Photodynamic efficacy of chlorin p6: a pH dependent study in aqueous and lipid environment

Photodynamic efficacy of chlorin p6, a potential candidate of photodynamic therapy (PDT), has been studied at pH 5.0, 6.0 and 7.6 in aqueous and lipid environment. Increased chlorin p6 mediated photodynamic bleaching of N,N-dimethyl-4-nitrosoaniline (RNO), a measure of singlet oxygen yield, was obtained at higher pH. Rate of photodynamic bleaching of RNO was also higher at higher pH and the rate decreased with lowering in pH of irradiated solution. Photodynamic oxidation of tryptophan was also found to be higher at higher pH. Diminished oxidation of RNO was obtained with decrease in pH of irradiated solution. Both, RNO bleaching and tryptophan oxidation was significantly reduced by sodium azide, a known quencher of singlet oxygen. At lower pH, chlorin p6 mediated photodynamic malondialdehyde (MDA) and lipid hydroperoxide formation in egg lecithin liposome was higher. At higher pH chlorin p6 was found to be photodynamically more effective in aqueous environment whereas at lower pH chlorin p6 was photodynamically more effective in hydrophobic environment.     

Proteomic analysis of carbonylated proteins in two-dimensional gel electrophoresis using avidin-fluorescein affinity staining

A method for detecting carbonylated proteins in two-dimensional electrophoresis (2-DE) was developed using biotinylation and avidin-fluorescein isothiocyanate (FITC) affinity staining. The method was used to examine oxidatively modified proteins associated with oxidative stress. Carbonyl formation in proteins was first examined in a model system by subjecting bovine serum albumin (BSA) and ribonuclease A (RNase A) to metal-catalyzed oxidation (MCO). Carbonyl group formation was found to occur at multiple sites along with a small amount of polypeptide chain cleavage. In vivo studies were conducted in yeast cell cultures using 5 mM hydrogen peroxide to induce oxidative stress. Biotinylation of yeast protein was accomplished during extraction at 4 degrees C in a lysis buffer containing 5 mM biotin-hydrazide. Biotin-hydrazide forms a Schiff base with a carbonyl group on an oxidized protein that is subsequently reduced before electrophoresis. Proteins were separated by either 2-DE or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Biotinylated species were detected using avidin-FITC affinity staining. Detection sensitivity with biotinylated proteins was five times higher than achieved by silver staining. The limit of detection with avidin-FITC staining approached 0.64 pmol of protein-associated carbonyls. Twenty carbonylated proteins were identified in the proteome of yeast following oxidative stress with hydrogen peroxide. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis of tryptic peptides was used to identify peptides extracted from gels. Aconitase, heat shock protein SSA1 and SSC1, pyruvate decarboxylase isozyme 1, pyruvate kinase 1, enolase 1 and 2, phosphoglycerate kinase, fructose-bisphosphate aldorase, and glyceraldehyde-3-phosphate dehydrogenase were among the major targets of oxidative stress.     

Fabrication,structure and surface charges of albumin-chitosan hybrids

Protein-polymer hybrids consisting of protein and natural polymers or synthetic polymers exhibit superior properties to unmodified proteins,generating a high demand for these materials in the fields of medicine,biotechnology,and nanotechnology.Herein,protein-polysaccharide hybrids were fabricated via the formation of an amide bond between bovine serum albumin(BSA) and chitosan(CS) using N-ethyl-N-(3-dimethylaminopropyl) carbodiimide hydrochloride(EDC) as the couple reagent.FTIR spectrum reveals that the carboxyl group of BSA conjugated with the amino group of chitosan backbone.The molecular weight of BSA-CS hybrids was identified by matrix-associated laser desorption ionization time of flight mass spectra(MALDI-TOF MS) and sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE).The maximum number of chitosan chains binding to each BSA molecule was estimated as 6,and the optimal number was estimated as 2.In addition,the secondary structure and surface property of BSA were dependent upon the number of polymer conjugating on protein.The secondary structure of BSA was not significantly changed,if a few chitosans were coupled with BSA.By further increasing the molar ratio of chitosan to BSA,the secondary structure of BSA was markedly damaged.The surface’s negative charges of modified BSA also decreased.The result of native polyacrylamide gel electrophoresis(native-PAGE) also demonstrated the changes in surface charges and molecular weight of BSA-CS hybrids.     

Extracellular pH influences the mode of cell death in human colon adenocarcinoma cells subjected to photodynamic treatment with chlorin p6

Effect of varying extracellular pH on mode of cell death induced by photodynamic action of chlorin p6 was investigated in human colon carcinoma (Colo-205) cells. At an extracellular pH of 7.4, compared to cells treated with chlorin p6 in dark, the photodynamically treated cells showed reduction in mitochondrial membrane potential, an increase in ADP/ATP ratio (1:2) and a large percentage of cells with chromatin condensation. In contrast, when photodynamic treatment and post irradiation incubation was carried out in acidic medium (pH 6.5), total loss of mitochondrial membrane potential, a marked increase in ADP/ATP ratio (1:33) and increased damage to plasma membrane were observed. Further, cells subjected to photodynamic treatment in a medium of pH 7.4 showed twofold increase in caspase-3 activity as compared to photodynamic treatment at pH 6.5. These results suggest that chlorin p6 mediated photodynamic action induces apoptotic cell death when extracellular pH is 7.4 whereas necrosis is more predominant under condition when extracellular pH is 6.5.     

Fluorescence Investigation of the Binding of Model PDT Drugs to Nonionic and Zwitterionic Surfactants

The binding of two model photodynamic therapy drugs, chlorin p ₆ and purpurin 18, with surfactants has been studied using steady-state and time-resolved fluorescence techniques. The surfactants used are amphiphilic nonionic surfactant (Tween 80 and Tween 40) and zwitterionic surfactant (HAPS). These have applications in drug delivery. The studies have been performed at pH 7 and 5 for chlorin p ₆ and at pH 7 for purpurin 18. The binding constants have been estimated from the change in fluorescence parameters and have been compared with those for Cremophor EL and human serum albumin. Chlorin p ₆ is found to bind to the surfactants to a greater extent at pH 5 than at pH 7. The same ionic species of chlorin p ₆ is found to exist at the maximum concentrations of the surfactants.     

Steady state and time-resolved fluorescence investigation of the specific binding of two chlorin derivatives with human serum albumin

The specific binding of two model drugs for photodynamic therapy, namely chlorin p6 and purpurin 18 in the vicinity of Sudlow's Site I of HSA has been investigated by monitoring the intrinsic fluorescence of single tryptophanyl residue and by competitive binding with warfarin. The distance from the tryptophanyl residue has been ascertained by FRET from Trp to the chlorins and has been found to indicate a binding to Sudlow's Site I. The principal driving force for the interaction is found to be the hydrophobic effect. The main mechanism of protein fluorescence quenching was static. Time-resolved fluorescence results of competitive binding with warfarin are found to confirm that they bind to the warfarin binding site.     

Changes in plasma membrane protein structure after photodynamic action in freshly isolated rat pancreatic acini. An FTIR study

Photodynamic action of a plasma membrane-specific photosensitizer sulphonated aluminium phthalocyanine (SALPC) has been found to regulate cellular signalling pathways. The present study aimed to investigate whether SALPC photodynamic action modulates the structure of plasma membrane proteins, and as control, of model proteins. To check the photodynamic effect, intrinsic fluorescence of model proteins bovine serum albumin (BSA), phospholipase A2 (PLA2), and calmodulin were monitored continuously during photodynamic action (SALPC 1 microM, light 14,000 1x at > 580 nm). Significant decrease in fluorescence intensity was observed in BSA and PLA2, whereas the fluorescence of calmodulin was not affected. Confirming a major change in protein structure, difference IR spectrum revealed a significant downward deflection after photodynamic action in both BSA and in pancreatic acinar cells, whereas SALPC alone or light illumination alone resulted in no major deflection. Quantitative FTIR analysis indicated that in BSA, photodynamic action decreased the content of alpha-helix, increased the content of beta-turn and random structures, whereas beta-sheet remained the same; in freshly isolated rat pancreatic acini, photodynamic action decreased the content of alpha-helix and beta-sheet, increased the content of 1-turn and random structures. Taken together the fact that under the present experimental conditions SALPC mainly localized at the plasma membrane, it is concluded that SALPC photodynamic action directly modulates plasma membrane protein structure.     

超声作用下稀土离子对纳米TiO2与牛血清白蛋白相互作用的影响

在模拟人体生理条件下,采用紫外光谱法、荧光光谱法和聚丙烯酰胺凝胶电泳(SDS-PAGE)研究了超声作用下三种稀土离子(La3+、Gd3+、Yb3+)存在的不同体系中,纳米TiO2与牛血清白蛋白(BSA)的相互作用.结果表明:在超声作用下,不同体系中纳米TiO2均导致BSA的内源荧光猝灭,其猝灭机制主要为静态猝灭和非辐射能量转移;凝胶电泳结果表明,不同体系中的纳米TiO2均未对BSA的结构造成明显的损伤,由热力学数据确定其作用力主要为氢键和范德华力.稀土离子的存在使纳米TiO2与BSA的结合常数、结合距离和热力学参数发生改变,但结合位点数和作用力类型基本不变.稀土离子对纳米TiO2与BSA的结合产生明显的"四分组效应",它们分别以"离子架桥"和"同位取代"方式参与纳米TiO2与BSA的结合过程.     

Routine diagnosis with PhastSystem compared to conventional electrophoresis: automated sodium dodecyl sulfate-polyacrylamide gel electrophoresis, silver staining and western blotting of urinary proteins

The recent introduction of the PhastSystem, an automatic electrophoresis and staining system with precast gradient-gels, allows rapid and reproducible analysis of proteinuria in patients suffering from renal injury. A routine method for sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis (SDS-PAGE) and silver staining of unconcentrated urine specimens in the PhastSystem is described and compared to our conventional "macro"-method with self-cast SDS-polyacrylamide gradient gels. The method described for the PhastSystem using 0.3 microL sample volumes and an 8-25% polyacrylamide gradient gel leads to highly reproducible results within 1.5 h. Before electrophoresis urine specimens were neither concentrated nor dialyzed. Samples with a protein concentration exceeding 5 mg/mL had to be diluted 1:5 (v/v). Analysis and documentation of PhastGels appeared as easy as with our conventional SDS-PAGE. Protein bands could reliably be identified by Western blotting. Urine and serum proteins, separated in PhastGels, were electrophoretically transferred to nitrocellulose and detected with specific antibodies against human albumin, transferrin, alpha-1-antitrypsin and IgG. Comparison of several standard kits for molecular weight determination revealed considerable differences concerning the quality of protein separation patterns. Availability of precast gels and automatization of SDS-PAGE and staining allows easy standardization of urine SDS-PAGE among clinical routine laboratories.     

Tissue distribution and in vivo photosensitizing activity of 13,15-[N-(3-hydroxypropyl)]cycloimide chlorin p6 and 13,15-(N-methoxy)cycloimide chlorin p6 methyl ester

Photosensitizers 13,15-[N-(3-hydroxypropyl)]cycloimide chlorin p6 (HPC) and 13,15-(N-methoxy)cycloimide chlorin p6 methyl ester (MMC) absorb at 711 nm and possess high photoinduced cytotoxicity in vitro. Here we report, that photodynamic therapy with HPC and MMC provide considerable antitumor effect in mice bearing subcutaneous P338 lymphoma. The highest antitumor effect was achieved at a dose of 4 micromol/kg when 1.5 h delay between dye injection and light irradiation (drug-light interval) was used. According to the confocal spectral imaging studies of tissue sections this drug-light interval corresponds to a maximum of tumor accumulation of MMC and HPC (tumor to skin accumulation ratio is 8-10). Short (15 min) drug-light interval can be used for efficient vasculature-targeted photodynamic therapy with HPC at a dose of 1 micromol/kg, whereas MMC is ineffective at the short drug-light interval. Relationships between the features of tissue distribution and efficacy of photodynamic therapy at different drug-light intervals are discussed for HPC and MMC.     

A photosensitizer-loaded zinc oxide-polydopamine core-shell nanotherapeutic agent for photodynamic and photothermal synergistic therapy of cancer cells

In clinical cancer research,it is quite promising to develop multimodal synergistic therapeutic strategies.Photodynamic and photothermal synergistic therapy is a very desirable multimodal therapy strategy.Herein,we report a facile and simple method to construct a nanotherapeutic agent for photodynamic and photothermal therapy.This nanotherapeutic agent(ZnO@Ce6-PDA) is composed of a ZnO nanoparticle core,an interlayer of photosensitizer chlorin e6(Ce6) and an outer layer of polydopamine(PDA).Due ...     

Glyco-Modification of Protein With O-Cyanate Chain-End Functionalized Glycopolymer via Isourea Bond Formation

Glycoengineering aimed at the addition of carbohydrates to proteins is an attractive approach to alter the pharmacokinetic properties of proteins, such as enhancing stability and prolonging the duration of action. We report a novel protein glyco-modification of BSA and recombinant thrombomodulin with O-cyanate chain-end functionalized glycopolymer via isourea bond formation. The protein glycoconjugates were confirmed by SDS-PAGE, western blot, and MALDI-TOF mass spectrometry. Protein C activation activity of the glyco-modified recombinant thrombomodulin was confirmed, proving no interference with activity from the glycopolymer modification. The isourea bond formation under mild conditions was demonstrated as an alternative method for protein modification with polymers.     

A systematic study of bovine serum albumin (BSA) and sodium dodecyl sulfate (SDS) interactions by surface tension and small angle X-ray scattering

Classical parameters obtained from surface tension technique coupled to small angle X-ray scattering (SAXS) measurements gave support to investigate conformational changes in the bovine serum albumin (BSA)-sodium dodecyl sulfate (SDS) complexes, as well as the size of the micelle-like clusters distributed along the polypeptide chain. The studied systems were composed of 1 wt% of BSA in the absence and presence of increasing SDS molar concentration up to 80 mM, under experimental conditions of low ionic strength and pH 5.40. At SDS concentrations below the critical aggregation concentration (cac) of 2.2 mM, SAXS results indicate that the detergent does not modify the native protein conformation. However, the beginning of protein unfolding, evidenced by SAXS through an increase in the values of radius of gyration Rg and protein maximum dimension Dmax, is coincident with the onset of SDS cooperative binding to BSA identified by the first breakpoint in the surface tension-SDS profile. Further SDS addition leads to the formation of micelle-like aggregates randomly distributed along the unfolded polypeptide chain, consistent to a necklace and bead model. The SAXS data also demonstrate that the SDS micelles grow in size up to 50 mM detergent. At 50 mM surfactant, the micelles stop growing. This concentration is near the BSA saturation binding by SDS measured by dialyzes and indicated by the second breakpoint in surface tension-SDS profile. The SAXS and surface tension data are also consistent with the formation of free micelles in equilibrium with BSA-SDS complexes for surfactant amount above the saturation.     

The Effects of Serum on Cellular Uptake and Phototoxicity of Mono-L-Aspartyl Chlorin e6 (NPe6) In Vitro

Abstract— Previous reports showed that the photosensitizer mono- l -aspartyl chlorin e6 (NPe6) binds to serum proteins. However, the influence of this binding on the cellular uptake and photodynamic therapy (PDT) phototoxicity of NPe6 is still undefined. In this paper, we studied how serum in medium affected the P388 cellular uptake and PDT phototoxicity of NPe6 in vitro. This was assessed by (1) detection of the red shift (654 nm Q band peak of absorption) induced by protein binding NPe6; (2) detection of intracellular concentration of NPe6 by HPLC and (3) measurements of the cell survival ratio after PDT by MTT assay. The 654 nm Q band peak of NPe6 shifted to 665 nm after binding of NPe6 and serum proteins. The protein-bound NPe6 cannot be uptaken by cells, thus there was no PDT phototoxicity. Nevertheless, phototoxicity recovered when the concentration of NPe6 excessed the serum protein binding ability or there was free serum protein in the medium. These data suggested that the cellular uptake of NPe6 is inhibited by serum components in the medium, and that only free NPe6 is accumulated by P388 cells even during relatively long incubations. The cytotoxicity of PDT mainly depends on the free NPe6 level in the medium.     

On the Pathomorphological Pattern of the Efficiency of Photodynamic Therapy of Murine Melanoma B16 Using a New Photosensitizer Based on Chlorin e6 Conjugate with a Prostate-Specific Membrane Antigen

Photodynamic therapy (PDT) is an effective treatment for a number of solid malignancies. In this work, the antitumor efficacy of photodynamic therapy for murine B16 melanoma with intravenous administration of a new photosensitizer (PS) based on the chlorin e₆ conjugate with a prostate-specific membrane antigen (PSMA) was studied in vivo. We have previously published the data obtained in the first part of the study: the dynamics of PS accumulation in the tumor and surrounding tissues and the antitumor efficacy of the photodynamic therapy, which was evaluated by the regression parameters and morphological characteristics of the tumors—including by the complete regression of the tumors, the absolute growth rate of the tumors among the mice with continued tumor growth, and an increase in life expectancy compared to the control. The criterion for a complete cure was the absence of signs of tumor recurrence within 90 days after therapy. The conducted studies demonstrated the high efficiency of the new photosensitizer for the photodynamic therapy of B16 melanoma. This article presents a continuation of this work, including histological studies of the zones exposed to laser irradiation on the 21st day after treatment and an assessment of the therapeutic potential of photodynamic therapy for the destruction of tumor cells. Pathological studies in the zones of photodynamic exposure revealed that the effectiveness of the PDT depended on the PS dose and the laser irradiation parameters.     

A versatile water soluble fluorescent probe for ratiometric sensing of Hg2+ and bovine serum albumin

A novel tetraazamacrocycle fluorescent sensor (6-(1-(dimethylamino)-5-naphthalene sulfonyl)-3,6,9,15-tetraazabicyclo[9.3.1] pentadeca-1(15),11,13-triene, 1) has been designed and prepared, which can be utilized for selective and ratiometric sensing of Hg(2+) and bovine serum albumin (BSA) with two different responsive modes in aqueous solution at physiological pH (50 mM Tris-HCl, pH 7.6). Above 0.5 ppb Hg(2+) can be discerned by coordination with 1 and the emission color changes enable 1 to be applied to a fast Hg(2+) test paper assay. Sensor 1 has also been demonstrated to be easily cell-penetrable and applicable for Hg(2+) imaging in living cells. Imaging of BSA in the gel using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) stained in the medium containing 1 verified that the binding of 1 and BSA was successful in the presence of nonprotein substances. The linear range of 1 towards BSA utilizing ratiometric fluorescent calibration via noncovalent interaction in solution is 0-100 μg mL(-1) with a detection limit of 1 μg mL(-1), and has been successfully employed to determine the albumin concentration in blood serum by means of ratiometric fluorescent measurements for the first time. Finally, sensor 1 behaves as a fluorescent molecular switch composed of triple logic gates upon chemical inputs of Hg(2+) and BSA, which potentially provides intelligent diagnostics for Hg(2+) contaminated serum on the nanoscale.     

In situ measurement of bovine serum albumin interaction with gold nanospheres

We present in situ observations of adsorption of bovine serum albumin (BSA) on citrate-stabilized gold nanospheres. We implemented scattering correlation spectroscopy as a tool to quantify changes in the nanoparticle brownian motion resulting from BSA adsorption onto the nanoparticle surface. Protein binding was observed as an increase in the nanoparticle hydrodynamic radius. Our results indicate the formation of a protein monolayer at similar albumin concentrations as those found in human blood. Additionally, by monitoring the frequency and intensity of individual scattering events caused by single gold nanoparticles passing the observation volume, we found that BSA did not induce colloidal aggregation, a relevant result from the toxicological viewpoint. Moreover, to elucidate the thermodynamics of the gold nanoparticle-BSA association, we measured an adsorption isotherm which was best described by an anticooperative binding model. The number of binding sites based on this model was consistent with a BSA monolayer in its native state. In contrast, experiments using poly(ethylene glycol)-capped gold nanoparticles revealed no evidence for adsorption of BSA.     

DETERMINANTS OF PHOTOSENSITIZATION BY MONO-L-ASPARTYL CHLORIN e6

The mono-N-aspartyl derivative of chlorin e6 (MACE) is a new photosensitizer being examined for use in anti-neoplastic photodynamic therapy. Studies were carried out to identify unique aspects of MACE localization by murine leukemia L1210 cells in vitro. Octanol/water partitioning studies were used to quantitate the hydrophobicity of MACE and two analogs, chlorin e6 and mesochlorin. Sites of cellular localization of these dyes were probed by fluorescence studies, and by examining loci of photodamage. These studies indicate that MACE, a hydrophilic dye, partitions to cytoplasmic loci. Data obtained with chlorin e6, a more hydrophobic dye, are consistent with binding at both membrane and cytoplasmic sites. A substantially more hydrophobic product, meso-chlorin, binds primarily to the cell membrane. While the tumor-localizing porphyrin product HPD binds to plasma LDL less than HDL, MACE and CE are predominantly bound to plasma protein and HDL. Patterns of distribution and localization of MACE differ substantially from those observed with HPD and other hydrophobic sensitizers. Phototoxic effects of MACE could not be specifically attributed to membrane or mitochondrial damage.     

Photodynamic therapy with chlorin e6. A morphologic study of tumor damage efficiency in experiment.

Morphological changes in rat sarcoma M-1 after photodynamic treatment with chlorin e6 were studied. The frequency of necrosis appearance and the depth of its spreading in tumor tissue were evaluated after intraperitoneal injection of chlorin e6 in doses of 1-10 mg kg-1 and subsequent irradiation by a krypton laser with light energy density 22.5-135 J cm-2, using the method of vital staining with Evans blue. It was found that the antitumoral effect of photodynamic treatment was strengthened by increasing the dose of the agent and light and reduced by increasing the time interval between chlorin e6 injection and light irradiation. The treatment being given in the parameters mentioned produced a depth of tumor necrosis which varied from 4.0 mm to 16.6 mm. The mechanisms of tumor tissue damage after photodynamic treatment in vivo are discussed.     

Photo-stabilising action of metal chelates in polypropylene—Part II: Photolysis versus photo-sensitised oxidation under monochromatic irradiation

The photo-stabilising action of three metal chelates in unprocessed and processed polypropylene is examined using normal and second order derivative ultraviolet and infra-red spectroscopic techniques and hydroperoxide analysis. The effects of photolysis with 254 nm light versus photo-sensitised oxidation with 365 nm light are compared. For each exposure condition the rate of carbonyl formation in the polymer is compared with the rate of decomposition of the metal complex. On photolysis, carbonyl growth commences well before the complete destruction of the complexes and none offers protection to the polymer. In fact, all three chelates behave as photo-sensitisers, indicating that stabiliser photolysis products are photo-active. On photo-sensitised oxidation, while the initial hydroperoxide concentration appears to control the onset of carbonyl growth in the polymer, the rate of decomposition of the complexes shows no dependence on hydroperoxide concentration. Solution experiments indicate that there are no dark reactions with hydroperoxides apart from one of the nickel chelates (Cyasorb UV 1084) at high concentrations (～ 10^?2m) only. Essentially, the metal chelates operate by scavenging macroalkyl radical species (P·) and not alkoxy (PO·) and hydroxy radicals (·OH) during photo-oxidation. They also inhibit hydroperoxide formation during processing and one of the nickel chelates (UV 1084) gives products during the early stages of photo-oxidation which appear to operate as effective stabilisers.