Difference in Endocytosis Pathways Used by Differentiated Versus Nondifferentiated Epithelial Caco-2 Cells to Internalize Nanosized Particles.

Understanding the internalization of nanosized particles by mucosal epithelial cells is essential in a number of areas including viral entry at mucosal surfaces, nanoplastic pollution, as well as design and development of nanotechnology-type medicines. Here, we report our comparative study on pathways of cellular internalization in epithelial Caco-2 cells cultured in vitro as either a polarized, differentiated cell layer or as nonpolarized, nondifferentiated cells. The study reveals a number of differences in the extent that endocytic processes are used by cells, depending on their differentiation status and the nature of applied nanoparticles. In polarized cells, actin-driven and dynamin-independent macropinocytosis plays a prominent role in the internalization of both positively and negatively charged nanoparticles, contrary to its modest contribution in nonpolarized cells. Clathrin-mediated cellular entry plays a prominent role in the endocytosis of positive nanoparticles and cholesterol inhibition in negative nanoparticles. However, in nonpolarized cells, dynamin-dependent endocytosis is a major pathway in the internalization of both positive and negative nanoparticles. Cholesterol depletion affects both nonpolarized and polarized cells' internalization of positive and negative nanoparticles, which, in addition to the effect of cholesterol-binding inhibitors on the internalization of negative nanoparticles, indicates the importance of membrane cholesterol in endocytosis. The data collectively provide a new contribution to understanding endocytic pathways in epithelial cells, particularly pointing to the importance of the cell differentiation stage and the nature of the cargo.

Probing milk extracellular vesicles for intestinal delivery of RNA therapies.

BACKGROUND: Oral delivery remains unattainable for nucleic acid therapies. Many nanoparticle-based drug delivery systems have been investigated for this, but most suffer from poor gut stability, poor mucus diffusion and/or inefficient epithelial uptake. Extracellular vesicles from bovine milk (mEVs) possess desirable characteristics for oral delivery of nucleic acid therapies since they both survive digestion and traverse the intestinal mucosa. RESULTS: Using novel tools, we comprehensively examine the intestinal delivery of mEVs, probing whether they could be used as, or inform the design of, nanoparticles for oral nucleic acid therapies. We show that mEVs efficiently translocate across the Caco-2 intestinal model, which is not compromised by treatment with simulated intestinal fluids. For the first time, we also demonstrate transport of mEVs in novel 3D 'apical-out' and monolayer-based human intestinal epithelial organoids (IEOs). Importantly, mEVs loaded with small interfering RNA (siRNA) induced (glyceraldehyde 3-phosphate dehydrogenase, GAPDH) gene silencing in macrophages. Using inflammatory bowel disease (IBD) as an example application, we show that administration of anti-tumour necrosis factor alpha (TNFα) siRNA-loaded mEVs reduced inflammation in a IBD rat model. CONCLUSIONS: Together, this work demonstrates that mEVs could either act as natural and safe systems for oral delivery or nucleic acid therapies, or inform the design of synthetic systems for such application.

Temperature-Responsive Methylcellulose-Hyaluronic Hydrogel as a 3D Cell Culture Matrix.

This study investigated the application of a temperature-responsive methylcellulose-hyaluronic acid (MC-HA) hydrogel to support 3D cell growth in vitro. Initial work focused on the preparation of hydrogels for 3D culture, followed by investigations of the biological compatibility of hydrogel components and optimization of the cell culture environment. Evaluation of viability and proliferation of HCT116 cells cultured in the MC-HA hydrogel was used to adjust the blend composition to design a hydrogel with optimal properties to support cell growth. Two important aspects in terms of the application of the proposed polymeric matrix in 3D cell culture were demonstrated: (i) 3D cultured cell aggregates can be released/recovered from the matrix via a gentle procedure that will preserve cell viability and (ii) the hydrogel matrix is amenable to application in a 96-well plate format as a standard approach employed in in vitro tissue culture tests. The work therefore shows that MC-HA hydrogels demonstrate potential for in vitro 3D cell culture as inexpensive and well-defined alternatives to animal-derived or complex synthetic systems.

Role of the Basement Membrane as an Intestinal Barrier to Absorption of Macromolecules and Nanoparticles.

Full understanding of the barrier property of mucosal tissues is imperative for development of successful mucosal drug delivery strategies, particularly for biologics and nanomedicines. The contribution of the mucosal basement membrane (BM) to this barrier is currently not fully appreciated. This work examined the role of the BM as a barrier to intestinal absorption of model macromolecules (5 and 10 kDa dextrans) and 100 nm polystyrene nanoparticles. Dextrans and nanoparticles were applied either directly to BM-coated inserts or to an intestinal model, namely, differentiated intestinal epithelial monolayers (Caco-2) cultured on BM-modified inserts. The work shows that the BM per se does not impact the diffusion of dextran macromolecules but severely hinders the movement of nanoparticles. However, importantly, Caco-2 monolayers cultured on BM-coated inserts, which show a remarkably different morphology, display a significantly larger barrier to the translocation of one dextran, as well as nanoparticle systems compared to cells cultured on unmodified inserts. Therefore, this work shows that, in addition to presenting a direct physical barrier to the movement of nanoparticles, the BM also exerts an indirect barrier effect, likely due to its influence on epithelial cell physiology. This work is important as it highlights the currently unmet need to consider and further study the barrier properties of the BM in mucosal delivery of biologics and nanomedicines.

Insight into the relationship between the cell culture model, cell trafficking and siRNA silencing efficiency.

Despite research efforts, cell uptake processes determining siRNA silencing efficiency remain unclear. Here, we examine the relationship between in vitro cell culture models, cellular trafficking and siRNA silencing efficiency to provide a mechanistic insight on siRNA delivery system design. Model siRNA-polyplexes, based on chitosan as a 'classical' condensing agent, were applied to a panel of lung epithelial cell lines, H1299, A549 and Calu-3 and cell internalization levels, trafficking pathways and gene silencing assessed on exposure to pharmacological inhibitors. The data reveal striking differences in the internalization behaviour and gene silencing efficiency in the tested cell lines, despite their common lung epithelial origins. The model system's silencing was lower where clathrin internalization pathway predominated in Calu-3, relative to silencing in H1299 cells where a non-clathrin internalization appears dominant. Increased silencing on endosomal disruption was apparent in Calu-3 cells, but absent when cellular internalization was not predominantly clathrin-mediated in A549 cells. This highlights that identifying cell trafficking pathways before incorporation of functional components to siRNA delivery systems (e.g. endosomolytic compounds) is crucial. The study hence stresses the importance of selection of appropriate cell culture model, relevant to in vivo target, to assess the gene silencing efficiency and decide which functionalities the 'stratified siRNA silencing vector' requires.

Live Imaging of Cellular Internalization of Single Colloidal Particle by Combined Label-Free and Fluorescence Total Internal Reflection Microscopy.

In this work we utilize the combination of label-free total internal reflection microscopy and total internal reflectance fluorescence (TIRM/TIRF) microscopy to achieve a simultaneous, live imaging of single, label-free colloidal particle endocytosis by individual cells. The TIRM arm of the microscope enables label free imaging of the colloid and cell membrane features, while the TIRF arm images the dynamics of fluorescent-labeled clathrin (protein involved in endocytosis via clathrin pathway), expressed in transfected 3T3 fibroblasts cells. Using a model polymeric colloid and cells with a fluorescently tagged clathrin endocytosis pathway, we demonstrate that wide field TIRM/TIRF coimaging enables live visualization of the process of colloidal particle interaction with the labeled cell structure, which is valuable for discerning the membrane events and route of colloid internalization by the cell. We further show that 500 nm in diameter model polystyrene colloid associates with clathrin, prior to and during its cellular internalization. This association is not apparent with larger, 1 µm in diameter colloids, indicating an upper particle size limit for clathrin-mediated endocytosis.

Mechanism of mucosal permeability enhancement of CriticalSorb® (Solutol® HS15) investigated in vitro in cell cultures.

PURPOSE: CriticalSorb™, with the principal component Solutol® HS15, is a novel mucosal drug delivery system demonstrated to improve the bioavailability of selected biotherapeutics. The intention of this study is to elucidate mechanism(s) responsible for the enhancement of trans-mucosal absorption of biological drugs by Solutol® HS15. METHODS: Micelle size and CMC of Solutol® HS15 were determined in biologically relevant media. Polarised airway Calu-3 cell layers were used to measure the permeability of a panel of biological drugs, and to assess changes in TEER, tight junction and F-actin morphology. The rate of cell endocytosis was measured in vitro in the presence of Solutol® HS15 using a membrane probe, FM 2-10. RESULTS: This work initially confirms surfactant-like behaviour of Solutol® HS15 in aqueous media, while subsequent experiments demonstrate that the effect of Solutol® HS15 on epithelial tight junctions is different from a 'classical' tight junction opening agent and illustrate the effect of Solutol® HS15 on the cell membrane (endocytosis rate) and F-actin cytoskeleton. CONCLUSION: Solutol® HS15 is the principle component of CriticalSorb™ that has shown an enhancement in permeability of medium sized biological drugs across epithelia. This study suggests that its mechanism of action arises primarily from effects on the cell membrane and consequent impacts on the cell cytoskeleton in terms of actin organisation and tight junction opening.

Basement membrane influences intestinal epithelial cell growth and presents a barrier to the movement of macromolecules.

This work examines the potential drug delivery barrier of the basement membrane (BM) by assessing the permeability of select macromolecules and nanoparticles. The study further extends to probing the effect of BM on intestinal epithelial cell attachment and monolayer characteristics, including cell morphology. Serum-free cultured Caco-2 cells were grown on BM-containing porous supports, which were obtained by prior culture of airway epithelial cells (Calu-3), shown to assemble and deposit a BM on the growth substrate, followed by decellularisation. Data overall show that the attachment capacity of Caco-2 cells, which is completely lost in serum-free culture, is fully restored when the cells are grown on BM-coated substrates, with cells forming intact monolayers with high electrical resistance and low permeability to macromolecules. Caco-2 cells cultured on BM-coated substrates displayed strikingly different morphological characteristics, suggestive of a higher level of differentiation and closer resemblance to the native intestinal epithelium. BM was found to notably hinder the diffusion of macromolecules and nanoparticles in a size dependent manner. This suggests that the specialised network of extracellular matrix proteins may have a significant impact on transmucosal delivery of certain therapeutics or drug delivery systems.

Mechanisms of nanoparticle internalization and transport across an intestinal epithelial cell model: effect of size and surface charge.

This study investigated the effect of nanoparticle size (50 and 100 nm) and surface charge on their interaction with Caco-2 monolayers as a model of the intestinal epithelium, including cell internalization pathways and the level of transepithelial transport. Initially, toxicity assays showed that cell viability and cell membrane integrity were dependent on the surface charge and applied mass, number, and total surface area of nanoparticles, as tested in two epithelial cell lines, colon carcinoma Caco-2 and airway Calu-3. This also identified suitable nanoparticle concentrations for subsequent cell uptake experiments. Nanoparticle application at doses below half maximal effective concentration (EC50) revealed that the transport efficiency (ratio of transport to cell uptake) across Caco-2 cell monolayers is significantly higher for negatively charged nanoparticles compared to their positively charged counterparts (of similar size), despite the higher level of internalization of positively charged systems. Cell internalization pathways were hence probed using a panel of pharmacological inhibitors aiming to establish whether the discrepancy in transport efficiency is due to different uptake and transport pathways. Vesicular trans-monolayer transport for both positively and negatively charged nanoparticles was confirmed via inhibition of dynamin (by dynasore) and microtubule network (via nocodazole), which significantly reduced the transport of both nanoparticle systems. For positively charged nanoparticles a significant decrease in internalization and transport (46% and 37%, respectively) occurred in the presence of a clathrin pathway inhibitor (chlorpromazine), macropinocytosis inhibition (42%; achieved by 5-(N-ethyl-N-isopropyi)-amiloride), and under cholesterol depletion (38%; via methyl-ß-cyclodextrin), but remained unaffected by the inhibition of lipid raft associated uptake (caveolae) by genistein. On the contrary, the most prominent reduction in internalization and transport of negatively charged nanoparticles (51% and 48%, respectively) followed the inhibition of lipid raft-associated pathway (caveolae inhibition by genistein) but was not significantly affected by the inhibition of clathrin pathway.

Development of an in vitro co-culture model using Caco-2 and J774A.1 cells to mimic intestinal inflammation.

In vitro models that mimic the pathophysiology in vivo are important tools to study mechanisms of disease and assess the pharmacology and toxicity of drugs. In this work, we report the development of a novel model of intestinal inflammation. This model is based on the co-culture of intestinal epithelial Caco-2 cells and murine J774A.1 macrophages. The model is shown to mimic the intestinal barrier in both healthy and inflamed state. In the healthy state, without external stimulation, Caco-2 and J774A.1 cells were co-cultured in one system without affecting the barrier integrity of intestinal epithelial cells and without inducing release of cytokines from macrophages. To mimic the inflamed intestine, Caco-2 cells were primed with an optimised cytokine cocktail (TNF-⍺, IFN-γ and IL-1ß) and J774A.1 cells were pre-exposed to lipopolysaccharide (LPS) and IFN-γ for 24 h before combining the two cell lines into co-culture. In these conditions, a significant disruption of the epithelial barrier and an increase in pro-inflammatory cytokine (TNF-⍺ and IL-6) levels released from macrophages were detected. The data also show that inflammation in the co-culture model was temporary and reversible upon the removal of the inflammatory stimulus. This new in vitro model could be a valuable tool for investigating the safety and efficacy of drugs in the context of intestinal inflammation and provides advantages over other reported co-culture models of intestinal inflammation in terms of cost and simplicity.

In Situ Self-Assembling Liver Spheroids with Synthetic Nanoscaffolds for Preclinical Drug Screening Applications.

Drug-induced liver injury (DILI) is one of the most common reasons for acute liver failure and a major reason for the withdrawal of medications from the market. There is a growing need for advanced in vitro liver models that can effectively recapitulate hepatic function, offering a robust platform for preclinical drug screening applications. Here, we explore the potential of self-assembling liver spheroids in the presence of electrospun and cryomilled poly(caprolactone) (PCL) nanoscaffolds for use as a new preclinical drug screening tool. This study investigated the extent to which nanoscaffold concentration may have on spheroid size and viability and liver-specific biofunctionality. The efficacy of our model was further validated using a comprehensive dose-dependent acetaminophen toxicity protocol. Our findings show the strong potential of PCL-based nanoscaffolds to facilitate in situ self-assembly of liver spheroids with sizes under 350 µm. The presence of the PCL-based nanoscaffolds (0.005 and 0.01% w/v) improved spheroid viability and the secretion of critical liver-specific biomarkers, namely, albumin and urea. Liver spheroids with nanoscaffolds showed improved drug-metabolizing enzyme activity and greater sensitivity to acetaminophen compared to two-dimensional monolayer cultures and scaffold-free liver spheroids. These promising findings highlight the potential of our nanoscaffold-based liver spheroids as an in vitro liver model for drug-induced hepatotoxicity and drug screening.

Nanoparticle transport in epithelial cells: pathway switching through bioconjugation.

The understanding and control of nanoparticle transport into and through cellular compartments is central to biomedical applications of nanotechnology. Here, it is shown that the transport pathway of 50 nm polystyrene nanoparticles decorated with vitamin B12 in epithelial cells is different compared to both soluble B12 ligand and unmodified nanoparticles, and this is not attributable to B12 recognition alone. Importantly, the study indicates that vitamin B12 -conjugated nanoparticles circumnavigate the lysosomal compartment, the destination of soluble vitamin B12 ligand. Whereas cellular trafficking of soluble B12 is confirmed to occur via the clathrin-mediated pathway, transport of B12 -conjugated nanoparticles appears to predominantly take place by a route that is perturbed by caveolae-specific inhibitors. This data suggests that, following its conjugation to nanoparticles, in addition to dramatically increasing the cellular uptake of nanoparticles, the normal cell trafficking of B12 is switched to an alternative pathway, omitting the lysosomal stage: a result with important implications for oral delivery of nanoparticulate diagnostics and therapeutics.

Polymersomes for protein drug delivery across intestinal mucosa.

The oral administration is the route preferred by patients due to its multiple advantages. In the case of biopharmaceuticals, due to their low stability and absorption in the intestine, these molecules must be administered by injectable routes. To circumvent these problems, several strategies have been studied, among which the use of nanosystems, such as polymersomes, can be highlighted. In this work the potential of poloxamer 401 polymersomes as a system for oral delivery of antibodies was evaluated. IgG-FITC-loaded poloxamer 401 polymerosomes were initially used to assess whether it improves intestinal epithelial permeation in Caco-2 cell monolayers. Subsequently, epithelial/macrophage co-culture model was used to evaluate the ability of poloxamer 401 polymersomes containing adalimumab to reduce proinflammatory cytokine levels. The data showed that polymersome-encapsulated IgG increased the transport across intestinal Caco-2 monolayers 2.7-fold compared to the antibody in solution. Also, when comparing the groups of blank polymersomes with polymersomes containing adalimumab, decreases of 1.5-, 5.5-, and 2.4-fold in TNF-α concentrations were observed for the polymersomes containing 1.5, 3.75, and 15 µg/mL of adalimumab, respectively. This could indicate a possibility for the oral administration of biopharmaceuticals which would revolutionize many conditions that require the systemic administration such as in inflammatory bowel disease.

Evaluation of calcium depletion as a strategy for enhancement of mucosal absorption of macromolecules.

Extracellular calcium is crucial for functioning of the epithelial barrier. Compounds that bind calcium, reducing its extracellular levels, have therefore been investigated as mucosal absorption enhancers. However, the conditions under which calcium reduction sufficiently modulates the epithelial barrier to result in meaningful improvements in mucosal drug absorption are unclear. Present work investigated the settings in which calcium depletion leads to optimal epithelial barrier-modulating effects. Using Calu-3 and Caco-2 cell layers and inducing calcium depletion site-specifically (apically, basolaterally or on both sides) we demonstrate that apical calcium removal produces a modest effect on the tight junctions (the extent of the effect being dependent on the duration of apical calcium unavailability), whilst basolateral calcium exhaustion leads to a prominent effect on the epithelial barrier. However, using polyacrylic acid as an example, we show that polymeric calcium-binding agents proposed as mucosal absorption-enhancing excipients alter calcium levels exclusively on the apical side of the epithelium, which explains their modest effect on epithelial barrier modulation (also demonstrated in our work). Therefore the use of calcium-depleting agents, especially those based on macromolecular polymers, is a relatively inefficacious strategy to promote mucosal absorption of macromolecules.

Enhanced permeation by amphiphilic surfactant is spatially heterogenous at membrane and cell level.

In the context of increased interest in permeability enhancement technologies to achieve mucosal delivery of drugs and biologics, we report our study on effects of the amphiphilic surfactant at cell membrane and cell population levels. Our results show that modulation in membrane order and fluidity initially occurs on insertion of individual surfactant molecules into the outer leaflet of membrane lipid bilayer; a process occurring at concentrations below surfactant's critical micellar concentration. The surfactant insertion, and consequent increase in membrane fluidity, are observed to be spatially heterogenous, i.e. manifested as 'patches' of increased membrane fluidity. At the cell population level, spatially heterogeneous activity of surfactant is also manifested, with certain cells displaying high permeability amongst a 'background' population. We propose that this heterogeneity is further manifested in a broad profile of intracellular and nuclear exposure levels to a model drug (doxorubicin) observed in cell population. The study points to heterogeneous nature of surfactant effects at cell membrane and cells in population levels.

Barrier characteristics of epithelial cultures modelling the airway and intestinal mucosa: a comparison.

The barrier characteristics of polarized layers of Calu-3 and Caco-2 cell lines, as commonly used in vitro models of intestinal and airway mucosa, respectively, were investigated by assessing the translocation of model macromolecules and nanoparticles. The barrier capacity of the cell layers towards the movement of macromolecules and nanoparticulates differed considerably between the cell lines. Permeability studies revealed the existence of a notably larger solute molecular weight limit for paracellular diffusion in Caco-2 monolayers compared to Calu-3 cells. Removal of mucus in Calu-3 cells resulted in cell layers exhibiting a larger macromolecular permeability, in addition to improved nanoparticle translocation. Microscopic examination of the tight junctions, as cellular features that play a major role in preventing transepithelial movement of macromolecules, revealed that the appearance of cell-cell boundaries was notably different in the two cell lines, which could explain the differences in macromolecular permeability. The data overall showed that epithelial layers of airway Calu-3 and intestinal Caco-2 cell cultures in vitro exhibit a different level of restrictiveness and this is due to the cell morphology and the presence of mucus.

Non-Invasive Drug Delivery Systems.

Non-invasive drug delivery generally refers to painless drug administration methods involving drug delivery across the biological barriers of the mucosal surfaces or the skin [...].

Exosome-based therapies for mucosal delivery.

Exosomes are membrane-bound extracellular nanovesicles secreted by most cells and found in multiple sources, including bodily fluids, plants, fruit, and bovine milk. They play an important role as mediators of intercellular communication, having a distinct ability to carry small molecules, proteins, and nucleic acids to recipient cells over large distances. Moreover, competency in crossing usually poorly permeable biological barriers has led to their promising use in diagnostics and in therapeutics, either as therapeutic entities on their own or as drug delivery vehicles, with superior stability, biocompatibility, circulation time and target specificity in comparison to conventional drug delivery systems. The aim of this review is to summarise and critically discuss the current literature on the use of exosomes in a therapeutic setting, with a particular focus on their use as drug delivery vehicles for mucosal drug delivery.

Effect of PEGylation on the toxicity and permeability enhancement of chitosan.

The aim of the present work is to investigate if conditions can be devised where PEGylation of chitosan would reduce its toxicity toward the nasal mucosa while maintaining its ability to open the cellular tight junctions and, consequently, produce an enhancement of macromolecular permeability. A series of mPEG-g-chitosan copolymers with varying levels of mPEG substitution, mPEG molecular weight, and chitosan molecular weight were synthesized by grafting carboxylic acid-terminated mPEGs (Mw 1.9 and 5.0 × 10(3) g mol(-1)) to chitosans (Mw 28.9 and 82.0 × 10(3) g mol(-1)) using a NHS/EDC coupling system. The synthesized mPEG-g-chitosans were fully characterized using a number of techniques, including FT-IR, (1)H NMR, and SEC-MALLS and their physicochemical properties were analyzed by TGA and DSC. Thereafter, the conjugates were tested for their cytotoxicity and tight junction modulating property in a relevant cell model, a mucus producing Calu-3 monolayer. mPEG-g-chitosan conjugates exhibited reduced toxicity toward cells, as compared to unmodified chitosan counterparts. Furthermore, the conjugates demonstrated a dramatic effect on cell monolayer transepithelial electrical resistance (TEER) and enhancement of permeability of model macromolecules. TEER and permeability-enhancing effects, as measurable indicators of tight junction modulation, were found to be pH-dependent and were notably more pronounced than those exhibited by unmodified chitosans. This work therefore demonstrates that conditions can be contrived where PEGylation improves the toxicity profile of chitosan, while preserving its effect on epithelial tight junctions in the nose.

Exploiting disease-induced changes for targeted oral delivery of biologics and nanomedicines in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic and progressive disorder with destructive inflammation in the gastrointestinal tract (GIT). Biologics have changed the management of IBD, but have serious limitations, which is associated with their systemic administration via injection. Oral administration is the most accepted route of drug administration. However, the physiological barriers of the GIT pose significant challenges for oral administration of biologics, making this route of administration currently unavailable. The status of tissue barriers to oral drug delivery is altered in IBD. This may bring more challenges, but also present opportunities for oral delivery of biologics. This article provides an overview of disease-induced alterations of GIT barriers in IBD and discusses challenges, opportunities and commonly-utilised strategies for oral delivery of complex therapeutics, including biologics and nanomedicines.