Simulation-based Reconstructed Diffusion unveils the effect of aging on protein diffusion in Escherichia coli.

We have developed Simulation-based Reconstructed Diffusion (SbRD) to determine diffusion coefficients corrected for confinement effects and for the bias introduced by two-dimensional models describing a three-dimensional motion. We validate the method on simulated diffusion data in three-dimensional cell-shaped compartments. We use SbRD, combined with a new cell detection method, to determine the diffusion coefficients of a set of native proteins in Escherichia coli. We observe slower diffusion at the cell poles than in the nucleoid region of exponentially growing cells, which is independent of the presence of polysomes. Furthermore, we show that the newly formed pole of dividing cells exhibits a faster diffusion than the old one. We hypothesize that the observed slowdown at the cell poles is caused by the accumulation of aggregated or damaged proteins, and that the effect is asymmetric due to cell aging.

Protein diffusion in Escherichia coli cytoplasm scales with the mass of the complexes and is location dependent.

We analyze the structure of the cytoplasm by performing single-molecule displacement mapping on a diverse set of native cytoplasmic proteins in exponentially growing Escherichia coli. We evaluate the method for application in small compartments and find that confining effects of the cell membrane affect the diffusion maps. Our analysis reveals that protein diffusion at the poles is consistently slower than in the center of the cell, i.e., to an extent greater than the confining effect of the cell membrane. We also show that the diffusion coefficient scales with the mass of the used probes, taking into account the oligomeric state of the proteins, while parameters such as native protein abundance or the number of protein-protein interactions do not correlate with the mobility of the proteins. We argue that our data paint the prokaryotic cytoplasm as a compartment with subdomains in which the diffusion of macromolecules changes with the perceived viscosity.

Boosting transfection efficiency: A systematic study using layer-by-layer based gene delivery platform.

Nowadays, the nanoparticle-based delivery approach is becoming more and more attractive in gene therapy due to its low toxicity and immunogenicity, sufficient packaging capacity, targeting, and straightforward, low-cost, large-scale good manufacturing practice (GMP) production. A number of research works focusing on multilayer structures have explored different factors and parameters that can affect the delivery efficiency of pDNA. However, there are no systematic studies on the performance of these structures for enhanced gene delivery regarding the gene loading methods, the use of additional organic components and cell/particle incubation conditions. Here, we conducted a detailed analysis of different parameters such as (i) strategy for loading pDNA into carriers, (ii) incorporating both pDNA and organic additives within one carrier and (iii) variation of cell/particle incubation conditions, to evaluate their influence on the efficiency of pDNA delivery with multilayer structures consisting of inorganic cores and polymer layers. Our results reveal that an appropriate combination of all these parameters leads to the development of optimized protocols for high transfection efficiency, compared to the non-optimized process (> 70% vs. < 7%), and shows a good safety profile. In conclusion, we provide the proof-of-principle that these multilayer structures with the developed parameters are a promising non-viral platform for an efficient delivery of nucleic acids.

Layer-by-Layer technique as a versatile tool for gene delivery applications.

Introduction: Gene therapy is a breakthrough medical field which focuses on the therapeutic delivery of recombinant nucleic acids in order to treat or prevent a broad spectrum of diseases. However, a number of important obstacles remain before its wide introduction into clinical practice can be envisaged. One of the biggest bottlenecks is the lack of efficient and safe delivery technologies, particularly, for in vivo distribution. Above and beyond standard requirements for carriers, the delivery systems for gene therapy ideally use a hit-and-run principle (to minimize off-target effect and display of immunogenic moieties). None of the currently used viral vectors fulfills all of these requirements. Therefore, the growing variety of non-viral delivery platforms represents a promising alternative.Areas covered: This review summarizes the Layer-by-Layer (LbL) approaches that can be effectively used for the gene delivery, considering various examples with the transfer of pDNA, mRNA, siRNA as well as genome-editing tools. Ex vivo gene modification of clinically relevant cells and clinical aspects for possible application of LbL systems in gene therapy are also underlined.Expert opinion: The LbL technique provides broad opportunities for the delivery of genetic material for various purposes and offers promise for future clinical application in gene therapy.