Enhanced Uptake and Endosomal Release of LbL Microcarriers Functionalized with Reversible Fusion Proteins.

The efficient application of smart drug-delivery systems requires further improvement of their cellular uptake and in particular their release from endolysosomal compartments into the cytoplasm of target cells. The usage of virus proteins allows for such developments, as viruses have evolved efficient entry mechanisms into the cell, mediated by their fusion proteins. In our investigations, the transferability of the glycoprotein G which is a fusion protein of the vesicular stomatitis virus (VSV-G) onto the surface of a layer-by-layer (LbL) designed microcarrier was investigated. The assembly of VSV-G as a reversible viral fusion protein onto LbL microcarriers indeed induced an enhanced uptake rate on Vero cells as well as a fast and efficient release of the intact carriers from endolysosomes into the cytoplasm. Additionally, neither virus-associated effects on cellular viability nor activation of an interferon response were detected. Our study emphasizes the suitability of VSV-G as an efficient surface functionalization of drug-delivery systems.

A New Genotype of Feline Morbillivirus Infects Primary Cells of the Lung, Kidney, Brain and Peripheral Blood.

Paramyxoviruses comprise a large number of diverse viruses which in part give rise to severe diseases in affected hosts. A new genotype of feline morbillivirus, tentatively named feline morbillivirus genotype 2 (FeMV-GT2), was isolated from urine of cats with urinary tract diseases. Whole genome sequencing showed about 78% nucleotide homology to known feline morbilliviruses. The virus was isolated in permanent cell lines of feline and simian origin. To investigate the cell tropism of FeMV-GT2 feline primary epithelial cells from the kidney, the urinary bladder and the lung, peripheral blood mononuclear cells (PBMC), as well as organotypic brain slice cultures were used for infection experiments. We demonstrate that FeMV-GT2 is able to infect renal and pulmonary epithelial cells, primary cells from the cerebrum and cerebellum, as well as immune cells in the blood, especially CD4⁺ T cells, CD20⁺ B cells and monocytes. The cats used for virus isolation shed FeMV-GT2 continuously for several months despite the presence of neutralizing antibodies in the blood. Our results point towards the necessity of increased awareness for this virus when clinical signs of the aforementioned organs are encountered in cats which cannot be explained by other etiologies.

Reversible Fusion Proteins as a Tool to Enhance Uptake of Virus-Functionalized LbL Microcarriers.

For the efficient treatment of an increasing number of diseases the development of new therapeutics as well as novel drug delivery systems is essential. Such drug delivery systems (DDS) must not only consider biodegradability and protective packaging but must also target and control the release of active substances, which is one of the most important points in DDS application. We highlight the improvement of these key aspects, the increased interaction rate of Layer-by-Layer (LbL) designed microcarriers as a promising DDS after functionalization with vesicular stomatitis virus (VSV). We make use of the unique conformational reversibility of the fusion protein of VSV as a surface functionalization of LbL microcarriers. This reversibility allows for VSV to be used both as a tool for assembly onto the DDS and as an initiator for an efficient cellular uptake. We could show that the evolutionary optimized viral fusion machinery can be successfully combined with a biophysical DDS for optimization of its cellular interaction.

Enhanced cytoplasmic release of drug delivery systems: chloroquine as a multilayer and template constituent of layer-by-layer microcarriers.

Nano- and microcarriers as vehicles for active agents are applied to support them to reach their target in a defined, specific, and protected way. This implies not only a safe transport of agents towards the desired cell type or tissue but also the intracellular processing of the carrier: in particular, release of the incorporated carriers into the cytoplasm is a prerequisite for the successful subsequent delivery of most active agents and is often impeded by endolysosomal degenerative enzymes. We address this issue by using the layer-by-layer strategy of carrier assembly offering the opportunity to independently integrate and carry active agents but also specific agents preventing endolysosomal acidification. The weak base chloroquine (CQ) was investigated as a multilayer, template and capsule constituent regarding its ability to delay endolysosomal acidification and prolong the tolerable time frame in endolysosomes, which allows the carrier to finally escape into the cytoplasm. As a model and reporter active agent, plasmid encoding enhanced green fluorescent protein was used as a multilayer-assembly component to illustrate the cytoplasmic release of the intact carrier by final expression of the green fluorescent protein. Integrating CQ into the carrier, GFP expression could be strongly increased and a transfection efficiency of up to 20% could be obtained. This represents a very high transfection rate for a drug delivery system reached by only one additional reagent that has no further influence on the activity of the transported drug and cell viability, offering a significantly enhanced delivery efficiency.

Specific Uptake of Lipid-Antibody-Functionalized LbL Microcarriers by Cells.

The modular construction of Layer-by-Layer biopolymer microcarriers facilitates a highly specific design of drug delivery systems. A supported lipid bilayer (SLB) contributes to biocompatibility and protection of sensitive active agents. The addition of a lipid anchor equipped with PEG (shielding from opsonins) and biotin (attachment of exchangeable outer functional molecules) enhances the microcarrier functionality even more. However, a homogeneously assembled supported lipid bilayer is a prerequisite for a specific binding of functional components. Our investigations show that a tightly packed SLB improves the efficiency of functional components attached to the microcarrier's surface, as illustrated with specific antibodies in cellular application. Only a low quantity of antibodies is needed to obtain improved cellular uptake rates independent from cell type as compared to an antibody-functionalized loosely packed lipid bilayer or directly assembled antibody onto the multilayer. A fast disassembly of the lipid bilayer within endolysosomes exposing the underlying drug delivering multilayer structure demonstrates the suitability of LbL-microcarriers as a multifunctional drug delivery system.

Polyacrylamide gel substrates that simulate the mechanical stiffness of normal and malignant neuronal tissues increase protoporphyin IX synthesis in glioma cells.

Protoporphyrin IX (PPIX) produced following the administration of exogenous 5d-aminolevulinic acid is clinically approved for photodynamic therapy and fluorescence-guided resection in various jurisdictions around the world. For both applications, quantification of PPIX forms the basis for accurate therapeutic dose calculation and identification of malignant tissues for resection. While it is well established that the PPIX synthesis and accumulation rates are subject to the cell's biochemical microenvironment, the effect of the physical microenvironment, such as matrix stiffness, has received little attention to date. Here we studied the proliferation rate and PPIX synthesis and accumulation in two glioma cell lines U373 and U118 cultured under five different substrate conditions, including the conventional tissue culture plastic and polyacrylamide gels that simulated tissue stiffness of normal brain (1 kPa) and glioblastoma tumors (12 kPa). We found that the proliferation rate increased with substrate stiffness for both cell lines, but not in a linear fashion. PPIX concentration was significantly higher in cells cultured on tissue-simulating gels than on the much stiffer tissue culture plastic for both cell lines. These findings, albeit preliminary, suggest that the physical microenvironment might be an important determinant of tumor aggressiveness and PPIX synthesis in glioma cells.

Synthesis and characterisation of lanthanide-hydroporphyrin dyads.

Fluorescence spectroscopy is in many ways the ideal tool for the interrogation of complex biological systems, as it is non-invasive, sensitive, and offers high spatiotemporal resolution. For biomedical imaging luminescent probes absorbing and emitting in the red-to-near infrared (NIR) region are best suited to maximise tissue penetration and minimise damage to cellular components. NIR-emitting lanthanides (Ln) sensitised with red-absorbing antennae are promising candidates for these applications, assuming the challenges of poor photophysical properties and tedious syntheses of the complexes are overcome. Chlorins are porphyrin-type tetrapyrroles with intense red absorption. Recently chlorins have been shown to sensitise Yb and Nd emission when incorporated into Ln-complexes. Here we expand on our previous work, and explore the effect of chlorin structure, metallation state, chlorin-Ln-complex linker length and mode of attachment on the properties of chlorin-Ln complexes. As chlorin absorption bands are ∼20 nm fwhm and readily tunable, a deeper understanding of structure-property relationships would enable the use of chlorin-Ln complexes in multicolour imaging using antenna-specific excitation. A detailed description of antenna and complex syntheses and photophysical characterisation is given. A number of challenges were identified, which will have to be addressed in future studies to enable multicolour imaging using the NIR-emitting lanthanides.