Research paperEstablishment of a triple co-culture in vitro cell models to study intestinal absorption of peptide drugs
Graphical abstract
Normal-oriented and inverted-oriented Caco-2/HT29/Raji C co-culture intestinal model for permeability studies
Introduction
Oral administration of therapeutics is the preferred route for drug delivery. Consequently, the transport of drugs across the intestinal epithelium is a major determinant of in vivo bioavailability. Numerous in vitro methods have been used in the drug selection process for assessing the intestinal absorption potential of drug candidates [1]. In vitro techniques for the assessment of permeability are less laborious and less expensive as compared to in vivo animal studies. In addition, the 3R’s policy encourages the development of alternatives to animal use. However, their successful application in predicting drug absorption across the intestinal mucosa depends on how closely the in vitro model mimics the characteristics of the in vivo intestinal epithelium [2]. It is clear that in vitro tools for screening and accurately predicting the potential success of a drug candidate are necessary. Cell culture models are becoming a routine part of drug development within the pharmaceutical industry and proven to be a more cost-effective method for initial permeability screening than in vivo studies [3]. Although cell cultures are limited models, being unable to predict multifactorial processes such as bioavailability or immune responses, advantages of using in vitro models in the initial stages of oral drug delivery research are obvious regarding the absence of laboratory animals and the ease to study the mechanisms of action [4]. Therefore, it is important to improve the existing cellular models for drug absorption studies.
Various cell monolayer models that mimic the human intestinal epithelial barrier have been developed and currently benefit from widespread popularity. Unlike enterocytes, human immortalized cells grow rapidly into confluent monolayers that exhibit several characteristics of differentiated epithelial cells. Therefore, the cell culture model provides an ideal system for the rapid assessment of the intestinal permeability of drug candidates [5]. Among the cell culture models, the Caco-2 cell line is the most widely used and best characterized system. Caco-2 cells are originated from human colon carcinoma and during in vitro growth form differentiated monolayers with microvillous structure, tight junctions, hydrolyzing enzymes and carrier-mediated transport systems for sugars, amino acids, and several drugs [6]. However, the Caco-2 monolayer model still lacks important properties that make further refinement desirable [5], [7]. For one, the tightness of the monolayer resembles more colonic than small intestinal tissue, resulting in poor permeability for hydrophilic compounds via the aqueous paracellular pathway. On the other hand, Caco-2 model is composed of solely absorptive cells, whereas the intestinal epithelium is a conglomerate of absorptive enterocytes and other cells such as goblet cells, endocrine cells, and M cells, with the mucus secreting goblet cells representing the second most frequent cell type [7]. A third limitation of the pure Caco-2 cell system is the frequently discussed potential overexpression of P-glycoprotein which may lead to higher secretion rates and consequently lower permeability in the absorptive direction [8]. Finally, it is well known that permeability of compounds that are transported via carrier-mediated absorptive pathways are lower in Caco-2 cell system as compared to the human small intestine, probably reflecting the colonic origin of this cell line [7]. Also, due to the lack of goblet cells, there is no mucus layer lining the cellular monolayer [3].
Several modifications and improvements of Caco-2 cells need to be investigated to generate a more predictable cell model. Thus, co-culture of Caco-2 cells with goblet and mucus-secreting cells characteristics such as HT29 cells has been proposed in order to solve this problem [3], [7], [9], [10], [11], [12]. The co-culture with Raji B lymphocytes is another interesting strategy for achieving a cell model that more closely reproduce the intestinal epithelium. Caco-2 cells when cultured with Raji B lymphocytes have the ability to acquire M cell phenotype with the main characteristic of being able of transporting particulate matters [13], [14], [15], [16], [17]. Thus far, no investigation has been reported on the use of the three cellular types above mentioned at the same time.
In this paper, we describe for the first time the development of cellular model of the intestinal epithelium based on Caco-2, HT29, and Raji B lymphocytes triple co-cultures. The model, proposed in two different growing conditions, was developed based on the individual characteristics of each cell type and the hypothesis that the three types of cell lines together may reproduce more closely the intestinal epithelium. Caco-2 cells partially reproduce the characteristics of intestinal enterocytes; mucus-secreting goblet cells (HT29) represent the mucus layer and M-cells (induced by Raji B) play an important role in the immune systems and have the unique ability of facilitating antigen uptake from the gut lumen to the gut associated lymphoid tissue and to deliver antigen via transcytosis to antigen presenting cells and lymphocytes located in a pocket-like structure to induce immune response [10], [14], [18].
Another part of the present work relates to the study of nanoparticles as enhancers of oral drug permeability. Nanoparticles are under consideration for oral delivery of proteins like insulin by stabilizing, ensuring biological activity during transit through the gastrointestinal tract, and facilitating absorption and delivery to the target site [19]. Chitosan, a weak cationic polysaccharide produced by deacetylation of the natural polymer chitin, has many useful biological properties, such as biocompatibility, biodegradability, and bioactivity [20]. Dextran sulfate is a biodegradable and biocompatible polyanion similar to heparin, with a branched carbohydrate backbone and negatively charged sulfate groups. Our group developed dextran sulfate/chitosan nanoparticles produced by polyelectrolyte complexation between dextran sulfate and chitosan and assessed their potential as insulin carriers [21], [22].
Section snippets
Materials and cells
Low molecular weight (MW) chitosan (≈50 kDa), degree of acetylation 15%, was purchased from Sigma (Sintra, Portugal), high MW dextran sulfate (500 kDa) was obtained from PKC (Copenhagen, Denmark). Dextran sulfate stock solutions were prepared with deionized water (Milli-Q) overnight under magnetic stirring, and chitosan was dissolved in 1% acetic acid solution in deionized water followed by filtration using a Millipore #2 paper filter and stored at 4 °C. Human crystalline zinc insulin (Lot RS0325,
Morphology of in vitro co-culture models
The intestinal epithelium is composed of different cell types with complex absorptive and secretory characteristics. The widely used Caco-2 cell based model has been established as a tool for in vitro investigations of epithelial transport processes because of its growth characteristics [26]. However, their characteristics are not similar enough to the human intestinal epithelium, and so there is a need to improve this model in order to achieve one that better represent the in vivo situation.
Conclusions
One of the most important factors in defining oral drug absorption should be drug permeability across the intestinal membrane [43]. Therefore, it is crucial to improve in vitro cell models in order to obtain a better correlation with in vivo data, which will be set in our ongoing research work. In this study, we reported the development of a new in vitro cell model, normally and inverted seeded, for drug and nanoparticle permeability assessment, based on Caco-2, HT29, and Raji B lymphocytes.
Acknowledgments
The authors gratefully acknowledge Fundação para a Ciência e a Tecnologia (FCT), Portugal, for financial support (PTDC/SAU-FCF/70651/2006 and SFRH/BPD/35996/2007).
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