Porcine Intestinal Mucus Research Articles

Porcine ex-vivo intestinal mucus has age-dependent blocking activity against transmissible gastroenteritis virus

Transmissible gastroenteritis virus (TGEV) causes high mortality in young piglets (< 3 days of age). With aging, the susceptibility/morbidity/mortality rates drop. We previously hypothesized that the age-related changes in the intestinal mucus could be responsible for this resistance. Hence, this study investigated the effect of porcine intestinal mucus from 3-day and 3-week-old pigs on the free mobility of the virulent TGEV Miller strain, and on the infection in swine testicle (ST) cells. Single particle tracking (SPT) revealed that TGEV had significantly higher diffusion coefficients in 3-day mucus compared to 3-week mucus. TGEV and charged and uncharged control nanoparticles diffused freely in 3-day mucus but were hindered by 3-week mucus in the diffusion model; TGEV mimicked the diffusion behavior of negatively charged carboxylated particles. Inoculation of ST cells with TGEV in the presence of 3-week mucus resulted in a significantly lower average number of infected cells (30.9 ± 11.9/5 fields) compared with 3-day mucus (84.6 ± 16.4/5 fields). These results show that 3-week mucus has a significant TGEV-blocking activity compared to 3-day mucus in free diffusion and infection of the underlying susceptible cells. Additionally, a label-free proteomics analysis revealed an increased expression of mucin 13, known for negatively regulating the tight junctions in intestinal epithelium, in 3-day-old pigs. In 3-week-old pigs, a higher expression of mucin 2, a type of secreted mucin which is known for inhibiting coronavirus infection, was observed. Concludingly, this study demonstrated a protective effect of 3-week mucus against viral infections.

Effect of beta glucan coating on controlled release, bioaccessibility, and absorption of β-carotene from loaded liposomes.

Recently, the use of biopolymers as coating material to stabilise phospholipid-based nanocarriers has increased. One such class of biopolymers is the dietary fibre beta-glucan (βG). In this study, we developed and characterized beta-carotene (βC) loaded βG coated nanoliposomes (GNLs) to investigate the effect of βG coating on the stability, controlled release, bioaccessibility, diffusion and subsequent absorption of the lipophilic active agent. The size, charge (Z-potential), and FTIR spectra were measured to determine the physicochemical stability of GNLs. βG coating reduced the bioaccessibility, provided prolonged release and improved the antioxidant activity of the nanoliposomes. Multiple particle tracking (MPT) data suggested that βC-GNLs were less diffusive in porcine intestinal mucus (PIM). Additionally, the microviscosity of the PIM treated with GNLs was observed to be higher (0.04744 ± 0.00865 Pa s) than the PIM incubated with uncoated NLs (0.015 ± 0.0004 Pa s). An Ex vivo experiment was performed on mouse jejunum to measure the absorption of beta-carotene from coated (βC-GNLs) and uncoated nanoliposomes (βC-NLs). Data showed that after 2 hours, 27.7 ± 1.3 ng mL-1 of βC encapsulated in GNLs and 61.54 ± 3 ng mL-1 of the βC encapsulated in uncoated NLs was absorbed by mouse intestinal mucosa. These results highlight that coating with βG stabilise NLs during gastrointestinal digestion and provides more sustained release of βC from nanoliposomes.

Milk exosomes anchored with hydrophilic and zwitterionic motifs enhance mucus permeability for applications in oral gene delivery.

Exosomes have emerged as a promising tool for the delivery of drugs and genetic materials, owing to their biocompatibility and non-immunogenic nature. However, challenges persist in achieving successful oral delivery due to their susceptibility to degradation in the harsh gastrointestinal (GI) environment and impeded transport across the mucus-epithelium barrier. To overcome these challenges, we have developed high-purity bovine milk exosomes (mExo) as a scalable and efficient oral drug delivery system, which can be customized by incorporating hydrophilic and zwitterionic motifs on their surface. In our study, we observed significantly improved transport rates by 2.5-4.5-fold in native porcine intestinal mucus after the introduction of hydrophilic and zwitterionic surface modifications, as demonstrated by transwell setup and fluorescence recovery after photobleaching (FRAP) analysis. Remarkably, mExo functionalized by a block peptide (BP), consisting of cationic and anionic amino acids arranged in blocks at the two ends, demonstrated superior tolerability in the acidic gastric environment (with a protein recovery rate of 84.8 ± 7.7%) and exhibited a 2.5-fold increase in uptake by intestinal epithelial cells. Furthermore, both mExo and mExo-BP demonstrated successful intracellular delivery of functional siRNA, resulting in up to 65% suppression of the target green fluorescence protein (GFP) gene expression at a low dose of siRNA (5 pmol) without causing significant toxicity. These findings highlight the immense potential of modifying mExo with hydrophilic and zwitterionic motifs for effective oral delivery of siRNA therapies.

Ex vivo models for intestinal translocation studies of cellulose nanocrystals

PurposeCellulose nanocrystals (CNC) play a promising role in the development of new advanced materials. The growing demand of CNC-containing products in the food industry will lead to an increased human exposure through oral uptake. To date, there is a dearth of studies reporting on the risks which CNC pose to human health following ingestion. In vitro models, which lack physiological accuracy, are often used to justify animal experiments in the field of nanosafety assessment. Nevertheless, ex vivo models of the intestine pose promising alternatives to in vivo experiments.MethodsTwo ex vivo models, a microfluidic chip based on porcine intestinal mucus and the Ussing chamber apparatus with tissue from abattoirs, which aim to complement in vitro models, are characterized by investigating the transport and toxicity of CNC through them in comparison to an in vitro triple co-culture model. Silver nanoparticles were included in this study as well-known and characterized nanomaterials for comparative purposes.ResultsStudy results show that CNC cross the intestinal mucus layer but do not pass the intestinal tissue barrier ex vivo and in vitro; furthermore, no toxic effects were observed under exposure conditions tested.ConclusionThese ex vivo models present complementary methods to the existing standardized in vitro and in silico methods to support data generation under physiologically relevant conditions without the use of animals. This multi-model approach offers an enhanced understanding of the complex interaction between new materials and human tissue and aligns with the flexible approach of IATA (Integrated Approaches to Testing and Assessment) and NAMs (New Approach Methods) for chemical and drug safety assessment.

Peptide Antibiotic-Polyphosphate Nanoparticles: A Promising Strategy to Overcome the Enzymatic and Mucus Barrier of the Intestine.

The aim of this study was to develop peptide antibiotic-polyphosphate nanoparticles that are able to overcome the enzymatic and mucus barriers providing a targeted drug release directly on the intestinal epithelium. Polymyxin B-polyphosphate nanoparticles (PMB-PP NPs) were formed via ionic gelation between the cationic peptide and the anionic polyphosphate (PP). The resulting NPs were characterized by particle size, polydispersity index (PDI), zeta potential, and cytotoxicity on Caco-2 cells. The protective effect of these NPs for incorporated PMB was evaluated via enzymatic degradation studies with lipase. Moreover, mucus diffusion of NPs was investigated with porcine intestinal mucus. Isolated intestinal alkaline phosphatase (IAP) was employed to trigger the degradation of NPs and consequent drug release. PMB-PP NPs exhibited an average size of 197.13 ± 14.13 nm, a PDI of 0.36, a zeta potential of -11.1 ± 3.4 mV and a concentration and time-dependent toxicity. They provided entire protection toward enzymatic degradation and exhibited significantly (p < 0.05) higher mucus permeating properties than PMB. When incubated with isolated IAP for 4 h, monophosphate and PMB were constantly released from PMB-PP NPs and zeta potential raised up to -1.9 ± 0.61 mV. According to these findings, PMB-PP NPs are promising delivery systems to protect cationic peptide antibiotics against enzymatic degradation, to overcome the mucus barrier and to provide drug release directly at the epithelium.

Abstract 1750: Removal of mucin glycans by Bifidobacterium bifidum enhances chemotherapy-mediated cell death in mucinous adenocarcinomas

Abstract Background: Mucinous colorectal adenocarcinoma, a subtype of colorectal cancer, is characterized by mucin production and increased resistance to several chemotherapeutic interventions; resulting in a poorer prognosis compared to non-mucus secreting cancers. Advances in treatment are stymied by the limited number of efficacious chemotherapeutic agents. The most common drug used to treat colorectal cancer is 5-Fluorouracil (5-FU), but less than 10% of mucinous tumors respond to this drug. The production of mucus has been theorized to limit the uptake of chemotherapeutic agents via charge repulsion and size exclusion, thereby promoting chemo-resistance and driving poor outcomes. Hypothesis: We predict that mucin-degrading microbes secrete glycosyl hydrolase enzymes which cleave mucin glycans and promote the uptake of chemotherapeutic agents and enhance cell death. Methods &amp; Results: We queried the human gut microbiota and identified multiple diverse microbes which encode enzymes to degrade mucin glycans. Among the mucin-degrading bacteria, we found that Bifidobacterium bifidum harbored one of the most extensive repertoires of mucin-degrading enzymes. B. bifidum was grown in a chemically defined medium with or without porcine intestinal mucus and the secreted products were then added to mucus-producing adenocarcinoma cell lines: HT29-MTX and T84 cells. Supernatant from cultures with mucus removed sialic acid residues and enhanced 5-FU uptake after 48 hrs in both cell lines. Supernatant without mucus also promoted 5-FU mediated cell death; although to a lesser degree than supernatant from cultures incubated with mucus. Gene expression analysis and immunostaining revealed decreased MUC13 gene expression and protein in B. bifidum metabolite treated cell lines compared to media controls. In contrast, we found that secreted products from B. dentium, which is unable to degrade mucus, had no effect on 5-FU induced cell death. Conclusions: Our data suggests that B. bifidum elicits chemo-sensitivity via the production of mucin-degrading enzymes, which cleave negatively charged sialic acid, increasing the activity of 5-FU in these cancers as well as by lowering the expression of adherent mucins. This work points to a novel role of mucin-degrading microbes to promote 5-FU penetration of mucinous colorectal adenocarcinoma. This strategy has the potential to provide unique treatment strategies for patients with mucinous colorectal cancer. Citation Format: Leah K. Stripe, Amy C. Engevik, Mindy A. Engevik. Removal of mucin glycans by Bifidobacterium bifidum enhances chemotherapy-mediated cell death in mucinous adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1750.

Mucoadhesive Dendrons Conjugated to Mesoporous Silica Nanoparticles as a Drug Delivery Approach for Orally Administered Biopharmaceuticals.

Biological drugs are increasingly important for patients and industry due to their application in the treatment of common and potentially life-threatening diseases such as diabetes, cancer, and obesity. While most marketed biopharmaceuticals today are injectables, the potential of mucoadhesive delivery systems based on dendron-coated mesoporous silica nanoparticles for oral delivery of biological drugs is explored in this project. We hypothesize that specifically designed dendrons can be employed as mucoadhesive excipients and used to decorate the surface of nanoparticles with properties to embed a drug molecule. We initially tested a novel synthesis method for the preparation of dendrons, which was successfully validated by the chemical characterization of the compounds. The interaction between dendrons and mucin was studied through isothermal titration calorimetry and quartz crystal microbalance with dissipation monitoring and proved to be spontaneous and thermodynamically favorable. Dendrons were conjugated onto 244.4 nm mesoporous silica nanoparticles and characterized for chemical composition, size, and surface charge, which all showed a successful conjugation. Finally, dynamic light scattering was used to study the interaction between nanoparticles and porcine gastric mucin, whereas the interaction between nanoparticles and porcine intestinal mucus was characterized by rheological measurements. This study shows a deeper biophysical understanding of the interaction between nanoparticles and mucin or native porcine intestinal mucus, further leveraging the current understanding of how dendrons can be used as excipients to interact with mucin. This will provide knowledge for the potential development of a new generation of mucoadhesive nanoformulations for the oral delivery of biopharmaceuticals.

Microscopic Cascading Devices for Boosting Mucus Penetration in Oral Drug Delivery-Micromotors Nesting Inside Microcontainers.

In the case of macromolecules and poorly permeable drugs, oral drug delivery features low bioavailability and low absorption across the intestinal wall. Intestinal absorption can be improved if the drug formulation could be transported close to the epithelium. To achieve this, a cascade delivery device comprising Magnesium-based Janus micromotors (MMs) nesting inside a microscale containers (MCs) has been conceptualized. The device aims at facilitating targeted drug delivery mediated by MMs that can lodge inside the intestinal mucosa. Loading MMs into MCs can potentially enhance drug absorption through increased proximity and unidirectional release. The MMs will be provided with optimal conditions for ejection into any residual mucus layer that the MCs have not penetrated. MMS confined inside MCs propel faster in the mucus environment as compared to non-confined MMs. Upon contact with a suitable fuel, the MM-loaded MC itself can also move. An in vitro study shows fast release profiles and linear motion properties in porcine intestinal mucus compared to more complex motion in aqueous media. The concept of dual-acting cascade devices holds great potential in applications where proximity to epithelium and deep mucus penetration are needed.

Physical and barrier changes in gastrointestinal mucus induced by the permeation enhancer sodium 8-[(2-hydroxybenzoyl)amino]octanoate (SNAC).

Drug delivery systems (DDS) for oral delivery of peptide drugs contain excipients that facilitate and enhance absorption. However, little knowledge exists on how DDS excipients such as permeation enhancers interact with the gastrointestinal mucus barrier. This study aimed to investigate interactions of the permeation enhancer sodium 8-[(2-hydroxybenzoyl)amino]octanoate (SNAC) with ex vivo porcine intestinal mucus (PIM), ex vivo porcine gastric mucus (PGM), as well as with in vitro biosimilar mucus (BM) by profiling their physical and barrier properties upon exposure to SNAC. Bulk mucus permeability studies using the peptides cyclosporine A and vancomycin, ovalbumin as a model protein, as well as fluorescein-isothiocyanate dextrans (FDs) of different molecular weights and different surface charges were conducted in parallel to mucus retention force studies using a texture analyzer, rheological studies, cryo-scanning electron microscopy (cryo-SEM), and single particle tracking of fluorescence-labelled nanoparticles to investigate the effects of the SNAC-mucus interaction. The exposure of SNAC to PIM increased the mucus retention force, storage modulus, viscosity, increased nanoparticle confinement within PIM as well as decreased the permeation of cyclosporine A and ovalbumin through PIM. Surprisingly, the viscosity of PGM and the permeation of cyclosporine A and ovalbumin through PGM was unaffected by the presence of SNAC, thus the effect of SNAC depended on the regional site that mucus was collected from. In the absence of SNAC, the permeation of different molecular weight and differently charged FDs through PIM was comparable to that through BM. However, while bulk permeation of neither of the FDs through PIM was affected by SNAC, the presence of SNAC decreased the permeation of FD4 and increased the permeation of FD150 kDa through BM. Additionally, and in contrast to observations in PIM, nanoparticle confinement within BM remained unaffected by the presence of SNAC. In conclusion, the present study showed that SNAC altered the physical and barrier properties of PIM, but not of PGM. The effects of SNAC in PIM were not observed in the BM in vitro model. Altogether, the study highlights the need for further understanding how permeation enhancers influence the mucus barrier and illustrates that the selected mucus model for such studies should be chosen with care.

Characterizing the mucin-degrading capacity of the human gut microbiota

Mucin-degrading microbes are known to harbor glycosyl hydrolases (GHs) which cleave specific glycan linkages. Although several microbial species have been identified as mucin degraders, there are likely many other members of the healthy gut community with the capacity to degrade mucins. The aim of the present study was to systematically examine the CAZyme mucin-degrading profiles of the human gut microbiota. Within the Verrucomicrobia phylum, all Akkermansia glycaniphila and muciniphila genomes harbored multiple gene copies of mucin-degrading GHs. The only representative of the Lentisphaerae phylum, Victivallales, harbored a GH profile that closely mirrored Akkermansia. In the Actinobacteria phylum, we found several Actinomadura, Actinomyces, Bifidobacterium, Streptacidiphilus and Streptomyces species with mucin-degrading GHs. Within the Bacteroidetes phylum, Alistipes, Alloprevotella, Bacteroides, Fermenitomonas Parabacteroides, Prevotella and Phocaeicola species had mucin degrading GHs. Firmicutes contained Abiotrophia, Blautia, Enterococcus, Paenibacillus, Ruminococcus, Streptococcus, and Viridibacillus species with mucin-degrading GHs. Interestingly, far fewer mucin-degrading GHs were observed in the Proteobacteria phylum and were found in Klebsiella, Mixta, Serratia and Enterobacter species. We confirmed the mucin-degrading capability of 23 representative gut microbes using a chemically defined media lacking glucose supplemented with porcine intestinal mucus. These data greatly expand our knowledge of microbial-mediated mucin degradation within the human gut microbiota.

Klebsiella pneumoniae utilizes intestinal mucus to increase fitness in the gastrointestinal tract

BackgroundKlebsiella pneumoniae is a growing concern in the healthcare setting, as carbapenem‐resistant Enterobacteriaceae are considered an urgent health threat by the Center of Disease Control (CDC). K. pneumoniae is found in the mucosa of the human gastrointestinal tract, but upon intestinal disruption K. pneumoniae can migrate into the blood stream and cause infection. One of the barriers against bacterial translocation is the intestinal mucus layer. Mucus proteins are covered with branching glycans which can serve as a fuel source for microbes that harbor mucus‐specific glycosyl hydrolases.HypothesisWe hypothesized that K. pneumoniae could degrade and utilize mucus as an energy source to increase its fitness in the GI tract.Methods &amp; ResultsUsing the Carbohydrate‐Active enZYme Database (CAZY), we identified that 274 K. pneumoniae genomes harbored at least 3 mucin‐degrading glycosyl hydrolase families. To confirm the ability of K. pneumoniae to degrade mucus, we grew five commercially available K. pneumoniae strains and six clinical isolates of K. pneumoniaein a chemically defined minimal media (CDMM) lacking glucose supplemented with mucin‐associated oligosaccharides or dialyzed porcine intestinal MUC2 mucus. All K. pneumoniae strains grew well is sialic acid, GalNAc, fucose and galactose. Interestingly, K. pneumoniae strains exhibited robust growth during log phase with glucose, but were unable to sustain long term growth. Surprisingly, some clinical isolates showed similar growth with GluNAC as with glucose, while other strains could not utilize GluNAC as an energy source. All but one strain of K. pneumoniae could grow with dialyzed mucus as the sole carbon source, indicating that K. pneumoniae can enzymatically degrade mucus and utilize the liberated sugars for growth. To determine how mucus influenced the pathogenesis of K. pneumoniae, we grew one commercially available and one clinical isolate of K. pneumoniae in CDMM with fucose or mucus, isolated the RNA and examined the expression of genes associated with K. pneumoniae virulence by qPCR. We examined virulence factors related to iron uptake (iroD), nucleic acid metabolism (allS), endotoxin production (clbA), fatty acid biosynthesis (clbB), type 1‐ fimbriae (fimH), and flagella (fliC). Both K. pneumoniae strains had increased iroD expression in the presence of mucus. Interestingly, we observed that the clinical isolate, but not the commercially available strain of K. pneumoniae had increased expression of virulence genes clbB, allS and fliC when grown with mucus when compared to being grown with fucose. K. pneumoniae is traditionally considered a nonmotile bacterium, however, we confirmed the expression of flagella in our clinical isolate by performing a chemotaxis assay using mucus‐associated sugars and mucus. We indeed saw that our commercially available strain was unable to migrate towards the mucus or mucus‐associated sugars. However, our clinical isolate was able to swim towards mucus‐associated sugars.ConclusionsThese results suggest that K. pneumoniae utilizes intestinal mucus as an energy source in the GI tract, which in turn influences the pathogenicity of clinical isolates of K. pneumoniae.

Barrier properties of ex vivo porcine intestinal mucus are highly independent of isolation and storage conditions

Porcine intestinal mucus (PIM) is often utilized as an ex vivo mucus model in mucus interaction studies. However, numerous isolation procedures and storage conditions for PIM are reported, yet their potential impact on preserving the critical properties of PIM remains unknown. This study investigated the effect of isolation procedures (rinsing and anatomical site of mucus isolation) and storage conditions (−20 °C, −80 °C, snap frozen in li-quid nitrogen prior to storage at −80 °C, or freeze-dried followed by storage at room temperature and reconstitution) of PIM in regard to the permeation of fluorescein-isothiocyanate-labelled dextran (FD) macromolecules of 4, 40 and 150 kDa, rheological properties as well as pH, osmolality, protein and water content. Rinsing intestines with tap water or phosphate-buffered saline as well as isolating PIM from different regions of the first five meters of the proximal jejunum did not affect the pH or osmolality of isolated PIM. The permeation of FD4, FD40 and FD150 through stored PIM was similar to permeation through fresh PIM. The rheological properties of stored PIM were similar to properties of fresh PIM. Osmolality, protein and water content were similar in stored and fresh PIM whereas pH decreased with 0.3 unit for all stored PIM. Overall, PIM samples stored at −20 °C, −80 °C, snap frozen or freeze-dried were found to have similar properties to freshly isolated PIM and can all be consi-dered good alternatives to fresh PIM for mucus studies.

Arcobacter vandammei sp. nov., isolated from the rectal mucus of a healthy pig

A study on the polyphasic taxonomic classification of an Arcobacter strain, R-73987T, isolated from the rectal mucus of a porcine intestinal tract, was performed. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the strain could be assigned to the genus Arcobacter and suggested that strain R-73987T belongs to a novel undescribed species. Comparative analysis of the rpoB gene sequence confirmed the findings. Arcobacter faecis LMG 28519T was identified as its closest neighbour in a multigene analysis based on 107 protein- encoding genes. Further, whole-genome sequence comparisons by means of average nucleotide identity and in silico DNA-DNA hybridization between the genome of strain R-73987T and the genomes of validly named Arcobacter species resulted in values below 95-96 and 70 %, respectively. In addition, a phenotypic analysis further corroborated the conclusion that strain R-73987T represents a novel Arcobacter species, for which the name Arcobacter vandammei sp. nov. is proposed. The type strain is R-73987T (=LMG 31429T=CCUG 75005T). This appears to be the first Arcobacter species recovered from porcine intestinal mucus.

Enhanced oral bioavailability of rutin by a self-emulsifying drug delivery system of an extract of calyces from Physalis peruviana

The aim of this study was to develop and optimize a self-emulsifying drug delivery system (SEDDS) containing an extract of calyces from Physalis peruviana with high mucus permeating properties to increase the bioavailability and the hypoglycemic activity of the active metabolites of the extract. The oil, surfactant, and co-solvent were selected based on solubility studies. The formulation was optimized using Box-Behnken statistical experimental design (BBD). Optimized conditions were Labrafac as oil 10% (w/w), Solutol HS 15 as surfactant 45% (w/w), propylene glycol as co-solvent 32% (w/w) and PDMSHEPMS 13% (w/w). The extract was loaded into SEDDS to 45% (w/w). The optimized SEDDS exhibited a mean droplet size of 19.5 nm, PDI of 0.20, and negative zeta potential. Furthermore, SEDDS permeating properties were investigated in porcine intestinal mucus using the Transwell® method. Extract-loaded SEDDS showed a 2.6-fold increase in mucus permeation compared to the unformulated extract. A pharmacokinetic study showed a nearly 6-fold increase in rutin oral bioavailability in comparison with the unformulated extract. Moreover, the hypoglycemic activity of the extract-loaded SEDDS was higher than that of the unformulated extract. These results indicate that incorporating an extract of calyces from P. peruviana into SEDDS is a promising strategy to improve the oral bioavailability of the active flavonoids in the extract and the hypoglycemic activity.

Alkaline pH Increases Swimming Speed and Facilitates Mucus Penetration for Vibrio cholerae.

Intestinal mucus is the first line of defense against intestinal pathogens. It acts as a physical barrier between epithelial tissues and the lumen that enteropathogens must overcome to establish a successful infection. We investigated the motile behavior of two Vibrio cholerae strains (El Tor C6706 and Classical O395) in mucus using single-cell tracking in unprocessed porcine intestinal mucus. We determined that V. cholerae can penetrate mucus using flagellar motility and that alkaline pH increases swimming speed and, consequently, improves mucus penetration. Microrheological measurements indicate that changes in pH between 6 and 8 (the physiological range for the human small intestine) had little effect on the viscoelastic properties of mucus. Finally, we determined that acidic pH promotes surface attachment by activating the mannose-sensitive hemagglutinin (MshA) pilus in V. cholerae El Tor C6706 without a measurable change in the total cellular concentration of the secondary messenger cyclic dimeric GMP (c-di-GMP). Overall, our results support the hypothesis that pH is an important factor affecting the motile behavior of V. cholerae and its ability to penetrate mucus. Therefore, changes in pH along the human small intestine may play a role in determining the preferred site for V. cholerae during infection.IMPORTANCE The diarrheal disease cholera is still a burden for populations in developing countries with poor sanitation. To develop effective vaccines and prevention strategies against Vibrio cholerae, we must understand the initial steps of infection leading to the colonization of the small intestine. To infect the host and deliver the cholera toxin, V. cholerae has to penetrate the mucus layer protecting the intestinal tissues. However, the interaction of V. cholerae with intestinal mucus has not been extensively investigated. In this report, we demonstrated using single-cell tracking that V. cholerae can penetrate intestinal mucus using flagellar motility. In addition, we observed that alkaline pH improves the ability of V. cholerae to penetrate mucus. This finding has important implications for understanding the dynamics of infection, because pH varies significantly along the small intestine, between individuals, and between species. Blocking mucus penetration by interfering with flagellar motility in V. cholerae, reinforcing the mucosa, controlling intestinal pH, or manipulating the intestinal microbiome will offer new strategies to fight cholera.

Comparing the permeability of human and porcine small intestinal mucus for particle transport studies

The gastrointestinal mucus layer represents the last barrier between ingested food or orally administered pharmaceuticals and the mucosal epithelium. This complex gel structure plays an important role in the process of small intestinal absorption. It provides protection against hazardous particles such as bacteria but allows the passage of nutrients and drug molecules towards the intestinal epithelium. In scientific research, mucus from animal sources is usually used to simulate difficult-to-obtain human small intestinal mucus for investigating the intramucus transport of drug delivery systems or food nanoparticles. However, there is a lack of evidence the human mucus can be reliably substituted by animal counterparts for human-relevant transport models. In this report, a procedure for collecting human mucus has been described. More importantly, the permeability characteristics of human and porcine small intestinal mucus secretions to sub-micron sized particles have been compared under simulated intestinal conditions. Negatively charged, 500 nm latex beads were used in multiple-particle tracking experiments to examine the heterogeneity and penetrability of mucus from different sources. Diffusion of the probe particles in adult human ileal mucus and adult pig jejunal and ileal mucus revealed no significant differences in microstructural organisation or microviscosity between the three mucus types (P > 0.05). In contrast to this interspecies similarity, the intraspecies comparison of particle diffusivity in the mucus obtained from adult pigs vs. 2-week old piglets showed better penetrability of the piglet mucus. The mean Stokes–Einstein viscosity of the piglet jejunal mucus was approx. two times lower than the viscosity of the pig jejunal mucus (P < 0.05). All mucus structures were also visualised by scanning electron microscopy. This work validates the use of porcine small intestinal mucus collected from fully-grown pigs for studying colloidal transport of sub-micron sized particles in mucus under conditions mimicking the adult human small intestinal environment.

Colloidal transport of lipid digesta in human and porcine small intestinal mucus

Small intestinal mucus transport of food-derived particulates has not been extensively studied, despite mucus being a barrier nutrients need to cross before absorption. We used complex dispersions of digesta obtained from simulated, dynamic gastrointestinal digestion of yogurt to examine the penetrability of human and porcine mucus to the particles formed of lipolysis products. Quantitative, time-lapse confocal microscopy revealed a sieve-like behaviour of the pig jejunal and ileal mucus. The digesta diffusivity decreased significantly over the first 30 min of mucus penetration, and then remained constant at ca. 5 × 10-12 m2 s−1 (approx. 70% decrease from initial values). A non-significantly different penetrability was recorded for the ileal mucus of adult humans. The digesta diffusion rates in neonatal, jejunal mucus of 2 week old piglets were 5–8 times higher than in the three different types of adult mucus. This is the first report that validates the mucus of fully-grown pigs as a human-relevant substitute for mucus permeation studies of nutrients/bio-actives and/or complex colloidal dispersions (e.g., post-digestion food particulates, orally-administrated delivery systems).

Synthesis and in vitro characterization of a preactivated thiolated acrylic acid/acrylamide-methylpropane sulfonic acid copolymer as a mucoadhesive sprayable polymer.

Development of a preactivated thiomer as sprayable excipient for mucoadhesive formulations. CG4500 (acrylic acid/acrylamide-methyl propane sulfonic acid copolymer) was thiolated by conjugation with L-cysteine and preactivated by further modification with 2-mercaptonicotinic acid (MNA) in a two-step synthesis and characterized regarding degree of modification and cytotoxicity on Caco-2 cells. The mucoadhesive properties of this novel thiomer were evaluated via rheological synergism, tensile and mucosal residence time studies. Furthermore, the sprayability of the thiomer was evaluated. The newly synthesized derivatives CG4500-SH and CG4500-S-S-MNA showed mean coupling rates of 651µmol thiol groups and 264µmol MNA per gram polymer, respectively. Even for the unmodified polymer a rheological synergism was observed with isolated porcine intestinal mucus, which was 2.81-fold higher in case of the preactivated thiomer. Mucoadhesion studies on freshly excised porcine intestinal mucosa confirmed these results via a 2.43-fold higher total work of adhesion and a 2.31-fold higher mucosal residence time of the preactivated thiomer. In sprayability tests it was shown that solutions of the preactivated thiomer could be sprayed in concentrations up to 12% (m/V). The novel polymer CG4500-S-S-MNA is a promising sprayable excipient for mucoadhesive formulations.

Oral Immunogenicity in Mice and Sows of Enterotoxigenic Escherichia Coli Outer-Membrane Vesicles Incorporated into Zein-Based Nanoparticles.

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of illness and death in neonatal and recently weaned pigs. The immune protection of the piglets derives from maternal colostrum, since this species does not receive maternal antibodies through the placenta. In the present study, outer membrane vesicles (OMVs) obtained from main ETEC strains involved in piglet infection (F4 and F18 serotypes), encapsulated into zein nanoparticles coated with Gantrez®® AN-mannosamine conjugate, were used to orally immunize mice and pregnant sows. Loaded nanoparticles were homogeneous and spherical in a shape, with a size of 220–280 nm. The diffusion of nanoparticles through porcine intestinal mucus barrier was assessed by a Multiple Particle Tracking technique, showing that these particles were able to diffuse efficiently (1.3% diffusion coefficient), validating their oral use. BALB/c mice were either orally immunized with free OMVs or encapsulated into nanoparticles (100 µg OMVs/mouse). Results indicated that a single dose of loaded nanoparticles was able to elicit higher levels of serum specific IgG1, IgG2a and IgA, as well as intestinal IgA, with respect to the free antigens. In addition, nanoparticles induced an increase in levels of IL-2, IL-4 and IFN-γ with respect to the administration of free OMVs. Orally immunized pregnant sows with the same formulation elicited colostrum-, serum- (IgG, IgA or IgM) and fecal- (IgA) specific antibodies and, what is most relevant, offspring suckling piglets presented specific IgG in serum. Further studies are needed to determine the infection protective capacity of this new oral subunit vaccine

A novel microfluidic mucus-chip for studying the permeation of compounds over the mucus barrier

A major challenge of drug discovery is to overcome the mucus barrier, which is lined the surface of the gastrointestinal tract. The ability to overcome this barrier must be tested in preclinical in vitro models, prior to in vivo studies. Therefore, a microfluidic chip was developed to examine the possibility of compounds to overcome a mucus barrier. Native porcine intestinal mucus was used as a mucus model. The mucus was loaded inside the chip comprising of a microporous membrane and a fluidic compartment. To validate the system, caffeine as a hydrophilic positive compound, fluorescein isothiocyanate–dextran (FITC-dextran) as high molecular negative compound and diclofenac sodium as poorly permeating compound were tested. Test samples were applied on the surface of the mucus and samples were collected every 30 min from the fluidic compartment underneath the membrane. The permeation was measured over 3 h and the permeated compound was detected by high-pressure liquid chromatography (HPLC) and a microplate fluorescence reader. The results show that caffeine and diclofenac sodium are permeating over the mucus layer proportionally to the time. FITC-dextran, chosen as non-permeating compound, displays equivalently no permeation. The validation provides the functionality of the developed mucus-chip system.