The influence of amphiphilic carbosilane dendrons on lipid model membranes.

Amphiphilic dendrons represent a relatively novel class of molecules which may show many unique properties suitable for applications in a field of molecular biology and nanomedicine. They were frequently studied as platforms suitable for drug delivery systems as were, e.g. polymersomes or hybrid lipid-polymer nanoparticles. Recently, natural extracellular lipid vesicles (EVs), called exosomes (EXs), were shown to be a promising candidate in drug delivery applications. Formation of hybrid exosome-dendron nanovesicles could bring benefits in their simple conjugation with selective targeting moieties. Unfortunately, the complex architecture of biological membranes, EXs included, makes obstacles in elucidating the important parameters and mechanisms of interaction with the artificial amphiphilic structures. The aim of the presented work was to study the interaction of two types of amphiphilic carbosilane dendritic structures (denoted as DDN-1 and DDN-2) suitable for further modification with streptavidin (DDN-1) or using click-chemistry approach (DDN-2), with selected neutral and negatively charged lipid model membranes, partially mimicking the basic properties of natural EXs biomembranes. To meet the goal, a number of biophysical methods were used for determination of the degree and mechanisms of the interaction. The results showed that the strength of interactions of amphiphilic dendrons with liposomes was related with surface charge of liposomes. Several steps of interactions were disclosed. The initialization step was mainly coupled with amphiphilic dendrons - liposomes surface interaction resulting in destabilization of large self-assembled amphiphilic dendrons structures. Such destabilization was more significant with liposomes of higher negative charge. With increasing concentration of amphiphilic dendrons in a solution the interactions were taking place also in the hydrophobic part of bilayer. Further increase of nanoparticle concentration resulted in a gradual dendritic cluster formation in a lipid bilayer structure. Due to high affinity of amphiphilic dendrons to model lipid bilayers the conclusion can be drawn that they represent promising platforms also for decoration of exosomes or other kinds of natural lipid vehicles. Such organized hybrid dendron-lipid biomembranes may be advantageous for their subsequent post-functionalization with small molecules, large biomacromolecules or polymers suitable for targeted drug-delivery or theranostic applications.

Immunocapturing rare cells from blood: A simple and robust microsystem approach.

Cell immunocapture microsystems are a fast-emerging field with several potential medical diagnostic applications. Isolation and quantification of circulating rare cells (CRCs) show great importance in the early stages of disease diagnostics and prognostics. Here, we present a simple and robust stop-flow microsystem (fabricated by a combination of glass microblasting and 3D printing) based on a planar antibody-coated surface that is effective in the immunocapture of the model as well as naturally occurring rare cells. A chip with a planar immunocapture channel working in the so-called stop-flow dynamic regime was designed to enable monitoring the efficiency of the cell capture by fluorescence microscopy. Up to 90% immunocapture efficiency of MCF-7 cells spiked into whole blood on CD326 antibody-coated planar surfaces was achieved. We discuss the role of the planar surface modifications, the influence of the set stop-flow dynamic conditions, and medium complexity on the efficiency of cell immunocapture. The presented results could be further employed in the design of microsystems for cell-size-independent isolation and identification of rare cells from blood.

Mucus-derived exosome-like vesicles from the Spanish slug (Arion vulgaris): taking advantage of invasive pest species in biotechnology.

The slug Arion vulgaris has attracted major attention as one of the worst invasive herbivore pests in Europe and is renowned for the stiff mucus it secretes for locomotion. In this study we focused on the isolation and characterisation of extracellular vesicles, specifically exosomes and exosome-like vesicles, from Arion secretions. We developed a method for slug mucus collection and subsequent vesicle isolation by ultracentrifugation. The isolated vesicles with an average diameter of ~ 100 nm carry abundant proteins and short RNAs, as well as adhesion molecules similar to mammalian galectins. We demonstrated that the slug extracellular vesicles are internalised by plant cells and human cancer cells in in vitro assays and are loadable by bioactive compounds, which makes them an interesting tool for utilisation in biotechnology.

Conventional and Nonconventional Sources of Exosomes-Isolation Methods and Influence on Their Downstream Biomedical Application.

Despite extensive study of extracellular vesicles (EVs), specifically exosomes (EXs) as biomarkers, important modulators of physiological or pathological processes, or therapeutic agents, relatively little is known about nonconventional sources of EXs, such as invertebrate or plant EXs, and their uses. Likewise, there is no clear information on the overview of storage conditions and currently used isolation methods, including new ones, such as microfluidics, which fundamentally affect the characterization of EXs and their other biomedical applications. The purpose of this review is to briefly summarize conventional and nonconventional sources of EXs, storage conditions and typical isolation methods, widely used kits and new "smart" technologies with emphasis on the influence of isolation techniques on EX content, protein detection, RNA, mRNA and others. At the same time, attention is paid to a brief overview of the direction of biomedical application of EXs, especially in diagnostics, therapy, senescence and aging and, with regard to the current situation, in issues related to Covid-19.

Cell immunocapture microfluidic chip based on high-affinity recombinant protein binders.

Cell immunocapture microfluidic devices represent a rapidly developing field with many potential applications in medical diagnostics. The core of such approach lies in the cell binding to antibody coated surfaces through their surface receptors. Here we show, that the small recombinant protein binders (PBs) can be used for this purpose as well, with the advantage of their constructional flexibility, possibility of fusion with range of tags and cheap mass production. For this purpose, two different PBs derived from Albumin Binding Domain (ABDwt) of streptococcal protein G, so called REX and ARS ligands with proved high affinity and selectivity to the human interleukin-23 (IL-23R) and IL-17 receptor A were used. Four PBs variants recognizing two different epitopes on two different receptors and two PBs variants binding to the same epitope on one receptor but having different peptide spacer with Avitag sequence necessary for their immobilization on sensor surface were tested for cell-capture efficiency. The glass microfluidic Y-system with planar immunocapture channel working in so-called stop-flow dynamic regime was designed. Up to 60-74% immunocapture efficiency of model THP-1 cells on REX/ARS surfaces and practically no cell binding on control ABDwt surfaces was achieved. Moreover, the specific immunocapture of THP-1 cells from mixture with IL-17RA negative DU-145 cells was demonstrated. We discuss the role of the epitope, affinity and immobilization spacer of PBs as well as the influence of stop-flow dynamic regime on the effectivity of THP-1 cell immunocapture. Results can be further exploited in design of microfluidic devices for rare cells immunocapture.

Carbosilane dendrimers with phosphonium terminal groups are low toxic non-viral transfection vectors for siRNA cell delivery.

Non-viral gene delivery vectors studied in the gene therapy applications are often designed with the cationic nitrogen containing groups necessary for binding and cell release of nucleic acids. Disadvantage is a relatively high toxicity which restricts the in vivo use of such nanoparticles. Here we show, that the 3rd generation carbosilane dendrimers possessing (trimethyl)phosphonium (PMe3) groups on their periphery were able to effectively deliver the functional siRNA into the cells (B14, Cricetulus griseus), release it into the cytosol and finally to achieve up to 40% gene silencing of targeted gene (glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) with the comparable or, in some cases, even better effectivity as their ammonium counterparts. Moreover, such cationic dendrimers show relatively low in vivo toxicity as compared to their ammonium analogues when analyzed by standard fish embryo test (FET) on Danio rerio in vivo model, with LD50 = 6.26 µM after 48 h of incubation. This is more than 10-fold improvement as compared to published values for various other types of cationic dendrimers. We discuss the potential of further increase of the transfection efficiency, endosomal escape and decrease of toxicity of such non-viral vectors, based on the systematic screening of different types of substituents on central phosphonium atom.

A simple approach for fabrication of optical affinity-based bioanalytical microsystem on polymeric PEN foils.

Herein, we report a novel concept of low-cost flexible platform for fluorescence-based biosensor. The surface of polyethylene naphthalate (PEN) foil was exposed to KrF excimer laser through a photolitographic contact mask. Laser initiated surface modification resulted in micro-patterned areas with surface functional groups available for localized covalent immobilization of biotin. High affinity binding protein (albumin-binding domain (ABD) of protein G, Streptococcus G148) recognizing human serum albumin (HSA), genetically fused with streptavidin (SA-ABDwt), was immobilized on the micro-patterned surface through biotin-streptavidin coupling. Fluorescently labelled HSA analyte was detected in several blocking environments, in 1% bovine serum albumin (BSA) and 6% fetal serum albumin (FBS), respectively. We conclude that the presented novel concept enabled us to micropattern functional biosensing layers on the surface of PEN foil in a fast and easy way. It brings all necessary aspects for continuous roll-to-roll fabrication of low-cost optical bioanalytical devices.

Biocompatible Size-Defined Dendrimer-Albumin Binding Protein Hybrid Materials as a Versatile Platform for Biomedical Applications.

For the design of a biohybrid structure as a ligand-tailored drug delivery system (DDS), it is highly sophisticated to fabricate a DDS based on smoothly controllable conjugation steps. This article reports on the synthesis and the characterization of biohybrid conjugates based on noncovalent conjugation between a multivalent biotinylated and PEGylated poly(amido amine) (PAMAM) dendrimer and a tetrameric streptavidin-small protein binding scaffold. This protein binding scaffold (SA-ABDwt) possesses nM affinity toward human serum albumin (HSA). Thus, well-defined biohybrid structures, finalized by binding of one or two HSA molecules, are available at each conjugation step in a controlled molar ratio. Overall, these biohybrid assemblies can be used for (i) a controlled modification of dendrimers with the HSA molecules to increase their blood-circulation half-life and passive accumulation in tumor; (ii) rendering dendrimers a specific affinity to various ligands based on mutated ABD domain, thus replacing tedious dendrimer-antibody covalent coupling and purification procedures.

Direct immobilization of biotin on the micro-patterned PEN foil treated by excimer laser.

Polymers with functionalized surfaces have attracted a lot of attention in the last few years. Due to the progress in the techniques of polymer micro-patterning, miniaturized bioanalytical assays and biocompatible devices can be developed. In the presented work, we performed surface modification of polyethylene naphthalate (PEN) foil by an excimer laser beam through a photolithographic contact mask. The aim was to fabricate micro-patterned areas with surface functional groups available for localized covalent immobilization of biotin. It was found out that depending on the properties of the laser scans, a polymer surface exhibits different degrees of modification and as a consequence, different degrees of surface biotinylation can be achieved. Several affinity tests with optical detection of fluorescently labeled streptavidin were successfully performed on biotinylated micro-patterns of a PEN foil. The polymer surface properties were also evaluated by electrokinetic analysis, Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The results have shown that PEN foils can be considered suitable substrates for construction of micro-patterned bioanalytical affinity assays.

A new concept in the separation of polyethyleneglycol-modified PAMAM dendrons by liquid chromatography.

In this work, a unique high-performance liquid chromatographic method was developed and applied for monitoring the synthesis of polyethyleneglycol surface modified poly(amidoamine) cystamine core dendrimers (PEG-PAMAMs) and PEG-PAMAM-alkynes with a single alkyne moiety attached to the core of a dendron through a unique sulfhydryl group. The separation of the products was performed on a column with a pentafluorphenylpropyl stationary phase, allowing multiple mechanisms of selectivity. More than 50 peaks were separated in one run, reflecting the degree of dendrimer PEGylation (PEG average molecular mass: 3,000). Moreover, modification of PAMAM with a single alkyne group could be distinguished. The developed method can be used for the general characterization and separation of PAMAM derivatives, in which the degree of modification is critical for final applications.

Intrinsically disordered enamel matrix protein ameloblastin forms ribbon-like supramolecular structures via an N-terminal segment encoded by exon 5.

Tooth enamel, the hardest tissue in the body, is formed by the evolutionarily highly conserved biomineralization process that is controlled by extracellular matrix proteins. The intrinsically disordered matrix protein ameloblastin (AMBN) is the most abundant nonamelogenin protein of the developing enamel and a key element for correct enamel formation. AMBN was suggested to be a cell adhesion molecule that regulates proliferation and differentiation of ameloblasts. Nevertheless, detailed structural and functional studies on AMBN have been substantially limited by the paucity of the purified nondegraded protein. With this study, we have developed a procedure for production of a highly purified form of recombinant human AMBN in quantities that allowed its structural characterization. Using size exclusion chromatography, analytical ultracentrifugation, transmission electron, and atomic force microscopy techniques, we show that AMBN self-associates into ribbon-like supramolecular structures with average widths and thicknesses of 18 and 0.34 nm, respectively. The AMBN ribbons exhibited lengths ranging from tens to hundreds of nm. Deletion analysis and NMR spectroscopy revealed that an N-terminal segment encoded by exon 5 comprises two short independently structured regions and plays a key role in self-assembly of AMBN.

Novel high-affinity binders of human interferon gamma derived from albumin-binding domain of protein G.

Recombinant ligands derived from small protein scaffolds show promise as robust research and diagnostic reagents and next generation protein therapeutics. Here, we derived high-affinity binders of human interferon gamma (hIFNγ) from the three helix bundle scaffold of the albumin-binding domain (ABD) of protein G from Streptococcus G148. Computational interaction energy mapping, solvent accessibility assessment, and in silico alanine scanning identified 11 residues from the albumin-binding surface of ABD as suitable for randomization. A corresponding combinatorial ABD scaffold library was synthesized and screened for hIFNγ binders using in vitro ribosome display selection, to yield recombinant ligands that exhibited K(d) values for hIFNγ from 0.2 to 10 nM. Molecular modeling, computational docking onto hIFNγ, and in vitro competition for hIFNγ binding revealed that four of the best ABD-derived ligands shared a common binding surface on hIFNγ, which differed from the site of human IFNγ receptor 1 binding. Thus, these hIFNγ ligands provide a proof of concept for design of novel recombinant binding proteins derived from the ABD scaffold.