Hydrophobic Porphyrin Research Articles

Luminescence properties of the porphyrin/porous silicon composites

AbstractA study of luminescence properties of water‐soluble and hydrophobic porphyrin and chlorin molecules immobilized in oxidized (i.e. non‐luminescent) and as‐prepared (i.e. luminescent) porous silicon (porSi) layers is carried out. It is found that the oxidized porSi matrix itself does not introduce any quenching effect on the porphyrin fluorescence, but the fluorescence is subjected to the concentration quenching. At maximum porphyrin/chlorin concentration used (ca. 10–3 M in impregnation solutions) the quenching is strong in case of hydrophobic porphyrins and sufficiently moderate for water‐soluble ones, especially for cationic porphyrin TMPyP4. A concentration of the latter in porSi layer is determined by direct measurements. Both hydrophobic and water‐soluble porphyrins immobilized on the surface of as‐prepared porSi are found to quench the luminescence of the latter. In case of hydrophobic porphyrins the quenched porSi luminescence is regained almost to the initial value on the time scale of tens of hours, whereas in case of water‐soluble porphyrins the fluorescence spectrum of the latter builds up in course of time. Possible mechanisms responsible for these effects are discussed. (© 2007 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Spectroscopic and self-association behavior of a porphyrin-β-cyclodextrin conjugate

A novel porphyrin–cyclodextrin conjugate, 5[4-(6-O-β-cyclodextrin)-phenyl],10,15,20-tri(4-hydroxyphenyl)-porphyrin (CD-THPP), has been synthesized in good yield (30%) starting from 6-O-tosyl-β-cyclodextrin using 5,10,15,20-tetra(4-hydroxyphenyl)porphyrin (p-THPP) as a nucleophile. The spectroscopic properties of CD-THPP have been investigated by combining NMR, circular dichroism, UV-vis absorption, fluorescence and laser flash photolysis techniques, allowing us to gaining insights into the self-association mode of the conjugate. In neat methanol, CD-THPP is present in a monomeric form and exhibits spectroscopic features virtually identical to those of the model compound p-THPP. The role of the CD macrocycle is essential in differentiating the behavior of the conjugate with respect to the non-modified porphyrin in the mixture water : methanol, 9 : 1 (v/v). Under these conditions, CD-THPP shows a marked tendency to self-arrange as a dimer, whose formation is strongly encouraged by stacking interactions between the aromatic rings, in addition to intermolecular hydrophobic porphyrin–cyclodextrin interactions. This supramolecular species exhibits a strong exciton coupling and, contrary to the unmodified p-THPP, shows a good response to light excitation, as confirmed by the considerable fluorescence emission and triplet–triplet transient absorption. It is also demonstrated that incorporation of suitable guests into the CD cavity triggers a new rearrangement of the dimer with a higher degree of overlap between the porphyrin chromophores, facilitating the occurrence of intramolecular photoinduced electron transfer between the guest and the porphyrin unit.

Porphyrin Nanotubes Based on Self-Assembly of Mo(V)−Dodecaphenylporphyrin Complexes and Inclusion of Mo−Oxo Clusters: Synthesis and Characterization by X-ray Crystallography and Transmission Electron Microscopy

A saddle-distorted molybdenum(V)−dodecaphenylporphyrin (H2DPP) complex, [Mo(DPP)(O)(OMe)] (1), was converted to [Mo(DPP)(O)(H2O)]+ (2) in the course of recrystallization from toluene with vapor diffusion of methanol. The complex 2 was organized via self-assembly to form a porphyrin nanotube having a diameter of 1 nm, concomitant with size-selective inclusion of three kinds of novel tetranuclear Mo(VI)−oxo clusters in the nanotube. This porphyrin nanotube exhibits amphiphilic characteristics, that is, a hydrophobic porphyrin surface and a hydrophilic inner-sphere made of aquo ligands to include ionic and hydrophilic entities. Each nanotube has no direct interaction; however, the toluene molecules of crystallization linked the nanotubes together with intermolecular π−π and CH/π interactions. Transmission electron microscopy measurements and energy-dispersive X-ray analysis revealed that the diameters of porphyrin nanotubes could be altered by the size of the Mo−oxo clusters included and also a porphyrin nanoring could be obtained. These results indicate that a variety of porphyrin nanotubular structures can be fabricated by using DPP complexes via cooperatively template-assisted self-assembly.

Coupling of acetonitrile deproteinization and salting‐out extraction with acetonitrile stacking for biological sample clean‐up and the enrichment of hydrophobic compounds (porphyrins) in capillary electrophoresis

A new sample pretreatment approach in CE was developed for concurrent biological sample clean-up and the concentration of hydrophobic compounds based on the combination of ACN deproteinization with salting-out extraction. Further enhancement in concentration detection sensitivity was achieved by coupling (offline) salting-out extraction with an online CE sample enrichment technique known as "ACN stacking". By optimizing the pH of salting-out extraction, a number of model compounds (hydrophobic porphyrins with clinical significances), i.e. zinc-protoporphyrin, protoporphyrin, and coproporphyrin (CP) III and I, can be efficiently extracted from the aqueous sample into a smaller volume organic solvent (ACN) phase and an enrichment factor of ca. 100 can be obtained. The pressure injection of the enriched ACN phase (containing ca.1% NaCl) into the CE capillary at 10% capillary volume resulted in additional concentration of the various hydrophobic porphyrins, allowing for a combined enrichment factor of ca.1000 to be obtained. Calibration curves obtained for the determination of a pair of positional isomers with significant diagnostic value, urinary CPIII and CPI, were found to be linear between 10-300 ng/mL (with R2 = 0.999), and LODs (absorbance detection at 400 nm) were ca. 0.8 ng/mL (1.1 nmol/L of CPIII or CPI). Based on a single salting-out extraction, intraday precisions (nine consecutive injections) for both CPIII and CPI (at spiked concentrations of 10-300 ng/mL into urine) in terms of migration time and peak area were found to be within the range of 0.2-0.5 and 0.8-2.9%, respectively.

Nano-structure of polyimide and poly(isobutyl methacrylate) monolayer films studied by interlayer electron transfer between porphyrin and pyromellitic diimide moieties

Hetero-multilayers composed of porphyrin-containing poly(isobutyl methacrylate) (PiBMA) and polyimide (PI) monolayers were prepared by using the Langmuir-Blodgett technique, and the interlayer electron transfer process from photo-excited porphyrins to pyromellitic diimide moieties in PI layers was observed by fluorescence spectroscopy. The efficiency of the electron transfer showed strong dependence on the number of PI layers, suggesting the presence of defects in the PI plane, which were filled with PI after the successive deposition of PI monolayers. The three-dimensional and two-dimensional active sphere models (Perrin model) well explained the interlayer electron transfer phenomena. We discussed the nano-structures of the multilayer films in terms of the thickness of each layer and the location of hydrophobic porphyrins in the PiBMA monolayer.

Peptide-Mediated Cell Transport of Water Soluble Porphyrin Conjugates

Five new porphyrin-peptide conjugates bearing a nuclear localizing sequence SV40 or a fusogenic peptide (HIV-1Tat 40-60 or octa-arginine) linked by low molecular weight poly(ethylene glycol) have been synthesized. In vitro studies using human HEp2 cells show that the cellular uptake of the conjugates depends significantly on the nature and sequence of amino acids in the peptide and on the nature of the substituents on the porphyrin macrocycle. The fusogenic peptide sequences HIV-1Tat 40-60 and octa-arginine were the most effective in delivering the conjugates to the cells. The subcellular distribution of the conjugates was found to be dependent on the nature of substituents on the porphyrin macrocycle. The conjugates bearing a hydrophobic porphyrin localized preferentially in the endoplasmic reticulum and were significantly more phototoxic to HEp2 cells than the carboxylic acid functionalized porphyrin conjugates, which localized mainly in the lysosomes.

Improved photodynamic activity of porphyrin loaded into nanoparticles: an in vivo evaluation using chick embryos

Hydrophobic porphyrins are potentially interesting molecules for the photodynamic therapy (PDT) of solid cancers or ocular vascularization diseases. Their pharmaceutical development is, however, hampered by their lipophilicity, which renders formulation difficult especially when intravenous administration is needed. Encapsulation of a lipophilic derivative of porphyrin, the meso-tetra( p-hydroxyphenyl)porphyrin ( p-THPP), into polymeric biodegradable poly( d, l-lactide- co-glycolide) (PLGA) nanoparticles proved to enhance its photodynamic activity against mammary tumour cells when compared to free drug. In order to further investigate these carriers, the efficacy of the encapsulated drug was assessed on the chick embryo chorioallantoic membrane (CAM) model. First, we identified a suitable solvent for the drug in terms of p-THPP solubility and tolerability by chick embryos. This solution was used as a reference. Then, the fluorescence pharmacokinetics and the photodynamic effects of the porphyrin on CAM vessels were evaluated after intravenous administration of either a p-THPP solution (free drug) or the drug loaded into nanoparticles. The results showed that: (i) the drug remained longer in the vascular compartment when incorporated into nanoparticles and (ii) vascular effects of p-THPP after light irradiation were enhanced with nanoparticle carriers. These results are discussed taking into account the extravasation of intravascular circulating photosensitizers and its influence on PDT performance.

Incorporation of hydrophobic porphyrins into liposomes: characterization and structural requirements

The ability of photosensitisers to give reactive oxygenated products is considered decisive for photodynamic applications, but the hydrophobic nature of many porphyrins makes necessary to obtain suitable pharmaceutical formulations. This paper reports the structural photosensitiser features that allow the preparation of stable liposomal formulations. Metallated and non-metallated TPPs and TPyPs and different lipid/porphyrin ratios were considered in order to procure liposomal preparations containing porphyrin concentrations adequate to necessary doses. The results show that the incorporation of porphyrins into liposomes can be related with their ability to form aggregates in a watery media. Thus, ZnTPP, which structural properties avoid the formation of aggregates, was efficiently incorporated into stable liposomes. Moreover, the efficient generation of singlet oxygen by ZnTPP liposomal suspensions has been shown. Because of this, the synthesis of hydrophobic porphyrin derived structures or other sensitisers, which do not aggregate in a watery media and with Q-bands shifted to higher λ values than ZnTPP, will be efficiently incorporated into liposomes and useful for clinical applications.

Isolable Micelles with a Fluid Core Made of Kanamycin A 6‘-Stearoylamide

Kanamycin, a pseudo-trisaccharide bearing four amino groups, was monoamidated with stearic acid. This amphiphile with a broad headgroup capable of forming +NH3−OH hydrogen bonds in aqueous solution and a narrow hydrophobic moiety produces micelles. They dissolve a hydrophobic porphyrin in water, and they are stable in the dry form. TEM and AFM pictures of these first isolable micelles with a fluid core are discussed.

Thermodynamics of porphyrin binding to human serum albumin using affinity capillary electrophoresis

The interaction thermodynamics of heptacarboxylporphyrin (HCP) and protoporhyrin (PP) with human serum albumin (HSA) was studied by affinity capillary electrophoresis (ACE) over the temperature range of 25–50°C, where HCP and PP bound to HSAvia 1:1 molecular association. The binding equilibrium constants (pH 7.4, phosphate buffer) for the binding of HCP with HSA were found to decrease with an increase in temperature, whereas the binding constants of the PP/HSA system appeared to be independent of temperature changes over the range studied. The van’t Hoff relationship (25–50°C) was found to be linear for the interaction of either HCP or PP with HSA. However, the interaction thermodynamics for both of these porphyrins with HSA were found to be quite different. In particular, the interaction of HCP (a hydrophilic porphyrin) with HSA appeared to be based on an enthalpy-driven process, whereas the binding between PP (a hydrophobic porphyrin) and HSA driven by a favorable change in entropy. The ability of using ACE to evaluate the interaction thermodynamics of serum proteins (e.g., HSA) with ligands (e.g., porphyrins and related compounds) should aid in the development of new and more effective photosensitizers in the photodynamic therapy of cancer.

Chromatographic behaviour of some porphyrins and their complexes with Zn, Pd(II) and Cu(II)

In this work the influence of the porphyrin structure and of the nature of the mobile phase upon retention parameters is examined by means of reversed phase high-performance liquid chromatography (HPLC). Acetonitrile–ethyl acetate and other mixtures were used as eluents. An increase in the retention of azo- and benzosubstituted porphyrins as well as of those containing a large number of carbon atoms as substituents of macrocycles may be noted. A variation in the polarity of the mobile phase affects the retention of the ligands more than that of their zinc complexes. The retention of the most hydrophobic compounds may be well described in coordinates lg k'– lg M. For less hydrophobic porphyrins these dependences are close to linear only within limited intervals of mobile phase ethyl acetate concentration. The best separation of zinc complexes was achieved with acetonitrile as the eluent. The detection limit of porphyrin ligands and complexes with metals is n × 10-8 M.

Determination of Fluorescence Yields, Singlet Lifetimes and Singlet Oxygen Yields of Water‐Insoluble Porphyrins and Metalloporphyrins in Organic Solvents and in Aqueous Media

Abstract— Fluorescence quantum yields and singlet lifetimes for a wide range of hydrophilic to hydrophobic porphyrins and metalloporphyrins have been determined in toluene, methanol or acetone. Photosensitized singlet oxygen yields have been determined in the same solvents. For some porphyrins, the same quantities were determined in an aqueous medium, through use of an amphiphilic polymer to solubilize the porphyrin sensitizer and target molecule, 1, 3‐diphenylisobenzofuran. Because rate constants for the deactivation of singlet oxygen (kd) and for its reaction with a target molecule (ka) are unknown in such aqueous polymer systems, a new method was developed for evaluating yields of singlet oxygen formation that also provides a value for the ratio kd/ka. A variation observed in quantum yield of singlet oxygen production for the aqueous polymer system with variation in initial concentration of the target molecule is discussed.

Determination of Fluorescence Yields, Singlet Lifetimes and Singlet Oxygen Yields of Water-Insoluble Porphyrins and Metalloporphyrins in Organic Solvents and in Aqueous Media

Abstract— Fluorescence quantum yields and singlet lifetimes for a wide range of hydrophilic to hydrophobic porphyrins and metalloporphyrins have been determined in toluene, methanol or acetone. Photosensitized singlet oxygen yields have been determined in the same solvents. For some porphyrins, the same quantities were determined in an aqueous medium, through use of an amphiphilic polymer to solubilize the porphyrin sensitizer and target molecule, 1, 3-diphenylisobenzofuran. Because rate constants for the deactivation of singlet oxygen (kd) and for its reaction with a target molecule (ka) are unknown in such aqueous polymer systems, a new method was developed for evaluating yields of singlet oxygen formation that also provides a value for the ratio kd/ka. A variation observed in quantum yield of singlet oxygen production for the aqueous polymer system with variation in initial concentration of the target molecule is discussed.

Self-organized monolayer assemblies of octopusporphyrins: photoinduced electron transfer and O2 coordination in aqueous media

Bolaamphiphilic tetraresorcinolporphyrins with eight long side chains (octopusporphyrins) and their metal complexes form monolayered assemblies in bulk aqueous solution. The nano-structure, the photoinduced electron transfers and the O2 coordination of these octopusporphyrin assemblies are described. In the micellar fibers of 1a and 1b, a unique spherical arrangement of eight methyl groups on both sides of the porphyrin ring plane provides hydrophobic porphyrin centers which align in a string of pearls. Exciton calculations indicated a tilt stacking porphyrins arrangement with a separation of 11 Å. These fibers fluoresced strongly; electron transfer reaction was therefore observed between the porphyrin center and hydrophobic quenchers as well as hydrophilic quenchers. The fibers were also active as photocatalysts in the reduction of dimethylviologen by triethanolamine. Octopusporphyrins with different metal centers can also produce fibrous aggregates, for example, H2P/Zn(II)P and Zn(II)P/Fe(III)P couples. The fluorescence quenching of Zn(II)P in the Zn(II)P/Fe(III)P hybrid fibers can be ascribed to the intermolecular electron transfer within the fibers. In H2P/Zn(II)P couple, excitation energy transfer from excited Zn1*P to H2P occurred after photoexcitation. Octopusporphyrin with four dialkylglycerophosphocholine groups on both sides of the ring plane (2b) forms spherical unilamellar vesicles. Based on cryomicroscopy, a white line was observed with a diameter of 15 Å in the middle of the membrane which are obviously a porphyrin layer with low molecular packing. The octopusheme (2c) vesicles prepared in a similar manner with 20-fold excess molar coexistence of 1-dodecyl-2-methylimidazole (DMIm) can bind and release oxygen reversibly at 25°C. Moreover, water-soluble octopusporphyrin (3a) produced fluorescent and non-fluorescent monolayer assemblies by anion exchange of the head groups, e.g. 3a with sodium perchlorate showed planar sheets. An exciton calculation is consistent with a two-dimensional arrangement with porphyrin separations of 25.6 and 17.4 Å in the x- and y-directions, respectively. External addition of negatively charged electron acceptors, naphtoquinone sulfonate and anthraquinone sulfonate, led to partial quenching of the fluorescence of the central porphyrin layer. The results have been evaluated using equations derived for this special quenching. © 1998 John Wiley & Sons, Ltd.

Mixed Fluorocarbon/Hydrocarbon Surfactant Vesicles as Carriers of Metalloproteins: Scattering and Magnetic Resonance Experiments

Spontaneously formed mixed vesicles were prepared with n-dodecylbetaine and the ammonium salt of perfluoropolyether carboxylate, and they were loaded with the water-soluble beef heart cytochrome c. The resulting dispersions were characterized with light and neutron scattering patterns, UV -vis spectra, and magnetic resonances (NMR and ESR). Proteins-containing vesicles showed scattering patterns that were interpreted in terms of a decrease of the membrane curvature with a resultant increase of the vesicle size that ranged from 83 to 113 nm, depending on the cytochrome c concentration in the dispersion. The interactions between surfaces of protein and of vesicle membrane, which could be responsible for the decreased curvature, did not alter either the optical properties of the heme unit of the protein (from UV -vis) or the dynamics of the vesicle membrane (from 1 H NMR of betaine and ESR of membrane-soluble doxylstearic acids). Cytochrome c was therefore suggested to be surrounded by a shell of water according to the so-called watershell model. A comparison was made with the behavior of hydrophobic porphyrins introduced in the same vesicle system, as previously reported from the authors.

Determination of Porphyrins by High Performance Liquid Chromatography with Chemiluminescence Detection

The peroxyoxalate chemiluminescence reaction, based on the oxidation of bis (2, 4 dinitrophenyl) oxalate with hydrogen peroxide, was developed as a new detection method for the determination of porphyrins after separation by reverse phase high performance liquid chromatography. To optimize signal to noise ratio, experimental parameters such as design of the detection flow-cell and the effects of pH and organic solvents were investigated. Significant enhancements in detectability, selectivity and linearity were demonstrated for the determination of various hydrophilic and hydrophobic porphyrins when compared to absorbance and fluorescence methods (e.g., the detection limits (S/N = 3) for uroporphyrin and coprophorphyrin were about 40 and 5 fmol, respectively). Application of this method for the sensitive and selective determination of urinary porphyrins was also demonstrated.

Wild type and mutant forms of recombinant horseradish peroxidase C expressed inEscherichia coli

Two horseradish peroxidase C (HRPC) mutants with substitutions in the active center, i.e., Phe41-->His and Phe143-->Glu, were compared to the wild-type recombinant enzyme expressed in Escherichia coli in terms of the enzymatic activity and stability under irradiation. Both mutations caused a significant decrease in activity, but it was still possible to follow the effect of mutations on the key steps of the reaction mechanism. Phe41 can be considered a nonpolar barrier separating histidine residues in the active center and providing a firm noncovalent binding with the highly hydrophobic porphyrin ring. The replacement of Phe41 with the ionizable His residue destabilizes the enzyme. The Phe143-->Glu replacement creates a negative charge at the entrance of the heme-binding pocket, and protects the latter from both donor substrates and free radicals. The radiolytic inactivation of the wild-type and mutant forms of recombinant HRP suggested different binding sites for iodide, 2,2'-bis(3-ethylbenzothiasoline-6-sulfonate (ABTS), guaiacol, and O-phenylene diamine. The study of kinetics and inactivation is in agreement with the direct binding of iodide to the heme porphyrin ring. The results also suggest that the ABTS binding site is less accessible than that for O-phenylene diamine.

Inhibition of the ATPase activity of P-glycoprotein by porphyrin photosensitization of multidrug-resistant cells in vitro.

The effectiveness of photodynamic therapy against P-glycoprotein ATPase activity in multidrug-resistant cells was studied. Chinese hamster ovary AUXB1 (drug-sensitive) and CR1R12 (multidrug-resistant) cell lines were compared with respect to uptake of 14C-polyhematoporphyrin and porphyrin photosensitization. Phototoxicity of Photofrin was similar in both cell lines, and no major differences in uptake or efflux of 14C-polyhematoporphyrin were observed. Porphyrin photosensitization in vitro of CR1R12 cells or isolated plasma membranes from these cells caused inhibition of P-glycoprotein ATPase activity. Application of porphyrin photosensitization at a sublethal level to CR1R12 cells resulted in a small but significant increase in adriamycin-induced cytotoxicity. The hydrophobic "picket-fence" porphyrin, meso-tetrakis-(o-propionamidophenyl)porphyrin, alpha,alpha,alpha,beta-isomer, was more inhibitory toward P-glycoprotein ATPase activity than the two less hydrophobic porphyrins tetraphenylporphine tetrasulfonate and Photofrin.

Photophysical and photobiological activities of a porphyrin peptide fraction derived from haemoglobin

In a previous study, we described the preparation of a porphyrin peptide hydrolysate from haemoglobin, its isolation and its analysis by high performance liquid chromatography (HPLC) and fast atom bombardment (FAB) mass spectrometry. The purpose of the present paper is to test the photosensitizing activity of this fraction. We determined the singlet oxygen quantum yield (Φ Δ) in order to quantify the efficiency of the porphyrin peptide fraction. The quantum yield is about Φ( 1O 2)=0.06. An analysis of the phototoxic effect on tumour cells in culture was performed and compared with haematoporphyrin derivative (HpD), the only photosensitizer in clinical use at present. The phototoxicity of the porphyrin peptide fraction is weaker than that of HpD. However, for a porphyrin dose of 50 μg ml −1, the difference in phototoxicity is low, and in the absence of irradiation porphyrin peptides are less toxic than HpD. These results suggest that porphyrin peptides could be potent photosensitizers; moreover, they are of great interest since they allow the solubilization of hydrophobic porphyrins and could be applied in the future as insoluble photosensitizer carriers.

Structural characterization of mixed Langmuir—Blodgett multilayer assemblies consisting of amphiphilic zinc phthalocyanine and hydrophobic porphyrin by Fourier—transform IR (FTIR) spectroscopy

A new amphiphilic zinc phthalocyanine (AmZnPc), containing long alkyl chains covalently linked to the positively charged macroring with the counter-anion iodide as electron donor, was synthesized. Multilayers of AmZnPc mixed with hydrophobic porphyrin were successfully fabricated on CaF 2 plates. From Fourier-transform IR (FTIR) transmission and polarized transmission spectroscopic studies, it was found that the long alkyl chains of AmZnPc exhibit hexagonal subcell packing and are oriented nearly perpendicular to the AmZnPc macroring. The AmZnPc macroring is oriented almost parallel to the substrate surface, whereas the porphyrin is embedded in the cavity between the long alkyl chains of AmZnPc and is tilted somewhat from the substrate surface.