Orally Delivered Stimulus-Sensitive Nanomedicine to Harness Teduglutide Efficacy in Inflammatory Bowel Disease.

Inflammatory Bowel Disease (IBD) is a chronic inflammatory condition affecting the gastrointestinal tract (GIT). Glucagon-like peptide-2 (GLP-2) analogs possess high potential in the treatment of IBD by enhancing intestinal repair and attenuating inflammation. Due to the enzymatic degradation and poor intestinal absorption, GLP-2 analogs are administered parenterally, which leads to poor patient compliance. This work aims to develop IBD-targeted nanoparticles (NPs) for the oral delivery of the GLP-2 analog, Teduglutide (TED). Leveraging the overproduction of Reactive Oxygen Species (ROS) in the IBD environment, ROS-sensitive NPs are developed to target the intestinal epithelium, bypassing the mucus barrier. PEGylation of NPs facilitates mucus transposition, but subsequent PEG removal is crucial for cellular internalization. This de-PEGylation is possible by including a ROS-sensitive thioketal linker within the system. ROS-sensitive NPs are established, with the ability to fully de-PEGylate via ROS-mediated cleavage. Encapsulation of TED into NPs resulted in the absence of absorption in 3D in vitro models, potentially promoting a localized action, and avoiding adverse effects due to systemic absorption. Upon oral administration to colitis-induced mice, ROS-sensitive NPs are located in the colon, displaying healing capacity and reducing inflammation. Cleavable PEGylated NPs demonstrate effective potential in managing IBD symptoms and modulating the disease's progression.

Trends in 3D models of inflammatory bowel disease.

Inflammatory bowel disease (IBD) encompasses a set of chronic inflammatory conditions, namely Crohn's disease and ulcerative colitis. Despite all advances in the management of IBD, a definitive cure is not available, largely due to a lack of a holistic understanding of its etiology and pathophysiology. Several in vitro, in vivo, and ex vivo models have been developed over the past few decades in order to abbreviate remaining gaps. The establishment of reliable and predictable in vitro intestinal inflammation models may indeed provide valuable tools to expedite and validate the development of therapies for IBD. Three-dimensional (3D) models provide a more accurate representation of the different layers of the intestine, contributing to a stronger impact on drug screening and research on intestinal inflammation, and bridging the gap between in vitro and in vivo research. This work provides a critical overview on the state-of-the-art on existing 3D models of intestinal inflammation and discusses the remaining challenges, providing insights on possible pathways towards achieving IBD mimetic models. We also address some of the main challenges faced by implementing cell culture models in IBD research while bearing in mind clinical translational aspects.

All layers matter: Innovative three-dimensional epithelium-stroma-endothelium intestinal model for reliable permeability outcomes.

Drug development is an ever-growing field, increasingly requesting reliable in vitro tools to speed up early screening phases, reducing the need for animal experiments. In oral delivery, understanding the absorption pattern of a new drug in the small intestine is paramount. Classical two-dimensional (2D) in vitro models are generally too simplistic and do not accurately represent native tissues. The main goal of this work was to develop an advanced three-dimensional (3D) in vitro intestinal model to test absorption in a more reliable manner, by better mimicking the native environment. The 3D model is composed of a collagen-based stromal layer with embedded fibroblasts mimicking the intestinal lamina propria and providing support for the epithelium, composed of enterocytes and mucus-secreting cells. An endothelial layer, surrogating the absorptive capillary network, is also present. The cellular crosstalk between the different cells present in the model is unveiled, disclosing key players, namely those involved in the contraction of collagen by fibroblasts. The developed 3D model presents lower levels of P-glycoprotein (P-gp) and Multidrug Resistance Protein 2 (MRP2) efflux transporters, which are normally overexpressed in traditional Caco-2 models, and are paramount in the absorption of many compounds. This, allied with transepithelial electrical resistance (TEER) values closer to physiological ranges, leads to improved and more reliable permeability outcomes, which are observed when comparing our results with in vivo data.

Mucus-producing 3D cell culture models.

In vitro cell-based models have been used for a long time since they are normally easily obtained and have an advantageous cost-benefit. Besides, they can serve a variety of ends, from studying drug absorption and metabolism to disease modeling. However, some in vitro models are too simplistic, not accurately representing the living tissues. It has been shown, mainly in the last years, that fully mimicking a tissue composition and architecture can be paramount for cellular behavior and, consequently, for the outcomes of the studies using such models. Because of this, 3D in vitro cell models have been gaining much attention, since they are able to better replicate the in vivo environment. In this review we focus on 3D models that contain mucus-producing cells, as mucus can play a pivotal role in drug absorption. Being frequently overlooked, this viscous fluid can have an impact on drug delivery. Thus, the aim of this review is to understand to which extent can mucus affect mucosal drug delivery and to provide a state-of-the-art report on the existing 3D cell-based mucus models.

3D tumor spheroids as in vitro models to mimic in vivo human solid tumors resistance to therapeutic drugs.

Three-dimensional cell culture models, such as spheroids, can be used in the process of the development of new anticancer agents because they are able to closely mimic the main features of human solid tumors, namely their structural organization, cellular layered assembling, hypoxia, and nutrient gradients. These properties imprint to the spheroids an anticancer therapeutics resistance profile, which is similar to that displayed by human solid tumors. In this review, an overview of the drug resistance mechanisms observed in 3D tumor spheroids is provided. Furthermore, comparisons between the therapeutics resistance profile exhibited by spheroids, and 2D cell cultures are presented. Finally, examples of the therapeutic approaches that have been developed to surpass the drug resistance mechanisms exhibited by spheroids are described.

Establishment of 2D Cell Cultures Derived From 3D MCF-7 Spheroids Displaying a Doxorubicin Resistant Profile.

In vitro 3D cancer spheroids generally exhibit a drug resistance profile similar to that found in solid tumors. Due to this property, these models are an appealing for anticancer compounds screening. Nevertheless, the techniques and methods aimed for drug discovery are mostly standardized for cells cultured in 2D. The development of 2D cell culture models displaying a drug resistant profile is required to mimic the in vivo tumors, while the equipment, techniques, and methodologies established for conventional 2D cell cultures can continue to be employed in compound screening. In this work, the response of 3D-derived MCF-7 cells subsequently cultured in 2D in medium supplemented with glutathione (GSH) (antioxidant agent found in high levels in breast cancer tissues and a promoter of cancer cells resistance) to Doxorubicin (DOX) is evaluated. These cells demonstrated a resistance toward DOX closer to that displayed by 3D spheroids, which is higher than that exhibited by standard 2D cell cultures. In fact, the 50% inhibitory concentration (IC50 ) of DOX in 3D-derived MCF-7 cell cultures supplemented with GSH is about eight-times higher than that obtained for conventional 2D cell cultures (cultured without GSH), and is only about two-times lower than that attained for 3D MCF-7 spheroids (cultured without GSH). Further investigation revealed that this improved resistance of 3D-derived MCF-7 cells may result from their increased P-glycoprotein (P-gp) activity and reduced production of intracellular reactive oxygen species (ROS).

Comparative study of the therapeutic effect of Doxorubicin and Resveratrol combination on 2D and 3D (spheroids) cell culture models.

The assessment of drug-combinations for pancreatic cancer treatment is usually performed in 2D cell cultures. In this study, the therapeutic effect and the synergistic potential of a particular drug-combination towards 2D and 3D cell cultures of pancreatic cancer were compared for the first time. Thus, the effect of Doxorubicin:Resveratrol (DOX:RES) combinations (at molar ratios ranging from 5:1 to 1:5) in the viability of PANC-1 cells cultured as 2D monolayers and as 3D spheroids was analyzed. The results showed that the cells' viability was more affected when DOX:RES combinations containing higher contents of RES (1:2-1:5 molar ratios) were used. This can be explained by the ability of RES to reduce the P-glycoprotein (P-gp)-mediated efflux of DOX. Further, it was also revealed that the synergic effect of this drug combination was different in 2D and in 3D cell cultures. In fact, despite of the 1:4 and 1:5 DOX:RES ratios being both synergistic for both types of PANC-1 cell cultures, their Combination Indexes (CI) in the monolayers were lower than those attained in spheroids. Overall, the obtained results revealed that the DOX:RES combination is promising for pancreatic cancer treatment and corroborate the emergent need to evaluate drug combinations in 3D cell cultures.