WP1 aimed to develop the detection technology for the measurement of chemotherapy drug concentrations. First, the optical characterisation of the anthracycline compounds was done. The fluorescent spectrum of doxorubicin, pegylated liposomal doxorubicin, epirubicin and mitoxantrone was analysed using a Tecan Spark spectrofluorometer in 96-well plates. The results showed that the concentration of anthracyclines in different solvents (dimethyl sulfoxide, fetal bovine serum and cell culture media) can be followed with high specificity and precision. Then, the same measurements were performed in the microfluidic environment using a simple microfluidic cartridge. The encouraging results proved the usability of microfluidics in detecting fluorescent drugs in liquid samples. Finally, the sensitivity and limit of the proposed approach was tested. Published plasma drug concentrations found in patients was used as reference to identify the limit of detection. Comparing with real-life drug concentrations, the results showed that the POC-TDM technology can detect relevant drug levels in liquid samples.
The main goal of WP2 was to design and manufacture a prototype microfluidic cartridge able to separate plasma from whole blood and transport the sample into the measurement chamber. The Research Fellow learned how to design and fabricate microfluidics using the CleWin software and the host institute specialized infrastructure. At first, a simple, sample transport microfluidic cassette was established to test the fluorescent detection method in microvolumes. After it was proved that fluorescent compounds can be measured in a plate reader-compatible microfluidic system, plasma filtration methods were tested which could be combined with the sample transport microfluidic cartridge. The design was tested with both human and mouse blood samples, and it was able to separate the plasma and transport it to the detection chamber.
The testing of the microfluidics and the fluorescents detection method using blood samples from mice took place in WP3. First, to provide evidence that the POC-TDM approach will work using real samples. a small amount of blood (~100 µl) was drawn from the tail vein of healthy animals and spiked with various concentrations of doxorubicin and pegylated liposomal doxorubicin. The plasma separation was done by centrifugation and the supernatant plasma was removed and measured. The results were satisfactory, and limit of detection was similar to doxorubicin concentrations found in cancer patients. In the next step, two groups of healthy mice were treated with different doses (6 and 0.6 mg/kg) of pegylated liposomal doxorubicin through the tail vein and blood samples were taken before administration and after 5, 15, 30, 60, 180, 360, 1440, 2880 minutes. The plasma concentrations were measured with the POC-TDM method and, simultaneously, using mass spectrometry. The pharmacokinetics, while greatly differ based on the injected dose, were almost identical for the two methods. The same experiment was repeated on Brca1 and p53 double knock-out mouse mammary tumour-bearing animals. Surprisingly, despite the identical genetic background (inbred mice) and virtually indistinguishable tumour (genetic inheritance) the peak plasma drug concentrations were significantly varied, suggesting that personal pharmacokinetics of individuals could even more altered than we thought. The experiment was repeated again on mice inoculated with Lewis lung carcinoma (LLC) cells through the tail vein. These rapidly growing cells quickly colonize the lungs of the animal, forming lung tumours. The results were also identical, proving the POC-TDM approach viability in critical conditions.
At the final stage of the project (WP4), the POC-TDM methodology was tested in the veterinary oncology environment. By collaborating with the Veterinary Haematology and Oncology Center (VHOC) and the University of Veterinary Medicine (Budapest, Hungary), the anthracycline-based therapy of ten veterinary oncology patient (six dogs and 4 cats) was monitored. Blood samples were taken via the saphenous vein before administration and after 30, 60, 150 and 270 minutes. The drug concentrations were measured by both the POC-TDM system and mass spectrometry and found to be comparable. This experiment ultimately proved that the POC-TDM approach is working in the relevant clinical setting
