Imaging therapeutic peptide transport across intestinal barriers

Larsen, Jørgen Hannover; Taebnia, Nayere; Dolatshahi-Pirouz, Alireza; Eriksen, Anne Zebitz; Hjørringgaard, Claudia U.; Kristensen, Kasper; Larsen, Nanna Wichmann; Larsen, Niels Bent; Marie, Rodolphe; Mündler, Ann Kathrin; Parhamifar, Ladan; Urquhart, Andrew J.; Weller, Arjen; Mortensen, Kim I.; Flyvbjerg, Henrik; Andresen, Thomas Lars

doi:10.1039/d1cb00024a

Cited by 11 publications

(11 citation statements)

References 316 publications

(467 reference statements)

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“…Drug screening platforms compatible with live-cell imaging could offer the possibility to directly track how biologics cross the intestinal epithelial barrier and hereby offer mechanistic insights facilitating the rational design of new and improved oral administrated drugs (Gumbleton, 2005;Watson, 2005;Mechanism matters, 2010;Time to deliver, 2014;Sahay et al, 2010;Larsen et al, 2021). Here we developed a fully polarized and differentiated in vitro model from mono-and coculture epithelial tubules in the microfluidic OrganoPlate system.…”

Section: Discussionmentioning

confidence: 99%

“…To identify and understand the mode of action employed by drug formulations for crossing the intestinal barrier, assays compatible with live cell imaging techniques for monitoring the drug-cell barrier penetration in real-time would be ideal (Gumbleton, 2005;Watson, 2005;Larsen et al, 2021). The most popular and established in vitro platform for studying drug transport has been the Transwell system (TW), where cells are cultivated on a rigid membrane that separates two mediumcontaining chambers under static conditions (Hidalgo et al, 1989;Hubatsch et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

“…Expanding and validating models for use in transport studies is non-trivial, as highlighted by recent accounts showing that it is paramount to perform a comprehensive validation as they often are limited by poor abilities to predict the in vivo level of drug transport (Dahlgren et al, 2015). Together this highlights how there are still room for new methodologies facilitating the study of in vitro drug transport in validated, easy-to-use and reproducible platforms (Gleeson and McCartney, 2019), especially compatible with real-time fluorescent imaging (Wong et al, 2020;Halamoda-Kenzaoui et al, 2021;Larsen et al, 2021;Yilmaz et al, 2022).…”

Section: Introductionmentioning

confidence: 99%

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Quantifying the transport of biologics across intestinal barrier models in real-time by fluorescent imaging

Weller

Hansen

Marie

et al. 2022

Front. Bioeng. Biotechnol.

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Unsuccessful clinical translation of orally delivered biological drugs remains a challenge in pharmaceutical development and has been linked to insufficient mechanistic understanding of intestinal drug transport. Live cell imaging could provide such mechanistic insights by directly tracking drug transport across intestinal barriers at subcellular resolution, however traditional intestinal in vitro models are not compatible with the necessary live cell imaging modalities. Here, we employed a novel microfluidic platform to develop an in vitro intestinal epithelial barrier compatible with advanced widefield- and confocal microscopy. We established a quantitative, multiplexed and high-temporal resolution imaging assay for investigating the cellular uptake and cross-barrier transport of biologics while simultaneously monitoring barrier integrity. As a proof-of-principle, we use the generic model to monitor the transport of co-administrated cell penetrating peptide (TAT) and insulin. We show that while TAT displayed a concentration dependent difference in its transport mechanism and efficiency, insulin displayed cellular internalization, but was restricted from transport across the barrier. This illustrates how such a sophisticated imaging based barrier model can facilitate mechanistic studies of drug transport across intestinal barriers and aid in vivo and clinical translation in drug development.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Quantifying the transport of biologics across intestinal barrier models in real-time by fluorescent imaging

Weller

Hansen

Marie

et al. 2022

Front. Bioeng. Biotechnol.

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“…Orally administered drugs are mainly absorbed in the small intestine, calling for reliable in vitro intestinal models as a tool to engineer drug uptake and assess toxicity. , Existing models, scalable to industrially relevant numbers, have been restricted to two-dimensional (2D) monolayers of a human colorectal carcinoma cell line (Caco-2) cultured on standard Transwell inserts, that is, microtiter plate inserts with a microporous membrane allowing for diffusive transport between separate liquid compartments. , While the simple and robust Transwell models have provided important biological insights, such 2D models fail to recapitulate the complex three-dimensional (3D) microenvironment of the small intestine with the associated limited predictive value of drug uptake and transport. , The small intestine luminal surface presents a dense array of “crypt-villus” units. Villi are finger-like protrusions maximizing the surface area for absorption, while crypts are well-like invaginations at the base of each villus accommodating intestinal stem cells.…”

Section: Introductionmentioning

confidence: 99%

Dual-Material 3D-Printed Intestinal Model Devices with Integrated Villi-like Scaffolds

Taebnia

Zhang

Kromann

et al. 2021

ACS Appl. Mater. Interfaces

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In vitro small intestinal models aim to mimic the in vivo intestinal function and structure, including the villi architecture of the native tissue. Accurate models in a scalable format are in great demand to advance, for example, the development of orally administered pharmaceutical products. Widely used planar intestinal cell monolayers for compound screening applications fail to recapitulate the three-dimensional (3D) microstructural characteristics of the intestinal villi arrays. This study employs stereolithographic 3D printing to manufacture biocompatible hydrogel-based scaffolds with villi-like micropillar arrays of tunable dimensions in poly(ethylene glycol) diacrylates (PEGDAs). The resulting 3D-printed microstructures are demonstrated to support a month-long culture and induce apicobasal polarization of Caco-2 epithelial cell layers along the villus axis, similar to the native intestinal microenvironment. Transport analysis requires confinement of compound transport to the epithelial cell layer within a compound diffusion-closed reservoir compartment. We meet this challenge by sequential printing of PEGDAs of different molecular weights into a monolithic device, where a diffusion-open villus-structured hydrogel bottom supports the cell culture and mass transport within the confines of a diffusion-closed solid wall. As a functional demonstrator of this scalable dual-material 3D micromanufacturing technology, we show that Caco-2 cells seeded in villi-wells form a tight epithelial barrier covering the villi-like micropillars and that compound-induced challenges to the barrier integrity can be monitored by standard high-throughput analysis tools (fluorescent tracer diffusion and transepithelial electrical resistance).

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“…NP localization in the tissue was identified, and the number of nanoparticles taken up by the tissue was quantified using super-resolution microscopy (structured illumination microscopy, SIM). 22 The investigations in human jejunum were complemented with studies in situ in rat intestinal loops, where NP–tissue interactions as well as pharmacokinetics and pharmacodynamics (PKPD) of the insulin cargo and its effect on blood glucose were investigated. 21 , 23 , 24 The workflow of our study is illustrated in Figure 1 B.…”

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confidence: 99%

Barriers to the Intestinal Absorption of Four Insulin-Loaded Arginine-Rich Nanoparticles in Human and Rat

et al. 2022

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Peptide drugs and biologics provide opportunities for treatments of many diseases. However, due to their poor stability and permeability in the gastrointestinal tract, the oral bioavailability of peptide drugs is negligible. Nanoparticle formulations have been proposed to circumvent these hurdles, but systemic exposure of orally administered peptide drugs has remained elusive. In this study, we investigated the absorption mechanisms of four insulin-loaded arginine-rich nanoparticles displaying differing composition and surface characteristics, developed within the pan-European consortium TRANS-INT. The transport mechanisms and major barriers to nanoparticle permeability were investigated in freshly isolated human jejunal tissue. Cytokine release profiles and standard toxicity markers indicated that the nanoparticles were nontoxic. Three out of four nanoparticles displayed pronounced binding to the mucus layer and did not reach the epithelium. One nanoparticle composed of a mucus inert shell and cell-penetrating octarginine (ENCP), showed significant uptake by the intestinal epithelium corresponding to 28 ± 9% of the administered nanoparticle dose, as determined by super-resolution microscopy. Only a small fraction of nanoparticles taken up by epithelia went on to be transcytosed via a dynamin-dependent process. In situ studies in intact rat jejunal loops confirmed the results from human tissue regarding mucus binding, epithelial uptake, and negligible insulin bioavailability. In conclusion, while none of the four arginine-rich nanoparticles supported systemic insulin delivery, ENCP displayed a consistently high uptake along the intestinal villi. It is proposed that ENCP should be further investigated for local delivery of therapeutics to the intestinal mucosa.

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Imaging therapeutic peptide transport across intestinal barriers

Abstract: Oral delivery is a highly preferred method for drug administration due to high patient compliance. However, oral administration is intrinsically challenging for pharmacologically interesting drug classes, in particular pharmaceutical peptides,...

Cited by 11 publications

References 316 publications

Quantifying the transport of biologics across intestinal barrier models in real-time by fluorescent imaging

Quantifying the transport of biologics across intestinal barrier models in real-time by fluorescent imaging

Dual-Material 3D-Printed Intestinal Model Devices with Integrated Villi-like Scaffolds

Barriers to the Intestinal Absorption of Four Insulin-Loaded Arginine-Rich Nanoparticles in Human and Rat

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