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Liposomes and lipid-based supramolecular systems have been used in clinical practice for more than 30 years as drug carriers and vaccines for the treatment of oncological diseases and infections [...].
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Herein, we describe the synthesis of pH-sensitive lipophilic colchicine prodrugs for liposomal bilayer inclusion, as well as preparation and characterization of presumably stealth PEGylated liposomes with above-mentioned prodrugs. These formulations liberate strongly cytotoxic colchicinoid derivatives selectively under slightly acidic tumor-associated conditions, ensuring tumor-targeted delivery of the compounds. The design of the prodrugs is addressed to pH-triggered release of active compounds in the slight acidic media, that corresponds to tumor microenvironment, while keeping sufficient stability of the whole formulation at physiological pH. Correlations between the structure of the conjugates, their hydrolytic stability, colloidal stability, ability of the prodrug retention in the lipid bilayer are described. Several formulations were found promising for further development and in vivo investigations.
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Previously, we showed in the human umbilical vein endothelial cells (HUVECs) model that a liposome formulation of melphalan lipophilic prodrug (MlphDG) decorated with selectin ligand tetrasaccharide Sialyl Lewis X (SiaLeX) undergoes specific uptake by activated cells and in an in vivo tumor model causes a severe antivascular effect. Here, we cultured HUVECs in a microfluidic chip and then applied the liposome formulations to study their interactions with the cells in situ under hydrodynamic conditions close to capillary blood flow using confocal fluorescent microscopy. The incorporation of 5 to 10% SiaLeX conjugate in the bilayer of MlphDG liposomes increased their consumption exclusively by activated endotheliocytes. The increase of serum concentration from 20 to 100% in the flow resulted in lower liposome uptake by the cells. To elucidate the possible roles of plasma proteins in the liposome-cell interactions, liposome protein coronas were isolated and analyzed by shotgun proteomics and immunoblotting of selected proteins. Proteomic analysis showed that a gradual increase in SiaLeX content correlated with the overall enrichment of the liposome-associated proteins with several apolipoproteins, including the most positively charged one, ApoC1, and serum amyloid A4, associated with inflammation, on the one hand, and a decrease in the content of bound immunoglobulins, on the other. The article discusses the potential interference of the proteins in the binding of liposomes to selectins of endothelial cells.
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To assess the stability and efficiency of liposomes carrying a phospholipase A2-sensitive phospholipid-allocolchicinoid conjugate (aC-PC) in the bilayer, egg phosphatidylcholine and 1-palmitoyl-2-oleoylphosphatidylglycerol-based formulations were tested in plasma protein binding, tubulin polymerization inhibition, and cytotoxicity assays. Liposomes L-aC-PC10 containing 10 mol. % aC-PC in the bilayer bound less plasma proteins and were more stable in 50% plasma within 4 h incubation, according to calcein release and FRET-based assays. Liposomes with 25 mol. % of the prodrug (L-aC-PC25) were characterized by higher storage stability judged by their hydrodynamic radius evolution yet enhanced deposition of blood plasma opsonins on their surface according to SDS-PAGE and immunoblotting. Notably, inhibition of tubulin polymerization was found to require that the prodrug should be hydrolyzed to the parent allocolchicinoid. The L-aC-PC10 and L-aC-PC25 formulations demonstrated similar tubulin polymerization inhibition and cytotoxic activities. The L-aC-PC10 formulation should be beneficial for applications requiring liposome accumulation at tumor or inflammation sites.
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Alcaloides/farmacologia , Antineoplásicos/farmacologia , Colchicina/análogos & derivados , Lipossomos/química , Fosfolipases A2/metabolismo , Fosfolipídeos/química , Alcaloides/síntese química , Alcaloides/química , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Estabilidade de Medicamentos , Transferência Ressonante de Energia de Fluorescência , Humanos , Polimerização/efeitos dos fármacos , Pró-Fármacos , Tubulina (Proteína)/metabolismoRESUMO
Phospholipase A2 (PLA2) exerts a wide range of biological effects and attracts a lot of attention of researchers. Two sites are involved in manifestation of PLA2 enzymatic activity: catalytic site responsible for substrate binding and fatty acid cleavage from the sn-2 position of a glycerophospholipid, and interface binding site (IBS) responsible for the protein binding to lipid membrane. IBS is formed by positively charged and hydrophobic amino acids on the outer surface of the protein molecule. Understanding the mechanism of PLA2 interaction with the lipid membrane is the most challenging step in biochemistry of this enzyme. We used a combination of experimental and computer simulation techniques to clarify molecular details of bee venom PLA2 interaction with lipid bilayers formed by palmitoyloleoylphosphatidylcholine or dipalmitoylphosphatidylcholine. We found that after initial enzyme contact with the membrane, a network of hydrogen bonds was formed. This led to deformation of the interacting leaflet and dint formation. The bilayer response to the deformation depended on its phase state. In a gel-phase bilayer, diffusion of lipids is restricted therefore chain melting occurred in both leaflets of the bilayer. In the case of a fluid-phase bilayer, lateral diffusion is possible, and lipid polar head groups were excluded from the contact area. As a result, the bilayer became thinner and a large hydrophobic area was formed. We assume that relative ability of a bilayer to come through lipid redistribution process defines the rate of initial stages of the catalysis.
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Venenos de Abelha/enzimologia , Abelhas/enzimologia , Proteínas de Insetos/química , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Fosfolipases A2/química , Animais , Hidrólise , Interações Hidrofóbicas e HidrofílicasRESUMO
Enzyme-responsive liposomes release their cargo in response to pathologically increased levels of enzymes at the target site. We report herein an assembly of phospholipase A2-responsive liposomes based on colchicinoid lipid prodrugs incorporated into lipid bilayer of the nanosized vesicles. The liposomes were constructed to addresses two important issues: (i) the lipid prodrugs were designed to fit the structure of the enzyme binding site; and (ii) the concept of lateral pressure profile was used to design lipid prodrugs that introduce almost no distortions into the lipid bilayer packing, thus ensuring that corresponding liposomes are stable. The colchicinoid agents exhibit antiproliferative activity in subnanomolar range of concentrations.
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Colchicina/química , Lipossomos , Fosfolipídeos/química , Pró-Fármacos/química , Fenômenos Biofísicos , Proliferação de Células/efeitos dos fármacos , Colchicina/farmacologia , Fluoresceínas/química , Humanos , Bicamadas Lipídicas , Fosfolipases A2/metabolismoRESUMO
In a previous study, a formulation of methotrexate (MTX) incorporated in the lipid bilayer of 100-nm liposomes in the form of diglyceride ester (MTX-DG, lipophilic prodrug) was developed. In this study, first, the interactions of MTX-DG liposomes with various human and mouse tumor cell lines were studied using fluorescence techniques. The liposomes composed of egg phosphatidylcholine (PC)/yeast phosphatidylinositol/MTX-DG, 8:1:1 by mol, were labeled with fluorescent analogs of PC and MTX-DG. Carcinoma cells accumulated 5 times more MTX-DG liposomes than the empty liposomes. Studies on inhibitors of liposome uptake and processing by cells demonstrated that the formulation used multiple mechanisms to deliver the prodrug inside the cell. According to the data from the present study, undamaged liposomes fuse with the cell membrane only 1.5-2 hours after binding to the cell surface, and then, the components of liposomal bilayer enter the cell separately. The study on the time course of plasma concentration in mice showed that the area under the curve of MTX generated upon intravenous injection of MTX-DG liposomes exceeded that of intact MTX 2.5-fold. These data suggested the advantage of using liposomal formulation to treat systemic manifestation of hematological malignancies. Indeed, the administration of MTX-DG liposomes to recipient mice bearing T-cell leukemic lymphoma using a dose-sparing regimen resulted in lower toxicity and retarded lymphoma growth rate as compared with MTX.
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Antimetabólitos Antineoplásicos/administração & dosagem , Lipossomos/administração & dosagem , Linfoma de Células T/tratamento farmacológico , Metotrexato/administração & dosagem , Pró-Fármacos/administração & dosagem , Animais , Antimetabólitos Antineoplásicos/química , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Injeções Intravenosas , Leucemia/tratamento farmacológico , Leucemia/patologia , Bicamadas Lipídicas/química , Lipossomos/química , Lipossomos/metabolismo , Linfoma de Células T/patologia , Metotrexato/química , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Pró-Fármacos/químicaRESUMO
In the study, MCF-7 human breast adenocarcinoma cells were used to study cytotoxicity of novel anticancer nanosized formulations, such as docetaxel-loaded nanoemulsion and liposomal formulation of a lipophilic methotrexate (MTX) prodrug. In vitro study of cytotoxicity was carried out in 2 models, namely using 3D in vitro model based on multicellular tumor spheroids (MTS) and 2D monolayer culture. MTS were generated by tumor cell cultivation within alginate-oligochitosan microcapsules. In the case of the monolayer culture, cell viability was found to be 25, 18 and 12% for the samples containing nanoemulsion at concentrations 20, 300 and 1000 nM of docetaxel, respectively, after 48 hs incubation. For MTS these values were higher, namely 33, 23 and 18%, respectively. Cytotoxicity of liposomal MTX prodrug-based formulation with final concentration of 1, 2, 10, 50, 100 and 1000 nM in both models was also studied. MTX liposomal formulation demonstrated lower cytotoxicity on MTS compared to intact MTX. Moreover, MTS were also more resistant to both liposomal formulation and intact MTX than the monolayer culture. Thus, at 1000 nM MTX in the liposomal form, cell viability in MTS was 1.4-fold higher than that in the monolayer culture. MTS could be proposed as a promising tool to test novel anticancer nanosized formulations in vitro.
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Adenocarcinoma/tratamento farmacológico , Antimetabólitos Antineoplásicos , Neoplasias da Mama/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Metotrexato , Nanopartículas/química , Esferoides Celulares , Taxoides , Adenocarcinoma/patologia , Antimetabólitos Antineoplásicos/química , Antimetabólitos Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Células Imobilizadas , Docetaxel , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Metotrexato/química , Metotrexato/farmacologia , Taxoides/química , Taxoides/farmacologiaRESUMO
Earlier we showed that liposome formulation of DL-melphalan lipophilic prodrug bearing tetrasaccharide Sialyl Lewis X (SiaLeX) caused prolonged therapeutic effect on mammary cancer in mice. Here, we compare antivascular effect of SiaLeX-liposomes loaded with diglyceride ester of melphalan (Mlph) against SiaLeX-free formulation in Lewis lung carcinoma model. METHODS: Liposomes of egg phosphatidylcholine/yeast phosphatidylinositol/1,2-dioleoyl glycerol (DOG) conjugate of Mlph/±SiaLeX-PEG8-15-DOG, 8:1:1:0.2 by mol, were prepared by standard extrusion. After two intravenous injections with Mlph or liposomes under either standard or delayed treatment protocols, vascular-disrupting effects of the preparations were evaluated basing on tumour section histomorphology, lectin perfusion assay and immunohistochemistry (anti-CD31 staining) data. Also, untreated mice were administered with fluorescently-labelled liposomes to assess their distribution in tumour sections with confocal laser scanning microscopy. RESULTS: Two injections of SiaLeX-liposomes reproducibly caused severe injuries of tumour vessels. SiaLeX-liposomes co-localized with CD31 marker on vascular endothelium while the non-targeted formulation extravasated into tumour. DISCUSSION: Cytotoxic SiaLeX-liposomes exhibit superior vascular-disrupting properties compared to non-targeted liposomes, yet the effect starts to transform into gain in tumour growth inhibition only under delayed treatment regimen. CONCLUSION: SiaLeX-ligand provides targeting of cytotoxic liposomes to tumour endothelium and subsequent antivascular effect.
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A panel of in vitro tests intended for evaluation of the nano-sized drug delivery systems' compliance with human blood was applied to liposomal formulations of anticancer lipophilic prodrugs incorporated into the lipid bilayer. Liposomes on the basis of natural phosphatidylcholine (PC) and phosphatidylinositol (PI), 8:1 (mol) were loaded with 10 mol% of either methotrexate or melphalan 1,2-dioleoylglyceride esters (MTX-DOG and Mlph-DOG respectively) and either decorated with 2 mol% of sialyl Lewis X/A (SiaLe(X/A)) tetrasaccharide ligand or not. Hemolysis rate, red blood cells and platelets integrity and size distribution, complement (C) activation, and coagulation cascade functioning were analyzed upon the material incubation with whole blood. Both formulations were negatively charged with the zeta potential value being higher in the case of MTX-DOG liposomes, which also were larger than Mlph-DOG liposomes and more prone to aggregation. Accordingly, in hemocompatibility tests Mlph-DOG liposomes did not provoke any undesirable effects, while MTX-DOG liposomes induced significant C activation and abnormal coagulation times in a concentration-dependent manner. Reactivity of the liposome surface was not affected by the presence of SiaLe(X/A) or PI. Decrease in liposome loading with MTX-DOG from 10 to 2.5% resulted in lower surface charge density, smaller liposome size and considerably reduced impact on C activation and coagulation cascades.