Pdots, a new type of nanoparticle, bind to mTHPC via their lipid modified surface and exhibit very high FRET efficiency between the core and the sensitizer.

Pdots are a new type of nanoparticle which exhibit strong potential for future applications in biophysics and cell biology. They are composed of organic chromophoric polymers, whose surfaces can be modified with different amphiphilic polymers, such as PEGylated lipids to make them very stable as colloids in water. We demonstrate in this manuscript that the lipid nano-coating around the Pdot can bind very efficiently to amphiphilic molecules, such as photosensitizers e.g. meso-tetrahydroxyphenylchlorin (mTHPC). As a result the sensitizer is brought into very close contact with the cores of the Pdots, and resonance energy transfer from the core to the sensitizer is very efficient; in some cases it is close to 1. We show the spectroscopic properties of two types of Pdots; their sizes, which are in the 13-47 nm range, depend on the kind of polymer and the length of the PEGylated lipid chains that wrap it. We measured the efficiency of FRET by investigating the decrease in donor intensity or its lifetime upon binding with mTHPC. We also show the relative yields of singlet oxygen that are obtained via two pathways: by exciting the Pdots which transfer the energy to the attached sensitizer, or by exciting the sensitizer directly. This methodology could be used to enhance the use of a photosensitizer by employing both pathways in parallel.

Size and ability do matter! Influence of acidity and pore size on the synthesis of hindered halogenated meso-phenyl porphyrins catalysed by porous solid oxides.

The rationalisation of the influence of acidity and pore size of several solid oxides so that they selectively act as supports for preparation of encapsulated porphyrin hybrid materials or as catalysts for synthesis of porphyrins in solution is discussed. Encapsulated porphyrin yields are dependent on both the acidity and the material pore size, Al-MCM-41 being the best fitting solid, with Lewis acidity of 120 µmol Py per g and a pore size 30 Å. On the other hand, when the goal is the synthesis of hindered mesoarylporphyrins in solution, the best solid porous catalyst is NaY, with Lewis acidity of 510 µmol Py per g and a pore size 14 Å. This method provides an appealing efficient, reusable and scalable catalyst alternative for one-pot synthesis of meso-arylporphyrins in high yields.

Photodynamic mechanisms induced by a combination of hypericin and a chlorin based-photosensitizer in head and neck squamous cell carcinoma cells.

The aim of this study was to elucidate photodynamic therapy (PDT) effects mediated by hypericin and a liposomal meso-tetrahydroxyphenyl chlorin (mTHPC) derivative, with focus on their 1:1 mixture, on head and neck squamous cell carcinoma cell lines. Absorption, excitation and photobleaching were monitored using fluorescence spectrometry, showing the same spectral patterns for the mixture as measured for single photosensitizers. In the mixture mTHPC showed a prolonged photo-stability. Singlet oxygen yield for light-activated mTHPC was Φ(Δ) = 0.66, for hypericin Φ(Δ) = 0.25 and for the mixture Φ(Δ) = ~0.4. A linear increase of singlet oxygen yield for mTHPC and the mixture was found, whereas hypericin achieved saturation after 35 min. Reactive oxygen species fluorescence was only visible after hypericin and mixture-induced PDT. Cell viability was also more affected with these two treatment options under the selected conditions. Examination of death pathways showed that hypericin-mediated cell death was apoptotic, with mTHPC necrotic and the 1:1 mixture showed features of both. Changes in gene expression after PDT indicated strong up-regulation of selected heat-shock proteins. The application of photosensitizer mixtures with the features of reduced dark toxicity and combined apoptotic and necrotic cell death may be beneficial in clinical PDT. This will be the focus of our future investigations.

Improvement of meta-tetra(hydroxyphenyl)chlorin-like photosensitizer selectivity with folate-based targeted delivery. synthesis and in vivo delivery studies.

The cell membrane folate receptor (FR) is a molecular target for tumor-selective drug delivery, including delivery of photosensitizers for anticancer photodynamic therapy (PDT). Tumor selectivity of meta-tetra(hydroxyphenyl)chlorin ( m-THPC), a photosensitizer used in PDT clinical trials, demonstrates a low tumor-to-normal epithelial uptake ratio. We report on the synthesis and on the photophysical properties of a m-THPC-like photosensitizer 1 conjugated to folic acid (compound 8). A comparative study of the accumulation of photosensitizers 1 and 8 is described. Nude mice were xenografted with FR-alpha-positive KB or HT-29 cells lacking FR-alpha as a negative control. Using optical fiber fluorimetry, we demonstrated that conjugate 8 exhibited enhanced accumulation in KB tumors compared to 1 4 h after injection. No significant difference between KB and HT-29 tumors was observed in case of compound 1. Tumor-to-normal tissue ratio exhibited a very interesting selectivity for conjugate 8 (5:1) in KB tumors 4 h postinjection.

Temoporfin-loaded invasomes: development, characterization and in vitro skin penetration studies.

Temoporfin (mTHPC) is a highly hydrophobic second generation photosensitizer with low percutaneous penetration. In order to enhance its percutaneous penetration it was necessary to develop a mTHPC-loaded drug carrier system for enhanced skin delivery. mTHPC-loaded invasomes were developed, characterized and investigated for the in vitro percutaneous penetration of mTHPC into abdominal human skin using Franz diffusion cells. mTHPC-loaded invasomes were prepared using non-hydrogenated soybean lecithin (10% w/v), ethanol (3.3% w/v) and a mixture of terpenes (0.5 and 1% w/v). The invasomes obtained were of a sufficiently small particle size (<150 nm) and polydispersity index (<0.3). The particle size of invasomes increased following an increase in the amount of terpenes in the invasomes. All invasomes possessed a negative surface charge. The vesicles appeared to be unilamellar and oligolamellar, spherical and oval in shape. An interesting phenomenon was the finding that with increasing the amount of terpenes, the number of deformed vesicles in the dispersion increased. In vitro skin penetration data revealed that the invasome dispersion with 1% of the mixture of terpenes showed a significantly enhanced deposition (p<0.05) of the drug in the SC compared to liposomes without terpenes and the ethanolic solution.

[Synthesis of lipophilic tetraphenylporphyrins to design lipid-porphyrin ensembles].

Synthesis of symmetrical meso-arylsubstituted porphyrins with long chain hydrophobic substituents in the phenyl rings was achieved. The lipoporphyrins can be used to design supramolecular lipid ensembles of nanometer size.

Contrast enhanced MRI-guided photodynamic therapy for site-specific cancer treatment.

Photodynamic therapy (PDT) is a minimally invasive and effective approach for cancer treatment. It is potentially useful for treating tumors that are not accessible to surgery, radiation, or destructive ablations, and are resistant to chemotherapy. Efficacious treatment of interstitial tumors with PDT requires efficient delivery of photosensitizers and accurate location of tumor tissues for effective light irradiations. In this study we performed contrast-enhanced (CE) MRI-guided PDT with a bifunctional polymer conjugate containing both a magnetic resonance imaging (MRI) contrast agent and a photosensitizer, poly(L-glutamic acid) (PGA)-(Gd-DO3A)-mesochlorin e(6) (Mce(6)). The efficacy of the bifunctional conjugate in cancer CE-MRI and cancer treatment was evaluated in athymic nude mice bearing MDA-MB-231 human breast carcinoma xenografts, with PGA-(Gd-DO3A) used as a control. The polymer conjugates preferentially accumulated in the solid tumor due to the hyperpermeability of the tumor vasculature, resulting in significant tumor enhancement for accurate tumor detection and localization by MRI. Significant therapeutic response was observed for PDT with the bifunctional conjugate as compared to the control. CE-MRI-guided PDT with the bifunctional conjugate is effective for tumor detection and minimally invasive cancer treatment.

Human serum albumin bearing covalently attached iron(II) porphyrins as O2-coordination sites.

Tetrakis{(alpha,alpha,alpha,alpha-o-pivalamido)phenyl}porphinatoiron(II) with a bifunctional tail possessing an axially coordinated imidazolyl group and a protein attachable succinimidyl(glutamyl) group (FeP-GluSu) has been synthesized. It can efficiently react with the lysine residues of recombinant human serum albumin (rHSA), giving a new albumin-heme conjugate [rHSA(FeP-Glu)]. MALDI-TOFMS showed a distinct molecular ion peak at m/z 70 643, which indicates that three FeP-Glu molecules were covalently linked to the rHSA scaffold. The binding number of FeP-Glu is approximately three (mol/mol) and independent of the mixing ratio. The CD spectrum and Native PAGE revealed that the albumin structure remained unaltered after the covalent bonding of the hemes. This rHSA(FeP-Glu) conjugate can bind and release O2 reversibly under physiological conditions (pH 7.3, 37 degrees C) in the same manner as hemoglobin and myoglobin. The O2-adduct complex had a remarkably long lifetime (tau(1/2): 5 h). The O2-binding affinity [P(1/2)O2: 27 Torr] was identical to that of human red cells. Laser flash photolysis experiments gave the O2- and CO-association rate constants and suggested that there are two different geometries of the imidazole binding to the central ion.

The embedding of meta-tetra(hydroxyphenyl)-chlorin into silica nanoparticle platforms for photodynamic therapy and their singlet oxygen production and pH-dependent optical properties.

This study relates to nanoparticle (NP) platforms that attach to tumor cells externally and only deliver singlet oxygen for photodynamic therapy (PDT) while conserving the embedded photosensitizers (PS). As a model, we demonstrate the successful embedding of the PS meta-tetra(hydroxyphenyl)-chlorin (m-THPC) in NP that are based on a sol-gel silica matrix and also show its positive effect on the singlet oxygen production. The embedding of m-THPC inside silica NP is accomplished by a modified Stöber sol-gel process, in which (3-aminopropyl)-triethoxysilane is introduced during the reaction. Singlet oxygen delivery by the targetable photodynamic NP exceeds that from free PS molecules. In the physiological pH range, there is no significant pH-induced decrease in the fluorescence of m-THPC embedded in silica NP, which might otherwise affect the efficiency of PDT.

Syntheses and preliminary biological studies of four meso-Tetra[(nido-carboranylmethyl)phenyl]porphyrins.

Two meso-tetra[(nido-carboranylmethyl)phenyl]porphyrins (para- and meta-regioisomers) and their corresponding Zn(II) complexes have been synthesized with the aim of studying the effect of carborane distribution and metalation on the biological properties of this series of compounds. In vitro cell toxicity, uptake/efflux, and subcellular localization using rat 9L, mouse B16 and/or human U-373MG cells were evaluated. All four amphiphilic porphyrins display very low cytotoxicities and time- and concentration-dependent uptake by cells, which is influenced by serum proteins. Preliminary subcellular localization studies suggest that one of these compounds localizes in close proximity to the cell nucleus. All four nido-carboranylporphyrins show promise as boron-carriers for the boron neutron capture therapy of cancers, particularly the metal-free nido-carboranylporphyrins 5 and 12, which are able to deliver higher amount of boron to cells in vitro than the corresponding zinc complexes.

Design and synthesis of porphyrins bearing rigid hydrogen bonding motifs: highly versatile building blocks for self-assembly of polymers and discrete arrays.

Two aldehydes, 2,6-diacetamido-4-formylpyridine (7) and 1-butyl-6-formyluracil (11), are used to synthesize five pyridyl and four uracyl meso-subsituted porphyrins. With these complementary porphyrin building blocks, it is possible to build various types of multi-porphyrin supramolecules with different spatial relationships in predefined geometries. The formation and properties of self-complementary dimers and a closed tetrameric square are presented as a basis of comparison to the latter system in the solid state. An X-ray structure of 5,10-bis(4-tert-butylphenyl)-15,20-bis(3,5-diacetamido-4-pyridyl)porphyrin confirms its molecular structure and reveals a hydrogen-bonded supramolecular organization mediated by water molecules.

A series of meso-tris (N-methyl-pyridiniumyl)-(4-alkylamidophenyl) porphyrins: synthesis, interaction with DNA and antibacterial activity.

A series of meso-5,10,15-tris(N-methyl-4-pyridiniumyl)-20-(4-alkylamidophen yl) porphyrins were synthesized by derivatizing the amino group on the phenyl ring with the following hydrophobic groups: -C(O)C7F15, -C(O)CH=CH2, C(O)CH3, -C(O)C7H15, and -C(O)C15H31. The cationic tris-pyridiumyl porphyrin core serves as a DNA binding motif and a photosensitizer to photomodify DNA molecules. The changes of the UV-Vis absorption spectra during the titration of these porphyrins with calf thymus DNA revealed a large bathochromic shift (up to 14 nm) and a hypochromicity (up to 55%) of the porphyrins Soret bands, usually considered as proof of porphyrin intercalation into DNA. Association constants (K) calculated according to the McGhee and von Hippel model, were in the range of 10(6)-10(7) M(-1). An increase in hydrophobicity of the substituents at the 20-meso-position produced higher binding affinity. These porphyrins caused photomodification of the supercoiled plasmid DNA when a green laser beam at 532 nm was applied. Those with higher surface activity acted more efficiently as DNA photomodifiers. The porphyrin with a perfluorinated alkyl chain (-COC7F15) at the meso-20-position inhibited the growth of gram-positive bacteria (S. aureus, or S. epidermidis). Other porphyrins exhibited moderate activity against both gram-negative and gram-positive organisms.

Inhibition of protohaem ferro-lyase by N-substituted porphyrins. Structural requirements for the inhibitory effect.

N-Methyl mesoporphyrin was a powerful inhibitor of protohaem ferro-lyase in vitro, whereas N-ethyl mesoporphyrin and N-methyl coproporphyrin were not and neither was the newly described green pigment produced by giving rats ethylene. This suggests that the size of the substituent at a pyrrole nitrogen and also the number of carboxylic acid side chains of the substituted porphyrin are important for the inhibitory effect. Evidence that N-methyl mesoporphyrin inhibited the enzyme, whereas the ethylene-derived pigment did not, was also obtained in vivo.