Athena Kasselouri

Glycodendrimeric phenylporphyrins as new candidates for retinoblastoma PDT: Blood carriers and photodynamic activity in cells

Photodynamic therapy (PDT) has recently been proposed as a possible indication in the conservative treatment of hereditary retinoblastoma. In order to create photosensitizers with enhanced targeting ability toward retinoblastoma cells, meso-tetraphenylporphyrins bearing one glycodendrimeric moiety have been synthesized. The binding properties to plasma proteins and photodynamic activity of two monodendrimeric porphyrins bearing three mannose units via monoethylene glycol (1) or diethylene glycol (2) linkers have been compared to that of the non-dendrimeric tri-substituted derivative [TPP(p-Deg-O-α-ManOH)(3)]. The dendrimeric structure was found to highly increase the binding affinity to plasma proteins and to modify to some extent plasma distribution. HDL and to a lesser extent LDL have been shown to be the main carriers of dendrimeric and non-dendrimeric compounds. The phototoxicity observed for the two glycodendrimers (1) and (2) (LD(50)=0.5 μM) in Y79 cells is of the same order of magnitude that for TPP(p-Deg-O-α-ManOH)(3) (LD(50)=0.7 μM), with a similar cellular uptake level for (1) and a lower for (2). A serum content increase from 2% to 20% (v/v) in the incubation medium was found to inhibit both cellular uptake and photoactivity of dendrimeric derivatives, whereas those of TPP(p-Deg-O-α-ManOH)(3) remained little affected. Specificities of glycodendrimeric porphyrins, combining a lower cellular uptake together with a higher affinity toward plasma proteins, make these derivatives possible candidates for a vascular targeting PDT.

Speciation of New Tri- and Tetra-Glucoconjugated Tetrapyrrolic Macrocycles (Porphyrins and Chlorins): An Electronic Molecular Spectroscopy Study

In this work, we study the physicochemical properties of some newly developed glycoconjugated photosensitizers that can be used in photodynamic therapy (PDT) of cancers: meso-tri-and tetra-(meta-O-β-d-glucosyloxyphenyl)porphyrins and meso-, tri-, and tetra-(meta-O-β-d-glucosyloxyphenyl)chlorins. Their properties are compared to the non-glycosylated hydroxylated parent compounds meso-tetra-(meta-hydroxyphenyl) porphyrin and meso-tetra-(meta-hydroxyphenyl)chlorin. It was found that at the ground state, all porphyrins present, independent of the substitution, have the same mean ionization constant (pKa = 2.7), corresponding to two indistiguishable steps of protonation of tetrapyrrolic nitrogens. On the other hand, in the case of chlorins, one proton process can be observed and the corresponding nitrogen exhibits a slightly superior basicity (pKa = 3.0) with respect to porphyrins. Hydroxylated compounds present a second transition at high pH corresponding to the ionization of phenol groups (pKa = 10.5). Consequently, all photosensitizers are not charged at physiological pH (≈7.4), and so the ionization process does not influence their activity in biological media. Ionization induces very important variations in photosensitizer absorption and emission spectra. For example, absorption in the red region (band V), one of the most important characteristics of a good photosensitizer, is only important for diprotonated porphyrins and neutral chlorins. As far as fluorescence emission is concerned, neutral chlorins are almost six times more fluorescent than the corresponding neutral porphyrins (Φchlorin/Φporphyrin ≈ 6). It should be emphasized that the spectra modifications induced by pH variations can find interesting applications in the optimization of visible and fluorescence detection in high-performance liquid chromatography (HPLC) as well as in the development of direct, rapid fluorimetric analytical methods.

Enhancement of 5,10,15,20-Tetra(m-Hydroxyphenyl)chlorin Fluorescence Emission by Inclusion in Natural and Modified Cyclodextrins

Fluorescence properties of 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (m-THPC), a sensitizer used in photodynamic therapy (PDT), were studied in the presence of three native cyclodextrins (CDs) -α, -β, and γ-CD—and two modified cyclodextrins—heptakis (2,6-di-O-methyl) β-CD (Me-β-CD) and heptakis (2-O-hydroxy-propyl) β-CD (HP-β-CD). The CDs studied have been shown to undergo the formation of an inclusion complex with m-THPC, leading to a large enhancement (up to 300 times) of m-THPC fluorescence intensity depending on the CD used. Stoichiometry and association constants have been determined on the basis of the variation of fluorescence intensity. A 1:2 stoichiometry has been found for m-THPC complexes with γ-CD, Me-β-CD, and HP-β-CD. Association constants as high as K = 9.5 × 108 M−2 in the case of m-THPC/2Me-β-CD and K = 2.7 × 109 M−2 in the case of m-THPC/ 2HP-β-CD complex were determined, whereas a lower value (K = 2.4 × 104 M−2) was found in the case of m-THPC/2γ-CD. Because of the highest fluorescence enhancement (a factor of about 300) observed with Me-β-CD, this compound would be the most suitable to improve the detection of m-THPC in further pharmacokinetics and biodistribution studies.

Plasma distribution of tetraphenylporphyrin derivatives relevant for Photodynamic Therapy: importance and limits of hydrophobicity.

In the course of a Photodynamic Therapy (PDT) protocol, disaggregation of the sensitizer upon binding to plasma proteins and lipoproteins is one of the first steps following intravenous administration. This step governs its subsequent biodistribution and has even been evoked as possibly orientating mechanism of tumor destruction. It is currently admitted as being mainly dependent on sensitizers hydrophobicity. In this context, as far as glycoconjugation of meso-tetraphenylporphyrin (TPP) macrocycle, a promising strategy to improve targeting of retinoblastoma cells confers to the sensitizer an amphiphilic character, we have studied the effect of this strategy on binding to plasma proteins and lipoproteins. With the exception of the majoritary protein binding (more than 80%) of more hydrophilic para-tetraglycoconjugated derivatives, high density lipoproteins (HDL) appear as main plasma carriers of the other amphiphilic glycoconjugated photosensitizers. This HDL-binding is a combined result of binding affinities (logKa ranging from 4.90 to 8.77 depending on the carrier and the TPP derivative considered) and relative plasma concentrations of the different carriers. Evaluation of binding affinities shows that if hydrophobicity can account for LDL- and HDL-affinities, it is not the case for albumin-affinity. Molecular docking simulations show that, if interactions are mainly of hydrophobic nature, polar interactions such as hydrogen bonds are also involved. This combination of interaction modalities should account for the absence of clear relationship between albumin-affinity and hydrophobicity. Taken together, our findings clarify the importance, but also the limits, of hydrophobicitys role in structure-plasma distribution relationship.

A direct sensitized fluorimetric determination of 5,10,15,20-tetra(m-hydroxyphenyl)chlorin [m-THPC (Foscan)] in human plasma using a cyclodextrin inclusion complex.

The 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (m-THPC) (Foscan) is a photosensitizer used in the photodynamic therapy (PDT) of cancers which is currently under clinical trial. The formation of a m-THPC inclusion complex with dimethyl-beta-cyclodextrin (Me-beta-CD) in solution was demonstrated on the basis of circular dichroism experiments. A 1:2 complex stoichiometry was found and an inclusion constant beta 2 = 2.8(+/- 0.4) x 10(10) M-2 was determined. The formation of such a complex was shown to enhance the m-THPC fluorescence intensity. It could be exploited to improve the sensitivity of the direct m-THPC detection in human plasma. Optimization of the operating conditions shows that the best results were obtained by the addition of 100 microL of a concentrated Me-beta-CD solution (3.2 x 10(-2) M) to 1 mL plasma samples. Compared to the standard conditions, a 90% increase in sensitivity was obtained. The proposed analytical method which showed a linear response function [0-300 ng mL-1 (440 pM)] and a low limit of detection [1.5 ng mL-1 (2 pM) (S/N = 3)] appears, especially due to the absence of metabolism, a simple and specific method suitable for pharmacokinetics studies in patients.

Artificial plasma membrane models based on lipidomic profiling.

Phospholipid monolayers are often described as membrane models for analyzing drug-lipid interactions. In many works, a single phosphatidylcholine is chosen, sometimes with one or two additional components. Drug penetration is studied at 30mN/m, a surface pressure considered as corresponding to the pressure in bilayers, independently of the density of lipid molecular packing. In this work, we have extracted, identified, and quantified the major lipids constituting the lipidome of plasma and mitochondrial membranes of retinoblastoma (Y79) and retinal pigment epithelium cells (ARPE-19), using liquid chromatography coupled to high-resolution mass spectrometry (LC-MS/MS). The results obtained from this lipidomic analysis were used in an attempt to build an artificial lipid monolayer with a composition mimicking that of the plasma membrane of Y79 cells, better than a single phospholipid. The variety and number of lipid classes and species in cell extracts monolayers exceeding by far those of the phospholipids chosen to mimic them, the π-A isotherms of model monolayers differed from those of lipid extracts in shape and apparent packing density. We propose a model monolayer based on the most abundant species identified in the extracts, with a surface compressional modulus at 30mN/m close to the one of the lipid extracts.

Design and Synthesis of Tubulin and Histone Deacetylase Inhibitor Based on iso-Combretastatin A-4

Designing multitarget drugs have raised considerable interest due to their advantages in the treatment of complex diseases such as cancer. Their design constitutes a challenge in antitumor drug discovery. The present study reports a dual inhibition of tubulin polymerization and HDAC activity. On the basis of 1,1-diarylethylenes ( isoCA-4) and belinostat, a series of hybrid molecules was successfully designed and synthesized. In particular compounds, 5f and 5h were proven to be potent inhibitors of both tubulin polymerization and HDAC8 leading to excellent antiproliferative activity.

Retinoblastoma membrane models and their interactions with porphyrin photosensitisers: An infrared microspectroscopy study

Fourier Transform Infrared (FTIR) microspectroscopy was used to highlight the interactions between two photosensitisers (PS) of different geometries, TPPmOH4 and a glycoconjugated analogous, TPPDegMan, and lipid bilayers modelling retinoblastoma cell membranes. Retinoblastoma is a rare disease occurring in young infants, for whom conservative treatments may present harmful side-effects. Photodynamic therapy (PDT) is expected to induce less side-effects, as the photosensitiser is only activated when the tumour is illuminated. Since efficiency of the treatment relies on photosensitiser penetration in cancer cells, bilayers with three lipid compositions - pure SOPC, SOPC/SOPE/SOPS/Chol (56:23:11:10) and SOPC/SOPE/SOPS/Chol/CL (42:32:9:8:6) - were used as plasma and mitochondria model membranes. FTIR spectra showed that the interaction of the PSs with the lipid bilayers impacted the lipid organization of the latter, causing significant spectral variations. Both studied photosensitisers inserted at the level of lipid hydrophobic chains, increasing chain fluidity and disorder. This was confirmed by surface pressure measurements. Photosensitisers - TPPmOH4 more than TPPDegMan - also interacted with the polar region of the bilayer, forming hydrogen bonds with phosphate groups that induced major shifts of phosphate absorption bands. This difference in PS interaction with moieties in the polar region was more pronounced with the models with complex lipid composition.