RESUMO
Liposomes are one of the most important drug delivery vectors, nowadays used in clinics. In general, polyethylene glycol (PEG) is used to ensure the stealth properties of the liposomes. Here, we have employed hydrophilic, biocompatible and highly non-fouling N-(2-hydroxypropyl) methacrylamide (HPMA)-based copolymers containing hydrophobic cholesterol anchors for the surface modification of liposomes, which were prepared by the method of lipid film hydration and extrusion through 100 nm polycarbonate filters. Efficient surface modification of liposomes was confirmed by transmission electron microscopy, atomic force microscopy, and gradient ultracentrifugation. The ability of long-term circulation in the vascular bed was demonstrated in rabbits after i.v. application of fluorescently labelled liposomes. Compared to PEGylated liposomes, HPMA-based copolymer-modified liposomes did not induce specific antibody formation and did not activate murine and human complement. Compared with PEGylated liposomes, HPMA-based copolymer-modified liposomes showed a better long-circulating effect after repeated administration. HPMA-based copolymer-modified liposomes thus represent suitable new candidates for a generation of safer and improved liposomal drug delivery platforms.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , Lipossomos , Polietilenoglicóis , Propriedades de Superfície , Animais , Coelhos , Camundongos , Polietilenoglicóis/química , Humanos , Ativação do Complemento/efeitos dos fármacos , Acrilamidas/química , Colesterol/química , Colesterol/sangue , Sistemas de Liberação de Medicamentos , Masculino , Polímeros/químicaRESUMO
In this contribution, we focused on a fundamental study targeting the interaction of water-soluble [6]helicene derivative 1 (1-butyl-3-(2-methyl[6]helicenyl)-imidazolium bromide) with double-stranded (ds) DNA. A synthetic 30-base pair duplex, plasmid, chromosomal calf thymus and salmon DNA were investigated using electrochemistry, electrophoresis and spectroscopic tools supported by molecular dynamics (MD) and quantum mechanical approaches. Both experimental and theoretical work revealed the minor groove binding of 1 to the dsDNA. Both the positively charged imidazole ring and hydrophobic part of the side chain contributed to the accommodation of 1 into the dsDNA structure. Neither intercalation into the duplex DNA nor the stable binding of 1 to single-stranded DNA were found in topoisomerase relaxation experiments with structural components of 1, i.e. [6]helicene (2) and 1-butyl-3-methylimidazolium bromide (3), nor by theoretical calculations. Finally, the binding of optically pure enantiomers (P)-1 and (M)-1 was studied using circular dichroism spectroscopy, isothermal titration calorimetry and UV Resonance Raman (UVRR) methods. Using MD and quantum mechanical methods, minor groove and semi-intercalation were proposed for compound 1 as the predominant binding modes. From the UVRR findings, we also can conclude that 1 tends to preferentially interact with adenine and guanine residues in the structure of dsDNA.
RESUMO
Diseases with the highest burden for society such as stroke, myocardial infarction, pulmonary embolism, and others are due to blood clots. Preclinical and clinical techniques to study blood clots are important tools for translational research of new diagnostic and therapeutic modalities that target blood clots. In this study, we employed a three-dimensional (3D) printed middle cerebral artery model to image clots under flow conditions using preclinical imaging techniques including fluorescent whole-body imaging, magnetic resonance imaging (MRI), and computed X-ray microtomography (microCT). Both liposome-based, fibrin-targeted, and non-targeted contrast agents were proven to provide a sufficient signal for clot imaging within the model under flow conditions. The application of the model for clot targeting studies and thrombolytic studies using preclinical imaging techniques is shown here. For the first time, a novel method of thrombus labeling utilizing barium sulphate (Micropaque®) is presented here as an example of successfully employed contrast agents for in vitro experiments evaluating the time-course of thrombolysis and thus the efficacy of a thrombolytic drug, recombinant tissue plasminogen activator (rtPA). Finally, the proof-of-concept of in vivo clot imaging in a middle cerebral artery occlusion (MCAO) rat model using barium sulphate-labelled clots is presented, confirming the great potential of such an approach to make experiments comparable between in vitro and in vivo models, finally leading to a reduction in animals needed.
RESUMO
Introduction of microfluidic mixing technique opens a new door for preparation of the liposomes and lipid-based nanoparticles by on-chip technologies that are applicable in a laboratory and industrial scale. This study demonstrates the role of phospholipid bilayer fragment as the key intermediate in the mechanism of liposome formation by microfluidic mixing in the channel with "herring-bone" geometry used with the instrument NanoAssemblr. The fluidity of the lipid bilayer expressed as fluorescence anisotropy of the probe N,N,N-Trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl) was found to be the basic parameter affecting the final size of formed liposomes prepared by microfluidic mixing of an ethanol solution of lipids and water phase. Both saturated and unsaturated lipids together with various content of cholesterol were used for liposome preparation and it was demonstrated, that an increase in fluidity results in a decrease of liposome size as analyzed by DLS. Gadolinium chelating lipids were used to visualize the fine structure of liposomes and bilayer fragments by CryoTEM. Experimental data and theoretical calculations are in good accordance with the theory of lipid disc micelle vesiculation.
Assuntos
Lipossomos/síntese química , Fluidez de Membrana , Microfluídica/métodos , Nanoestruturas , Materiais Biocompatíveis/metabolismo , Resina de Colestiramina/metabolismo , Polarização de Fluorescência , Dispositivos Lab-On-A-Chip , Microfluídica/instrumentaçãoRESUMO
Development of tools for direct thrombus imaging represents a key step for diagnosis and treatment of stroke. Nanoliposomal carriers of contrast agents and thrombolytics can be functionalized to target blood thrombi by small protein binders with selectivity for fibrin domains uniquely formed on insoluble fibrin. We employed a highly complex combinatorial library derived from scaffold of 46 amino acid albumin-binding domain (ABD) of streptococcal protein G, and ribosome display, to identify variants recognizing fibrin cloth in human thrombus. We constructed a recombinant target as a stretch of three identical fibrin fragments of 16 amino acid peptide of the Bß chain fused to TolA protein. Ribosome display selection followed by large-scale Enzyme-Linked ImmunoSorbent Assay (ELISA) screening provided four protein variants preferentially binding to insoluble form of human fibrin. The most specific binder variant D7 was further modified by C-terminal FLAG/His-Tag or double His-tag for the attachment onto the surface of nanoliposomes via metallochelating bond. D7-His-nanoliposomes were tested using in vitro flow model of coronary artery and their binding to fibrin fibers was demonstrated by confocal and electron microscopy. Thus, we present here the concept of fibrin-targeted binders as a platform for functionalization of nanoliposomes in the development of advanced imaging tools and future theranostics.
RESUMO
Nanodiamonds (ND), especially fluorescent NDs, represent potentially applicable drug and probe carriers for in vitro/in vivo applications. The main purpose of this study was to relate physical-chemical properties of carboxylated NDs to their intracellular distribution and impact on membranes and cell immunity-activation of inflammasome in the in vitro THP-1 cell line model. Dynamic light scattering, nanoparticle tracking analysis, and microscopic methods were used to characterize ND particles and their intracellular distribution. Fluorescent NDs penetrated the cell membranes by both macropinocytosis and mechanical cutting through cell membranes. We proved accumulation of fluorescent NDs in lysosomes. In this case, lysosomes were destabilized and cathepsin B was released into the cytoplasm and triggered pathways leading to activation of inflammasome NLRP3, as detected in THP-1 cells. Activation of inflammasome by NDs represents an important event that could underlie the described toxicological effects in vivo induced by NDs. According to our knowledge, this is the first in vitro study demonstrating direct activation of inflammasome by NDs. These findings are important for understanding the mechanism(s) of action of ND complexes and explain the ambiguity of the existing toxicological data.
Assuntos
Inflamassomos/efeitos dos fármacos , Microscopia Intravital/métodos , Lisossomos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nanodiamantes/administração & dosagem , Catepsina B/imunologia , Catepsina B/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Difusão Dinâmica da Luz , Fluorescência , Humanos , Inflamassomos/imunologia , Inflamassomos/metabolismo , Lisossomos/imunologia , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Microscopia de Força Atômica , Microscopia Confocal , Microscopia Eletrônica , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Nanodiamantes/química , Pinocitose , Células THP-1RESUMO
New synthetic aminooxy lipid was designed and synthesized as a building block for the formulation of functionalised nanoliposomes (presenting onto the outer surface of aminooxy groups) by microfluidic mixing. Orthogonal binding of cellular mannan (Candida glabrata (CCY 26-20-1) onto the outer surface of functionalised nanoliposomes was modified by orthogonal binding of reducing termini of mannans to oxime lipids via a click chemistry reaction based on aminooxy coupling (oxime ligation). The aminooxy lipid was proved as a suitable active component for preparation of functionalised nanoliposomes by the microfluidic mixing method performed with the instrument NanoAssemblr™. This "on-chip technology" can be easily scaled-up. The structure of mannan-liposomes was visualized by transmission and scanning electron microscopy, including immunogold staining of recombinant mannan receptor bound onto mannosylated-liposomes. The observed structures are in a good correlation with data obtained by DLS, NTA, and TPRS methods. In vitro experiments on human and mouse dendritic cells demonstrate selective internalisation of fluorochrome-labelled mannan-liposomes and their ability to stimulate DC comparable to lipopolysaccharide. We describe a potentially new drug delivery platform for mannan receptor-targeted antimicrobial drugs as well as for immunotherapeutics. Furthermore, the platform based on mannans bound orthogonally onto the surface of nanoliposomes represents a self-adjuvanted carrier for construction of liposome-based recombinant vaccines for both systemic and mucosal routes of administration.
Assuntos
Células Dendríticas/imunologia , Lectinas Tipo C/imunologia , Lipossomos/imunologia , Mananas/imunologia , Lectinas de Ligação a Manose/imunologia , Nanopartículas/química , Receptores de Superfície Celular/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Antígenos de Superfície/metabolismo , Candida glabrata/química , Química Click , Humanos , Hidroxilaminas/síntese química , Hidroxilaminas/química , Lipídeos/síntese química , Lipídeos/química , Lipossomos/química , Lipossomos/farmacologia , Mananas/química , Mananas/farmacologia , Receptor de Manose , Camundongos Endogâmicos BALB C , Microfluídica/métodos , Tamanho da PartículaRESUMO
New synthetic aminoxy lipids are designed and synthesized as building blocks for the formulation of functionalized nanoliposomes by microfluidization using a NanoAssemblr. Orthogonal binding of hyaluronic acid onto the outer surface of functionalized nanoliposomes via aminoxy coupling ( N-oxy ligation) is achieved at hemiacetal function of hyaluronic acid and the structure of hyaluronic acid-liposomes is visualized by transmission electron microscopy and cryotransmission electron microscopy. Observed structures are in a good correlation with data obtained by dynamic light scattering (size and ζ-potential). In vitro experiments on cell lines expressing CD44 receptors demonstrate selective internalization of fluorochrome-labeled hyaluronic acid-liposomes, while cells with down regulated CD44 receptor levels exhibit very low internalization of hyaluronic acid-liposomes. A method based on microfluidization mixing was developed for preparation of monodispersive unilamellar liposomes containing aminoxy lipids and orthogonal binding of hyaluronic acid onto the liposomal surface was demonstrated. These hyaluronic acid-liposomes represent a potentially new drug delivery platform for CD44-targeted anticancer drugs as well as for immunotherapeutics and vaccines.
Assuntos
Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/química , Lipídeos/síntese química , Lipossomos/química , Linhagem Celular , Endocitose , Corantes Fluorescentes , Humanos , Receptores de Hialuronatos/análise , Ácido Hialurônico/metabolismo , Lipossomos/uso terapêutico , Microfluídica , Microscopia Eletrônica de Transmissão , Neoplasias/tratamento farmacológicoRESUMO
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
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
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
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.