Peptide-induced membrane leakage by lysine derivatives of gramicidin A in liposomes, planar bilayers, and erythrocytes.

Introducing a charged group near the N-terminus of gramicidin A (gA) is supposed to suppress its ability to form ion channels by restricting its head-to-head dimerization. The present study dealt with the activity of [Lys1]gA, [Lys3]gA, [Glu1]gA, [Glu3]gA, [Lys2]gA, and [Lys5]gA in model membrane systems (planar lipid bilayers and liposomes) and erythrocytes. In contrast to the Glu-substituted peptides, the lysine derivatives of gA caused non-specific liposomal leakage monitored by fluorescence dequenching of lipid vesicles loaded with carboxyfluorescein or other fluorescent dyes. Measurements of electrical current through a planar lipid membrane revealed formation of giant pores by Lys-substituted analogs, which depended on the presence of solvent in the bilayer lipid membrane. The efficacy of unselective pore formation in liposomes depended on the position of the lysine residue in the amino acid sequence, increasing in the row: [Lys2]gA<[Lys5]gA<[Lys1]gA<[Lys3]gA. The similar series of potency was exhibited by the Lys-substituted gA analogs in facilitating erythrocyte hemolysis, whereas the Glu-substituted analogs showed negligible hemolytic activity. Oligomerization of the Lys-substituted peptides is suggested to be involved in the process of nonselective pore formation.

Effect of photodynamic therapy supplemented with quercetin in HEp-2 cells.

Photodynamic therapy (PDT) is a technique that can be used as a complementary therapy in cancer treatment combined with other therapeutic modalities. Quercetin (QCT) is known to be effective in the treatment of cancer, by reducing the cell viability of different cancer cell lines. This study aimed to evaluate the influence of different concentrations of QCT in PDT on the viability, mitochondrial membrane potential and induction of apoptosis/necrosis in the human larynx carcinoma cells (HEp-2). The HEp-2 cells were treated with aluminum phthalocyanine tetrasulfonate (AlPcS4) and QCT and subsequently irradiated with a diode laser light (685 nm, 35 mW, 4.5 J/cm(2)). The results demonstrated that treatment of HEp-2 cells with high concentrations of QCT (at least 50 µM) reduced cell viability. This response was enhanced in cells subjected to PDT supplemented with high concentrations of QCT. In addition, was observed decrease in the mitochondrial membrane potential and characteristics of late apoptosis and/or initial necrosis process. QCT at concentrations from 50 µM improves PDT-induced cytotoxicity by significantly reducing cell viability by apoptosis and/or necrosis, and mitochondrial membrane potential of Hep-2 cells.

Lysosome-targeted photodynamic treatment induces primary keratinocyte differentiation.

Photodynamic therapy is an attractive technique for various skin tumors and non-cancerous skin lesions. However, while the aim of photodynamic therapy is to target and damage only the malignant cells, it unavoidably affects some of the healthy cells surrounding the tumor as well. However, data on the effects of PDT to normal cells are scarce, and the characterization of the pathways activated after the photodamage of normal cells may help to improve clinical photodynamic therapy. In our study, primary human epidermal keratinocytes were used to evaluate photodynamic treatment effects of photosensitizers with different subcellular localization. We compared the response of keratinocytes to lysosomal photodamage induced by phthalocyanines, aluminum phthalocyanine disulfonate (AlPcS2a) or aluminum phthalocyanine tetrasulfonate (AlPcS4), and cellular membrane photodamage by m-tetra(3-hydroxyphenyl)-chlorin (mTHPC). Our data showed that mTHPC-PDT promoted autophagic flux, whereas lysosomal photodamage induced by aluminum phthalocyanines evoked differentiation and apoptosis. Photodamage by AlPcS2a, which is targeted to lysosomal membranes, induced keratinocyte differentiation and apoptosis more efficiently than AlPcS4, which is targeted to lysosomal lumen. Computational analysis of the interplay between these molecular pathways revealed that keratin 10 is the coordinating molecular hub of primary keratinocyte differentiation, apoptosis and autophagy.

AlPcS4-PDT for gastric cancer therapy using gold nanorod, cationic liposome, and Pluronic® F127 nanomicellar drug carriers.

PURPOSE: As a promising photodynamic therapy (PDT) agent, Al(III) phthalocyanine chloride tetrasulfonic acid (AlPcS4) provides deep penetration into tissue, high quantum yields, good photostability, and low photobleaching. However, its low delivery efficiency and high binding affinity to serum albumin cause its low penetration into cancer cells, further limiting its PDT effect on gastric cancer. In order to improve AlPcS4/PDT effect, the AlPcS4 delivery sys tems with different drug carriers were synthesized and investigated. MATERIALS AND METHODS: Gold nanorods, cationic liposomes, and Pluronic® F127 nanomicellars were used to formulate the AlPcS4 delivery systems. The anticancer effect was evaluated by CCK-8 assay and colony formation assay. The delivery efficiency of AlPcS4 and the binding affinity to serum proteins were determined by fluorescence intensity assay. The apoptosis and necrosis ability, reactive oxygen species and singlet oxygen generation, mitochondrial transmembrane potential and ([Ca2+]i) concentration were further measured to evaluate the mechanism of cell death. RESULTS: The series of synthesized AlPcS4 delivery systems with different drug carriers improve the limited PDT effect in varying degrees. In contrast, AlPcS4 complex with gold nanorods has significant anticancer effects because gold nanorods are not only suitable for AlPcS4 delivery, but also exhibit enhanced singlet oxygen generation effect and photothermal effect to induce cell death directly. Moreover, AlPcS4 complex with cationic liposomes shows the potent inhibition effect because of its optimal AlPcS4 delivery efficiency and ability to block serum albumin. In addition, AlPcS4 complex with Pluronic F127 exhibits inferior PDT effect but presents lower cytotoxicity, slower dissociation rate, and longer retention time of incorporated drugs; thus, F127-AlPcS4 is used for prolonged gastric cancer therapy. CONCLUSION: The described AlPcS4 drug delivery systems provide promising agents for gastric cancer therapy.

DNA analysis of cattle parasitic protozoan Tritrichomonas foetus after photodynamic therapy.

Photodynamic therapy (PDT) is a modality of therapy that involves the activation of photosensitive substances and the generation of cytotoxic oxygen species and free radicals to promote the selective destruction of target tissues. This study analyzed the application of PDT to Tritrichomonas foetus, a scourged and etiological agent of bovine trichomoniasis, a sexually transmitted infectious disease. As it is an amitochondrial and aerotolerant protozoan, it produces energy under low O2 tension via hydrogenosome. T. foetus from an axenic culture was incubated with photosensitizer tetrasulfonated aluminium phthalocyanine and then irradiated with a laser source (InGaAIP) at a density of 4.5Jcm-2. The DNA integrity of the control and treated group parasites was analyzed by conventional gel electrophoresis and comet assay techniques. In previous results, morphological changes characterized by apoptotic cell death were observed after T. foetus was submitted to PDT treatment. In the treated groups, T. foetus DNA showed a higher concentration of small fragments, about 200pb, in gel electrophoresis after PDT. In the comet assay, the DNA tail percentage was significantly higher in the treated groups. These results demonstrate that PDT leads to DNA fragmentation with changes in nuclear morphology and apoptotic features.

Stability enhanced polyelectrolyte-coated gold nanorod-photosensitizer complexes for high/low power density photodynamic therapy.

Photodynamic therapy (PDT) is a promising treatment modality for cancer and other malignant diseases, however safety and efficacy improvements are required before it reaches its full potential and wider clinical use. Herein, we investigated a highly efficient and safe photodynamic therapy procedure by developing a high/low power density photodynamic therapy mode (high/low PDT mode) using methoxypoly(ethylene glycol) thiol (mPEG-SH) modified gold nanorod (GNR)-AlPcS4 photosensitizer complexes. mPEG-SH conjugated to the surface of simple polyelectrolyte-coated GNRs was verified using Fourier transform infrared spectroscopy; this improved stability, reduced cytotoxicity, and increased the encapsulation and loading efficiency of the nanoparticle dispersions. The GNR-photosensitizer complexes were exposed to the high/low PDT mode (high light dose = 80 mW/cm(2) for 0.5 min; low light dose = 25 mW/cm(2) for 1.5 min), and a high PDT efficacy leads to approximately 90% tumor cell killing. Due to synergistic plasmonic photothermal properties of the complexes, the high/low PDT mode demonstrated improved efficacy over using single wavelength continuous laser irradiation. Additionally, no significant loss in viability was observed in cells exposed to free AlPcS4 photosensitizer under the same irradiation conditions. Consequently, free AlPcS4 released from GNRs prior to cellular entry did not contribute to cytotoxicity of normal cells or impose limitations on the use of the high power density laser. This high/low PDT mode may effectively lead to a safer and more efficient photodynamic therapy for superficial tumors.

Assessment of Leishmania major and Leishmania braziliensis promastigote viability after photodynamic treatment with aluminum phthalocyanine tetrasulfonate (AlPcS4)

Cutaneous leishmaniasis is an infectious disease caused by protozoans of the genus Leishmania, which is transmitted through the bite of hematophagous insects of the genus Lutzomyia. This study aimed at testing in vitro the phototoxic effect of aluminum phthalocyanine tetrasulfonate (AlPcS4) on the viability of Leishmania major and Leishmania braziliensis. Stationary phase promastigote forms were treated with AlPcS4 at 1.0 µM and 10.0 µM and incubated for one hour. Then 659 nm laser was applied at 5 and 10 J/cm². Parasite viability was determined by differential count using the trypan blue dye exclusion method and by monitoring growth curves for nine days. Trypan blue exclusion assay showed a significant reduction of viable parasites compared to controls, L. major seemed more sensitive to the toxic effects of AlPcS4 in the dark. The most effective photodynamic therapy (PDT) was obtained with AlPcS4 at 10.0 µM and 10 J/cm² whereas L. braziliensis showed the highest mortality rate after treatment.