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BackgroundEndogenously expressed microRNAs (miRNAs) have attracted attention as important regulators in post-transcriptionally controlling gene expression of various physiological processes. As miRNA dysregulation is often associated with various disease patterns, such as obesity, miRNA-27a might therefore be a promising candidate for miRNA mimic replacement therapy by inhibiting adipogenic marker genes. However, application of naked nucleic acids faces some limitations concerning poor enzymatic stability, bio-membrane permeation and cellular uptake. To overcome these obstacles, the development of appropriate drug delivery systems (DDS) for miRNAs is of paramount importance.MethodsIn this work, a triple combination of atomic force microscopy (AFM), brightfield (BF) and fluorescence microscopy was used to trace the cellular adhesion of N-TER peptide-nucleic acid complexes followed by time-dependent uptake studies using confocal laser scanning microscopy (cLSM). To reveal the biological effect of miRNA-27a on adipocyte development after transfection treatment, Oil-Red-O (ORO)- staining was performed to estimate the degree of in lipid droplets accumulated ORO in mature adipocytes by using light microscopy images as well as absorbance measurements.ResultsThe present findings demonstrated that amphipathic N-TER peptides represent a suitable DDS for miRNAs by promoting non-covalent complexation through electrostatic interactions between both components as well as cellular adhesion of the N-TER peptide – nucleic acid complexes followed by uptake across cell membranes and intracellular release of miRNAs. The anti-adipogenic effect of miRNA-27a in 3T3-L1 cells could be detected in mature adipocytes by reduced lipid droplet formation.ConclusionThe present DDS assembled from amphipathic N-TER peptides and miRNAs is capable of inducing the anti-adipogenic effect of miRNA-27a by reducing lipid droplet accumulation in mature adipocytes. With respect to miRNA mimic replacement therapies, this approach might provide new therapeutic strategies to prevent or treat obesity and obesity-related disorders.

Micro RNA (miRNA)-based medicines have attracted attention as new therapeutic strategies to treat genetic diseases and metabolic and immunological disorders. MiRNAs have emerged as key mediators of metabolic processes fulfilling regulatory functions in maintaining physiological conditions, while altered miRNA expression profiles are often associated with genetic diseases. However, naked miRNAs exhibit poor enzymatic stability, biomembrane permeation, and cellular uptake. To overcome these limitations, the development of appropriate drug delivery systems (DDS) is necessary. Herein, a DDS is characterized being assembled from miRNA-27a (negative regulator in fat metabolism) and the amphipathic N-TER peptide. Dynamic light scattering (DLS), electrophoretic light scattering, and atomic force microscopy (AFM) are used to investigate physicochemical properties (i.e., size, shape, and charge) of the DDS. Although surface charges should provide decent stabilization, the AFM results confirm a state of agglomeration, which is also suggested by DLS. Furthermore, AFM studies reveal adhesion on hydrophilic as well as hydrophobic substrates, which is related to the amphipathic properties of the N-TER peptide. Physicochemical properties of DDS are important parameters, which have an impact on cell internalization/uptake and have to be taken into account for in vitro studies to develop a successful peptide-based DDS for miRNA replacement therapy in metabolic diseases, such as obesity and others.

Apolipoprotein B-100 (apo B-100) is the protein moiety of both low-and verylow-density lipoproteins, whose role is crucial to cholesterol and triglyceride transport. Aiming at the molecular dynamics' details of apo B-100, scarcely studied, we performed elastic and quasi-elastic incoherent neutron scattering (EINS, QENS) experiments combining different instruments and time scales. Similar to classical membrane proteins, the solubilization results in remaining detergent, here Nonidet P-40 (NP40). Therefore, we propose a framework for QENS studies of protein−detergent complexes, with the introduction of a combined model, including the experimental apo B-100/NP40 ratio. Relying on the simultaneous analysis of all QENS amplitudes, this approach is sensitive enough to separate both contributions. Its application identified two points: (i) apo B-100 slow dynamics and (ii) the acceleration of NP40 dynamics in the presence of apo B-100. Direct translation of the exposed methodology now makes the investigation of more membrane proteins by neutron spectroscopy achievable.