RESUMO
To address the lack of high-resolution electron ionisation mass spectral libraries (HR-[EI+]-MS) for environmental chemicals, a retention-indexed HR-[EI+]-MS library has been constructed following analysis of authentic compounds via GC-Orbitrap MS. The library is freely provided alongside a compound database of predicted physicochemical properties. Currently, the library contains over 350 compounds from 56 compound classes and includes a range of legacy and emerging contaminants. The RECETOX Exposome HR-[EI+]-MS library expands the number of freely available resources for use in full-scan chemical exposure studies and is available at: https://doi.org/10.5281/zenodo.4471217.
Assuntos
Expossoma , Gerenciamento de Dados , Cromatografia Gasosa-Espectrometria de MassasRESUMO
Fatty acyl analogues of muramyldipeptide (MDP) (abbreviated N-L18 norAbuGMDP, N-B30 norAbuGMDP, norAbuMDP-Lys(L18), norAbuMDP-Lys(B30), norAbuGMDP-Lys(L18), norAbuGMDP-Lys(B30), B30 norAbuMDP, L18 norAbuMDP) are designed and synthesized comprising the normuramyl-l-α-aminobutanoyl (norAbu) structural moiety. All new analogues show depressed pyrogenicity in both free (micellar) state and in liposomal formulations when tested in rabbits in vivo (sc and iv application). New analogues are also shown to be selective activators of NOD2 and NLRP3 (inflammasome) in vitro but not NOD1. Potencies of NOD2 and NLRP3 stimulation are found comparable with free MDP and other positive controls. Analogues are also demonstrated to be effective in stimulating cellular proliferation when the sera from mice are injected sc with individual liposome-loaded analogues, causing proliferation of bone marrow-derived GM-progenitors cells. Importantly, vaccination nanoparticles prepared from metallochelation liposomes, His-tagged antigen rOspA from Borrelia burgdorferi, and lipophilic analogue norAbuMDP-Lys(B30) as adjuvant, are shown to provoke OspA-specific antibody responses with a strong Th1-bias (dominance of IgG2a response). In contrast, the adjuvant effects of Alum or parent MDP show a strong Th2-bias (dominance of IgG1 response).
Assuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Adjuvantes Imunológicos/farmacologia , Antígenos de Superfície/farmacologia , Proteínas da Membrana Bacteriana Externa/farmacologia , Vacinas Bacterianas/farmacologia , Borrelia burgdorferi/imunologia , Lipoproteínas/farmacologia , Acetilmuramil-Alanil-Isoglutamina/química , Acetilmuramil-Alanil-Isoglutamina/imunologia , Adjuvantes Imunológicos/química , Animais , Formação de Anticorpos , Antígenos de Superfície/química , Antígenos de Superfície/imunologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/química , Vacinas Bacterianas/imunologia , Feminino , Células HEK293 , Humanos , Imunização , Lipoproteínas/química , Lipoproteínas/imunologia , Doença de Lyme/imunologia , Doença de Lyme/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Proteína 3 que Contém Domínio de Pirina da Família NLR/agonistas , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Células RAW 264.7RESUMO
Nanofibre-based mucoadhesive films were invented for oromucosal administration of nanocarriers used for delivery of drugs and vaccines. The mucoadhesive film consists of an electrospun nanofibrous reservoir layer, a mucoadhesive film layer and a protective backing layer. The mucoadhesive layer is responsible for tight adhesion of the whole system to the oral mucosa after application. The electrospun nanofibrous reservoir layer is intended to act as a reservoir for polymeric and lipid-based nanoparticles, liposomes, virosomes, virus-like particles, dendrimers and the like, plus macromolecular drugs, antigens and/or allergens. The extremely large surface area of nanofibrous reservoir layers allows high levels of nanoparticle loading. Nanoparticles can either be reversibly adsorbed to the surface of nanofibres or they can be deposited in the pores between the nanofibres. After mucosal application, nanofibrous reservoir layers are intended to promote prolonged release of nanoparticles into the submucosal tissue. Reversible adsorption of model nanoparticles as well as sufficient mucoadhesive properties were demonstrated. This novel system appears appropriate for the use in oral mucosa, especially for sublingual and buccal tissues. To prove this concept, trans-/intramucosal and lymph-node delivery of PLGA-PEG nanoparticles was demonstrated in a porcine model. This system can mainly be used for sublingual immunization and the development of "printed vaccine technology".
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Nanofibras/química , Preparações Farmacêuticas/administração & dosagem , Vacinas/administração & dosagem , Adesivos/química , Administração Bucal , Administração Sublingual , Animais , Lipossomos/química , Linfonodos/metabolismo , Camundongos , Mucosa Bucal/metabolismo , Nanopartículas/química , Polietilenoglicóis/química , Poliglactina 910/química , Suínos , Vacinação/métodosRESUMO
Lyme disease, Borrelia burgdorferi-caused infection, if not recognized and appropriately treated by antibiotics, may lead to chronic complications, thus stressing the need for protective vaccine development. The immune protection is mediated by phagocytic cells and by Borrelia-specific complement-activating antibodies, associated with the Th1 immune response. Surface antigen OspC is involved in Borrelia spreading through the host body. Previously we reported that recombinant histidine tagged (His-tag) OspC (rOspC) could be attached onto liposome surfaces by metallochelation. Here we report that levels of OspC-specific antibodies vary substantially depending upon whether rOspC possesses an N' or C' terminal His-tag. This is the case in mice immunized: (a) with rOspC proteoliposomes containing adjuvants MPLA or non-pyrogenic MDP analogue MT06; (b) with free rOspC and Montanide PET GEL A; (c) with free rOspC and alum; or (d) with adjuvant-free rOspC. Stronger responses are noted with all N'-terminal His-tag rOspC formulations. OspC-specific Th1-type antibodies predominate post-immunization with rOspC proteoliposomes formulated with MPLA or MT06 adjuvants. Further analyses confirmed that the structural features of soluble N' and C' terminal His-tag rOspC and respective rOspC proteoliposomes are similar including their thermal stabilities at physiological temperatures. On the other hand, a change in the position of the rOspC His-tag from N' to C' terminal appears to affect substantially the immunogenicity of rOspC arguably due to steric hindrance of OspC epitopes by the C' terminal His-tag itself and not due to differences in overall conformations induced by changes in the His-tag position in rOspC variants.
Assuntos
Adjuvantes Imunológicos , Anticorpos Antibacterianos/imunologia , Formação de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Borrelia burgdorferi/imunologia , Proteínas Recombinantes de Fusão/imunologia , Animais , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/química , Proteínas da Membrana Bacteriana Externa/administração & dosagem , Proteínas da Membrana Bacteriana Externa/química , Ensaio de Imunoadsorção Enzimática , Imunização , Doença de Lyme/imunologia , Camundongos , Modelos Animais , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteolipídeos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Pseudorabies virus (PrV), a causative agent of Aujeszky's disease, is deadly to most mammals with the exception of higher primates and men. This disease causes serious economic loses among farm animals, especially pigs, yet many European countries are today claimed to be Aujeszky's disease free because of the discovery of an efficient vaccination for pigs. In reality, the virus is still present in wild boar. Current vaccines are neither suitable for dogs nor are there anti-PrV drugs approved for veterinary use. Therefore, the disease still represents a high threat, particularly for expensive hunting dogs that can come into close contact with infected boars. Here we report on the anti-PrV activities of a series of synthetic diaminopurine-based acyclic nucleoside phosphonate (DAP-ANP) analogues. Initially, all synthetic DAP-ANPs under investigation are shown to exhibit minimal cytotoxicity by MTT and XTT tests (1-100µM range). Thereafter in vitro infection models are established using PrV virus SuHV-1, optimized on PK-15 and RK-13 cell lines. Out of the six DAP-ANP analogues tested, analogue VI functionalized with a cyclopropyl group on the 6-amino position of the purine ring proves the most effective antiviral DAP-ANP analogue against PrV infection, aided by sufficient hydrophobic character to enhance bioavailability to its cellular target viral DNA-polymerase. Four other DAP-ANP analogues with functional groups introduced to the C2'position are shown ineffective against PrV infection, even with favourable hydrophobic properties. Cidofovir(®), a drug approved against various herpesvirus infections, is found to exert only low activity against PrV in these same in vitro models.
Assuntos
Antivirais/farmacologia , Herpesviridae/efeitos dos fármacos , Organofosfonatos/farmacologia , 2-Aminopurina/análogos & derivados , 2-Aminopurina/química , 2-Aminopurina/farmacologia , Animais , Antivirais/química , Linhagem Celular , Replicação do DNA/efeitos dos fármacos , Cães , Técnicas In Vitro , Células Madin Darby de Rim Canino , Microscopia Eletrônica de Transmissão , Organofosfonatos/química , Pseudorraiva/tratamento farmacológico , Células VeroRESUMO
Several plasminogen activators (PAs) have been found effective in treating different thromboembolic diseases. However, administration of conventional thrombolytic therapy is limited by a low efficacy of present formulations of PAs. Conventional treatments using these therapeutic proteins are associated with several limitations including rapid inactivation and clearance, short half-life, bleeding complications or non-specific tissue targeting. Liposome-based formulations of PAs such as streptokinase, tissue-plasminogen activator and urokinase have been developed to improve the therapeutic efficacy of these proteins. Resulting liposomal formulations were found to preserve the original activity of PAs, promote their selective delivery and improve thrombus targeting. Therapeutic potential of these liposome-based PAs has been demonstrated successfully in various pre-clinical models in vivo. Reductions in unwanted side effects (e.g., hemorrhage or immunogenicity) as well as enhancements of efficacy and safety were achieved in comparison to currently existing treatment options based on conventional formulations of PAs. This review summarizes present achievements in: (i) preparation of liposome-based formulations of various PAs, (ii) development of PEGylated and targeted liposomal PAs, (iii) physico-chemical characterization of these developed systems, and (iv) testing of their thrombolytic efficacy. We also look to the future and the imminent arrival of theranostic liposomal formulations to move this field forward.
Assuntos
Fibrinolisina/administração & dosagem , Fibrinolíticos/administração & dosagem , Lipossomos/química , Metaloendopeptidases/administração & dosagem , Estreptoquinase/administração & dosagem , Ativador de Plasminogênio Tecidual/administração & dosagem , Ativador de Plasminogênio Tipo Uroquinase/administração & dosagem , Animais , Fibrinolisina/uso terapêutico , Fibrinolíticos/uso terapêutico , Humanos , Lipossomos/ultraestrutura , Metaloendopeptidases/uso terapêutico , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Estreptoquinase/uso terapêutico , Tromboembolia/tratamento farmacológico , Terapia Trombolítica/métodos , Ativador de Plasminogênio Tecidual/uso terapêutico , Ativador de Plasminogênio Tipo Uroquinase/uso terapêuticoRESUMO
Pro-apoptotic analogues of vitamin E (VE) exert selective anti-cancer effect on various animal cancer models. Neither suitable formulation of α-tocopheryl succinate (α-TOS), representative semi-synthetic VE analogue ester, nor suitable formulations of the other VE analogues for clinical application have been reported yet. The major factor limiting the use of VE analogues is their low solubility in aqueous solvents. Due to the hydrophobic character of VE analogues, liposomes are predetermined as suitable delivery system. Liposomal formulation prevents undesirable side effects of the drug, enhances the drug biocompatibility, and improves the drug therapeutic index. Liposomal formulations of VE analogues especially of α-TOS and α-tocopheryl ether linked acetic acid (α-TEA) have been developed. The anti-cancer effect of these liposomal VE analogues has been successfully demonstrated in pre-clinical models in vivo. Present achievements in: (i) preparation of liposomal formulations of VE analogues, (ii) physico-chemical characterization of these developed systems and (iii) testing of their biological activity such as induction of apoptosis and evaluation of anti-cancer effect are discussed in this review.
Assuntos
Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Lipídeos/química , Neoplasias/tratamento farmacológico , Vitamina E/administração & dosagem , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Química Farmacêutica , Humanos , Lipossomos , Neoplasias/patologia , Solubilidade , Vitamina E/análogos & derivados , Vitamina E/química , alfa-Tocoferol/administração & dosagemRESUMO
PURPOSE: The aim of this work was to demonstrate an immunostimulatory and adjuvant effect of new apyrogenic lipophilic derivatives of norAbuMDP and norAbuGMDP formulated in nanoliposomes. METHODS: Nanoliposomes and metallochelating nanoliposomes were prepared by lipid film hydration and extrusion methods. The structure of the liposomal formulation was studied by electron microscopy, AF microscopy, and dynamic light scattering. Sublethal and lethal γ-irradiation mice models were used to demonstrate stimulation of innate immune system. Recombinant Hsp90 antigen (Candida albicans) bound onto metallochelating nanoliposomes was used for immunisation of mice to demonstrate adjuvant activities of tested compounds. RESULTS: Safety and stimulation of innate and adaptive immunity were demonstrated on rabbits and mice. The liposomal formulation of norAbuMDP/GMDP was apyrogenic in rabbit test and lacking any side effect in vivo. Recovery of bone marrow after sublethal γ-irradiation as well as increased survival of mice after lethal irradiation was demonstrated. Enhancement of specific immune response was demonstrated for some derivatives incorporated in metallochelating nanoliposomes with recombinant Hsp90 protein antigen. CONCLUSIONS: Liposomal formulations of new lipophilic derivatives of norAbuMDP/GMDP proved themselves as promising adjuvants for recombinant vaccines as well as immunomodulators for stimulation of innate immunity and bone-marrow recovery after chemo/radio therapy of cancer.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Imunidade Adaptativa/efeitos dos fármacos , Adjuvantes Imunológicos/farmacologia , Portadores de Fármacos/química , Imunidade Inata/efeitos dos fármacos , Acetilmuramil-Alanil-Isoglutamina/administração & dosagem , Acetilmuramil-Alanil-Isoglutamina/química , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Acetilmuramil-Alanil-Isoglutamina/uso terapêutico , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/uso terapêutico , Animais , Anticorpos Antifúngicos/sangue , Antígenos de Fungos/imunologia , Feminino , Proteínas de Choque Térmico HSP90/imunologia , Lipossomos , Camundongos , Camundongos Endogâmicos ICR , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Estrutura Molecular , Nanopartículas , Coelhos , Lesões Experimentais por Radiação/imunologia , Lesões Experimentais por Radiação/prevenção & controle , Proteínas Recombinantes/imunologia , Análise de SobrevidaRESUMO
Specific contrast ultrasound is widely applied in diagnostic procedures on humans but remains underused in veterinary medicine. The objective of this study was to evaluate the use of microbubble-based contrast for rapid ultrasonographic diagnosis of thrombosis in small animals, using male New Zealand white rabbits (average weight about 3.5 kg) as a model. It was hypothesized that the use of microbubble-based contrast agents will result in a faster and more precise diagnosis in our model of thrombosis. A pro-coagulant environment had been previously established by combining endothelial denudation and external vessel wall damage. Visualization of thrombi was achieved by application of contrast microbubbles [sterically stabilized, phospholipid-based microbubbles filled with sulfur hexafluoride (SF6) gas] and ultrasonography. As a result, rapid and clear diagnosis of thrombi in aorta abdominalis was achieved within 10 to 30 s (mean: 17.3 s) by applying microbubbles as an ultrasound contrast medium. In the control group, diagnosis was not possible or took 90 to 180 s. Therefore, sterically stabilized microbubbles were found to be a suitable contrast agent for the rapid diagnosis of thrombi in an experimental model in rabbits. This contrast agent could be of practical importance in small animal practice for rapid diagnosis of thrombosis.
L'échographie par contraste spécifique est une procédure diagnostique couramment utilisée chez les humains mais demeure sous-utilisée chez les animaux. L'objectif de la présente étude était d'évaluer l'utilisation du contraste basée sur les micro-bulles pour le diagnostic échographique rapide de thrombose chez les petits animaux, en utilisant comme modèle le lapin blanc de Nouvelle-Zélande mâle (poids moyen de 3,5 kg). L'hypothèse a été émise que l'utilisation d'agents de contraste à base de micro-bulles résulterait en un diagnostic plus rapide et plus précis dans notre modèle de thrombose. Un environnement pro-coagulant a préalablement été établi en combinant le dénudement endothélial et du dommage à la paroi externe du vaisseau. La visualisation des thrombi a été obtenue par application de micro-bulles de contraste [micro-bulles à base de phospholipides remplies d'hexafluorure de soufre (SF6) stabilisées stériquement] et échographie. L'application de micro-bulles comme milieu de contraste pour l'échographie résulta en un diagnostic rapide et clair de thrombi dans l'aorte abdominale en 10 à 30 secondes (moyenne de 17,3 s). Dans le groupe témoin, le diagnostic n'était pas possible ou prenait de 90 à 180 s. Ainsi, des micro-bulles stabilisées stériquement ont été trouvées comme étant un agent de contraste convenable pour le diagnostic rapide de thrombi dans un modèle expérimental chez les lapins. Cet agent de contraste pourrait être d'importance concrète en pratique des petits animaux pour le diagnostic rapide de thromboses.(Traduit par Docteur Serge Messier).
Assuntos
Aorta Abdominal/patologia , Doenças do Gato/patologia , Microbolhas/veterinária , Tromboembolia/veterinária , Ultrassonografia Doppler/veterinária , Animais , Aorta Abdominal/diagnóstico por imagem , Doenças do Gato/diagnóstico por imagem , Gatos , Meios de Contraste , Modelos Animais de Doenças , Masculino , Coelhos , Estatísticas não Paramétricas , Hexafluoreto de Enxofre , Tromboembolia/diagnóstico por imagem , Tromboembolia/patologia , Ultrassonografia Doppler/métodosRESUMO
α-Tocopheryl succinate (α-TOS) is a promising anti-cancer agent due to its selectivity for cancer cells. It is important to understand whether long-term exposure of tumour cells to the agent will render them resistant to the treatment. Exposure of the non-small cell lung carcinoma H1299 cells to escalating doses of α-TOS made them resistant to the agent due to the upregulation of the ABCA1 protein, which caused its efflux. Full susceptibility of the cells to α-TOS was restored by knocking down the ABCA1 protein. Similar resistance including ABCA1 gene upregulation was observed in the A549 lung cancer cells exposed to α-TOS. The resistance of the cells to α-TOS was overcome by its mitochondrially targeted analogue, MitoVES, that is taken up on the basis of the membrane potential, bypassing the enhanced expression of the ABCA1 protein. The in vitro results were replicated in mouse models of tumours derived from parental and resistant H1299 cells. We conclude that long-term exposure of cancer cells to α-TOS causes their resistance to the drug, which can be overcome by its mitochondrially targeted counterpart. This finding should be taken into consideration when planning clinical trials with vitamin E analogues.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , alfa-Tocoferol/uso terapêutico , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , CamundongosRESUMO
A device for continuous infusion of microbubbles (MBs) 'Infucon' has been designed, constructed and tested on rabbits. The device prevents MBs from flotation and accumulation in the layer directly below the surface in the syringe injection during i.v. application. Homogenous i.v. application of MBs was tested on 16 male New Zealand White rabbits (average weight about 3.5 kg). Two sorts of MBs were used - a set of commercial SonoVue diagnostic microbubbles (Bracco) and pegylated DPPC microbubbles (PegMBs), which had been prepared in our laboratory. Sulphur hexafluoride was used as a filling gas. The application of MBs by continuous infusion via Infucon prolonged the ultrasound signal period in the heart of the rabbit to 12 min in comparison to about 1 min observed in bolus application. No adverse effects were observed on the tested rabbits after the MB application via Infucon. The principle employed in the prototype device Infucon could be used for development of the device intended for clinical applications.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Microbolhas , Polietilenoglicóis/química , Ultrassonografia/métodos , Animais , Infusões Intravenosas , Masculino , Fosfolipídeos , Coelhos , Hexafluoreto de Enxofre , Fatores de TempoRESUMO
Over the past three decades, taxanes represent one of the most important new classes of drugs approved in oncology. Paclitaxel (PTX), the prototype of this class, is an anti-cancer drug approved for the treatment of breast and ovarian cancer. However, notwithstanding a suitable premedication, present-day chemotherapy employing a commercial preparation of PTX (Taxol®) is associated with serious side effects and hypersensitivity reactions. Liposomes represent advanced and versatile delivery systems for drugs. Generally, both in vivo mice tumor models and human clinical trials demonstrated that liposomal PTX formulations significantly increase a maximum tolerated dose (MTD) of PTX which outperform that for Taxol®. Liposomal PTX formulations are in various stages of clinical trials. LEP-ETU (NeoPharm) and EndoTAG®-1 (Medigene) have reached the phase II of the clinical trials; Lipusu® (Luye Pharma Group) has already been commercialized. Present achievements in the preparation of various liposomal formulations of PTX, the development of targeted liposomal PTX systems and the progress in clinical testing of liposomal PTX are discussed in this review summarizing about 30 years of liposomal PTX development.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Paclitaxel/administração & dosagem , Animais , Antineoplásicos Fitogênicos/química , Humanos , Lipossomos , Neoplasias/tratamento farmacológico , Paclitaxel/químicaRESUMO
Lyme disease caused by spirochete Borrelia burgdorferi sensu lato, is a tick-born illness. If the infection is not eliminated by the host immune system and/or antibiotics, it may further disseminate and cause severe chronic complications. The immune response to Borrelia is mediated by phagocytic cells and by Borrelia-specific complement-activating antibodies associated with Th1 cell activation. A new experimental vaccine was constructed using non-lipidized form of recombinant B. burgdorferi s.s. OspC protein was anchored by metallochelating bond onto the surface of nanoliposomes containing novel nonpyrogenic lipophilized norAbuMDP analogues denoted MT05 and MT06. After i.d. immunization, the experimental vaccines surpassed Alum with respect to OspC-specific titers of IgG2a, IgG2b isotypes when MT06 was used and IgG3, IgM isotypes when MT05 was used. Both adjuvants exerted a high adjuvant effect comparable or better than MDP and proved themselves as nonpyrogenic.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/química , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Borrelia burgdorferi/imunologia , Quelantes/química , Portadores de Fármacos/química , Vacinas contra Doença de Lyme/imunologia , Nanopartículas/química , Acetilmuramil-Alanil-Isoglutamina/toxicidade , Animais , Varredura Diferencial de Calorimetria , Quelantes/toxicidade , Portadores de Fármacos/toxicidade , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Luz , Lipossomos , Vacinas contra Doença de Lyme/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Nanopartículas/toxicidade , Espalhamento de Radiação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
We designed and synthesised a series of new cationic lipids based on spermine linked to various hydrophobic anchors. These lipids could be potentially useful for the preparation of stable cationic liposomes intended for the construction of drug targeting systems applicable in the field of anticancer/antiviral therapy, vaccine carriers, and vectors for the gene therapy. Low in vitro toxicity was found for these compounds, especially for LD1, in several cell lines. The delivery of both a fluorescence marker (calcein) and antiviral drugs into cells has been achieved owing to a large extent of internalization of cationic liposomes (labelled by Lyssamine-Rhodamine PE or fluorescein-PE) as demonstrated by fluorescent microscopy and quantified by flow cytometry. The bovine herpes virus type 1 (BHV-1) virus infection in vitro model using MDBK cells was employed to study the effect of the established antiviral drug HPMPC (Cidofovir®) developed by Prof. A. Holý. Inhibition of BHV-1 virus replication was studied by quantitative RT-PCR and confirmed by both Hoffman modulation contrast microscopy and transmission electron microscopy. We found that in vitro antiviral activity of HPMPC was significantly improved by formulation in cationic liposomes, which decreased the viral replication by about 2 orders of magnitude.
Assuntos
Antivirais/farmacologia , Citosina/análogos & derivados , Portadores de Fármacos/química , Herpesvirus Bovino 1/efeitos dos fármacos , Lipídeos/química , Organofosfonatos/farmacologia , Animais , Antivirais/administração & dosagem , Cátions , Bovinos , Técnicas de Cultura de Células , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cidofovir , Efeito Citopatogênico Viral , Citosina/administração & dosagem , Citosina/farmacologia , Herpesvirus Bovino 1/fisiologia , Rim/citologia , Rim/virologia , Lipossomos , Microscopia de Fluorescência , Organofosfonatos/administração & dosagem , Reação em Cadeia da Polimerase em Tempo Real , Replicação Viral/efeitos dos fármacosRESUMO
The histidine-metallochelating lipid complex is one of the smallest high affinity binding units used as tools for rapid noncovalent binding of histidine tagged molecules, especially recombinant proteins. The advantage of metallochelating complex over protein-ligand complexes (e.g., streptavidine-biotin, glutathiontransferase-glutathion) consists in its very low immunogenicity, if any. This concept for the construction of surface-modified metallochelating microbubbles was proved with recombinant green fluorescent protein (rGFP) containing 6His-tag. This protein is easy to be detected by various fluorescence techniques as flow cytometry and confocal microscopy. Microbubbles (MB) composed of DPPC with various contents of metallochelating lipid DOGS-NTA-Ni were prepared by intensive shaking of the liposome suspension under the atmosphere of sulfur hexafluoride. For this purpose, the instrument 3M ESPE CapMix was used. Various techniques (static light scattering, flow cytometry, and optical microscopy) were compared and used for the measurements of the size distribution of MB. All three methods demonstrated that the prepared MB were homogeneous in their size, and the mean diameter of the MB in various batches was within the range of 2.1-2.8 µm (the size range of 1-10 µm). The presence of large MB (8-10 µm) was marginal. Counting of MB revealed that the average amount of MB prepared of 10 mg of phospholipid equaled approximately 10(9) MB/mL. Lyophilized MB were prepared with saccharose as a cryoprotectant. These MB were shown to be stable both in vitro (the estimated half-live of the MB in bovine serum at 37 °C was 3-7 min) and in vivo (mouse). The stability of the MB was affected by molar content of DOGS-NTA-Ni. DPPC-based metallochelating MB provided a clear and very contrast image of the ventricular cavity soon after the injection. Site selective and stable binding of rGFP-HisTag (as a model of His-tagged protein) onto the surface of metallochelating MB was demonstrated by confocal microscopy.
Assuntos
Quelantes/química , Proteínas de Fluorescência Verde/metabolismo , Lipossomos/metabolismo , Microbolhas , Animais , Sítios de Ligação , Histidina , Metais , Modelos Biológicos , Ligação Proteica , Proteínas RecombinantesRESUMO
Hsp90-CA is present in cell wall of Candida pseudohyphae or hyphae-typical pathogenic morphotype for both systemic and mucosal Candida infections. Heat shock protein from Candida albicans (hsp90-CA) is an important target for protective antibodies during disseminated candidiasis of experimental mice and human. His-tagged protein rHsp90 was prepared and used as the antigen for preparation of experimental recombinant liposomal vaccine. Nickel-chelating liposomes (the size around 100nm, PDI≤0.1) were prepared from the mixture of egg phosphatidyl choline and nickel-chelating lipid DOGS-NTA-Ni (molar ratio 95:5%) by hydration of lipid film and extrusion methods. New non-pyrogenic hydrophobised derivative of MDP (C18-O-6-norAbuMDP) was incorporated into liposomes as adjuvans. rHsp90 was attached onto the surface of metallochelating liposomes by metallochelating bond and the structure of these proteoliposomes was studied by dynamic light scattering, AF microscopy, TEM and GPC. The liposomes with surface-exposed C18-O-6-norAbuMDP were well recognised and phagocyted by human dendritic cells in vitro. In vivo the immune response towards this experimental vaccine applied in mice (i.d.) demonstrated both TH1 and TH2 response comparable to FCA, but without any side effects. Metallochelating liposomes with lipophilic derivatives of muramyl dipeptide represent a new biocompatible platform for construction of experimental recombinant vaccines and drug-targeting systems.
Assuntos
Antígenos de Fungos/imunologia , Quelantes/metabolismo , Proteínas de Choque Térmico HSP90/imunologia , Imunidade Celular , Níquel/metabolismo , Animais , Antígenos de Fungos/metabolismo , Candida/imunologia , Células Cultivadas , Quelantes/química , Materiais Revestidos Biocompatíveis/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lipossomos , Camundongos , Camundongos Endogâmicos BALB C , Níquel/imunologia , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/metabolismoRESUMO
Liposomes represent a biocompatible platform for the construction of self-assembling proteoliposomes using nickel or zinc metallochelation. Potential applications of such structures consist in the development of new biocompatible vaccination nanoparticles and drug delivery nanoparticle systems. Here, we describe the design and construction of a flow-through ultrafiltration cell suitable for the preparation of monodisperse liposomes enabled for metallochelation and, hence, the formation of proteoliposomes. The linkage of the cell with a fast protein liquid chromatography system facilitates automation of the procedure, which fits the criteria for upscaling. Proof-of-concept experiments are performed using a mixture of egg phosphatidyl choline and nickel-chelating lipid DOGS-NTA-Ni (1,2-dioleoyl-sn-glycero-3-{[N(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl}(nickel salt)) to formulate proteoliposomes with proteins attached by metallochelation, including histidine (His)-tagged recombinant green fluorescent protein and rgp120 (derived from HIV-1 Env). These model proteoliposomes are characterized by gel permeation chromatography and by dynamic light scattering. Transmission electron microscopy and immunogold staining are used to characterize surface-bound proteins, revealing the tendency of rgp120 to form microdomains on liposome surfaces. These microdomains possess a two-dimensional crystal-like structure that is seen more precisely by atomic force microscopy.
Assuntos
Quelantes/química , Proteínas Imobilizadas/química , Lipossomos/química , Níquel/química , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Histidina/química , Histidina/genética , Histidina/metabolismo , Humanos , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Micelas , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Proteolipídeos/química , Ultrafiltração/métodosRESUMO
alpha-Tocopheryl succinate (alpha-TOS) is a semisynthetic analogue of alpha-tocopherol with selective toxicity to the cancer cells and anticancer activity in vivo. Yet, no suitable formulation of alpha-TOS for medical application has been reported. Various formulations, for example, solutions in organic solvents, oil emulsions and vesicules prepared by spontaneous vesiculation, polyethylene glycol conjugates and liposomes of various compositions have been tested. We developed and characterised a stable lyophilised liposome-based alpha-TOS formulation. alpha-TOS (15 mol%) was incorporated into large oligolamellar vesicles (OLVs) composed of soy phosphatidylcholine (SPC) by the method of lipid film hydration followed by extrusion through polycarbonate filters. Stabilised liposomal formulation was prepared by lyophilisation in the presence of sucrose (molar ratio lipid/sucrose, 1:5). The size distribution of the liposomes (130-140 nm, polydispersity index 0.14) as well as the stable lipid and alpha-TOS contents were preserved during storage in the lyophilised form at 2-8 degrees C for at least 6 months. The data indicate good physical and chemical stability of the lyophilised preparation of alpha-TOS liposomes that can be used in clinical medicine.
Assuntos
Antineoplásicos/administração & dosagem , Composição de Medicamentos/métodos , Lipídeos/química , alfa-Tocoferol/administração & dosagem , Antineoplásicos/química , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Liofilização , Peróxido de Hidrogênio/química , Lipossomos , Lisofosfolipídeos/química , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Propriedades de Superfície , alfa-Tocoferol/químicaRESUMO
Paclitaxel (PTX) is approved for the treatment of ovarian and breast cancer. The commercially available preparation of PTX, Cremophor EL(R) is associated with hypersensitivity reactions in spite of a suitable premedication. In general, the developed liposomal PTX formulations are troubled with low PTX encapsulation capacity (maximal content, 3 mol%) and accompanied by PTX crystallisation. The application of "pocket-forming" lipids significantly increased the encapsulation capacity of PTX in the liposomes up to 10 mol%. Stable lyophilised preparation of PTX (7 mol%) encapsulated in the liposomes composed of SOPC/POPG/MOPC (molar ratio, 60:20:20) doped with 5 mol% vitamin E had the size distribution of 180-190 nm (PDI, 0.1) with zeta-potential of -31 mV. Sucrose was found to be a suitable cryoprotectant at the lipid:sugar molar ratios of 1:5-1:10. This liposomal formulation did not show any evidence of toxicity in C57BL/6 mice treated with the highest doses of PTX (100 mg/kg administered as a single dose and 150 mg/kg as a cumulative dose applied in three equivalent doses in 48-h intervals). A dose-dependent anticancer effect was found in both hollow fibre implants and syngenic B16F10 melanoma mouse tumour models.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Paclitaxel/administração & dosagem , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/uso terapêutico , Antineoplásicos Fitogênicos/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Composição de Medicamentos , Estabilidade de Medicamentos , Liofilização , Lipídeos/química , Lipossomos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nanotecnologia , Paclitaxel/química , Paclitaxel/uso terapêutico , Paclitaxel/toxicidade , Tamanho da Partícula , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The vitamin E analogue alpha-tocopheryl succinate (alpha-TOS) is an efficient anti-cancer drug. Improved efficacy was achieved through the synthesis of alpha-tocopheryl maleamide (alpha-TAM), an esterase-resistant analogue of alpha-tocopheryl maleate. In vitro tests demonstrated significantly higher cytotoxicity of alpha-TAM towards cancer cells (MCF-7, B16F10) compared to alpha-TOS and other analogues prone to esterase-catalyzed hydrolysis. However, in vitro models demonstrated that alpha-TAM was cytotoxic to non-malignant cells (e.g. lymphocytes and bone marrow progenitors). Thus we developed lyophilized liposomal formulations of both alpha-TOS and alpha-TAM to solve the problem with cytotoxicity of free alpha-TAM (neurotoxicity and anaphylaxis), as well as the low solubility of both drugs. Remarkably, neither acute toxicity nor immunotoxicity implicated by in vitro tests was detected in vivo after application of liposomal alpha-TAM, which significantly reduced the growth of cancer cells in hollow fiber implants. Moreover, liposomal formulation of alpha-TAM and alpha-TOS each prevented the growth of tumours in transgenic FVB/N c-neu mice bearing spontaneous breast carcinomas. Liposomal formulation of alpha-TAM demonstrated anti-cancer activity at levels 10-fold lower than those of alpha-TOS. Thus, the liposomal formulation of alpha-TAM preserved its strong anti-cancer efficacy while eliminating the in vivo toxicity found of the free drug applied in DMSO. Liposome-based targeted delivery systems for analogues of vitamin E are of interest for further development of efficient and safe drug formulations for clinical trials.