An improved in vitro model of human M cells as a useful tool to study nanoparticle transport across the intestinal epithelium
(2006)
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- Recently, the progresses in the field of biotechnologies have led to the development of many drugs based on peptides and proteins. These new drugs have been traditionally delivered by parenteral administration. However, aiming to improve the patient quality of life, as well as the compliance, more and more efforts have been dedicated to the development of mucosal formulations as an alternative to parenteral administration. The most popular mucosal route is the oral administration. The intestinal mucosa is composed of an epithelium which consists in an effective barrier against foreign material (particles, microorganisms) and Organized Mucosa Associated Lymphoid Tissues (O-MALT) called also GALT (Gut Associated Lymphoid Tissues) which are specialized antigen sampling sites. The GALT function is mainly performed by membranous epithelial M cells. The most interesting characteristic of these M cells is their increased ability to transcytose particles. The objective of this thesis was to develop a more robust and reliable M cell model and to study how nanoparticles with different surface characteristics and a therapeutic peptide in free and nanoparticulate form were transported in this model as compared to a reference model of absorptive cells of the intestinal epithelium. An in vitro model of M like cells in the Follicle Associated Epithelium (FAE) was used. The importance of the nanoparticle size, concentration, surface properties, as well as the influence of proteins in the apical medium has been first enlightened. The optimal nanoparticle size is about 200 nm, and hydrophobic nanoparticles are better transported than less hydrophobic ones. The transport is concentration-dependent and the presence of some proteins in the apical medium seems to inhibit the nanoparticle transport. Then, the in vitro model has been improved, being more physiological (presence of B lymphocytes within the cell monolayers), more reproducible and more robust (90 % of model functional). The presence of M-like cells has been demonstrated, using different techniques and the percentage of M-like cells in cell monolayers has been estimated, for the first time in an in vitro model of the FAE, at 15-30%. Mechanistic studies performed with 0.2 µm polystyrene model nanoparticles allowed to conclude that the transport of these nanoparticles occurred via a transcellular, energy-dependent mechanism, probably by macropinocytosis. The transport of helodermin, a model therapeutic peptide, through intestinal barrier was investigated. Encapsulation in PEG-PLA:PLGA {poly(ethylene glycol)-co-poly(lactic acid): ploy(lactic-co-glycolic acid)} nanoparticles protected the peptide from degradation. M cells increased the transport of nanoparticles containing helodermin. At last, the transport of different type of nanoparticles by M cells was compared. Chitosan (CS) nanoparticles appeared as the most efficient delivery system but were not M cells specific. Although less efficient than CS nanoparticles, pegylated nanoparticles were more transported by M-like cells than PLGA nanoparticles. Therefore, even it remains difficult to elicit the optimal formulation for M cells uptake, pegylated nanoparticles seem the optimal choice. In conclusion, this thesis has contributed to produce, settle and characterize a functional and reliable in vitro model of the human FAE, providing a tool to evaluate the role of M cells on drug delivery. This model has allowed to underline the influence of various parameters on the nanoparticle transport by M-like cells, as well as to compare transcytosis of different carrier. It has also put in evidence the influence of M-like cells on the delivery of therapeutic peptide across the epithelial barrier. In the future, this model could be used in the context of local drug delivery and oral vaccination studies.
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des Rieux, A. (2006). An improved in vitro model of human M cells as a useful tool to study nanoparticle transport across the intestinal epithelium. https://hdl.handle.net/2078.5/112183