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Intestinal organoids: assessing nanoparticle uptake in an in vivo relevant model

Terrmin

HT24

Beskrivning

Aim
To validate intestinal organoids as a reliable model for studying nanoparticle uptake, focusing on major internalization pathways, and to benchmark their efficacy against established cell lines commonly used in drug delivery research.
Background
Nano-sized formulations hold promise for targeted oral drug delivery, providing a patient-friendly alternative to predominantly parenteral treatments. However, assessing these advanced delivery systems requires more comprehensive intestinal models. Current in vitro models, primarily utilizing human colon carcinoma epithelial (Caco-2) cultures or human colorectal adenocarcinoma (HT-29) cells, lack differentiation into various intestinal epithelial cell types and proper polarity. To bridge this gap, 3D organoids offer a closer mimicry of normal human intestinal epithelium, partially recapitulating cellular phenotypes. By reversing the polarity of organoids to an apical-out orientation, direct access to the apical surface of the epithelium is achieved, enhancing the evaluation of drug delivery system efficacy. Furthermore, intestinal organoids are expected to express proteins involved in nanoparticle cell uptake at levels comparable to those in the human body. However, further validation is necessary to establish intestinal organoids as an effective model for nanoparticle uptake studies.
Methods
We will compare organoids with current in vitro models based on Caco-2 and HT-29 cells. Cells are maintained in medium and regularly passaged. To understand the distribution of key proteins involved in cell uptake pathways, such as clathrin-mediated endocytosis and macropinocytosis, expression will be analyzed using mass spectrometry-based proteomics. Optimizing incubation times and concentrations of validated cargo for each pathway will allow us to verify pathway functionality and compare uptake kinetics between cell lines and organoids. Cell viability will be assessed using a cytotoxicity assay, with experiments repeated twice. Cargo will be fluorescently labeled, and uptake quantified using a plate reader or flow cytometry. Uptake of validated cargo for each cell uptake pathway in Caco-2 and HT-29 cell lines and intestinal organoids will be quantified over time, and uptake kinetics compared between cell lines and organoids.
My task
To culture Caco-2 and HT-29 cell lines and prepare cells for uptake experiments. To assist in sample preparation for proteomics analysis and data analysis. To plan and perform optimization of incubation time (5 time-points) and cargo concentration (5 concentrations, 4 cargos) in cell lines (Caco-2 and HT-29) and conduct uptake experiments under optimal conditions.
Expected results:
This project aims to establish uptake kinetics of cargo for major internalization pathways in established cell lines, serving as the "gold standard" for comparison. By systematically assessing nanoparticle uptake in Caco-2 and HT-29 cell lines, it will provide crucial data to benchmark the effectiveness of intestinal organoids as a model for studying nanoparticle uptake.

Huvudområde

Läkemedelsutveckling

Ämne

Läkemedelsformulering och Molekylär galenisk farmaci

Typ

Laborativ studie

Företag

Uppsala universitet

Ort/Plats

Uppsala

Handledarens namn

Madlen Hubert

Handledarens e-post

madlen.hubert@farmaci.uu.se

Institution

Institutionen för farmaci

Program

Masterprogram i läkemedelsutveckling

Kurs

Degree project in Drug Discovery and Development 45 c - 3FK044

Omfattning/hp

45hp