Glycosaminoglycans compositional analysis of Urodele axolotl (Ambystoma mexicanum) and Porcine Retina.

Retinal degenerative diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), are major causes of blindness worldwide. Humans cannot regenerate retina, however, axolotl (Ambystoma mexicanum), a laboratory-bred salamander, can regenerate retinal tissue throughout adulthood. Classic signaling pathways, including fibroblast growth factor (FGF), are involved in axolotl regeneration. Glycosaminoglycan (GAG) interaction with FGF is required for signal transduction in this pathway. GAGs are anionic polysaccharides in extracellular matrix (ECM) that have been implicated in limb and lens regeneration of amphibians, however, GAGs have not been investigated in the context of retinal regeneration. GAG composition is characterized native and decellularized axolotl and porcine retina using liquid chromatography mass spectrometry. Pig was used as a mammalian vertebrate model without the ability to regenerate retina. Chondroitin sulfate (CS) was the main retinal GAG, followed by heparan sulfate (HS), hyaluronic acid, and keratan sulfate in both native and decellularized axolotl and porcine retina. Axolotl retina exhibited a distinctive GAG composition pattern in comparison with porcine retina, including a higher content of hyaluronic acid. In CS, higher levels of 4- and 6- O-sulfation were observed in axolotl retina. The HS composition was greater in decellularized tissues in both axolotl and porcine retina by 7.1% and 15.4%, respectively, and different sulfation patterns were detected in axolotl. Our findings suggest a distinctive GAG composition profile of the axolotl retina set foundation for role of GAGs in homeostatic and regenerative conditions of the axolotl retina and may further our understanding of retinal regenerative models.

Integrated gut/liver microphysiological systems elucidates inflammatory inter-tissue crosstalk.

A capability for analyzing complex cellular communication among tissues is important in drug discovery and development, and in vitro technologies for doing so are required for human applications. A prominent instance is communication between the gut and the liver, whereby perturbations of one tissue can influence behavior of the other. Here, we present a study on human gut-liver tissue interactions under normal and inflammatory contexts, via an integrative multi-organ platform comprising human liver (hepatocytes and Kupffer cells), and intestinal (enterocytes, goblet cells, and dendritic cells) models. Our results demonstrated long-term (>2 weeks) maintenance of intestinal (e.g., barrier integrity) and hepatic (e.g., albumin) functions in baseline interaction. Gene expression data comparing liver in interaction with gut, versus isolation, revealed modulation of bile acid metabolism. Intestinal FGF19 secretion and associated inhibition of hepatic CYP7A1 expression provided evidence of physiologically relevant gut-liver crosstalk. Moreover, significant non-linear modulation of cytokine responses was observed under inflammatory gut-liver interaction; for example, production of CXCR3 ligands (CXCL9,10,11) was synergistically enhanced. RNA-seq analysis revealed significant upregulation of IFNα/ß/γ signaling during inflammatory gut-liver crosstalk, with these pathways implicated in the synergistic CXCR3 chemokine production. Exacerbated inflammatory response in gut-liver interaction also negatively affected tissue-specific functions (e.g., liver metabolism). These findings illustrate how an integrated multi-tissue platform can generate insights useful for understanding complex pathophysiological processes such as inflammatory organ crosstalk. Biotechnol. Bioeng. 2017;114: 2648-2659. © 2017 Wiley Periodicals, Inc.

Mathematical model of intestinal lipolysis of a long-chain triglyceride.

Lipids are an important component of food and oral drug formulations. Upon release into gastrointestinal fluids, triglycerides, common components of foods and drug delivery systems, form emulsions and are digested into simpler amphiphilic lipids (e.g., fatty acids) that can associate with intestinal bile micelles and impact their drug solubilization capacity. Digestion of triglycerides is dynamic and dependent on lipid quantity and type, and quantities of other components in the intestinal environment (e.g., bile salts, lipases). The ability to predict lipid digestion kinetics in the intestine could enhance understanding of lipid impact on the fate of co-administered compounds (e.g., drugs, nutrients). In this study, we present a kinetic model that can predict the lipolysis of emulsions of triolein, a model long-chain triglyceride, as a function of triglyceride amount, droplet size, and quantity of pancreatic lipase in an intestinal environment containing bile micelles. The model is based on a Ping Pong Bi Bi mechanism coupled with quantitative analysis of partitioning of lipolysis products in colloids, including bile micelles, in solution. The agreement of lipolysis model predictions with experimental data suggests that the mechanism and proposed assumptions adequately represent triglyceride digestion in a simulated intestinal environment. In addition, we demonstrate the value of such a model over simpler, semi-mechanistic models reported in the literature. This lipolysis framework can serve as a basis for modeling digestion kinetics of different classes of triglycerides and other complex lipids as relevant in food and drug delivery systems.

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.

Cationic-motif-modified exosomes for mRNA delivery to retinal photoreceptors.

Topical treatment of vitreoretinal diseases remains a challenge due to slow corneal uptake and systemic clearance. Exosomes are emerging nanocarriers for drug delivery due to biocompatibility and cellular targeting properties. To apply them for retinal targeting via the topical route, exosomes must traverse various ocular barriers including the cornea, lens, vitreous humor (VH), and the retina itself. Here we engineered high-purity milk-derived exosomes by anchoring arginine-rich cationic motifs via PEG2000 lipid insertion on their surface. Modification enabled exosomes to use weak-reversible electrostatic interactions with anionic glycosaminoglycan (GAG) and water content of the tissue to enhance their transport rate and retention. Addition of cationic motifs neutralized the anionic surface charge of exosomes (-24 to -2 mV) without impacting size or morphology. Cationic-motif-modified exosomes exhibited two-fold faster steady state diffusivity through bovine corneas compared to unmodified exosomes. Fluorescence recovery after photobleaching confirmed that cationic-motif-modified exosomes can diffuse through VH without steric hindrance. In healthy VH, cationic-motif-modified exosomes demonstrated stronger binding resulting in three-fold lower average diffusivity that enhanced by six-fold in 50% GAG-depleted VH recapitulating advanced liquefaction. Cationic-motif-modified exosomes penetrated through the full-thickness of porcine retinal explants resulting in ten-fold higher uptake in photoreceptors and three-fold greater transfection with encapsulated eGFP mRNA compared to unmodified exosomes. Cationic-motif-modified exosomes are safe to use as they did not adversely affect the mechanical swelling properties of the cornea or lens nor impact retinal cell viability. Cationic-motif-modified exosomes, therefore, offer themselves as a cell-free nanocarrier platform for gene delivery to retinal photoreceptors potentially via the topical route.

Model predicting impact of complexation with cyclodextrins on oral absorption.

Significant effort and resource expenditure is dedicated to enabling low-solubility oral drug delivery using solubilization technologies. Cyclodextrins (CD) are cyclic oligosaccharides which form inclusion complexes with many drugs and are often used as solubilizing agents. It is not clear prior to developing a drug delivery device with CD what level of absorption enhancement might be achieved; modeling can provide useful guidance in formulation and minimize resource intensive iterative formulation development. A model was developed to enable quantitative, dynamic prediction of the influence of CD on oral absorption of low solubility drug administered as a pre-formed complex. The predominant effects of CD considered were enhancement of dissolution and slowing of precipitation kinetics, as well as binding of free drug in solution. Simulation results with different parameter values reflective of typical drug and CD properties indicate a potential positive (up to five times increase in drug absorption), negative (up to 50% decrease in absorption) or lack of effect of CD. Comparison of model predictions with in vitro and in vivo experimental results indicate that a systems-based dynamic model incorporating CD complexation and key process kinetics may enable quantitative prediction of impact of CD delivered as a pre-formed complex on drug bioavailability.

Effect of ingested lipids on drug dissolution and release with concurrent digestion: a modeling approach.

PURPOSE: To mechanistically study and model the effect of lipids, either from food or self-emulsifying drug delivery systems (SEDDS), on drug transport in the intestinal lumen. METHODS: Simultaneous lipid digestion, dissolution/release, and drug partitioning were experimentally studied and modeled for two dosing scenarios: solid drug with a food-associated lipid (soybean oil) and drug solubilized in amodel SEDDS (soybean oil and Tween 80 at 1:1 ratio). Rate constants for digestion, permeability of emulsion droplets, and partition coefficients in micellar and oil phases were measured, and used to numerically solve the developed model. RESULTS: Strong influence of lipid digestion on drug release from SEDDS and solid drug dissolution into food-associated lipid emulsion was observed and predicted by the developed model. Ninety minutes after introduction of SEDDS, there was 9% and 70% drug release in the absence and presence of digestion, respectively. However, overall drug dissolution in the presence of food-associated lipids occurred over a longer period than without digestion. CONCLUSION: A systems-based mechanistic model incorporating simultaneous dynamic processes occurring upon dosing of drug with lipids enabled prediction of aqueous drug concentration profile. This model, once incorporated with a pharmacokinetic model considering processes of drug absorption and drug lymphatic transport in the presence of lipids, could be highly useful for quantitative prediction of impact of lipids on bioavailability of drugs.

Native gastrointestinal mucus: Critical features and techniques for studying interactions with drugs, drug carriers, and bacteria.

Gastrointestinal mucus plays essential roles in modulating interactions between intestinal lumen contents, including orally delivered drug carriers and the gut microbiome, and underlying epithelial and immune tissues and cells. This review is focused on the properties of and methods for studying native gastrointestinal mucus and its interactions with intestinal lumen contents, including drug delivery systems, drugs, and bacteria. The properties of gastrointestinal mucus important to consider in its analysis are first presented, followed by a discussion of different experimental setups used to study gastrointestinal mucus. Applications of native intestinal mucus are then described, including experimental methods used to study mucus as a barrier to drug delivery and interactions with intestinal lumen contents that impact barrier properties. Given the significance of the microbiota in health and disease, its impact on drug delivery and drug metabolism, and the use of probiotics and microbe-based delivery systems, analysis of interactions of bacteria with native intestinal mucus is then reviewed. Specifically, bacteria adhesion to, motility within, and degradation of mucus is discussed. Literature noted is focused largely on applications of native intestinal mucus models as opposed to isolated mucins or reconstituted mucin gels.

Recent Advances in Biocompatible Ionic Liquids in Drug Formulation and Delivery.

The development of effective drug formulations and delivery systems for newly developed or marketed drug molecules remains a significant challenge. These drugs can exhibit polymorphic conversion, poor bioavailability, and systemic toxicity, and can be difficult to formulate with traditional organic solvents due to acute toxicity. Ionic liquids (ILs) are recognized as solvents that can improve the pharmacokinetic and pharmacodynamic properties of drugs. ILs can address the operational/functional challenges associated with traditional organic solvents. However, many ILs are non-biodegradable and inherently toxic, which is the most significant challenge in developing IL-based drug formulations and delivery systems. Biocompatible ILs comprising biocompatible cations and anions mainly derived from bio-renewable sources are considered a green alternative to both conventional ILs and organic/inorganic solvents. This review covers the technologies and strategies developed to design biocompatible ILs, focusing on the design of biocompatible IL-based drug formulations and delivery systems, and discusses the advantages of these ILs in pharmaceutical and biomedical applications. Furthermore, this review will provide guidance on transitioning to biocompatible ILs rather than commonly used toxic ILs and organic solvents in fields ranging from chemical synthesis to pharmaceutics.

Engineered bacteria titrate hydrogen sulfide and induce concentration-dependent effects on the host in a gut microphysiological system.

Hydrogen sulfide (H2S) is a gaseous microbial metabolite whose role in gut diseases is debated, with contradictory results stemming from experimental difficulties associated with accurate dosing and measuring H2S and the use of model systems that do not accurately represent the human gut environment. Here, we engineer Escherichia coli to titrate H2S across the physiological range in a gut microphysiological system (chip) supportive of the co-culture of microbes and host cells. The chip is engineered to maintain H2S gas tension and enables visualization of co-culture in real time with confocal microscopy. Engineered strains colonize the chip and are metabolically active for 2 days, during which they produce H2S across a 16-fold range and induce changes in host gene expression and metabolism in an H2S-concentration-dependent manner. These results validate a platform for studying the mechanisms underlying microbe-host interactions by enabling experiments that are infeasible with current animal and in vitro models.

Interactions of microbicide nanoparticles with a simulated vaginal fluid.

The interaction with cervicovaginal mucus presents the potential to impact the performance of drug nanocarriers. These systems must migrate through this biological fluid in order to deliver their drug payload to the underlying mucosal surface. We studied the ability of dapivirine-loaded polycaprolactone (PCL)-based nanoparticles (NPs) to interact with a simulated vaginal fluid (SVF) incorporating mucin. Different surface modifiers were used to produce NPs with either negative (poloxamer 338 NF and sodium lauryl sulfate) or positive (cetyltrimethylammonium bromide) surface charge. Studies were performed using the mucin particle method, rheological measurements, and real-time multiple particle tracking. Results showed that SVF presented rheological properties similar to those of human cervicovaginal mucus. Analysis of NP transport indicated mild interactions with mucin and low adhesive potential. In general, negatively charged NPs underwent subdiffusive transport in SVF, i.e., hindered as compared to their diffusion in water, but faster than for positively charged NPs. These differences were increased when the pH of SVF was changed from 4.2 to 7.0. Diffusivity was 50- and 172-fold lower in SVF at pH 4.2 than in water for negatively charged and positively charged NPs, respectively. At pH 7.0, this decrease was around 20- and 385-fold, respectively. The estimated times required to cross a layer of SVF were equal to or lower than 1.7 h for negatively charged NPs, while for positively charged NPs these values were equal to or higher than 7 h. Overall, our results suggest that negatively charged PCL NPs may be suitable to be used as carriers in order to deliver dapivirine and potentially other antiretroviral drugs to the cervicovaginal mucosal lining. Also, they further reinforce the importance in characterizing the interactions of nanosystems with mucus fluids or surrogates when considering mucosal drug delivery.

An in vitro intestinal model captures immunomodulatory properties of the microbiota in inflammation.

Considerable effort has been put forth to understand mechanisms by which the microbiota modulates and responds to inflammation. Here, we explored whether oxidation metabolites produced by the host during inflammation, sodium nitrate and trimethylamine oxide, impact the composition of a human stool bacterial population in a gut simulator. We then assessed whether an immune-competent in vitro intestinal model responded differently to spent medium from bacteria exposed to these cues compared to spent medium from a control bacterial population. The host-derived oxidation products were found to decrease levels of Bacteroidaceae and overall microbiota metabolic potential, while increasing levels of proinflammatory Enterobacteriaceae and lipopolysaccharide in bacterial cultures, reflecting shifts that occur in vivo in inflammation. Spent microbiota media induced elevated intracellular mucin levels and reduced intestinal monolayer integrity as reflected in transepithelial electrical resistance relative to fresh medium controls. However, multiplexed cytokine analysis revealed markedly different cytokine signatures from intestinal cultures exposed to spent medium with added oxidation products relative to spent control medium, while cytokine signatures of cultures exposed to fresh media were similar regardless of addition of host-derived cues. Further, the presence of immune cells in the intestinal model was required for this differentiation of cytokine signatures. This study indicates that simple in vitro immune-competent intestinal models can capture bacterial-mammalian cross-talk in response to host-derived oxidation products and supports utility of these systems for mechanistic studies of interactions between the gut microbiome and host in inflammation.

Reactive oxygen species limit intestinal mucosa-bacteria homeostasis in vitro.

Interactions between epithelial and immune cells with the gut microbiota have wide-ranging effects on many aspects of human health. Therefore, there is value in developing in vitro models capable of performing highly controlled studies of such interactions. However, several critical factors that enable long term homeostasis between bacterial and mammalian cultures have yet to be established. In this study, we explored a model consisting of epithelial and immune cells, as well as four different bacterial species (Bacteroides fragilis KLE1958, Escherichia coli MG1655, Lactobacillus rhamnosus KLE2101, or Ruminococcus gnavus KLE1940), over a 50 hour culture period. Interestingly, both obligate and facultative anaerobes grew to similar extents in aerobic culture environments during the co-culture period, likely due to measured microaerobic oxygen levels near the apical surface of the epithelia. It was demonstrated that bacteria elicited reactive oxygen species (ROS) production, and that the resulting oxidative damage heavily contributed to observed epithelial barrier damage in these static cultures. Introduction of a ROS scavenger significantly mitigated oxidative damage, improving cell monolayer integrity and reducing lipid peroxidation, although not to control (bacteria-free culture) levels. These results indicate that monitoring and mitigating ROS accumulation and oxidative damage can enable longer term bacteria-intestinal epithelial cultures, while also highlighting the significance of additional factors that impact homeostasis in mammalian cell-bacteria systems.

Synergistic Action of Diclofenac with Endotoxin-Mediated Inflammation Exacerbates Intestinal Injury in Vitro.

Intestinal homeostasis is tightly regulated by the orchestrated actions of a multitude of cell types, including enterocytes, goblet cells, and immune cells. Disruption of intestinal barrier function can increase susceptibility to pathogen invasion and destabilize commensal microbial-epithelial-immune interaction, manifesting in various intestinal and systemic pathologies. However, a quantitative understanding of how these cell types communicate and collectively contribute to tissue function in health and disease is lacking. Here, we utilized a human intestinal epithelial-dendritic cell model and multivariate analysis of secreted factors to investigate the cellular crosstalk in response to physiological and/or pathological cues (e.g., endotoxin, nonsteroidal anti-inflammation drug (NSAID)). Specifically, we demonstrated that treatment with diclofenac (DCF), an NSAID commonly used to treat inflammation associated with acute infection and other conditions, globally suppressed cytokine secretion when dosed in isolation. However, the disruption of barrier function induced by DCF allowed for luminal lipopolysaccharide (LPS) translocation and activation of resident immune cells that overrode the anti-inflammatory influence of DCF. DCF-facilitated inflammation in the presence of LPS was in part mediated by upregulation of macrophage migration inhibitory factor (MIF), an important regulator of innate immunity. However, while neutralization of MIF activity normalized inflammation, it did not lead to intestinal healing. Our data suggest that systems-wide suppression of inflammation alone is insufficient to achieve mucosal healing, especially in the presence of DCF, the target of which, the COX-prostaglandin pathway, is central to mucosal homeostasis. Indeed, DCF removal postinjury enabled partial recovery of intestinal epithelium functions, and this recovery phase was associated with upregulation of a subset of cytokines and chemokines, implicating their potential contribution to intestinal healing. The results highlight the utility of an intestinal model capturing immune function, coupled with multivariate analysis, in understanding molecular mechanisms governing response to microbial factors, supporting application in studying host-pathogen interactions.

Coculture of primary human colon monolayer with human gut bacteria.

The presence of microbes in the colon impacts host physiology. Therefore, microbes are being evaluated as potential treatments for colorectal diseases. Humanized model systems that enable robust culture of primary human intestinal cells with bacteria facilitate evaluation of potential treatments. Here, we describe a protocol that can be used to coculture a primary human colon monolayer with aerotolerant bacteria. Primary human colon cells maintained as organoids are dispersed into single-cell suspensions and then seeded on collagen-coated Transwell inserts, where they attach and proliferate to form confluent monolayers within days of seeding. The confluent monolayers are differentiated for an additional 4 d and then cocultured with bacteria. As an example application, we describe how to coculture differentiated colon cells for 8 h with four strains of Bacteroides thetaiotaomicron, each engineered to detect different colonic microenvironments via genetically embedded logic circuits incorporating deoxycholic acid and anhydrotetracycline sensors. Characterization of this coculture system reveals that barrier function remains intact in the presence of engineered B. thetaiotaomicron. The bacteria stay close to the mucus layer and respond in a microenvironment-specific manner to the inducers (deoxycholic acid and anhydrotetracycline) of the genetic circuits. This protocol thus provides a useful mucosal barrier system to assess the effects of bacterial cells that respond to the colonic microenvironment, and may also be useful in other contexts to model human intestinal barrier properties and microbiota-host interactions.

Primary human colonic mucosal barrier crosstalk with super oxygen-sensitive Faecalibacterium prausnitzii in continuous culture.

BACKGROUND: The gut microbiome plays an important role in human health and disease. Gnotobiotic animal and in vitro cell-based models provide some informative insights into mechanistic crosstalk. However, there is no existing system for a long-term co-culture of a human colonic mucosal barrier with super oxygen-sensitive commensal microbes, hindering the study of human-microbe interactions in a controlled manner. METHODS: Here, we investigated the effects of an abundant super oxygen-sensitive commensal anaerobe, Faecalibacterium prausnitzii, on a primary human mucosal barrier using a Gut-MIcrobiome (GuMI) physiome platform that we designed and fabricated. FINDINGS: Long-term continuous co-culture of F. prausnitzii for two days with colon epithelia, enabled by continuous flow of completely anoxic apical media and aerobic basal media, resulted in a strictly anaerobic apical environment fostering growth of and butyrate production by F. prausnitzii, while maintaining a stable colon epithelial barrier. We identified elevated differentiation and hypoxia-responsive genes and pathways in the platform compared with conventional aerobic static culture of the colon epithelia, attributable to a combination of anaerobic environment and continuous medium replenishment. Furthermore, we demonstrated anti-inflammatory effects of F. prausnitzii through HDAC and the TLR-NFKB axis. Finally, we identified that butyrate largely contributes to the anti-inflammatory effects by downregulating TLR3 and TLR4. CONCLUSIONS: Our results are consistent with some clinical observations regarding F. prausnitzii, thus motivating further studies employing this platform with more complex engineered colon tissues for understanding the interaction between the human colonic mucosal barrier and microbiota, pathogens, or engineered bacteria.

Biocompatibility of plasma enhanced chemical vapor deposited poly(2-hydroxyethyl methacrylate) films for biomimetic replication of the intestinal basement membrane.

It is recognized that topographical features such as ridges and grooves can dramatically influence cell phenotype, motivating the development of substrates with precisely biomimetic topography for study of the influence on cultured cells. Intestinal basement membrane topography has been precisely replicated using plasma enhanced chemical vapor deposition (CVD) of poly(2-hydroxyethyl methacrylate) (pHEMA) on native tissue. The ability for CVD pHEMA to coat and retain the complex architecture of the intestinal basement membrane at the micrometer scale was demonstrated using electron microscopy and surface chemical analysis (XPS). The suitability of CVD pHEMA as a cell culture substrate was assessed. Caco-2 cells maintained a high (>85%) viability on CVD pHEMA. Cell attachment and proliferation on CVD pHEMA were similar to those observed on materials traditionally used for cell culture and microfabrication purposes. Results indicate that CVD pHEMA is useful for development of precise (micrometer-scale) topographically biomimetic substrates for cell culture.

Impact of Developmental Age, Necrotizing Enterocolitis Associated Stress, and Oral Therapeutic Intervention on Mucus Barrier Properties.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of incompletely understood pathophysiology predominantly affecting premature infants. While NEC is associated with microbial invasion of intestinal tissues, and mucus modulates interactions between microbes and underlying tissues, variations in mucus barrier properties with NEC-associated risk factors have not been investigated. This study explored differences in mucus composition (total protein, DNA, mucin content, sialic acid, and immunoregulatory proteins), as well as structural and transport properties, assessed by tracking of particles and bacteria (E. coli and E. cloacae) with developmental age and exposure to NEC stressors in Sprague Dawley rats. Early developmental age (5 day old) was characterized by a more permeable mucus layer relative to 21 day old pups, suggesting immaturity may contribute to exposure of the epithelium to microbes. Exposure to NEC stressors was associated with reduced mucus permeability, which may aid in survival. Feeding with breastmilk as opposed to formula reduces incidence of NEC. Thus, NEC-stressed (N-S) rat pups were orally dosed with breastmilk components lysozyme (N-S-LYS) or docosahexaenoic acid (N-S-DHA). N-S-LYS and N-S-DHA pups had a less permeable mucus barrier relative to N-S pups, which suggests the potential of these factors to strengthen the mucus barrier and thus protect against disease.

Intestinal mucus is capable of stabilizing supersaturation of poorly water-soluble drugs.

The utilization of polymers to stabilize drug supersaturation and enhance oral drug absorption has recently garnered considerable interest. The potential role of intestinal mucus in stabilizing drug supersaturation, however, has not been previously explored. The ability for intestinal mucus to stabilize drug supersaturation and delay drug precipitation is potentially useful in enhancing the absorption of orally dosed compounds from drug delivery systems that generate supersaturation within the gastrointestinal tract (e.g., solid dispersions, lipid-based drug delivery systems). This work aims to evaluate the precipitation-delaying abilities of intestinal mucus using carvedilol (CVDL) and piroxicam (PXM) as model drugs. In supersaturation-precipitation (S-P) experiments, CVDL and PXM supersaturation were induced in test media (0, 0.1, 0.2, 0.4%w/v mucin and 8%w/v native pig intestinal mucus (PIM)) via the solvent-shift method at supersaturation ratios (SSR) of 5 and 6, respectively. Time to drug precipitation was assessed using ion-selective electrodes and HPLC. The S-P experiments showed that increasing mucin concentration led to increasingly delayed CVDL precipitation, while PXM precipitation was prevented at all mucin concentrations studied. The ability of mucus-stabilized CVDL supersaturation to translate into enhanced CVDL absorption was evaluated in transport experiments using mucus-producing (90% Caco-2:10% HT29-MTX-E12 co-cultures) vs. non-mucus-producing intestinal monolayers (100% Caco-2 cultures). The absorption enhancement of CVDL (SSR = 5 relative to SSR = 1) was higher across mucus-producing than non-mucus-producing intestinal monolayers. This work demonstrates the potential for intestinal mucus to delay the precipitation and enhance the absorption of poorly water-soluble compounds, suggesting that drug supersaturation can be stabilized in close proximity to the absorptive site, thereby presenting a possible novel approach for targeted supersaturating drug delivery systems.