthalocyanine was used as internal standard. The results indicated that the methods were linear and selective for all matrices studied. Analysis of accuracy and precision showed adequate values, with variations lower than 10% in biological samples and lower than 2% in analytical samples. The recoveries were as high as 96% and 99% in the plasma and livers, respectively. The quantification limit of the analytical method was 1.12 ng/ml, and the limits of quantification of the bioanalytical method were 15 ng/ml and 75 ng/g for plasma and liver samples, respectively. The bioanalytical method developed was sensitive in the ranges of 15-100 ng/ml in plasma and 75-500 ng/g in liver samples and was applied to studies of biodistribution and pharmacokinetics of AlClPc. (C) 2011 Elsevier B.V. All rights reserved.

Validated spectrophotometric and spectrofluorimetric methods for determination of chloroaluminum phthalocyanine in nanocarriers

UV-VIS-Spectrophotometric and spectrofluorimetric methods have been developed and validated allowing the quantification of chloroaluminum phthalocyanine (CIAIPc) in nanocarriers. In order to validate the methods, the linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, and selectivity were examined according to USP 30 and ICH guidelines. Linearities range were found between 0.50-3.00 mu g.mL(-1) (Y=0.3829 X [CIAIPc, mu g.mL(-1)] + 0.0126; r=0.9992) for spectrophotometry, and 0.05-1.00 mu g.mL(-1) (Y=2.24 x 10(6) X [CIAIPc, mu g.L(-1)] + 9.74 x 10(4); r=0.9978) for spectrofluorimetry. In addition, ANOVA and Lack-of-fit tests demonstrated that the regression equations were statistically significant (p<0.05), and the resulting linear model is fully adequate for both analytical methods. The LOD values were 0.09 and 0.01 mu g.mL(-1), while the LOCI were 0.27 and 0.04 mu g.mL(-1) for spectrophotometric and spectrofluorimetric methods, respectively. Repeatability and intermediate precision for proposed methods showed relative standard deviation (RSD) between 0.58% to 4.80%. The percent recovery ranged from 98.9% to 102.7% for spectrophotometric analyses and from 94.2% to 101.2% for spectrofluorimetry. No interferences from common excipients were detected and both methods were considered specific. Therefore, the methods are accurate, precise, specific, and reproducible and hence can be applied for quantification of CIAIPc in nanoemulsions (NE) and nanocapsules (NC).

In Vitro Phototoxicity of Liposomes and Nanocapsules Containing Chloroaluminum Phthalocyanine on Human Melanoma Cell Line

In this study, the photodynamic action of liposomes (LP) and nanocapsules (NC) containing Chloroaluminum phthalocyanine (CIAIPc), on the human melanoma cell (WM 1552C), was assessed. The light source was setup at 672 nm, which corresponds to the maximum absorption wavelength of the CIAIPc. Both colloidal carriers presented size in nanometric scale as well as negative zeta potential. The cellular damage was light dose dependent ranging from 30% of cell death at 70 mJ.cm(-2) to 90% of death at 700 mJ.cm(-2). However, the photocytotoxic effect of LP at 70 mJ.cm(-2) was slightly more efficient to induce cellular death than NC formulation. At 140 mJ.cm(-2), and 700 mJ.cm(-2) both nanocarriers were equally efficient to induce cellular damage. Therefore, in the present work, the maximum phototoxic effect was obtained with 700 mJ.cm(-2) of light dose, in combination with 0.29 mu g.mL(-1) of CIAIPc encapsulated into LP and NC. The cells were also positive to annexin V, after the PDT treatment with LP and NC, showing that one of the mechanisms of cellular death involved is apoptosis. In summary, the potential of LP and NC as a drug delivery system, in Photodynamic Therapy (PDT) against melanoma, has been confirmed using a lower concentration of the photosensitizer and lower light doses than that applied in current protocols. This is an innovative proposal to treat melanoma cell lines that until now have not received the benefit of the PDT protocol for treatment.

Photophysical studies of zinc phthalocyanine and chloroaluminum phthalocyanine incorporated into liposomes in the presence of additives

The photophysical properties of zinc phthalocyanine (ZnPC) and chloroaluminum phthalocyanine (AlPHCl) incorporated into liposomes of dimyristoyl phosphatidylcholine in the presence and absence of additives such as cholesterol or cardiolipin were studied by time-resolved fluorescence, laser flash photolysis and steady-state techniques. The absorbance of the drugs changed linearly with drug concentration, at least up to 5.0 µM in homogeneous and heterogeneous media, indicating that aggregation did not occur in these media within this concentration range. The incorporation of the drugs into liposomes increases the dimerization constant by one order of magnitude (for ZnPC, 3.6 x 10(4) to 1.0 x 10(5) M-1 and for AlPHCl, 3.7 x 10(4) to 1.5 x 10(5) M-1), but this feature dose does not rule out the use of this carrier, since the incorporation of these hydrophobic drugs into liposomes permits their systemic administration. Probe location in biological membranes and predominant positions of the phthalocyanines in liposomes were inferred on the basis of their fluorescence and triplet state properties. Both phthalocyanines are preferentially distributed in the internal regions of the liposome bilayer. The additives affect the distribution of these drugs within the liposomes, a fact that controls their delivery when both are used in a biological medium, retarding their release. The addition of the additives to the liposomes increases the internalization of phthalocyanines. The interaction of the drugs with a plasma protein, bovine serum albumin, was examined quantitatively by the fluorescence technique. The results show that when the drugs were incorporated into small unilamellar liposomes, the association with albumin was enhanced when compared with organic media, a fact that should increase the selectivity of tumor targeting by these phthalocyanines (for ZnPC, 0.71 x 10(6) to 1.30 x 10(7) M-1 and for AlPHCl, 4.86 x 10(7) to 3.10 x 10(8) M-1).

Infrared to visible up-conversion in biocellulose yttrium vanadate nanoparticle composite membranes. Demonstration of chloroaluminum phthalocyanine light emission under up-converted light excitation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Development, characterization, and in vitro trials of chloroaluminum phthalocyanine-magnetic nanoemulsion to hyperthermia and photodynamic therapies on glioblastoma as a biological model

A glioblastoma multiforme (GBM) is the highest grade glioma tumor (grade IV) and is the most malignant form of astrocytomas. Grade IV tumors, which are the most malignant and aggressive, affect people between the ages of 45 and 70 years. A GBM exhibits remarkable characteristics that include excessive proliferation, necrosis, genetic instability, and chemoresistance. Because of these characteristics, GBMs are difficult to treat and have a poor prognosis with a median survival of less than one year. New methods to achieve widespread distribution of therapeutic agents across infiltrative gliomas significantly improve brain tumor therapy. Photodynamic therapy (PDT) and hyperthermia (HPT) are well-established tumor therapies with minimal side effects while acting synergistically. This study introduces a new promising nanocarrier for the synergistic application of PDT and magnetic hyperthermia therapy against human glioma cell line T98 G, with cellular viability reduction down to as low as 17% compared with the control. (C) 2012 American Institute of Physics. [doi:10.1063/1.3671775]

In Vitro Photodynamic Inactivation of Cryptococcus neoformans Melanized Cells with Chloroaluminum Phthalocyanine Nanoemulsion

The selection of fungi resistant to currently used fungicides and the emergence of new pathogenic species make the development of alternative fungus-control techniques highly desirable. Photodynamic antimicrobial chemotherapy (PACT) is a promising method which combines a nontoxic photosensitizer (PS) with visible light to cause selective killing of microbial cells. The development of PACT to treat mycoses or kill fungi in the environment depends on identifying effective PS for the different pathogenic species and delivery systems able to expand and optimize their use. In the present study, the in vitro susceptibility of Cryptococcus neoformans melanized cells to the photodynamic effects of the PS agent ClAlPc in nanoemulsion (ClAlPc/NE) was examined. Cells were killed in a PS concentration- and light dose-dependent manner. Treatment with ClAlPc/NE, using PS concentrations (e.g. 4.5 mu m) and light doses (e.g. 10 J cm-2) compatible with PACT, resulted in a reduction of up to 6 logs in survival. Washing the cells to remove unbound PS before light exposure did not inhibit fungal photodynamic inactivation. Internalization of ClAlPc by C.neoformans was confirmed by confocal fluorescence microscopy, and the degree of uptake was dependent on PS concentration.

Preparation and characterization of chloroaluminum phthalocyanine-loaded solid lipid nanoparticles by thermal analysis and powder X-ray diffraction techniques

Solid lipid nanoparticles (SLN) without drug and SLN loaded with chloroaluminum phthalocyanine (AlClPc) were prepared by solvent diffusion method in aqueous system and characterized by thermal analyses and X-ray diffraction (XRD) in this study. Determination of particle size, zeta potential (ZP), and encapsulation efficiency were also evaluated. SLN containing AlClPc of nanometer size with high encapsulation efficiency and ZP were obtained. The results indicated that the size of SLN loaded with AlClPc is larger than that of the inert particle, but ZP is not changed significantly with incorporation of the drug. In differential scanning calorimetry (DSC) curves, it was observed that the melting point of stearic acid (SA) isolated and in SLN occurred at 55 and 64 degrees C, respectively, suggesting the presence of different polymorphs. DSC also shows that the crystallinity state of SLN was much less than that of SA isolated. The incorporation of drug in SLN may have been favored by this lower crystallinity degree of the samples. XRD techniques corroborated with the thermal analytic techniques, suggesting the polymorphic modifications of stearic acid.

Analysis of Liquid Crystalline Nanoparticles by Small Angle X-Ray Diffraction: Evaluation of Drug and Pharmaceutical Additives Influence on the Internal Structure

The goal of this work was to study the liquid crystalline structure of a nanodispersion delivery system intended to be used in photodynamic therapy after loading with photosensitizers (PSs) and additives such as preservatives and thickening polymers. Polarized light microscopy and light scattering were performed on a standard nanodispersion in order to determine the anisotropy of the liquid crystalline structure and the mean diameter of the nanoparticles, respectively. Small angle X-ray diffraction (SAXRD) was used to verify the influence of drug loading and additives on the liquid crystalline structure of the nanodispersions. The samples, before and after the addition of PSs and additives, were stable over 90 days, as verified by dynamic light scattering. SAXRD revealed that despite the alteration observed in some of the samples analyzed in the presence of photosensitizing drugs and additives, the hexagonal phase still remained in the crystalline phase. (C) 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100: 2849-2857, 2011

Photodynamic therapy with aluminum-chloro-phtalocyanine induces necrosis and vascular damage in mice tongue tumors

In this paper we describe the efficacy of the liposomal-AlClPc (aluminum-chloro-phthalocyanine) formulation in PDT study against Ehrlich tumor cells proliferation in immunocompetent swiss mice tongue. Experiments were conduced in sixteen tumor induced mice that were divided in three control groups: (1) tumor without treatment; (2) tumor with 100 J/cm(2) laser (670 nm) irradiation; and (3) tumor with AlClPc peritumoral injection; and a PDT experimental group when tumors received AlClPc injection followed by tumor irradiation. Control groups present similar macroscopically and histological patterns after treatments, while PDT treatment induced 90% of Ehrlich tumor necrosis after 24 h of one single showing the efficacy of liposome-AlClPc (aluminum-chloro-phthalocyanine) mediated PDT application, on the treatment of oral cancer. (C) 2008 Elsevier B.V. All rights reserved.

Synergistic interaction between gold nanoparticles and nickel phthalocyanine in layer-by-layer (LbL) films: evidence of constitutional dynamic chemistry (CDC)

The concept of constitutional dynamic chemistry (CDC) based on the control of non-covalent interactions in supramolecular structures is promising for having a large impact on nanoscience and nanotechnology if adequate nanoscale manipulation methods are used. In this study, we demonstrate that the layer-by-layer (LbL) technique may be used to produce electroactive electrodes with ITO coated by tetrasulfonated nickel phthalocyanine (NiTsPc) alternated with poly(allylamine hydrochloride) (PAH) incorporating gold nanoparticles (AuNP), in which synergy has been achieved in the interaction between the nanoparticles and NiTsPc. The catalytic activity toward hydrogen peroxide (H(2)O(2)) in multilayer films was investigated using cyclic voltammetry, where oxidation of H(2)O(2) led to increased currents in the PAH-AuNP/NiTsPc films for the electrochemical processes associated with the phthalocyanine ring and nickel at 0.52 and 0.81 V vs. SCE, respectively, while for PAH/NiTsPc films (without AuNP) only the first redox process was affected. In control experiments we found out that the catalytic activity was not solely due to the presence of AuNP, but rather to the nanoparticles inducing NiTsPc supramolecular structures that favored access to their redox sites, thus yielding strong charge transfer. The combined effects of NiTsPc and AuNP, which could only be observed in nanostructured LbL films, point to another avenue to pursue within the CDC paradigm.

Photochemical production of nitric oxide from a nitrosyl phthalocyanine ruthenium complex by irradiation with light in the phototherapeutic window

The photochemical behavior of [Ru(NO)(NO)(2)pc] (pc = phthalocyanine) is reported in this paper. In addition to ligand localized absorption bands (lambda < 300 nm), the electronic spectrum of this complex in dichloromethane solution was dominated by an intense absorption at 640 nm characterized as Q-bands. Irradiation of [Ru(NO)(NO)(2)pc] at 366 and 660 nm led to the production of nitric oxide (NO) as detected by a NO-sensor. NO production by light irradiation at high energy involved excitation of d(pi)-pi* transition, while a photoinduced electron transfer occurred at long wavelength irradiation. The NO quantum yields varied from 1.4 x 10(-3) to 2.3 x 10(-2) mol einstein(-1), depending on oxygen concentration. (c) 2008 Elsevier B.V. All rights reserved.

Photocytotoxic activity of a nitrosyl phthalocyanine ruthenium complex - A system capable of producing nitric oxide and singlet oxygen

The synthesis, structural aspects, pharmacological assays, and in vitro photoinduced cytotoxic properties of [Ru(NO)(ONO)(pc)] (pc = phthalocyanine) are described. Its biological effect on the B16F10 cell line was studied in the presence and absence of visible light irradiation. At comparable irradiation levels, [Ru(NO) (ONO)(pc)] was more effective than [Ru(pc)] at inhibiting cell growth, suggesting that occurrence of nitric oxide release following singlet oxygen production upon light irradiation may be an important mechanism by which the nitrosyl ruthenium complex exhibits enhanced biological activity in cells. Following visible light activation, the [Ru(NO)(ONO)(pc)] complex displayed increased potency in B16F10 cells upon modifications to the photoinduced dose; indeed, enhanced potency was detected when the nitrosyl ruthenium complex was encapsulated in a drug delivery system. The liposome containing the [Ru(NO)(ONO)(pc)] complex was over 25% more active than the corresponding ruthenium complex in phosphate buffer solution. The activity of the complex was directly proportional to the ruthenium amount present inside the cell, as determined by inductively coupled plasma mass spectroscopy. Flow cytometry analysis revealed that the photocytotoxic activity was mainly due to apoptosis. Furthermore, the vasorelaxation induced by [Ru(NO)(ONO)(pc)], proposed as NO carrier, was studied in rat isolated aorta. The observed vasodilation was concentration-dependent. Taken together, the present findings demonstrate that the [Ru(NO)(ONO)(pc)] complex induces vascular relaxation and could be a potent anti-tumor agent. Nitric oxide release following singlet oxygen production upon visible light irradiation on a nitrosyl ruthenium complex produces two radicals and may elicit phototoxic responses that may find useful applications in photodynamic therapy. Crown Copyright (C) 2011 Published by Elsevier Inc. All rights reserved.