The present study is aimed at the development of drug-in-cyclodextrin-in-liposome (DCL) nanoconstruct by coupling two independent delivery systems: cyclodextrin/mTHPC inclusion complexes and liposomal vesicles to improve the transport of mTHPC to the target tissue and to strengthen its intra-tissue accumulation in the tumor. Liposomes offer an excellent opportunity to achieve selective drug, targeting what is expected to prevent local irritation and reduce drug toxicity. Сyclodextrins (CDs) have been utilized as independent carriers for improvement of pharmaceutical properties such as solubility, stability, and bioavailability of various drug molecules, including mTHPC. Therefore, we assumed that encapsulation of CD-complexed drug into liposomes might increase drug loading capacity, entrapment efficiency, may restrain the dissociation of drug-CD complexes and prolong its systemic circulation. DCL nanoparticles have been prepared with various compositions to optimize the structure aiming to alter more favorably the distribution of temoporfin in tumor tissue. To enhance the encapsulation efficiency, double loaded DCLs, which include mTHPC in lipid bilayer along with (CD-mTHPC) inclusion complexes in the inner aqueous lumen, were prepared. It was demonstrated that DCLs possessed higher serum stability compared with conventional mTHPC liposomes (Foslip). In fine, we showed that the presence of serum in the medium less affected cellular uptake of mTHPC delivered by double loaded MDCL compared with Foslip.
The objectives of the present work were to compare the kinetics of porphyrin exchange between different plasma lipoproteins and biological membranes. Isolated proteins were loaded by porphyrins (chlorin e6 derivatives and Zn- phthalocyanine) and the kinetics of pigment transfer from protein carrier to pigment-free proteins or biological membranes were analyzed by spectroscopic techniques. It was obtained that the rate of exchange depended strongly on the pigment polarity. A main trend was that the time required for equilibrium redistribution increased with decreasing dyes polarity. Its values changed from seconds for polar sensitizers to tens of minutes in the case of apolar dyes. Extremely slow redistribution (several hours) was observed when the recovery of apolar pigments (chlorin e6 trimethylester and Zn-phthalocyanine) from LDL was studied. The results clear indicate that the rate of pigment exchange between proteins and cells may contribute to the control of sensitizer distribution in blood and solid tissues.
Using fluorescence activated cell sorting we have compared the binding of a number of porphyrins with different polarity by blood cells. According to pigment level blood cells may be arranged in order granulocytes greater than or equal to monocytes greater than lymphocytes greater than erythrocytes. Cellular accumulation of selected porphyrins in blood cells was remarkably different. The equilibrium level of chlorin e6 dimethylester in blood cells was about 15 times higher compared with chlorin e6. As a result, the percentage of pigment binding by blood cells varied from 0% (of total amount) in the case of polar pigments to about 50% for moderately apolar porphyrins. The results obtained show that pigment binding to blood cells may be of certain value when the pharmacokinetic behavior of porphyrin sensitizer is analyzed.
Insertion of cholesterol into phosphatidylcholin vesicles modifies the kinetics of intermembrane porphyrin movement due to as its effects on the rate of transmembrane and intermembrane pigment transfer, as the change of pigment intramembrane distribution pattern. The cholesterol-induced alteration of porphyrin intra- and intermembrane redistributions depends strongly on pigment polarity. According to flow cytometric study results, the reduction of cellular cholesterol through the incubation of cells with phosphatidylcholin liposomes enhances the cells' affinity for porphyrins and at that time efficiently promoted pigments clearance from cells. The potential role for cholesterol and its disproportionation within the cellular membranes as determinants of porphyrin binding and retention by cells is suggested.
Using a fluorescence activated cell sorting, we investigated the dynamics of porphyrins in suspensions of tumor cells. In addition to direct studies of the incorporation and output of several porphyrins (hematoporphyrin, hematoporphyrin dimethyl ester, chlorin e6 and its mono-, di-, trimethyl esters) from cells, their transfer between cells was investigated. It was shown that the rate of pigment accumulation by cells correlated with the rate of porphyrin penetration across the plasma membrane. As a result, apolar chlorins and HpDME displayed enhanced staining capacity which was independent on the integrity of plasma membrane of cells. To estimate the rate of pigment redistribution between cells, the suspension of tumor cells loaded with porphyrin had been mixed with unloaded cells and the distribution of all cells according to porphyrin fluorescence was determined in different intervals of time. It was obtained that the highest rate of the pigment transfer between cells was exhibited in the case of moderately apolar pigment. Porphyrins with dominantly hydrophobic and hydrophilic properties had a decreased capacity to intercellular migration. The results of this study indicate that, depending on the photosensitizer used, the processes of its distribution in the bulk of tumor tissue mediated by intercellular exchange may occur with a different rate.
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.
The parameters of chlorin e6 and trimethylester of chlorin e6 incorporation and distribution in suspensions of unilamellar liposomes of DMPC, DPPC, and DSPC, as well as efficiency of the pigment redistribution from liposomes to cellular membranes have been studied. Determination of the fraction of pigments' fluorescence which is accessible to quenching by a watersoluble quencher indicates that for both chlorins the outer monolayer of the liposomal membrane is more populated than the inner one. Gel-liquid crystalline phase transition induces a shift of a part of the pigments' molecules toward the inner monolayer. By means of ultrafiltration technique it is shown that chlorins binding to liposomal membrane occurs as partitioning between water and lipid phases. The partition coefficient is affected strongly by the type of pigment, the phase state of the lipid bilayer. Similar results were obtained when the influence of the physical state of the lipid bilayer on the rate of chlorins redistribution from liposomes to cellular membrane was studied. These findings show that diffusive mobility of the sensitizer in suspensions of cellular and model membranes is a complex process which is dependent on structural features of both the pigment and its biological carriers.
The mechanisms of cellular uptake of the photosensitizers chlorin e6, chlorin e6 trimethylester and chlorin e6 ethylendiamid are reported in this paper. Each photosensitizer cellular uptake mechanism was determined by preferentially inhibiting the process of low-density lipoprotein (LDL) binding to cells and endocytosis by chilling cellular cultures or by the action of metabolic inhibitors or heparin. The results indicate that delivery with LDL plays a significant role in the process of pigment accumulation by tumor cells. At the same time chlorin e6 derivatives uptake is only partly mediated by receptor dependence endocytosis of LDL. The involvement of molecular diffusion exchange into the binding of apolar chlorin e6 derivatives to cells is suggested.
The photoinduced damage sensitized by chlorin e6 to obligate-pathogenic bacteria Klebsiella rhinoscleromatis in the presence of streptomycin has been studied. A significant synergic effect is observed resulting in both higher photoinactivation rates in the presence of streptomycin and reduction of the threshold of toxic antibiotic concentrations for bacteria, exposed to the sublethal photosensitized action. One may expect that the use of the combined action of a photosensitizer and visible light will allow one to decrease an effective antibiotic dose using combined photo- and chemotherapy of scleroma.