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.     

Characterization of Particle Translocation through Mucin Hydrogels

Biological functional entities surround themselves with selective barriers that control the passage of certain classes of macromolecules while rejecting others. A prominent example of such a selective permeability barrier is given by mucus. Mucus is a biopolymer-based hydrogel that lines all wet epithelial surfaces of the human body. It regulates the uptake of nutrients from our gastrointestinal system, adjusts itself with the menstrual cycle to control the passage of sperm, and shields the underlying cells from pathogens such as bacteria and viruses. In the case of drug delivery, the mucus barrier needs to be overcome for successful medical treatment. Despite its importance for both physiology and medical applications, the underlying principles which regulate the permeability of mucus remain enigmatic. Here, we analyze the mobility of microscopic particles in reconstituted mucin hydrogels. We show that electrostatic interactions between diffusing particles and mucin polymers regulate the permeability properties of reconstituted mucin hydrogels. As a consequence, various parameters such as particle surface charge and mucin density, and buffer conditions such as pH and ionic strength, can modulate the microscopic barrier function of the mucin hydrogel. Our findings suggest that the permeability of a biopolymer-based hydrogel such as native mucus can be tuned to a wide range of settings in different compartments of our bodies.     

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.     

Functionalized positive nanoparticles reduce mucin swelling and dispersion

Multi-functionalized nanoparticles (NPs) have been extensively investigated for their potential in household and commercial products, and biomedical applications. Previous reports have confirmed the cellular nanotoxicity and adverse inflammatory effects on pulmonary systems induced by NPs. However, possible health hazards resulting from mucus rheological disturbances induced by NPs are underexplored. Accumulation of viscous, poorly dispersed, and less transportable mucus leading to improper mucus rheology and dysfunctional mucociliary clearance are typically found to associate with many respiratory diseases such as asthma, cystic fibrosis (CF), and COPD (Chronic Obstructive Pulmonary Disease). Whether functionalized NPs can alter mucus rheology and its operational mechanisms have not been resolved. Herein, we report that positively charged functionalized NPs can hinder mucin gel hydration and effectively induce mucin aggregation. The positively charged NPs can significantly reduce the rate of mucin matrix swelling by a maximum of 7.5 folds. These NPs significantly increase the size of aggregated mucin by approximately 30 times within 24 hrs. EGTA chelation of indigenous mucin crosslinkers (Ca(2+) ions) was unable to effectively disperse NP-induced aggregated mucins. Our results have demonstrated that positively charged functionalized NPs can impede mucin gel swelling by crosslinking the matrix. This report also highlights the unexpected health risk of NP-induced change in mucus rheological properties resulting in possible mucociliary transport impairment on epithelial mucosa and related health problems. In addition, our data can serve as a prospective guideline for designing nanocarriers for airway drug delivery applications.     

Biochemical examination ofRana pipiens epithelial mucus

Summary The epithelial mucus ofRana pipiens is shown to be highly negative by histochemical procedures, uptake of tagged extracellular markers, equilibrium dialysis, and QAE-Sephadex G-25 ion exchange chromatography. The mucus is found to contain 0.4% (dry weight) sulfate, 16% (dry weight) protein and 9% (dry weight) neutral sugars, whereas no sialic acid is detected. A mucin charge of –40 equivalents mole^–1 is calculated by equilibrium dialysis using a molecular mass of 100,000 Dalton. An independent determination of in situ charge density by radionuclide uptake onto the frog surface (1.55 meq l^–1) suggests that this estimate of mucin charge is correct. Based on analysis of size-exclusion chromatography fractions, it is suggested that mucus contains a sulfated glyco-protein (100,000 Dalton) that forms aggregates of about one million Dalton.     

Serine Protease(s) Secreted by the Nematode Trichuris muris Degrade the Mucus Barrier

The polymeric mucin component of the intestinal mucus barrier changes during nematode infection to provide not only physical protection but also to directly affect pathogenic nematodes and aid expulsion. Despite this, the direct interaction of the nematodes with the mucins and the mucus barrier has not previously been addressed. We used the well-established Trichuris muris nematode model to investigate the effect on mucins of the complex mixture of immunogenic proteins secreted by the nematode called excretory/secretory products (ESPs). Different regimes of T. muris infection were used to simulate chronic (low dose) or acute (high dose) infection. Mucus/mucins isolated from mice and from the human intestinal cell line, LS174T, were treated with ESPs. We demonstrate that serine protease(s) secreted by the nematode have the ability to change the properties of the mucus barrier, making it more porous by degrading the mucin component of the mucus gel. Specifically, the serine protease(s) acted on the N-terminal polymerising domain of the major intestinal mucin Muc2, resulting in depolymerisation of Muc2 polymers. Importantly, the respiratory/gastric mucin Muc5ac, which is induced in the intestine and is critical for worm expulsion, was protected from the depolymerising effect exerted by ESPs. Furthermore, serine protease inhibitors (Serpins) which may protect the mucins, in particular Muc2, from depolymerisation, were highly expressed in mice resistant to chronic infection. Thus, we demonstrate that nematodes secrete serine protease(s) to degrade mucins within the mucus barrier, which may modify the niche of the parasite to prevent clearance from the host or facilitate efficient mating and egg laying from the posterior end of the parasite that is in intimate contact with the mucus barrier. However, during a T_H2-mediated worm expulsion response, serpins, Muc5ac and increased levels of Muc2 protect the barrier from degradation by the nematode secreted protease(s).     

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.     

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.     

Modeling the human intestinal Mucin (MUC2) C-terminal cystine knot dimer

Intestinal mucus, a viscous secretion that lines the mucosa, is believed to be a barrier to absorption of many therapeutic compounds and carriers, and is known to play an important physiological role in controlling pathogen invasion. Nevertheless, there is as yet no clear understanding of the barrier properties of mucus, such as the nature of the molecular interactions between drug molecules and mucus components as well as those that govern gel formation. Secretory mucins, large and complex glycoprotein molecules, are the principal determinants of the viscoelastic properties of intestinal mucus. Despite the important role that mucins play in controlling transport and in diseases such as cystic fibrosis, their structures remain poorly characterized. The major intestinal secretory mucin gene, MUC2, has been identified and fully sequenced. The present study was undertaken to determine a detailed structure of the cysteine-rich region within the C-terminal end of human intestinal mucin (MUC2) via homology modeling, and explore possible configurations of a dimer of this cysteine-rich region, which may play an important role in governing mucus gel formation. Based on sequence–structure alignments and three-dimensional modeling, a cystine knot tertiary structure homologous to that of human chorionic gonadotropin (HCG) is predicted at the C-terminus of MUC2. Dimers of this C-terminal cystine knot (CTCK) were modeled using sequence alignment based on HCG and TGF-beta, followed by molecular dynamics and simulated annealing. Results support the formation of a cystine knot dimer with a structure analogous to that of HCG.      

Coarse-grained modeling of mucus barrier properties

We designed a simple coarse-grained model of the glycocalyx layer, or adhesive mucus layer (AML), covered by mucus gel (luminal mucus layer) using a polymer lattice model and stochastic sampling (replica exchange Monte Carlo) for canonical ensemble simulations. We assumed that mucin MUC16 is responsible for the structural properties of the AML. Other mucins that are much smaller in size and less relevant for layer structure formation were not included. We further assumed that the system was in quasi-equilibrium. For systems with surface coverage and concentrations of model mucins mimicking physiological conditions, we determined the equilibrium distribution of inert nanoparticles within the mucus layers using an efficient replica exchange Monte Carlo sampling procedure. The results show that the two mucus layers penetrate each other only marginally, and the bilayer imposes a strong barrier for nanoparticles, with the AML layer playing a crucial role in the mucus barrier.     

Quantitative MUC5AC and MUC6 mucin estimations in gastric mucus by a least-squares minimization method

We have determined the molar proportions of the MUC5AC and MUC6 mucus glycoproteins (mucins) in mucus from the normal and pathological human gastric antrum using a least-squares minimization analysis applied to amino acid compositions. We noted that the content of MUC5AC mucin in mucus from individuals without gastroduodenal disease was very high, suggesting that the integrity and barrier properties of the adherent gastric mucus layer are normally maintained by building-block structures formed from this mucin alone. We observed that the molar content of MUC6 mucin doubled (without significance) in mucus from patients with duodenal ulcer, and increased five times (with high significance) in mucus from patients with gastric ulcer, when compared with that in mucus from individuals without gastroduodenal disease.     

Biodegradable nanoparticles meet the bronchial airway barrier: how surface properties affect their interaction with mucus and epithelial cells

Despite the wide interest raised by lung administration of nanoparticles (NPs) for the treatment of various diseases, little information is available on their effect toward the airway epithelial barrier function. In this study, the potential damage of the pulmonary epithelium upon exposure to poly(lactide-co-glycolide) (PLGA) NPs has been assessed in vitro using a Calu-3-based model of the bronchial epithelial barrier. Positively and negatively charged as well as neutral PLGA NPs were obtained by coating their surface with chitosan (CS), poloxamer (PF68), or poly(vinyl alcohol) (PVA). The role of NP surface chemistry and charge on the epithelial resistance and mucus turnover, using MUC5AC as a marker, was investigated. The interaction with mucin reduced the penetration of CS- and PVA-coated NPs, while the hydrophilic PF68-coated NPs diffused across the mucus barrier leading to a higher intracellular accumulation. Only CS-coated NPs caused a transient but reversible decrease of the trans-epithelial electrical resistance (TEER). None of the NP formulations increased MUC5AC mRNA expression or the protein levels. These in vitro results highlight the safety of PLGA NPs toward the integrity and function of the bronchial airway barrier and demonstrate the crucial role of NP surface properties to achieve a controlled and sustained delivery of drugs via the pulmonary route.     

Mucin-type glycoproteins.

Considerable advances have been made in recent years in our understanding of the biochemistry of mucin-type glycoproteins. This class of compounds is characterized mainly by a high level of O-linked oligosaccharides. Initially, the glycoproteins were solely known as the major constituents of mucus. Recent studies have shown that mucins from the gastrointestinal tract, lungs, salivary glands, sweat glands, breast, and tumor cells are structurally related to high-molecular-weight glycoproteins, which are produced by epithelial cells as membrane proteins. During mucin synthesis, an orchestrated sequence of events results in giant molecules of Mr 4 to 6 x 10(6), which are stored in mucous granules until secretion. Once secreted, mucin forms a barrier, not only to protect the delicate epithelial cells against the extracellular environment, but also to select substances for binding and uptake by these epithelia. This review is designed to critically examine relations between structure and function of the different compounds categorized as mucin glycoproteins.     

Action of reactive oxygen species on colonic mucus secretions

Reactive oxygen species (ROS) have been implicated in the pathogenesis of many colonic diseases. Mucus is the colon's first line of defence against luminal agents. This study has therefore characterised ROS action on colonic mucus secretions. ROS were produced using peroxide-based systems of different concentrations. The effects of these systems were tested on native colonic mucus gels, isolated colonic mucins, and in vivo models. Colonic mucus gels were resistant to ROS breakdown. Mucins were susceptible to ROS attack, causing loss of terminal sugars and protein and mucin fragmentation. The in vivo thickness of the mucus barrier was reduced by up to 50% by ROS (above 5 mM peroxide). A 5 mM peroxide caused a significant increase in resting mucus thickness of ca. 15%. All ROS-generating systems caused mucosal damage once the loosely adherent mucus had been removed. As native mucus gel is more resistant to ROS damage than purified mucin, nonmucin components of mucus may have extensive ROS-scavenging properties. Low levels of luminal colonic ROS increase the protection afforded by the mucus barrier in vivo. Higher levels of ROS significantly reduce this protection. In vitro modeling of mucus degradation by ROS does not necessarily correlate with the dynamic, in vivo situation.     

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.     

Physico-Chemical Characteristics of Lipoplexes Influence Cell Uptake Mechanisms and Transfection Efficacy

Background

Formulation of DNA/cationic lipid complexes (lipoplexes) designed for nucleic acid delivery mostly results in positively charged particles which are thought to enter cells by endocytosis. We recently developed a lipoplex formulation called Neutraplex that allows preparation of both cationic and anionic stable complexes with similar lipid content and ultrastructure.

Methodology/Principal Findings

To assess whether the global net charge could influence cell uptake and activity of the transported oligonucleotides (ON), we prepared lipoplexes with positive and negative charges and compared: (i) their physicochemical properties by zeta potential analysis and dynamic light scattering, (ii) their cell uptake by fluorescence microscopy and flow cytometry, and (iii) the biological activity of the transported ON using a splicing correction assay. We show that positively or negatively charged lipoplexes enter cells cells using both temperature-dependent and -independent uptake mechanisms. Specifically, positively charged lipoplexes predominantly use a temperature-dependent transport when cells are incubated OptiMEM medium. Anionic lipoplexes favour an energy-independent transport and show higher ON activity than cationic lipoplexes in presence of serum. However, lipoplexes with high positive global net charge and OptiMEM medium give the highest uptake and ON activity levels.

Conclusions

These findings suggest that, in addition to endocytosis, lipoplexes may enter cell via a temperature-independent mechanism, which could be mediated by lipid mixing. Such characteristics might arise from the specific lipoplex ultrastructure and should be taken into consideration when developing lipoplexes designed for in vivo or ex vivo nucleic acid transfer.     

Tea Derived Galloylated Polyphenols Cross-Link Purified Gastrointestinal Mucins

Polyphenols derived from tea are thought to be important for human health. We show using a combination of particle tracking microrheology and small-angle neutron scattering that polyphenols acts as cross-linkers for purified gastrointestinal mucin, derived from the stomach and the duodenum. Both naturally derived purified polyphenols, and green and black tea extracts are shown to act as cross-linkers. The main active cross-linking component is found to be the galloylated forms of catechins. The viscosity, elasticity and relaxation time of the mucin solutions experience an order of magnitude change in value upon addition of the polyphenol cross-linkers. Similarly small-angle neutron scattering experiments demonstrate a sol-gel transition with the addition of polyphenols, with a large increase in the scattering at low angles, which is attributed to the formation of large scale (>10 nm) heterogeneities during gelation. Cross-linking of mucins by polyphenols is thus expected to have an impact on the physicochemical environment of both the stomach and duodenum; polyphenols are expected to modulate the barrier properties of mucus, nutrient absorption through mucus and the viscoelastic microenvironments of intestinal bacteria.     

Lysine uptake and exchange in Corynebacterium glutamicum.

Resting cells of Corynebacterium glutamicum (ATCC 13032) accumulate [14C]lysine by a transport system with a relatively high affinity (10 microMs) and a low maximum velocity (0.15 nmol/min per mg [dry weight]). Uptake of lysine was not inhibited by uncouplers or by ionophores affecting the ion gradients and the energetic state of the cell. Analysis of intracellular amino acid concentrations during the transport reaction as well as kinetic studies revealed that the observed uptake of lysine in fact represents a homologous antiport between extracellular [14C]lysine and intracellular unlabeled lysine. Intracellular [14C]lysine could only be released by the addition of unlabeled lysine to the bacterial suspension. In contrast to this homologous antiport reaction, we observed net uptake of lysine in lysine-depleted cells of a lysine auxotrophic strain. This net uptake was found to be electrogenic and could also be observed as a heterologous antiport reaction in wild-type cells under particular conditions. In this case exchange was mediated between internal lysine and external alanine, isoleucine, or valine. This antiport was electrogenic, since the substrates differ in charge. The cells can switch between electroneutral homologous exchange and electrogenic heterologous antiport mode during fermentation because of changing metabolic conditions.     

Somatostatin stimulates colonic MUC2 expression through SSTR5-Notch-Hes1 signaling pathway

Colonic mucus barrier is regarded as the first defense line against bacteria and antigens from directly attaching to the epithelium, which would further lead to intestinal inflammation activation and pathological conditions. As MUC2 mucin is the predominant component of the mucus, understanding the regulatory mechanisms of MUC2 is important for mucus barrier protection. Somatostatin (SST) has been found to play a role in colon protection through various manners. However, whether SST involves in colonic mucus barrier regulation is still unclear. The aim of this study is to investigate the effects and potential mechanisms of SST on colonic MUC2 expression and mucus secretion. In vivo study, exogenous somatostatin (octreotide) administration effectively stimulated mice colonic MUC2 expression and mucus secretion. In human goblet-like cell LS174T cells, SST exposure also significantly stimulated MUC2 expression and mucus secretion. Further studies indicated that SST receptor 5 (SSTR5) was significantly activated by SST, whereas specific SSTR5 siRNA transfection of LS174T cells significantly blocked SST-induced increase in MUC2 expression and mucus secretion. In addition, SSTR5 agonist L817,818 also upregulated MUC2 expression and mucus secretion in LS174T cells. Mechanistic studies further demonstrated that SST/SSTR5-mediated MUC2 upregulation was dependent on Notch-Hes1 pathway suppression by detecting notch intracellular domain (NICD) and Hes1 proteins. Taken together, our findings suggested that SST could participate in colonic mucus barrier regulation through SSTR5-Notch-Hes1-MUC2 signaling pathway. These findings provide a deep insight into the role of SST on colonic mucus regulation under physiological conditions.