Selective Filtering of Particles by the Extracellular Matrix: An Electrostatic Bandpass

The transport of microscopic particles such as growth factors, proteins, or drugs through the extracellular matrix (ECM) is based on diffusion, a ubiquitous mechanism in nature. The ECM shapes the local distribution of the transported macromolecules and at the same time constitutes an important barrier toward infectious agents. To fulfill these competing tasks, the hydrogels have to employ highly selective filtering mechanisms. Yet, the underlying microscopic principles are still an enigma in cell biology and drug delivery. Here, we show that the extracellular matrix presents an effective electrostatic bandpass, suppressing the diffusive motion of both positively and negatively charged objects. This mechanism allows uncharged particles to easily diffuse through the matrix, while charged particles are effectively trapped. However, by tuning the strength of this physical interaction of the particles with the biopolymer matrix, the microscopic mobility of formerly trapped particles can be rescued on demand. Moreover, we identify heparan sulfate chains to be one important key factor for the barrier function of the extracellular matrix. We propose that localized charge patches in the ECM are responsible for its highly unspecific but strongly selective filtering effect. Such localized interactions could also account for the observed tunability and selectivity of many other important permeability barriers that are established by biopolymer-based hydrogels, e.g., the mucus layer of endothelial cells or the hydrogel in the nuclear core complex.     

Biological hydrogels as selective diffusion barriers

The controlled exchange of molecules between organelles, cells, or organisms and their environment is crucial for life. Biological gels such as mucus, the extracellular matrix (ECM), and the biopolymer barrier within the nuclear pore are well suited to achieve such a selective exchange, allowing passage of particular molecules while rejecting many others. Although hydrogel-based filters are integral parts of biology, clear concepts of how their barrier function is controlled at a microscopic level are still missing. We summarize here our current understanding of how selective filtering is established by different biopolymer-based hydrogels. We ask if the modulation of microscopic particle transport in biological hydrogels is based on a generic filtering principle which employs biochemical/biophysical interactions with the filtered molecules rather than size-exclusion effects.     

Spatial Configuration and Composition of Charge Modulates Transport into a Mucin Hydrogel Barrier

The mucus barrier is selectively permeable to a wide variety of molecules, proteins, and cells, and establishes gradients of these particulates to influence the uptake of nutrients, the defense against pathogens, and the delivery of drugs. Despite its importance for health and disease, the criteria that govern transport through the mucus barrier are largely unknown. Studies with uniformly functionalized nanoparticles have provided critical information about the relevance of particle size and net charge for mucus transport. However, these particles lack the detailed spatial arrangements of charge found in natural mucus-interacting substrates, such as certain viruses, which may have important consequences for transport through the mucus barrier. Using a novel, to our knowledge, microfluidic design that enables us to measure real-time transport gradients inside a hydrogel of mucins, the gel-forming glycoprotein component of mucus, we show that two peptides with the same net charge, but different charge arrangements, exhibit fundamentally different transport behaviors. Specifically, we show that certain configurations of positive and negative charges result in enhanced uptake into a mucin barrier, a remarkable effect that is not observed with either charge alone. Moreover, we show that the ionic strength within the mucin barrier strongly influences transport specificity, and that this effect depends on the detailed spatial arrangement of charge. These findings suggest that spatial charge distribution is a critical parameter to modulate transport through mucin-based barriers, and have concrete implications for the prediction of mucosal passage, and the design of drug delivery vehicles with tunable transport properties.     

FG/FxFG as well as GLFG repeats form a selective permeability barrier with self‐healing properties

The permeability barrier of nuclear pore complexes (NPCs) controls all nucleo‐cytoplasmic exchange. It is freely permeable for small molecules. Objects larger than ≈30 kDa can efficiently cross this barrier only when bound to nuclear transport receptors (NTRs) that confer translocation‐promoting properties. We had shown earlier that the permeability barrier can be reconstituted in the form of a saturated FG/FxFG repeat hydrogel. We now show that GLFG repeats, the other major FG repeat type, can also form highly selective hydrogels. While supporting massive, reversible importin‐mediated cargo influx, FG/FxFG, GLFG or mixed hydrogels remained firm barriers towards inert objects that lacked nuclear transport signals. This indicates that FG hydrogels immediately reseal behind a translocating species and thus possess ‘self‐healing’ properties. NTRs not only left the barrier intact, they even tightened it against passive influx, pointing to a role for NTRs in establishing and maintaining the permeability barrier of NPCs.     

Systematic analysis of barrier-forming FG hydrogels from Xenopus nuclear pore complexes

Nuclear pore complexes (NPCs) control the traffic between cell nucleus and cytoplasm. While facilitating translocation of nuclear transport receptors (NTRs) and NTR·cargo complexes, they suppress passive passage of macromolecules ?30 kDa. Previously, we reconstituted the NPC barrier as hydrogels comprising S. cerevisiae FG domains. We now studied FG domains from 10 Xenopus nucleoporins and found that all of them form hydrogels. Related domains with low FG motif density also substantially contribute to the NPC's hydrogel mass. We characterized all these hydrogels and observed the strictest sieving effect for the Nup98‐derived hydrogel. It fully blocks entry of GFP‐sized inert objects, permits facilitated entry of the small NTR NTF2, but arrests importin β‐type NTRs at its surface. O‐GlcNAc modification of the Nup98 FG domain prevented this arrest and allowed also large NTR·cargo complexes to enter. Solid‐state NMR spectroscopy revealed that the O‐GlcNAc‐modified Nup98 gel lacks amyloid‐like β‐structures that dominate the rigid regions in the S. cerevisiae Nsp1 FG hydrogel. This suggests that FG hydrogels can assemble through different structural principles and yet acquire the same NPC‐like permeability.     

The permeability of reconstituted nuclear pores provides direct evidence for the selective phase model

Nuclear pore complexes (NPCs) maintain a permeability barrier between the nucleus and the cytoplasm through FG-repeat-containing nucleoporins (Nups). We previously proposed a "selective phase model" in which the FG repeats interact with one another to form a sieve-like barrier that can be locally?disrupted by the binding of nuclear transport receptors (NTRs), but not by inert macromolecules, allowing selective passage of NTRs and associated cargo. Here, we provide direct evidence for this model in a physiological context. By using NPCs reconstituted from Xenopus laevis egg extracts, we show that Nup98 is essential for maintaining the permeability barrier. Specifically, the multivalent cohesion between FG repeats is required, including cohesive FG repeats close to the anchorage point to the NPC scaffold. Our data exclude alternative models that are based solely on an interaction between the FG repeats and NTRs and indicate that the barrier is formed by a sieve-like FG hydrogel.     

Spatial configuration of charge and hydrophobicity tune particle transport through mucus

Mucus is a selectively permeable hydrogel that protects wet epithelia from pathogen invasion and poses a barrier to drug delivery. Determining the parameters of a particle that promote or prevent passage through mucus is critical, as it will enable predictions about the mucosal passage of pathogens and inform the design of therapeutics. The effect of particle net charge and size on mucosal transport has been characterized using simple model particles; however, predictions of mucosal passage remain challenging. Here, we utilize rationally designed peptides to examine the integrated contributions of charge, hydrophobicity, and spatial configuration on mucosal transport. We find that net charge does not entirely predict transport. Specifically, for cationic peptides, the inclusion of hydrophobic residues and the position of charged and hydrophobic residues within the peptide impact mucosal transport. We have developed a simple model of mucosal transport that predicts how previously unexplored amino acid sequences achieve slow versus fast passage through mucus. This model may be used as a basis to predict transport behavior of natural peptide-based particles, such as antimicrobial peptides or viruses, and assist in the engineering of synthetic sequences with desired transport properties.     

Mucin biopolymers as broad-spectrum antiviral agents

Mucus is a porous biopolymer matrix that coats all wet epithelia in the human body and serves as the first line of defense against many pathogenic bacteria and viruses. However, under certain conditions viruses are able to penetrate this infection barrier, which compromises the protective function of native mucus. Here, we find that isolated porcine gastric mucin polymers, key structural components of native mucus, can protect an underlying cell layer from infection by small viruses such as human papillomavirus (HPV), Merkel cell polyomavirus (MCV), or a strain of influenza A virus. Single particle analysis of virus mobility inside the mucin barrier reveals that this shielding effect is in part based on a retardation of virus diffusion inside the biopolymer matrix. Our findings suggest that purified mucins may be used as a broad-range antiviral supplement to personal hygiene products, baby formula or lubricants to support our immune system.     

Altering Mucus Rheology to “Solidify” Human Mucus at the Nanoscale

The ability of mucus to function as a protective barrier at mucosal surfaces rests on its viscous and elastic properties, which are not well understood at length scales relevant to pathogens and ultrafine environmental particles. Here we report that fresh, undiluted human cervicovaginal mucus (CVM) transitions from an impermeable elastic barrier to non-adhesive objects sized 1 µm and larger to a highly permeable viscoelastic liquid to non-adhesive objects smaller than 500 nm in diameter. Addition of a nonionic detergent, present in vaginal gels, lubricants and condoms, caused CVM to behave as an impermeable elastic barrier to 200 and 500 nm particles, suggesting that the dissociation of hydrophobically-bundled mucin fibers created a finer elastic mucin mesh. Surprisingly, the macroscopic viscoelasticity, which is critical to proper mucus function, was unchanged. These findings provide important insight into the nanoscale structural and barrier properties of mucus, and how the penetration of foreign particles across mucus might be inhibited.     

Transport of Particles in Intestinal Mucus under Simulated Infant and Adult Physiological Conditions: Impact of Mucus Structure and Extracellular DNA

The final boundary between digested food and the cells that take up nutrients in the small intestine is a protective layer of mucus. In this work, the microstructural organization and permeability of the intestinal mucus have been determined under conditions simulating those of infant and adult human small intestines. As a model, we used the mucus from the proximal (jejunal) small intestines of piglets and adult pigs. Confocal microscopy of both unfixed and fixed mucosal tissue showed mucus lining the entire jejunal epithelium. The mucus contained DNA from shed epithelial cells at different stages of degradation, with higher amounts of DNA found in the adult pig. The pig mucus comprised a coherent network of mucin and DNA with higher viscosity than the more heterogeneous piglet mucus, which resulted in increased permeability of the latter to 500-nm and 1-µm latex beads. Multiple-particle tracking experiments revealed that diffusion of the probe particles was considerably enhanced after treating mucus with DNase. The fraction of diffusive 500-nm probe particles increased in the pig mucus from 0.6% to 64% and in the piglet mucus from ca. 30% to 77% after the treatment. This suggests that extracellular DNA can significantly contribute to the microrheology and barrier properties of the intestinal mucus layer. To our knowledge, this is the first time that the structure and permeability of the small intestinal mucus have been compared between different age groups and the contribution of extracellular DNA highlighted. The results help to define rules governing colloidal transport in the developing small intestine. These are required for engineering orally administered pharmaceutical preparations with improved delivery, as well as for fabricating novel foods with enhanced nutritional quality or for controlled calorie uptake.     

Breakdown of mucin as barrier to digestive enzymes in the ischemic rat small intestine

Loss of integrity of the epithelial/mucosal barrier in the small intestine has been associated with different pathologies that originate and/or develop in the gastrointestinal tract. We showed recently that mucin, the main protein in the mucus layer, is disrupted during early periods of intestinal ischemia. This event is accompanied by entry of pancreatic digestive enzymes into the intestinal wall. We hypothesize that the mucin-containing mucus layer is the main barrier preventing digestive enzymes from contacting the epithelium. Mucin breakdown may render the epithelium accessible to pancreatic enzymes, causing its disruption and increased permeability. The objective of this study was to investigate the role of mucin as a protection for epithelial integrity and function. A rat model of 30 min splanchnic arterial occlusion (SAO) was used to study the degradation of two mucin isoforms (mucin 2 and 13) and two epithelial membrane proteins (E-cadherin and toll-like receptor 4, TLR4). In addition, the role of digestive enzymes in mucin breakdown was assessed in this model by luminal inhibition with acarbose, tranexamic acid, or nafamostat mesilate. Furthermore, the protective effect of the mucin layer against trypsin-mediated disruption of the intestinal epithelium was studied in vitro. Rats after SAO showed degradation of mucin 2 and fragmentation of mucin 13, which was not prevented by protease inhibition. Mucin breakdown was accompanied by increased intestinal permeability to FITC-dextran as well as degradation of E-cadherin and TLR4. Addition of mucin to intestinal epithelial cells in vitro protected against trypsin-mediated degradation of E-cadherin and TLR4 and reduced permeability of FITC-dextran across the monolayer. These results indicate that mucin plays an important role in the preservation of the mucosal barrier and that ischemia but not digestive enzymes disturbs mucin integrity, while digestive enzymes actively mediate epithelial cell disruption.     

A saturated FG-repeat hydrogel can reproduce the permeability properties of nuclear pore complexes

The permeability barrier of nuclear pore complexes (NPCs) controls the exchange between nucleus and cytoplasm. It suppresses the flux of inert macromolecules > or = 30 kDa but allows rapid passage of even very large cargoes, provided these are bound to appropriate nuclear transport receptors. We show here that a saturated hydrogel formed by a single nucleoporin FG-repeat domain is sufficient to reproduce the permeability properties of NPCs. Importin beta and related nuclear transport receptors entered such hydrogel >1000x faster than a similarly sized inert macromolecule. The FG-hydrogel even reproduced import signal-dependent and importin-mediated cargo influx, allowing importin beta to accelerate the gel entry of a large cognate cargo more than 20,000-fold. Intragel diffusion of the importin beta-cargo complex occurred rapidly enough to traverse an NPC within approximately 12 ms. We extend the "selective phase model" to explain these effects.     

Fatty acid synthase modulates intestinal barrier function through palmitoylation of mucin 2

The intestinal mucus barrier prevents pathogen invasion and maintains host-microbiota homeostasis. We show that fatty acid synthase (FAS), an insulin-responsive enzyme essential for de novo lipogenesis, helps maintain the mucus barrier by regulating Mucin 2, the dominant mucin in the colon and a central component of mucus. Inducible Cre recombinase-directed inactivation of the FAS gene in the colonic epithelium of mice is associated with disruptions in the intestinal mucus barrier as well as increased intestinal permeability, colitis, systemic inflammation, and changes in gut microbial ecology. FAS deficiency blocked the generation of palmitoylated Mucin 2, which must be S-palmitoylated at its N terminus for proper secretion and function. Furthermore, a diabetic mouse model exhibited lower FAS levels and a decreased mucus layer, which could be restored with insulin treatment. Thus, the role of FAS in maintaining intestinal barrier function may explain the pathogenesis of intestinal inflammation in diabetes and other disorders.     

Mucin networks: Dynamic structural assemblies controlling mucus function

Contrary to first appearances, mucus structural biology is not an oxymoron. Though mucus hydrogels derive their characteristics largely from intrinsically disordered, heavily glycosylated polypeptide segments, the secreted mucin glycoproteins that constitute mucus undergo an orderly assembly process controlled by folded domains at their termini. Recent structural studies revealed how mucin complexes promote disulphide-mediated polymerization to produce the mucus gel scaffold. Additional protein–protein and protein-glycan interactions likely tune the mesoscale properties, stability, and activities of mucins. Evidence is emerging that even intrinsically disordered glycosylated segments have specific structural roles in the production and properties of mucus. Though soft-matter biophysical approaches to understanding mucus remain highly relevant, high-resolution structural studies of mucins and other mucus components are providing new perspectives on these vital, protective hydrogels.     

Particle Transport through Hydrogels Is Charge Asymmetric

Transport processes within biological polymer networks, including mucus and the extracellular matrix, play an important role in the human body, where they serve as a filter for the exchange of molecules and nanoparticles. Such polymer networks are complex and heterogeneous hydrogel environments that regulate diffusive processes through finely tuned particle-network interactions. In this work, we present experimental and theoretical studies to examine the role of electrostatics on the basic mechanisms governing the diffusion of charged probe molecules inside model polymer networks. Translational diffusion coefficients are determined by fluorescence correlation spectroscopy measurements for probe molecules in uncharged as well as cationic and anionic polymer solutions. We show that particle transport in the charged hydrogels is highly asymmetric, with diffusion slowed down much more by electrostatic attraction than by repulsion, and that the filtering capability of the gel is sensitive to the solution ionic strength. Brownian dynamics simulations of a simple model are used to examine key parameters, including interaction strength and interaction range within the model networks. Simulations, which are in quantitative agreement with our experiments, reveal the charge asymmetry to be due to the sticking of particles at the vertices of the oppositely charged polymer networks.     

MIND the gap: an astroglial perspective on barrier regulation

The blood-brain barrier (BBB) is a specialized tissue interface that provides an important homeostatic and immunosurveillance role in the CNS. Unlike most microvascular tissues, which readily promote paracellular passage of solutes and macromolecules, the BBB is more analogous to polarized mucosal epithelia that restrict such permeability in order to prevent disease onset. Recent transgenic ablation studies have demonstrated that the BBB and mucosal tissues also share a requirement for astroglial-regulated barrier integrity. This review highlights the emerging concept that astroglia regulate barrier function at markedly different tissue interfaces. It also explores possible lessons that might be learnt by adopting epithelial model paradigms of the BBB. For example, novel glial-derived S-nitrosylation signals that regulate intestinal permeability in the digestive tract might provide new mechanistic insights into the function of the BBB. A better understanding of such universal mechanisms for barrier regulation will facilitate novel therapeutic strategies that target permeability disorders at CNS and mucosal tissue interfaces.     

Biopolymer-based hydrogels as scaffolds for tissue engineering applications: a review

Hydrogels are physically or chemically cross-linked polymer networks that are able to absorb large amounts of water. They can be classified into different categories depending on various parameters including the preparation method, the charge, and the mechanical and structural characteristics. The present review aims to give an overview of hydrogels based on natural polymers and their various applications in the field of tissue engineering. In a first part, relevant parameters describing different hydrogel properties and the strategies applied to finetune these characteristics will be described. In a second part, an important class of biopolymers that possess thermosensitive properties (UCST or LCST behavior) will be discussed. Another part of the review will be devoted to the application of cryogels. Finally, the most relevant biopolymer-based hydrogel systems, the different methods of preparation, as well as an in depth overview of the applications in the field of tissue engineering will be given.     

Microbial adhesins to gastrointestinal mucus

The gastrointestinal tract (GIT) is lined by a layer of mucus formed by mucin glycoproteins. This layer constitutes a physical and chemical barrier between the intestinal contents and the underlying epithelia. In addition to this protective role, mucins harbor glycan-rich domains that provide preferential binding sites for pathogens and commensal bacteria. Although mucus-microbial interactions in the GIT play a crucial role in determining the outcome of relationships of both commensal and pathogens with the host, the adhesins and ligands involved in the interaction are poorly delineated. This review focuses on the current knowledge of microbial adhesins to gastrointestinal mucus and mucus components.     

Diffusion Regulation in the Vitreous Humor

The efficient treatment of many ocular diseases depends on the rapid diffusive distribution of solutes such as drugs or drug delivery vehicles through the vitreous humor. However, this multicomponent hydrogel possesses selective permeability properties, which allow for the diffusion of certain molecules and particles, whereas others are immobilized. In this study, we perform an interspecies comparison showing that the selective permeability properties of the vitreous are conserved across several mammalian species. We identify the polyanionic glycosaminoglycans hyaluronic acid and heparan sulfate as two key macromolecules that establish this selective permeability. We show that electrostatic interactions between the polyanionic macromolecules and diffusing solutes can be weakened by charge screening or enzymatic glycosaminoglycan digestion. Furthermore, molecule penetration into the vitreous is also charge-dependent and only efficient as long as the net charge of the molecule does not exceed a certain threshold.     

Molecular physiology and pathophysiology of tight junctions. IV. Regulation of tight junctions by extracellular stimuli: nutrients, cytokines, and immune cells

The epithelial lining of the gastrointestinal tract forms a regulated, selectively permeable barrier between luminal contents and the underlying tissue compartments. Permeability across the epithelium is, in part, determined by the rate-limiting barrier of the paracellular pathway-the most apical intercellular junction referred to as the tight junction (TJ). The TJ is composed of a multiprotein complex that affiliates with the underlying apical actomyosin ring. TJ structure and function, and therefore epithelial permeability, are influenced by diverse physiological and pathological stimuli; here we review examples of such stimuli that are detected at the cell surface. For example, luminal glucose induces an increase in paracellular permeability to small molecules. Similarly, but by other means, cytokines and leukocytes in the vicinity of the epithelium also regulate TJ structure and paracellular permeability by influencing the TJ protein complex and/or its association with the underlying actin cytoskeleton.