Fluorescent Molecularly Imprinted Polymers as sensors for anticancer drugs

Chemotherapy is a medical treatment mainly aimed at damaging solid and haematological tumors, by the administration of specific drugs able to target cancer cells. These drugs, however, often show many secondary effects and present variable inter-individual pharmacokinetics. Hence, the ideal optimization of the therapy would consist of continuously monitoring, in each patient, drug absorption in blood circulation, in order to adjust the daily dose regimen, decreasing therefore its side effects and improving the whole treatment. This methodology is known as Therapeutic Drug Monitoring (TDM) and requires the determination of drug concentration in various biological matrix, as blood, plasma, urine or saliva, and evaluation of these concentrations in terms of relevant clinical parameters. The main goal of TDM consists of individualization of therapeutic treatment of the patient. However, TDM application usually involves the support of specific instrumentations, as HPLC-MS or LC-MS/MS, that allow to perform an accurate and precise analysis of the samples, but they result time consuming, expensive and require trained personnel. Thanks to the recent technological developments, it is possible to miniaturize all of this, towards the design of specific Point-of-Care (POC) devices, able to perform a rapid quantification of the sample, without requirement of clinical support. The main advantages of using POC devices are portability, inexpensiveness and easiness to handle, hence to be used directly from patients themselves. This work is part of the project “Application of Advanced Nanotechnology in the development of innovative cancer diagnostic tools”, funded by Associazione Italiana Ricerca Cancro (AIRC) and coordinated by Centro di Riferimento Oncologico di Aviano (CRO); one of its aims consists of the development of Point-of-Care devices to be used for the Therapeutic Drug Monitoring of several anticancer drugs, included irinotecan and imatinib. This thesis project is focused, in particular, on the development of artificial receptors, named Molecularly Imprinted Polymers (MIPs), that can act as sensors for antitumor agents irinotecan and imatinib detection in human plasma samples. MIPs have been prepared by incorporating various fluorescent functional monomers in the polymer matrix, in order to obtain a fluorescent sensor, acting as recognition element and transducer at the same time. Different fluorescent 1,8-naphtalimide and a polymerisable EDANS derivatives have been synthesized; the interactions of these monomers and of fluorescein O-acrylate (commercially available) with each anticancer drug were investigated through NMR experiments. After selection of the best monomer-drug match, several MIPs have been prepared for each target molecule, following a high dilution radical polymerization protocol, and characterized by Dynamic Laser Light Scattering (DLS) and Transmission Electron Microscopy (TEM); nanoparticles with an average diameter of 15 nm were obtained. The MIPs rebinding capacity and specificity were studied through HPLC assays in water, and, exploiting their intrinsic fluorescence properties, it was possible to investigate on their rebinding capabilities in different media, by observing the eventual quenching of fluorescence upon binding. A MIP designed for irinotecan, in particular, containing a naphtalimide fluorescent dye, showed very promising results, as best specificity in water and an optimal drug sensitivity within its therapeutic concentrations range (17 nM – 17 μM), also in samples of plasma treated with acetonitrile (LOD 9.4 nM with within-run variability 10% and day to day variability 13%). The bes MIP for imatinib, instead, was obtained by incorporation of EDANS fluorophore in the polymerix matrix; at fluorescence measurements, MIP showed both a good specificity and rebinding capacity toward the imatinib in water (LOD of 1.7 μM and within-run variability 4.4%).