Preparation of Poly-Lactic-Co-Glycolic Acid Nanoparticles in a Dry Powder Formulation for Pulmonary Antigen Delivery.

One of the key requirements for successful vaccination via the mucosa is particulate antigen uptake. Poly-lactic-co-glycolic acid (PLGA) particles were chosen as well-known model carriers and ovalbumin (OVA) as the model antigen. Aiming at application to the respiratory tract, which allows direct interaction of the formulation with the mucosal immune system, this work focuses on the feasibility of delivering the antigen in a nanoparticulate carrier within a powder capable of pulmonary delivery. Further requirements were adequate antigen encapsulation in order to use the characteristics of the particulate carrier for (tunable) antigen release, and capability of the production process for industrialisation (realisation in industry). For an effective particulate antigen uptake, nanoparticles with a size of around 300 nm were prepared. For this, two production methods for nanoparticles, solvent change precipitation and the double emulsion method, were evaluated with respect to antigen incorporation, transfer to a dry powder formulation, redispersion and antigen release characteristics. A spray drying step was included in the production procedure in order to obtain a respirable powder with an aerodynamic particle size of between 0.5 and 5 µm. The dried products were characterised for particle size, dispersibility and aerodynamic behaviour, as well as for immune response and cytotoxicity in cell culture models. It could be shown that the double emulsion method is suitable to prepare nanoparticles (270 nm) and to incorporate the antigen. By modifying the production method to prepare porous particles, it was possible to obtain an acceptable antigen release while maintaining an antigen load of about 10%. By the choice of polyvinyl alcohol as a stabiliser, nanoparticles could be dried and redispersed without further excipients and the production steps were capable of realisation in industry. Aerodynamic characteristics were good with a mass median aerodynamic diameter of 3.3 µm upon dispersion from a capsule-based inhaler.

Synthesis of furo[3,2-b:4,5-b']diindoles and their optical and electrochemical properties.

An efficient two-step palladium catalyzed synthesis of furo[3,2-b:4,5-b']diindoles, a hitherto unknown symmetrical heterocyclic core structure, was developed. The synthesis is based on a regioselective Suzuki-Miyaura cross coupling reaction of tetrabromofuran and subsequent double N-arylation. Selected compounds were studied with regard to their optical and electrochemical properties. The compounds show fluorescence with high quantum yields and non-reversible oxidation events. The compounds possess similar HOMO-LUMO band gaps compared to their sulfur and nitrogen analogs. Variation of the substituents hardly affects the HOMO-LUMO gap, but allows for some fine-tuning of the electron affinity and ionization potential as well as quantum yields. The compounds prepared represent interesting candidates for the development of organic electronic materials.

A domino reaction of 3-chlorochromones with aminoheterocycles. Synthesis of pyrazolopyridines and benzofuropyridines and their optical and ecto-5'-nucleotidase inhibitory effects.

A new and efficient domino reaction of 3-chlorochromones with electron-rich aminoheterocycles was developed which allows for a convenient synthesis of a variety of pyrazolo[3,4-b]pyridines, pyrrolo[2,3-b]pyridines, pyrido[2,3-d]pyrimidines and benzofuro[3,2-b]pyridines. The products exhibit strong fluorescence. In addition, they exhibit significant ecto-5'-nucleotidase inhibition properties and cytotoxic behavior.

Autologous co-culture of primary human alveolar macrophages and epithelial cells for investigating aerosol medicines. Part I: model characterisation.

The development of new formulations for pulmonary drug delivery is a challenge on its own. New in vitro models which address the lung are aimed at predicting and optimising the quality, efficacy and safety of inhaled drugs, to facilitate the more rapid translation of such products into the clinic. Reducing the complexity of the in vivo situation requires that such models reproducibly reflect essential physiological factors in vitro. The choice of cell types, culture conditions and the experimental set-up, can affect the outcome and the relevance of a study. In the alveolar space of the lung, epithelial cells and alveolar macrophages are the most important cell types, forming an efficient cellular barrier to aerosols. Our aim was to mimic this barrier with primary human alveolar cells. Cell densities of alveolar macrophages and epithelial cells, isolated from the same human donor, were optimised, with a focus on barrier properties. The combination of 300,000 epithelial cells/cm² together with 100,000 macrophages/cm² showed a functional barrier (transepithelial electrical resistance > 500Ω.cm²). This cell model was combined with the Pharmaceutical Aerosol Deposition Device on Cell Cultures. The functionality of the in vitro system was investigated with spray-dried fluorescently labelled poly(lactic-co-glycolic) acid particles loaded with ovalbumin as a model drug.

Antigen delivery via hydrophilic PEG-b-PAGE-b-PLGA nanoparticles boosts vaccination induced T cell immunity.

Here, we evaluate the use of hydrophilic PEG-b-PAGE-b-PLGA (PPP) for the preparation of antigen loaded nanoparticles (NPs) as a platform for prophylactic vaccination. To investigate the suitability of PPP-NPs for antigen delivery, we used the double emulsion evaporation technique to prepare NPs of different sizes, antigen-loading efficiencies and -release kinetics for the model antigen Ovalbumin (OVA). Prior to applying the PPP-NPs in biological in vitro or in vivo models, all materials were tested for absence of cytotoxicity and endotoxins. While the uptake of NPs in antigen presenting cells was size but not polymer dependent, the efficiency of cross presentation of NP-associated antigen on MHC I molecules for CD8 T cell activation depended on the polymer type. T cell activation by antigen-presenting cells was significantly increased in vitro if antigen was delivered via PPP NPs compared to PLGA NPs or soluble OVA, although antigen content was the same in all tested formulations. Subcutaneous application of PPP-OVA-NPs even without adjuvants led to generation of potent CD8 T cell-mediated OVA-specific cytotoxicity in vivo that was more pronounced than after application of OVA alone or PLGA-OVA-NPs. Our data suggest that PPP-NPs can serve as platform for antigen-delivery in future vaccination formulations. Although PPP-NPs already bear intrinsic adjuvant-function, the complementation with TLR ligands loaded inside NPs may further strengthen the immune response to a point, where it might be possible to use it as a therapeutic vaccine to break immune tolerance in chronic disease states.

Efficient synthesis of α- and δ-carbolines by sequential Pd-catalyzed site-selective C-C and twofold C-N coupling reactions.

Two concise and efficient approaches were developed for the synthesis of α- and δ-carboline derivatives. The success of the synthesis relies on site-selective Suzuki-Miyaura reactions of 1-chloro-2-bromopyridine or 2,3-dibromopyridine with 2-bromophenylboronic acid and subsequent cyclization with amines which proceeds by twofold Pd-catalyzed C-N coupling reactions.