Multi-layered nanofibrous mucoadhesive films for buccal and sublingual administration of drug-delivery and vaccination nanoparticles - important step towards effective mucosal vaccines.

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".

The Position of His-Tag in Recombinant OspC and Application of Various Adjuvants Affects the Intensity and Quality of Specific Antibody Response after Immunization of Experimental Mice.

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.

Antiviral activities of 2,6-diaminopurine-based acyclic nucleoside phosphonates against herpesviruses: In vitro study results with pseudorabies virus (PrV, SuHV-1).

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.

Specific contrast ultrasound using sterically stabilized microbubbles for early diagnosis of thromboembolic disease in a rabbit model.

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).

A prototype 'Infucon' device for continuous infusion of microbubbles in vivo.

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.

Enhancement of immune response towards non-lipidized Borrelia burgdorferi recombinant OspC antigen by binding onto the surface of metallochelating nanoliposomes with entrapped lipophilic derivatives of norAbuMDP.

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.

Preparation of metallochelating microbubbles and study on their site-specific interaction with rGFP-HisTag as a model protein.

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.

Metallochelating liposomes with associated lipophilised norAbuMDP as biocompatible platform for construction of vaccines with recombinant His-tagged antigens: preparation, structural study and immune response towards rHsp90.

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.

Liposomes with high encapsulation capacity for paclitaxel: Preparation, characterisation and in vivo anticancer effect.

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.

Heterologous expression and molecular characterization of the NAD(P)H:acceptor oxidoreductase (FerB) of Paracoccus denitrificans.

FerB is a flavoenzyme capable of reducing quinones, ferric complexes and chromate. Its expression in Escherichia coli as a hexahistidine fusion resulted in a functional product only when the tag was placed on the C-terminus. The molecular mass values estimated by gel permeation chromatography were compatible with the existence of either dimer or trimer, whereas the light scattering data, together with cross-linking experiments that yielded exclusively monomer and dimer bands on dodecyl sulfate-polyacrylamide gels, strongly supported a dimeric nature of both native and tagged form of FerB. These two proteins also exhibited almost identical secondary structure as judged by Fourier transform infra red spectrometry. The presence of tag, however, shifted the temperature of thermal inactivation as well as the thermal denaturation curve towards lower temperatures. Despite somewhat lower thermal stability, the fusion protein is considered a better candidate for crystallization than the wild-type one due to a more negative value of its second optical viral coefficient.

Different elution modes and field programming in gravitational field-flow fractionation: field programming using density and viscosity gradients.

In previous papers, several approaches to programming of the resulting force field in GFFF were described and investigated. The experiments were dealing with flow-velocity and channel thickness, i.e. factors influencing hydrodynamic lift forces (HLF). The potential of density and viscosity of carrier liquid for field programming was predicted and demonstrated by preliminary experiments. This work is devoted to experimental verification of the influence of carrier liquid density and viscosity. Several carrier liquid density and simultaneously viscosity gradients using water-methanol mixtures are in this work implemented in the separation of a model silica mixture. Working with the water-methanol gradients, one is not able to separate the influence of density from the contribution of viscosity. However, we found experimental conditions to show the isolated effect of carrier liquid density (two water-methanol mixtures of equal viscosity differing in their densities). In order to demonstrate the isolated effect of viscosity, we implemented in this work a new system of (hydroxypropyl)methyl cellulose (HPMC) carrier liquids. Three different HPMC compositions enabled to vary the viscosity more than two times at almost constant density. With increasing carrier liquid viscosity, the focusing and elevating trend was clearly pronounced for 5 and 10 microm silica particles. By the isolated effect of increased viscosity, the centre of the 10 microm particle zone was elevated to the streamline at 16% of the channel height. These experiments have shown that the influence of carrier liquid viscosity on HLF should be taken into account even at higher levels above the channel bottom, i.e. beyond the near-wall region. Further, it is shown that higher value of carrier liquid viscosity improves the separation of the model mixture in terms of time and resolution.

Different elution modes and field programming in gravitational field-flow fractionation. IV. Field programming achieved with channels of non-constant cross-sections.

Force field programming provided increased speed of separation with an improved resolution and detection capability in many field-flow fractionation (FFF) techniques. Gravitational field-flow fractionation (GFFF) uses the Earth's gravitational field to cause the settlement of particles towards the channel accumulation wall. Although this field is constant and relatively weak, there are different ways to implement force field programming in GFFF. Because hydrodynamic lift forces (HLF) participate in the separation process in focusing (hyperlayer) elution mode, they can control the resulting force field acting on particles via changes in flow-velocity. These changes can be accomplished by a programmable pump or with channels of non-constant cross-sections. This work is focused on flow-velocity programming accomplished with channels of non-constant cross-sections. Three trapezoidal channels of decreasing breadth and two channels of decreasing height (along the longitudinal axis) are tested as tools for optimization of the separation of a model silica gel particle mixture. The trapezoidal channels yielded reduced separation times. However, taking into account both speed of separation and resolution, the optimization effect was lower compared with the flow-rate gradients generated by a programmable pump. The channels of non-constant height did not yield advantageous separations.