Hypericin supramolecular assembles: A way to increase the skin availability and photodynamic efficiency in tumor cells.

Cyclodextrins (CDs) are molecules approved by the FDA and show promise in increasing the solubility of hydrophobic molecules and making them more available to the skin. These CDs have been used to form complexes with some photosensitizers for Photodynamic Therapy (PDT), such as Hypericin (HY). HY is a lipophilic photosensitizer known for its exceptional fluorescence and singlet oxygen quantum yield generation of over 20 % under 590 nm irradiation. In this study, we found a six-fold increase in the release of HY in vitro after complexation with ß-CD. The ß-CDHY assembly also demonstrated better skin retention, which is crucial for the topical application of this photosensitizer. Furthermore, the ß-CD complexation led to a significant increase in the phototoxicity of HY at three different light doses (3, 6, and 10 J cm-2) due to its improved water solubility and higher in vitro accumulation (approximately two times compared with free HY) in HeLa and Vero cell lines.

Nitrofurantoin removal by the photo-Fenton process: degradation, mineralization, and biological inactivation.

Antibiotics may induce super-resistant bacteria if they are available in the environment. Therefore, the removal of aqueous nitrofurantoin (NFT), and more importantly, the removal of the remaining antimicrobial activity after treatment, by the photo-Fenton process, was herein studied. Degradation experiments were performed according to an experimental design (0.5% error; factors: concentrations of NFT, Fe3+, and H2O2). Degradation conditions were: 20 mg NFT L-1, 10 mg Fe3+ L-1, and 170 mg H2O2 L-1. Fixed parameters were: 100 mL of the NFT solution, pH 2.5, 15-min stirring, and 25.0 ± 0.5°C. The initial rate constant (k0) and the maximum oxidation capacity (MOC) of the system were 0.61 min-1 and 100%, respectively (R2 = 0.986). 97% of the NFT and 93% of the organic carbon initially present were removed. Five degradation products (DPs) were detected by HPLC-MS and their endpoints estimated by the ECOSAR (ECOlogical Structure-Activity Relationships) 2.0 software. NFT and its DPs presented no toxicity towards Lactuca sativa. The antimicrobial activity (Escherichia coli) of NFT and/or DPs was completely removed in 15 min. Structures were proposed for the detected DPs. In short, the tested advanced oxidation technology (AOP), besides being capable of removing and mineralizing aqueous NFT in a short time, 15 min, also rendered the treated water biologically inactive (no ecotoxicity, no antimicrobial activity).

Improved Hypericin solubility via ß-cyclodextrin complexation: Photochemical and theoretical study for PDT applications.

Hypericin (HY) is a lipophilic photosensitizer (PS) extensively employed for photodynamic therapy (PDT), presenting high absorption in the visible region, chemical and photostability, as well as a good triplet quantum yield. Supramolecular complexation of photosensitizers into cyclodextrins (CD) is promising to improve their poor solubility, compromising their bioavailability and upcoming applications in PDT. This research produced an inclusion complex between HY and ß-CD through the co-solvent method. HY became soluble after inclusion into ß-CD cavities, besides retaining its fluorescent and singlet oxygen quantum yields (ϕf =0.115 and ϕΔ= 0.23, respectively), which are essential parameters for PDT uses and are not reported in the literature. By the theoretical analysis, since ΔG < 0, it was easy to conclude that HY inclusion into ß-CD is a spontaneous process. Additionally, the complexes presented no changes in excited states after complexation. ß-CDHY was 27% more phototoxic than free HY when tested in MCF7 cells using 3 J cm-2 of irradiation, indicating a better cell uptake of HY. These outcomes suggest that the inclusion complex of HY into ß-CD has the potential for use in PDT.

Hypericin-loaded oil-in-water nanoemulsion synthesized by ultrasonication process enhances photodynamic therapy efficiency.

Hypericin (Hy) is a hydrophobic photosensitizer used in photodynamic therapy for cancer therapeutic. In this study, Hy-loaded oil-in-water (O/W) nanoemulsions (NEs) were produced by the ultrasonication method combing different biocompatible oils and surfactants to enhance Hy aqueous solubility and bioavailability. Experimental parameters were optimized by the characterization of droplet size, zeta potential, and physicochemical properties. In vitro studies based on the release profile, cytotoxicity, cell morphology, and Hy intracellular accumulation were assayed. Hy at 100 mg L-1 was incorporated into the low viscosity (~0.005 Pa s) NEs with spherical droplets averaging 20-40 nm in size and polydispersity index <0.02. Hy release from the NE was significantly higher (4-fold) than its suspension (p < 0.001). The NEs demonstrated good physical stability during storage at 5 °C for at least six months. The Hy-loaded NEs exhibited an IC50 value 6-fold lower than Hy suspension during PDT against breast cancer cell lines (MCF-7). Cell microscopy imaging confirmed the increased cytotoxic effects of Hy-loaded NEs, showing damaged and apoptotic cells. Confocal laser scanning microscopy evidenced greater Hy delivery through NE into MCF-7 cells followed by improved intracellular ROS generation. Our results suggest that the Hy-loaded NEs can improve hypericin efficacy and assist Hy-PDT's preclinical development as a cancer treatment.

Antileishmanial activity of amphiphilic chlorin derivatives mediated by photodynamic therapy.

Leishmaniasis is a serious and neglected disease that affects 14 million people around the World. The currently available drugs for treatment present several drawbacks such as low efficacy and severe side effects, contributing to patients' low compliance. Photodynamic therapy (PDT) is rising as a promising treatment of cutaneous leishmaniasis, mainly considering its topical administration that circumvents any potential adverse effects commonly related to oral/parenteral administration. PDT depends on the interaction between a light-sensitive compound (photosensitizer - PS), light and molecular oxygen. The reaction generates reactive oxygen species (ROS) which induce cell death by oxidative stress. The main goal of this study is to demonstrate the antileishmanial effect of three chlorin derivatives (CHL-OH-A, CHL-OH-B, CHL-TRISMA) using PDT, as well as to investigate their cell death pathway on Leishmania amazonensis promastigote forms after chlorin-PDT application. The chlorin derivatives herein studied did not exhibit aggregates in aqueous medium and showed fast accumulation in Leishmania acidic compartments. CHL-OH-A exhibited the highest antiparasitic activity at 24 h (0.33 µmol L-1) and 48 h (0.14 µmol L-1) after irradiation at 660 nm (6.0 Jcm-2). CHL-OH-A, CHL-OH-B and CHL-TRISMA molecules induced the cell death of parasites mainly by an apoptotic-like process in the presence of light. These chlorin derivatives are 80-fold more active against Leishmania when compared to other PSs reported in the literature. In this study, we have shown that these amphiphilic chlorins, and in particular, CHL-OH-A, exert an interesting leishmanicidal activity suggesting that the use of these PSs associated with PDT could be a promising strategy for treatment of cutaneous leishmaniasis.

Hypericin-glucamine antimicrobial photodynamic therapy in the progression of experimentally induced periodontal disease in rats.

OBJECTIVE: To evaluate the effect of antimicrobial photodynamic therapy (aPDT) using the photosensitizer hypericin-glucamine in the progression of experimentally induced periodontal disease (PD) in rats. MATERIAL AND METHODS: Subgingival ligatures were inserted around the upper second molars of 30 rats. After 7 days (Baseline), the animals were randomly distributed into 3 experimental (n = 5) groups: Hypericin-glucamine; LED (amber LED, 700 mA, 590 nm, 90 mW, 34.10 J/cm2); and aPDT (Hypericin-glucamine + LED). The treated hemimaxillae were randomly chosen. The periodontal disease progression was monitored without treatment interference in the opposite hemimaxillaes, which were used as the negative control of each animal. The euthanasia was programmed according to each experimental period, 7 or 15 days after the Baseline. Microtomographic, histometric and Tartrate Resistant Acid Phosphatase (TRAP) immunohistochemistry analyses were carried out. RESULTS: Computerized microtomography analyses indicated that the aPDT group had a significantly higher percentage of bone tissue when compared to the other 7 days experimental groups. This result was corroborated by the histometric evaluations of the furcal area. The LED-treated group presented the highest percentages of bone volume for the 15 days experimental groups, which is remarkably higher than the groups treated with Hy-g and aPDT. The histometric analyses demonstrated the control groups had greater bone loss in the proximal regions when compared to the treated groups. The aPDT led to a lower osteoclast activity at both 7 and 15 days. Thus, we can conclude that aPDT exhibits positive effects in PD treatment by promoting favorable conditions for periodontal repair.

Hypericin cytotoxicity in tumor and non-tumor cell lines: A chemometric study.

Hypericin (HY) is an excellent photoactive compound that has been investigated for the photodynamic treatment of cancer as well as for microorganism inactivation. In this study, chemometric analysis was applied for the first time on photodynamic assays to investigate the cytotoxicity of HY in tumor (HEp-2) and non-tumor (Vero and HUVEC) cell lines. The experimental planning was based on eight assays using the 23 full factorial design combining three important variables for PDT: photosensitizer concentrations, incubation time of cells in HY solutions and employed light dose (λ=590±10nm). The statistical data analysis evidenced the relative significance of such variables and the correlations among them on the cell death. The chemometric results suggested that long incubation time and a low HY concentration and/or light dose allow killing selectively tumor cells. The chemometric analysis could be a new useful empiric method to a previous prediction of the IC50. In this study, the estimated values were in agreement with the experimental IC50 values.

Electroporation enhances antimicrobial photodynamic therapy mediated by the hydrophobic photosensitizer, hypericin.

The effective transport of photosensitizers (PS) across the membrane and the intracellular accumulation of PS are the most crucial elements in antimicrobial photodynamic therapy (aPDT). However, due to the morphological complexity of Gram-negative bacteria the penetration of PS is limited, especially hydrophobic PS. Electroporation (EP) could increase the effectiveness of aPDT, by promoting the formation of transient pores that enhance the permeability of the bacterial membrane to PS. In this study we evaluated the combination of aPDT mediated by the hydrophobic PS, hypericin and EP (aPDT/EP) against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. These bacteria were exposed to light (590 nm) in the presence of hypericin (4 µM), following electroporation. The results showed that aPDT/EP inactivated 3.67 logs more E. coli and 2.65 logs more S. aureus than aPDT alone. Based on these results we suggest that EP can potentiate the aPDT effect.

Comparando inativação fotodinâmica e antimicrobianos / Photoinactivation versus antimicrobial agents

O aparecimento de uma grande variedade de micro-organismos patogênicos resistentes aos antimicrobianos tem resultado tanto no aumento do índice de doenças quanto no aumento do índice de mortalidade, ambos provocados por infecções facilmente tratadas no passado. Assim, tornou-se urgente a necessidade de desenvolver novos procedimentos de intervenção a fim de promover a inviabilização do crescimento microbiano. A Inativação Fotodinâmica de micro-organismos é uma alternativa promissora no combate de infecções localizadas de micro-organismos. Basicamente esse mecanismo envolve a combinação sinérgica de um fotossensibilizador, oxigênio molecular e luz visível de comprimento de onda adequado para produzir espécies reativas de oxigênio, que causam oxidação dos componentes da célula levando-a à morte. A principal vantagem dessa técnica é o fato de não haver desenvolvimento de resistência ao tratamento pelos micro-organismos, devido ao grande número de alvos possíveis por parte dos radicais de oxigênio. Este trabalho faz uma comparação entre inativação fotodinâmica de micro-organismos e a ação dos antimicrobianos.

The appearance of a large variety of antimicrobial-resistant pathogenic microorganisms has led to increased rates of disease and mortality caused by infections that were easily treated in the past. Hence,338Fotoinativação versus antimicrobianosRev Ciênc Farm Básica Apl., 2012;33(3):331-340there is an urgent need to develop new procedures to prevent microbial growth. Photodynamic inactivation of microorganisms is a promising alternative way to fight localized microbial infections. This technique, basically, involves the synergistic combination of a photosensitizer, molecular oxygen and visible light of an appropriate wavelength, to produce highly reactive oxygen species that lead to the oxidation of several cell components and to cell inactivation. The main advantage of the technique is that, given the existence of multiple targets, there is no development of resistance. This paper aims to compare the photodynamic inactivation of microorganisms and the action of antibiotics.