K.E. Carr

Morphological aspects of interactions between microparticles and mammalian cells: intestinal uptake and onward movement.

Uptake of ingested microparticles into small intestinal tissues and on to secondary organs has moved from being an anecdotal phenomenon to a recognised and quantifiable process, which is relevant to risk assessment of accidental exposure, treatment of multi-organ dysfunction syndrome and therapeutic uses of encapsulated drug or vaccine delivery. This review puts in context with the literature the findings of a morphological study of microparticle uptake, using two approaches. The first is a rat in vivo in situ model, appropriate to a study rooted in the exposure of human populations to microparticles. Latex microspheres 2 μm in diameter are the principal particle type used, although others are also investigated. Most data are based on microscopy, but analysis of macerated bulk tissue is also useful. Uptake occurs at early time points after a single dose and is shown to take place almost entirely at villous rather than Peyers patch sites: however, multiple feeding and therefore a longer time-span produces a higher proportion of particles associated with Peyers patches, albeit for very small total uptake at those later time points. Uptake is less affected by species, fasting and immunological competence than by age and reproductive status. The second approach uses in vitro methods to confirm the role of intercellular junctions in particle uptake. Particle-associated tight junction opening, in a Caco-2 monolayer, is reflected in changes in transepithelial resistance and particle uptake across the epithelial monolayer: Tight junction opening and particle uptake are both increased further by external irradiation, ethanol and sub-epithelial macrophages, but reduced by exposure to ice. An M cell model has looser tight junctions than Caco-2 cells, but a similar level of particle uptake. These results, along with the changes seen in junctional proteins after particle addition, confirm the role of tight junctions in uptake but suggest that adhering junctions are also important.

Macrophages increase microparticle uptake by enterocyte-like Caco-2 cell monolayers.

Caco‐2 cells form an enterocyte‐like monolayer that has been used to explore small intestinal microparticle uptake. They are a useful functional model for the investigation of in vivo drug delivery systems and the uptake of particulate environmental pollutants. The aim of this paper was to determine if the previously reported decrease in Caco‐2 transepithelial resistance following exposure to macrophages was matched by increased microparticle uptake, especially as macrophage phagocytosis simulates removal of particles from the subepithelial compartment. Caco‐2 cells were grown as a monoculture for 21 days on insert membranes. A compartmentalised model involved Caco‐2 cells in the upper compartment, with THP‐1‐derived macrophages adhering to the base of the underlying well, the two cell populations communicating only through the shared culture medium. Caco‐2 cells were also cultured in macrophage‐conditioned medium and all groups were exposed apically to 2 μm latex particles for 5 or 60 min. Parameters measured were: transepithelial resistance; cytokine levels; cell dimensions and the distribution of nuclei, actin and junctional proteins. Subepithelial particle numbers, defined as those located below the insert membrane, were also counted and were significantly increased in the Caco‐2/macrophage model, with over 90% associated with the macrophages. Other changes induced by the presence of macrophages included decreased transepithelial resistance levels, diffuse localisation of some junctional proteins, higher proinflammatory cytokine levels, disorganisation of cell shape and decreased cell height associated with actin reorganisation. Macrophage‐conditioned medium produced a smaller transepithelial resistance decrease than the Caco‐2/macrophage model and there were few other changes. In conclusion, culture of Caco‐2 cells with underlying macrophages produced a lower, less organised epithelium and greater microparticle uptake.

Culture conditions and treatments affect Caco-2 characteristics and particle uptake.

Small intestinal microparticle uptake via a paracellular route is relevant to oral drug delivery and environmental pollution. In vitro investigation uses latex microparticle passage across a confluent Caco-2 cell epithelium. This paper examines the influence of culture conditions on transepithelial resistance (TER); cell dimensions from confocal microscopy; and number of particles below the epithelium. Variables investigated include level of initial TER; multiple TER measurements; involvement of medium; cell source; and pretreatment with ethanol or a range of temperatures. Data were collected after exposure to 2 microm latex particles for 5-120 min: sham groups were exposed to pretreatment but not particles. The results highlight the importance of very precise control of the experimental environment; confirm the pattern of sequential-TER increase/decrease in groups exposed only to particles and show accompanying increases in cell dimensions. Greater particle uptake was associated with ethanol-induced decreased TER, decreased cell height and increased intercellular spaces, similar to previous findings for external irradiation. Low temperatures raised TER but, despite this, cooling did not alter particle uptake. In conclusion, culture microenvironment and sham treatment are crucial considerations in studies of epithelial microparticle uptake in vitro.

Changes produced by external radiation in parameters influencing intestinal permeability and microparticle uptake in vitro

Purpose: To determine the interaction between X-irradiation and in vitro intestinal microparticle uptake through Caco-2 epithelial cells. Methods: Caco-2 cells were cultured on 3 μm porous membranes for 21 days, X-irradiated with 2 Gy or sham-irradiated, then incubated for 5 or 30 min and exposed apically for 30 min to 2 μm latex microparticles. Measurements included cell dimensions, from confocal microscope ‘optical slices’; transepithelial resistance (TER) for tight junction (TJ) permeability; particle aggregation; and particle numbers on (adsorbed), in (intraepithelial) and through (submembranous) the epithelium. Results: Irradiation alone reduced TJ permeability more than sham-treatment, more so 5 min than 30 min after treatment. Irradiated epithelia were more permeable to particles than the equivalent sham-irradiated or previously untreated (particle only) groups: the latter two were similar. Irradiation altered adsorbed particle numbers and increased submembranous counts: particle uptake correlated best with cell height. Conclusions: 2 Gy X-irradiation increased particle uptake and translocation through the epithelium. This correlated well with the TJ opening seen after particle exposure in irradiated samples and changes in cell morphology. New data on cell dimensions underlined the similarity in particle uptake between this in vitro epithelium and that in an in vivo model, highlighting the translational significance of the work.

Roles of pre-treatment time and junctional proteins in Caco-2 cell microparticle uptake.

Microparticle uptake in the small intestine is relevant to both the delivery of pharmaceutics and exposure to environmental pollutants. The Caco-2 enterocyte model is a useful tool to study the parameters that affect epithelial microparticle permeability and the mechanisms controlling them. The current study used this model to explore further the different effects of 10% ethanol v/v or ice on transepithelial resistance (TER), microparticle uptake and immunofluorescent labelling of intercellular junctions. The same exposure times for both treatments were used, rather than those shown in the literature to produce demonstrable changes induced by each. The effects of both pre-treatments were greater after 60 min than after 15 min. Ethanol pre-treatment for 60 min decreased TER, increased particle uptake and was associated with a disorganisation of tight and adhering junctional proteins. Pre-treatment with ice for 60 min however, increased TER, decreased particle uptake and was associated with concentration of intercellular junctional proteins in a more constrained manner. These findings on the effects of pre-treatment with ethanol or ice for 60 min suggest that the extent of uptake is influenced by changes in the distribution of intercellular junctional proteins.