Quantification of internalized drug candidates and study of their intracellular distribution, localisation
Our aim is to synthesise and characterise drug-carrier bioconjugates and nanoconstructs – specific for cancer cells or host cells of intracellular pathogens – for selective targeting. It is important to investigate the mechanism of the cellular uptake and intracellular distribution and localisation of the active compounds, providing essential structure – activity relations.
In the framework of our study we will determine the quantity of the internalised drug candidates (small molecular weight compounds, oligopeptides, peptide conjugates, nanoconstructs) from the cellular extracts. The human disease related model cell cultures (tumour cells and pathogen host cells, etc.) will be treated with the compounds. After the treatment, extraction will be performed in order to isolate the intact active compounds and their possible metabolites. Several purification and centrifugation steps of the cellular extracts will be employed during the sample preparation process before RP-HPLC/MS analysis. It is necessary to work with a high number of samples simultaneously, in order to optimise a suitable method (calibration curves, experimental conditions, etc) for different drug candidates and compound families.
Prior to RP-HPLC/MS the cellular uptake and intracellular localisation will be studied using fluorescence microscopy. The simultaneous application of RP-HPLC/MS and fluorescence microscopy will allow us to compare data from quantitative analysis of the cellular extracts and the microscopic images related to biodistribution and localisation in order to define important structure–activity relations.
Gitta Schlosser, Rita Szabó Oláhné, Szilvia Bősze, Lilla Horváth