ABSTRACT
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).
Subject(s)
Acetylmuramyl-Alanyl-Isoglutamine/analogs & derivatives , Acetylmuramyl-Alanyl-Isoglutamine/pharmacology , Adjuvants, Immunologic/pharmacology , Antigens, Surface/pharmacology , Bacterial Outer Membrane Proteins/pharmacology , Bacterial Vaccines/pharmacology , Borrelia burgdorferi/immunology , Lipoproteins/pharmacology , Acetylmuramyl-Alanyl-Isoglutamine/chemistry , Acetylmuramyl-Alanyl-Isoglutamine/immunology , Adjuvants, Immunologic/chemistry , Animals , Antibody Formation , Antigens, Surface/chemistry , Antigens, Surface/immunology , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/immunology , Bacterial Vaccines/chemistry , Bacterial Vaccines/immunology , Female , HEK293 Cells , Humans , Immunization , Lipoproteins/chemistry , Lipoproteins/immunology , Lyme Disease/immunology , Lyme Disease/microbiology , Mice , Mice, Inbred BALB C , NLR Family, Pyrin Domain-Containing 3 Protein/agonists , NLR Family, Pyrin Domain-Containing 3 Protein/immunology , RAW 264.7 CellsABSTRACT
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".
Subject(s)
Drug Delivery Systems/methods , Nanofibers/chemistry , Pharmaceutical Preparations/administration & dosage , Vaccines/administration & dosage , Adhesives/chemistry , Administration, Buccal , Administration, Sublingual , Animals , Liposomes/chemistry , Lymph Nodes/metabolism , Mice , Mouth Mucosa/metabolism , Nanoparticles/chemistry , Polyethylene Glycols/chemistry , Polyglactin 910/chemistry , Swine , Vaccination/methodsABSTRACT
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.
Subject(s)
Adjuvants, Immunologic , Antibodies, Bacterial/immunology , Antibody Formation/immunology , Antibody Specificity/immunology , Antigens, Bacterial/immunology , Bacterial Outer Membrane Proteins/immunology , Borrelia burgdorferi/immunology , Recombinant Fusion Proteins/immunology , Animals , Antigens, Bacterial/administration & dosage , Antigens, Bacterial/chemistry , Bacterial Outer Membrane Proteins/administration & dosage , Bacterial Outer Membrane Proteins/chemistry , Enzyme-Linked Immunosorbent Assay , Immunization , Lyme Disease/immunology , Mice , Models, Animal , Protein Stability , Protein Structure, Secondary , Proteolipids , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purificationABSTRACT
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.
Subject(s)
Antineoplastic Agents/administration & dosage , Drug Delivery Systems/methods , Lipids/chemistry , Neoplasms/drug therapy , Vitamin E/administration & dosage , Animals , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Chemistry, Pharmaceutical , Humans , Liposomes , Neoplasms/pathology , Solubility , Vitamin E/analogs & derivatives , Vitamin E/chemistry , alpha-Tocopherol/administration & dosageABSTRACT
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.
Subject(s)
Acetylmuramyl-Alanyl-Isoglutamine/analogs & derivatives , Adaptive Immunity/drug effects , Adjuvants, Immunologic/pharmacology , Drug Carriers/chemistry , Immunity, Innate/drug effects , Acetylmuramyl-Alanyl-Isoglutamine/administration & dosage , Acetylmuramyl-Alanyl-Isoglutamine/chemistry , Acetylmuramyl-Alanyl-Isoglutamine/pharmacology , Acetylmuramyl-Alanyl-Isoglutamine/therapeutic use , Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/chemistry , Adjuvants, Immunologic/therapeutic use , Animals , Antibodies, Fungal/blood , Antigens, Fungal/immunology , Female , HSP90 Heat-Shock Proteins/immunology , Liposomes , Mice , Mice, Inbred ICR , Microscopy, Atomic Force , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , Molecular Structure , Nanoparticles , Rabbits , Radiation Injuries, Experimental/immunology , Radiation Injuries, Experimental/prevention & control , Recombinant Proteins/immunology , Survival AnalysisABSTRACT
The main objective of this study was to evaluate the influence of the formulation and process parameters on PLGA microparticles containing a practically insoluble model drug (ibuprofen) prepared by the o/w solvent evaporation method. Multivariate data analysis was used. The effects of altered stirring speed of a mechanical stirrer (600, 1000 rpm), emulsifier concentrations (PVA concentration 0.1 %, 1 %) and solvent selection (dichloromethane, ethyl acetate) on microparticle characteristics (encapsulation efficiency, drug loading, burst effect) were observed. It was found that with increased stirring speed, the PVA concentration or the use of ethyl acetate had a significantly negative effect on encapsulation efficiency. In addition, ethyl acetate had an adverse effect on the burst effect, while increased stirring speed had the opposite effect. Drug load was not affected by any particular variable, but rather by the interactions of evaluated variables.
Subject(s)
Drug Carriers/chemistry , Ibuprofen/administration & dosage , Lactic Acid/chemistry , Microspheres , Polyglycolic Acid/chemistry , Acetates/chemistry , Chemistry, Pharmaceutical/methods , Drug Compounding/methods , Excipients/chemistry , Ibuprofen/chemistry , Methylene Chloride/chemistry , Multivariate Analysis , Polylactic Acid-Polyglycolic Acid Copolymer , Polyvinyl Alcohol/chemistry , Solubility , Solvents/chemistryABSTRACT
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).
Subject(s)
Aorta, Abdominal/pathology , Cat Diseases/pathology , Microbubbles/veterinary , Thromboembolism/veterinary , Ultrasonography, Doppler/veterinary , Animals , Aorta, Abdominal/diagnostic imaging , Cat Diseases/diagnostic imaging , Cats , Contrast Media , Disease Models, Animal , Male , Rabbits , Statistics, Nonparametric , Sulfur Hexafluoride , Thromboembolism/diagnostic imaging , Thromboembolism/pathology , Ultrasonography, Doppler/methodsABSTRACT
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.
Subject(s)
1,2-Dipalmitoylphosphatidylcholine/chemistry , Microbubbles , Polyethylene Glycols/chemistry , Ultrasonography/methods , Animals , Infusions, Intravenous , Male , Phospholipids , Rabbits , Sulfur Hexafluoride , Time FactorsABSTRACT
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.
