Vladimir Zorin

Interaction of Liposomal Formulations of Meta-tetra(hydroxyphenyl)chlorin (Temoporfin) with Serum Proteins: Protein Binding and Liposome Destruction

mTHPC is a non polar photosensitizer used in photodynamic therapy. To improve its solubility and pharmacokinetic properties, liposomes were proposed as drug carriers. Binding of liposomal mTHPC to serum proteins and stability of drug carriers in serum are of major importance for PDT efficacy; however, neither was reported before. We studied drug binding to human serum proteins using size‐exclusion chromatography. Liposomes destruction in human serum was measured by nanoparticle tracking analysis (NTA). Inclusion of mTHPC into conventional (Foslip®) and PEGylated (Fospeg®) liposomes does not affect equilibrium serum protein binding compared with solvent‐based mTHPC. At short incubation times the redistribution of mTHPC from Foslip® and Fospeg® proceeds by both drug release and liposomes destruction. At longer incubation times, the drug redistributes only by release. The release of mTHPC from PEGylated vesicles is delayed compared with conventional liposomes, alongside with greatly decreased liposomes destruction. Thus, for long‐circulation times the pharmacokinetic behavior of Fospeg® could be influenced by a combination of protein‐ and liposome‐bound drug. The study highlights the modes of interaction of photosensitizer‐loaded nanovesicles in serum to predict optimal drug delivery and behavior in vivo in preclinical models, as well as the novel application of NTA to assess the destruction of liposomes.

Photodynamic therapy with intratumoral administration of Lipid-Based mTHPC in a model of breast cancer recurrence

Generalized skin sensitization is a main drawback of photodynamic therapy with systemic administration of photosensitizers. We have evaluated the potential use of an intratumoral injection of a liposomal formulation of mTHPC (Foslip) in a mouse model of local recurrence of breast cancer.

Photodynamic therapy with conventional and PEGylated liposomal formulations of mTHPC (temoporfin): comparison of treatment efficacy and distribution characteristics in vivo

A major challenge in the application of a nanoparticle-based drug delivery system for anticancer agents is the knowledge of the critical properties that influence their in vivo behavior and the therapeutic performance of the drug. The effect of a liposomal formulation, as an example of a widely-used delivery system, on all aspects of the drug delivery process, including the drug’s behavior in blood and in the tumor, has to be considered when optimizing treatment with liposomal drugs, but that is rarely done. This article presents a comparison of conventional (Foslip®) and polyethylene glycosylated (Fospeg®) liposomal formulations of temoporfin (meta-tetra[hydroxyphenyl]chlorin) in tumor-grafted mice, with a set of comparison parameters not reported before in one model. Foslip® and Fospeg® pharmacokinetics, drug release, liposome stability, tumor uptake, and intratumoral distribution are evaluated, and their influence on the efficacy of the photodynamic treatment at different light–drug intervals is discussed. The use of whole-tumor multiphoton fluorescence macroscopy imaging is reported for visualization of the in vivo intratumoral distribution of the photosensitizer. The combination of enhanced permeability and retention-based tumor accumulation, stability in the circulation, and release properties leads to a higher efficacy of the treatment with Fospeg® compared to Foslip®. A significant advantage of Fospeg® lies in a major decrease in the light–drug interval, while preserving treatment efficacy.

Unusual Photoinduced Response of mTHPC Liposomal Formulation (Foslip)

Liposomal formulations of meso‐tetra(hydroxyphenyl)chlorin (mTHPC) have already been proposed with the aim to optimize photodynamic therapy. Spectral modifications of these compounds upon irradiation have not yet been investigated. The objective of this study was to evaluate photobleaching properties of mTHPC encapsulated into dipalmitoylphosphatidylcholine (DPPC) liposomes, Foslip. Fluorescence measurements in DPPC liposomes with different DPPC:mTHPC ratios demonstrated a dramatic decrease in fluorescence anisotropy with increasing local mTHPC concentration, thus suggesting strong interactions between mTHPC molecules in lipid bulk medium. Exposure of Foslip suspensions to small light doses (<50 mJ/cm2) resulted in a substantial drop in fluorescence, which, however, was restored after addition to the sample of a non‐ionic surfactant Triton X‐100. We attributed this behavior to photoinduced fluorescence quenching. This effect depended strongly on the molar DPPC:mTHPC ratio and was revealed only for high local mTHPC concentrations. The results were interpreted supposing energy migration between closely located mTHPC molecules with its subsequent dissipation by the molecules of photoproduct acting as excitation energy traps. We further assessed the effect of photoinduced quenching in plasma protein solution. Relatively slow kinetics of photoinduced Foslip response during incubation in the presence of proteins was attributed to mTHPC redistribution from liposomal formulations to proteins. Therefore, changes in mTHPC distribution pattern in biological systems would be consistent with changes in photoinduced quenching and would provide valuable information on mTHPC interactions with a biological environment.

Redistribution of Foscan® from plasma proteins to model membranes

Photodynamic therapy is a comparatively novel modality of tumours treatment that includes simultaneous action of photosensitizers, light and oxygen. Photosensitizer redistribution between plasma proteins and biomembranes define photosensitizers interaction with cells, their intracellular localization and kinetics of sensitizers accumulation in the tumour. Present study investigates the kinetics of Foscan release from plasma proteins to model membranes using fluorescence resonance energy transfer (FRET) from label, covalently bound to protein, to sensitizer. We have demonstrated very slow kinetics of Foscan release from protein complexes with rate constants of (1.7 +/- 0.1) x 10(-3) s(-1) for albumin and (1.6 +/- 0.3) x 10(-4) s(-1) for high-density lipoproteins (HDL). Foscan redistributes by both collision and diffusion-mediated transfer from complexes with HDL, with bimolecular rate constant k(out) = (8.8 +/- 1.4) x 10(-2) M(-1) s(-1). Thermodynamic considerations proposed that sensitizer release from HDL into the aqueous medium is unfavourable and collision mechanism appeared to be a preferred mode of transfer in biological environment. Slow rates of Foscan redistribution from plasma proteins should be considered while planning dosimetry protocol of Foscan-PDT.

Inclusion complexation with β-cyclodextrin derivatives alters photodynamic activity and biodistribution of meta-tetra(hydroxyphenyl)chlorin

Application of meta-tetra(hydroxyphenyl)chorin (mTHPC) one of the most effective photosensitizer (PS) in photodynamic therapy of solid tumors encounters several complications resulting from its insolubility in aqueous medium. To improve its solubility and pharmacokinetic properties, two modified β-cyclodextrins (β-CDs) methyl-β-cyclodextrin (M-β-CD) and 2-hydroxypropyl-β-cyclodextrin (Hp-β-CD) were proposed. The aim of this work was to evaluate the effect of β-CDs on mTHPC behavior at various stages of its distribution in vitro and in vivo. For this purpose, we have studied the influence of the β-CDs on mTHPC binding to the serum proteins, its accumulation, distribution and photodynamic efficiency in HT29 cells. In addition, the processes of mTHPC biodistribution in HT29 tumor bearing mice after intravenous injection of PS alone or with the β-CDs were compared. Interaction of mTHPC with studied β-CDs leads to the formation of inclusion complexes that completely abolishes its aggregation after introduction into serum. It was demonstrated that the β-CDs have a concentration-dependent effect on the process of mTHPC distribution in blood serum. At high concentrations, β-CDs can form inclusion complexes with mTHPC in the blood that can have a significant impact on PS distribution out of the vascular system in solid tissues. Besides, the β-CDs increase diffusion movement of mTHPC molecules that can significantly accelerate the delivery of PS to the targets cells and tissues. In vivo study confirms the fact that the use of β-CDs allows to modify mTHPC distribution processes in tumor bearing animals that is reflected in the decreased level of PS accumulation in skin and muscles, as well as in the increased PS accumulation in tumor. Further studies are underway to verify the optimal protocols of mTHPC/β-CD formulation for photodynamic therapy.

Distribution of chlorin-e6 derivatives in biological systems: investigation of pH-effect

The importance of pH as a factor relating to porphyrin binding and distribution in different models was examined using for sensitizers, derivatives of chlorin e6 differing in their lateral substitutors. On the basis of experimental data obtained with the use of several different methods (spectral fluorescence, fluorescence quenching, ultrafiltration) we have investigated the influence of pH on pigments affinity to serum proteins (including serum albumin, high- and low-density lipoproteins), porphyrin intramembrane distribution pattern and its mobility across membrane and when membrane-membrane exchange of porphyrin molecules occurs. The affinity of chlorin e6 to serum proteins is very sensitive to the protonation of side carboxylic groups. A fraction of the dye that is bound to serum albumin decreases with decreasing pH. In contrast to serum proteins, there is a significant increase of chlorin e6- binding capacity to the model membrane when pH shifts from 7.0 to 5.5. In acid medium, deeper penetration of chlorin molecules into lipid is observed. As compared with neutral medium, more pigment molecules are localized in inner monolayer bulk. The pH dependence of the rate of chlorin e6 molecules exchange from outer lipid layer of donor vesicles to acceptor vesicles is markedly different from that of transmembrane movement. The rate of the latter, slower process increases greatly in acid medium, whereas the rate of intervesicle exchange decreases.

Drug delivery to solid tumors: the predictive value of the multicellular tumor spheroid model for nanomedicine screening

The increasing number of publications on the subject shows that nanomedicine is an attractive field for investigations aiming to considerably improve anticancer chemotherapy. Based on selective tumor targeting while sparing healthy tissue, carrier-mediated drug delivery has been expected to provide significant benefits to patients. However, despite reduced systemic toxicity, most nanodrugs approved for clinical use have been less effective than previously anticipated. The gap between experimental results and clinical outcomes demonstrates the necessity to perform comprehensive drug screening by using powerful preclinical models. In this context, in vitro three-dimensional models can provide key information on drug behavior inside the tumor tissue. The multicellular tumor spheroid (MCTS) model closely mimics a small avascular tumor with the presence of proliferative cells surrounding quiescent cells and a necrotic core. Oxygen, pH and nutrient gradients are similar to those of solid tumor. Furthermore, extracellular matrix (ECM) components and stromal cells can be embedded in the most sophisticated spheroid design. All these elements together with the physicochemical properties of nanoparticles (NPs) play a key role in drug transport, and therefore, the MCTS model is appropriate to assess the ability of NP to penetrate the tumor tissue. This review presents recent developments in MCTS models for a better comprehension of the interactions between NPs and tumor components that affect tumor drug delivery. MCTS is particularly suitable for the high-throughput screening of new nanodrugs.

The alteration of temoporfin distribution in multicellular tumor spheroids by β-cyclodextrins

To be effective anticancer drugs must penetrate tissue efficiently, reaching all target population of cancer cells in a concentration sufficient to exert a therapeutic effect. This study aimed to investigate the ability of methyl-β-cyclodextrin (Me-β-CD) and 2-hydroxypropyl-β-cyclodextrin (Hp-β-CD) to alter the penetration and diffusion of temoporfin (mTHPC) in HT29 multicellular tumor spheroids. mTHPC had а nonhomogenous distribution only on the periphery of spheroids. The presence of β-CDs significantly altered the distribution of mTHPC consisting in the increase of both the depth of photosensitizer penetration and accumulation in HT29 spheroids. We suggest that this improvement is related to the nanoshuttle mechanism of β-CD action, when β-CDs facilitate mTHPC transportation to the cells in the inner layers of spheroids. As a result of mTHPC distribution improvement, β-CDs enhance mTHPC photosensitizing activity towards HT29 multicellular tumor spheroids. The observed effects strongly depend on the type of β-CD. Thus, varying the type of β-CD we can finely tune the possibility of using mTHPC for diagnostic (delimitation of tumor margins) or therapeutic purposes.

Factors affecting the selectivity of nanoparticle-based photoinduced damage in free and xenografted chorioallantoïc membrane model.

Abstract Background: Photodynamic therapy (PDT) is a minimally invasive treatment modality for selective destruction of tumours. Critical anatomical structures, like blood vessels in close proximity to the tumour, could be harmed during PDT. Purpose: This study aims to discriminate the photoinduced response of normal and cancerous tissues to photodamage induced by liposomal formulations of meta-tetra(hydroxyphenyl)chlorin (mTHPC). Methods: Normal vascular and cancerous tissues were represented, respectively, by free and xenografted in vivo model of chick chorioallantoïc membrane (CAM). Eggs received an intravenous administration of plain (Foslip®) or stabilised formulations (Fospeg®). Drug release and liposome destruction were, respectively, determined by photoinduced quenching and nanoparticle tracking analysis. PDT was performed at different drug-light intervals (DLI) with further assessment of photothrombic activity, tumoritropism and photoinduced necrosis. Results: Compared to Foslip®, Fospeg® demonstrated significantly higher stability, slower drug release, better tumoricidal effect and lower damage to the normal vasculature at already 1 h DLI. Discussion: This work suggests that nanoparticle-based PDT selectivity could be optimised by analyzing the photoinduced damage of healthy and tumour tissues. Conclusion: In fine, Fospeg® appeared to be the ideal candidate in clinical context due to its potential to destroy tumours and reduce vascular damage to normal tissues at short DLI.