Nanomembranes in biotechnology: Separation of small and large biomolecules

Membranes are widely applied in many industrial areas. However, due to the thickness of the membranes the transport of biomolecules across a membrane is impeded by the relatively long transport times. Nanomembranes, in contrast, provide ultra-fast diffusion times and speed up the transfer of biomolecules significantly. Difficulties in production, the fragility, and hydrophobicity of current nanomembranes prevented their widespread use. However, the key characteristics of nanomembranes are very attractive for a broad variety of applications in e.g. biomedical applications, bioseparation technologies, biosensors, and membrane bioreactors. We have made from hydrophilic polymers in waterproof porous nanomembranes and they are therefore especially suited for use in aqueous media, such as used in biological systems. It can withstand more than tens of thousands time its own weight in water and can be used in a wide pH range and in the presence of a broad range of electrolytes. The porous hydrophilic nanomembranes surface is planar with a thickness between 50-150 nm only with uniform pores and a tensile strength of at least 0.1 MPa. Pore diameter can be adjusted according to application from 10 nm to 500 nm. Currently our porous hydrophilic nanomembranes can be produced on large scale from 1 to 250 cm2. It can additionally comprise (embedded or on the surface) bioactive substances, such as enzymes, substrate receptors, active drugs, etc.. The use of our porous hydrophilic nanomembranes allows for ultra-high speed diffusion of biomolecules, but also selective transport of compounds. Please click Additional Files below to see the full abstract

Conductive nanothick gold on hydrophilic polymeric nanomembranes

The properties of separation membranes have been predicted and proven to be outstanding when their thickness approaches the dimensions of the molecules being separated. Ultrafast diffusion and high selectivity of such nanomembranes promise significant economic benefits by fewer and shorter processes with lower pressures. However, their widespread and industrial application is commonly impaired by poor biocompatibility and laborious, costly fabrication of currently used materials. Here we present the fabrication of self-supporting, hydrophilic, permeable nanomembranes from a thermosetting resin. A facile spin-coating procedure is employed which can be altered to yield two different kinds of porosity: (i) diffusion channels intrinsic to the covalently crosslinked resin network allowing small molecule permeation and (ii) perforations of defined geometric shape and size suitable for biomacromolecule separations. We show that the permeability of type (i) can be tuned by adjusting the resin component concentrations whereas perforations in type (ii) are introduced by a phase separation approach. Their remarkable features make nanomembranes, in particular biocompatible ones, very attractive materials for fast (bio-)sensing or functional bio-composite materials. In this respect, we furthermore show that small molecule separation nanomembranes can be rendered electrically conductive by coating with a thin gold layer whilst permeability is preserved. Please click Additional Files below to see the full abstract

Integrating Nanomembrane Separation with Plasmonic Detection for Real-Time Cell Culture Monitoring

To further understand cellular responses to drug treatment the dynamics of a reduced secretome shall be investigated. Currently there is no method for the detection of secreted small molecules in real time, label-free and with a high resolution. We present a novel design, which integrates nanopore filtration technology with highly sensitive plasmonic detection that allows real time monitoring of filtered molecules with a high spatial resolution and label free. The cell culture chamber is separated from the site of detection only by our biocompatible nanomembrane filter with a thickness of less than 100 nm to exclude the majority of background signals from the cell culture. The fast filtration of the cell culture constituents through the nanomembrane to the detector allows the observation of the dynamics of secreted molecules during cell culture and/or drug application. The setup offers new possibilities for drug screening and cell assays and may reveal new insights into cell signaling and drug responses. This setup shall be used to monitor cell culture or tissue culture without the necessity of labeling. This can be particularly important for the very popular “organ-on-a-chip” or “patient-on-a-chip” approaches to monitor tissue reactions to drug treatments with a high spatial resolution. Please click Additional Files below to see the full abstract

Development and validation of a porcine artificial colonic mucus model reflecting the properties of native colonic mucus in pigs

Colonic mucus plays a key role in colonic drug absorption. Mucus permeation assays could therefore provide useful insights and support rational formulation development in the early stages of drug development. However, the collection of native colonic mucus from animal sources is labor-intensive, does not yield amounts that allow for routine experimentation, and raises ethical concerns. In the present study, we developed an in vitro porcine artificial colonic mucus model based on the characterization of native colonic mucus. The structural properties of the artificial colonic mucus were validated against the native secretion for their ability to capture key diffusion patterns of macromolecules in native mucus. Moreover, the artificial colonic mucus could be stored under common laboratory conditions, without compromising its barrier properties. In conclusion, the porcine artificial colonic mucus model can be considered a biorelevant way to study the diffusion behavior of drug candidates in colonic mucus. It is a cost-efficient screening tool easily incorporated into the early stages of drug development and it contributes to the implementation of the 3Rs (refinement, reduction, and replacement of animals) in the drug development process

Validity of an FFQ assessing the vitamin D intake of young Serbian women living in a region without food fortification: the method of triads model

Objective: The objective of the present study was to examine the external validity of an FFQ designed to estimate dietary vitamin D intake compared with a plasma biomarker and three repeated 24 h dietary recalls in women of reproductive age in Serbia, where there is no exposure to food fortified with vitamin D. The method of triads was applied. Design: In a cross-sectional study, 422 women completed the Women and Reproductive Health FFQ (WRH-FFQ) during the winter months. From a representative subgroup (n 44), three 24 h dietary recalls and anthropometric parameters were collected as well as a fasting blood sample for vitamin D biomarker analyses. Correlation coefficients were calculated between each of the dietary methods. Validity coefficients, as a correlation between the measured and estimated 'true' exposure, were calculated using the method of triads. Bland-Altman plots were also constructed. Setting: Three major universities in Serbia. Subjects: Healthy young women (n 422) aged 18-35 years. Results: The WRH-FFQ estimate of vitamin D intake for all participants was 4.0 (SD 3.3) mu g/d and 3.1 (SD 2.3) mu g/d for the subgroup. Bland-Altman plots for these intakes showed high agreement. Validity coefficients for the FFQ, 24 h recall and biomarker were. rho(QI) = 0.847 (95 % CI 0.564, 0.928), rho(RI) = 0.810 (95 % CI 0.537, 0.997) and rho(BI) = 0.499 (95 % CI 0.190, 0.840), while the correlation coefficients were 0.686, 0.422 and 0.404. Conclusions: The FFQ applied in the present study is a valid tool for assessing dietary vitamin D intake in women living in Serbia, a region without mandatory vitamin D food fortification

Physiological properties, composition and structural profiling of porcine gastrointestinal mucus

The gastrointestinal mucus is a hydrogel that lines the luminal side of the gastrointestinal epithelium, offering barrier protection from pathogens and lubrication of the intraluminal contents. These barrier properties likewise affect nutrients and drugs that need to penetrate the mucus to reach the epithelium prior to absorption. In order to assess the potential impact of the mucus on drug absorption, we need information about the nature of the gastrointestinal mucus. Today, most of the relevant available literature is mainly derived from rodent studies. In this work, we used a larger animal species, the pig model, to characterize the mucus throughout the length of the gastrointestinal tract. This is the first report of the physiological properties (physical appearance, pH and water content), composition (protein, lipid and metabolite content) and structural profiling (rheology and gel network) of the porcine gastrointestinal mucus. These findings allow for direct comparisons between the characteristics of mucus from various segments and can be further utilized to improve our understanding of the role of the mucus on region dependent drug absorption. Additionally, the present work is expected to contribute to the assessment of the porcine model as a preclinical species in the drug development process

Responsive Hyaluronic Acid-Ethylacrylamide Microgels Fabricated Using Microfluidics Technique

Volume changes of responsive microgels can probe interactions between polyelectrolytes and species of opposite charges such as peptides and proteins. We have investigated a microfluidics method to synthesize highly responsive, covalently crosslinked, hyaluronic acid microgels for such purposes. Sodium hyaluronate (HA), pre-modified with ethylacrylamide functionalities, was crosslinked in aqueous droplets created with a microfluidic technique. We varied the microgel properties by changing the degree of modification and concentration of HA in the reaction mixture. The degree of modification was determined by H-1 NMR. Light microscopy was used to investigate the responsiveness of the microgels to osmotic stress in aqueous saline solutions by simultaneously monitoring individual microgel species in hydrodynamic traps. The permeability of the microgels to FITC-dextrans of molecular weights between 4 and 250 kDa was investigated using confocal laser scanning microscopy. The results show that the microgels were spherical with diameters between 100 and 500 mu m and the responsivity tunable by changing the degree of modification and the HA concentration. Microgels were fully permeable to all investigated FITC-dextran probes. The partitioning to the microgel from an aqueous solution decreased with the increasing molecular weight of the probe, which is in qualitative agreement with theories of homogeneous gel networks

Microfluidics platform for studies of peptide - polyelectrolyte interaction

Subcutaneous injection is one of the most common approaches for administering biopharmaceuticals unsuitable for oral delivery. However, there is a lack of methods to predict the behavior of biopharmaceuticals within the extracellular matrix of the subcutaneous tissue. In this work, we present a novel miniaturized microfluidic-based in vitro method able to investigate interactions between drug molecules and the polymers of the subcutaneous extracellular matrix. To validate the method, microgels consisting of, respectively, covalently cross-linked hy-aluronic acid, polyacrylic acid, and commercially available DC BeadTM, were exposed to three model substances: cytochrome C, protamine sulfate and amitriptyline hydrochloride. These components were chosen to include systems with widely different physiochemical properties (charge, size, self-assembly, etc.) The experimental results were compared with theoretical predictions from a gel model developed earlier. The results show that the method is suitable as a rapid screening method for automated, large-scale, probing of interactions between biopolymers and drug molecules, with small consumption of material