Modulating tight junctions and the paracellular transport of different epithelial barriers using a novel chitosan derivativeOtrin, Katja (Avtor) Žakelj, Simon (Mentor) Schatzlein, Andreas G. (Komentor) paracellular transport tight junctions GCPQ nanoparticles transepithelial electrical resistance transport assaysOral administration is the most common route and also the most convenient choice for drug delivery. Although it offers good patient adherence due to its ease of application, formulations for oral administration are not always easy to develop. Formulation taken orally faces many obstacles on the path to the site of action and it has to reach its target on time and in sufficient concentration. The majority of newly discovered drugs that are not yet released on the market are hydrophobic and will have low oral bioavailability due to their poor solubility in the luminal fluids. One of the measures to improve their solubility is to encapsulate them in amphiphilic polymers. In the case of hydrophobic drugs, GCPQ nanoparticles form highly stable self-assemblies, providing a large potential diffusion gradient and surface area reservoir for faster drug dissolution, as well as promoting drug epithelial transport through nanoparticle adhesion to and invasion into the mucus. Tight junctions are required for the formation of functional epithelial and endothelial barriers that regulate the passage of cells and solutes through paracellular space. In the intestine, reduced paracellular barrier function results in disorders in which the paracellular flux of ions and molecules contributes to symptoms such as diarrhoea, malabsorption and intestinal protein loss. Tight junctions were believed to be very rigid structures, but recent discoveries show that they express different dynamic properties that allow them to reversibly open and close. In this research, the activity of tight junctions was observed in 2 different cell lines; MDCK and Caco-2. The main aim of this research was to investigate the sensitivity of tight junctions to GCPQ nanoparticles. Three varieties of GCPQ were used to determine the sensitivity of the tight junctions to hydrophobic/hydrophilic compounds. Of course, for the tight junction to open, a balance of hydrophobicity and hydrophilicity is needed, but it turns out that the polymer that was the most hydrophilic of all opened the tight junctions to an extent that resulted in the greatest increase in permeability in both cell lines. To determine the sensitivity of the tight junctions to GCPQ nanoparticles, transepithelial electrical resistance was measured and the most hydrophilic polymer resulted in the greatest increase in the flux of ions. The next polymer that opened the tight junctions enough to increase the permeability of FITC-dextrans, though not FITC-albumin, was the one with a medium degree of hydrophilicity and the most hydrophobic polymer did not open tight junctions enough to increase the permeability of fluorescently labelled probes. Although transepithelial electrical resistance showed an increase of ion flux, the pore for the permeation of fluorescently marked probes was not wide enough.Besides the sensitivity of the tight junctions, the upper size limit for permeation was investigated. For this, different sizes of fluorescently labelled probes were used. We used 4 kDa FITC-dextran, 20 kDa, 40 kDa, 70 kDa FITC-dextran and 65 kDa FITC-albumin. We measured the appearance of fluorescently labelled probes in the basolateral compartment after administering them to the apical compartment. The samples from the basolateral compartment were taken out every 30 minutes for 2 hours to observe the trend of permeation. The largest size that went through an open tight junction in a significant amount was 65 kDa and that was fluorescently labelled albumin. 65 kDa FITC-albumin has a hydrodynamic radius of 3.5 nm, which is also the upper size limit of an open tight junction. We have proven that GCPQ opens tight junctions to an extent that allows the increased permeability of molecules up to 65 kDa and does not damage the cells irreversibly. [K. Otrin]20182020-09-17 11:47:24Magistrsko delo/naloga120270UDK: 620.3:661.12(043.3)COBISS_ID: 4537713sl