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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%).

Fluorescent, imprinted nanosized polymers for the detection of irinotecan have been synthesised using a napthalimide polymerisable derivative (2-allyl-6-[2-(aminoethyl)-amino] napthalimide) as functional monomer. The imprinted polymers contain ethylene glycol dimethacrylate (EGDMA) as a cross-linker and were prepared by high dilution radical polymerisation in dimethylsulphoxide (DMSO). The material was able to rebind irinotecan up to 18 nmol/mg with good specificity. Fluorescence emission at 525 nm (excitation at 448 nm) was quenched by increasing concentrations of irinotecan via a static mechanism and also in analytically useful environments as mixtures of human plasma and organic solvents. This allowed the direct detection of irinotecan (in the 10&ndash, 30 &mu, M range) in human plasma treated with acetonitrile, the limit of detection (LOD) was 9.4 nM, with within-run variability of 10% and day-to-day variability of 13%.

Several fluorescent molecularly imprinted nanogels for the detection of the anticancer drug sunitinib were synthesized and characterised. A selection of functional monomers based on different aminoacids and coumarin allowed isolation of polymers with very good rebinding properties and sensitivities. The direct detection of sunitinib in human plasma was successfully demonstrated by fluorescence quenching of the coumarin-based nanogels. The plasma sample simply diluted in DMSO allowed the recovery of various amounts of sunitib, as determined by an averaged calibration curve. The LOD was 400 nM, with within-run variability < 9%, day to day variability < 5%, and good accuracy in the recovery of sunitinib from spiked samples.