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Point-of-care microfluidic device for quantification of chemotherapeutic drugs in small body fluid samples by highly selective nanoparticle extraction and liquid crystal detection

Periodic Reporting for period 4 - DiaChemo (Point-of-care microfluidic device for quantification of chemotherapeutic drugs in small body fluid samples by highly selective nanoparticle extraction and liquid crystal detection)

Reporting period: 2019-05-01 to 2020-10-31

The aim of the project is the development of point-of-care devices for the measurement of chemotherapeutic drugs in the blood of patients in real-time. The necessity of a fast and reliable bedside measurement is that the individual metabolic modification of the drug in the patient can influence significantly the outcome for the patient in terms of overall survival because if non-efficient drug concentrations are reached tumour continue to grow or even develop drug resistance and quality of life due to severe side effects by intoxication.

The decision-making in chemotherapy nowadays depends on standard methods that are liquid chromatography followed by mass spectrometry or capillary electrophoresis. Both are labour- and cost-intensive and can be performed only in dedicated hospitals or specialized laboratories. Especially labour-intensive, error-sensitive blood sampling is a major source of imprecision in particular for the intrinsically instable doxorubicin. This lead to a minimal therapeutic drug monitoring in patients and hence that 30-60% of drugs are administered without clinical benefits.

We propose to develop a point-of-care device for quantification of chemotherapeutic drugs in small body fluid samples in real-time at the bed-side of the patient by highly selective nanoparticle extraction and liquid crystal detection incorporated in a microfluidic optofluidics based lab-on-chip device. With a device which allows a fast and automatic blood sampling and fast analysis with a sensitivity high enough to measure clinically relevant concentrations the medical doctor will be given to adjust the drug dose to every individual patient depending on his/her metabolism even during treatment. This will improve the therapeutic outcome and reduce health care costs.
In the first half the project, an emphasis was placed to induce a fast degradation of Doxorubicin to a more stable molecule. Intrinsic instability of Doxorubicin and its modification after sampling is one of the main obstacles of an efficient and handy quantification of the drug nowadays. A deeper insight in the degradation mechanism, which was patented before the project as functionalized gold nanoparticles in combination with a liquid system as detection system, allowed to develop a protocol for the separation and quantitative transfer of Doxorubicin into a stable form. This protocol allowed the implementation of a simplified fluorescence-based approach was translated into the a prototype for a point-of-care device. A very similar approach can be used for an even more simplified point-of-care device to measure the concentration of SN-38, the active drug of Irinotecan in blood. In both cases, the focus was on simple, cheap and easy-to-use systems with a clinically significant detection limit to measure the drugs with a minimum of preparative effort.

In the context of concluding experiments related to the patented approach with drug-loaded nanoparticles and phase transfer into the liquid crystal matrix it was determined that no gold nanoparticle is able to enter into the liquid crystal phase and therefore initial approach of using the phase transfer of drug-loaded nanoparticles for detection had to be abandoned.

In the second half of the project, the main focus of the work was to implement the detection protocols for Irinotecan and doxorubicin developed in laboratory scale into an automatized sequence of steps handled by the prototype of the drug related modular DiaChemo device by the engineering industrial partner. The respective modules for a point-of-care devices were either build or commercially available solutions were identified by the consortium.

Finally, selective colour-changing sensor gold nanoparticles have been developed for Paclitaxel and its metabolite basing on molecularly imprinted polymer coated gold nanoparticles were developed by IRCCS de Bellis. The technology was patented and will be published in a scientific journal soon. The required modules for the read-out such as the LioniX based microfluidic absorbance chip as well as the mixing chip were already tested and implemented in the Paclitaxel device from Vermes but was not tested in clinical settings.

Ligand monolayer based gold nanoparticles were modelled binding selectively either methotrexate, the rescue drug Leucovorin or the naturally present folic acid. The particle preparation and proof-of-concept will be finalized and published after the end of the project.
A new chemical technique transferring the instable Doxorubicin into a stable form in around 5 minutes was developed by UCL. Moreover, a protocol was developed to distinguish doxorubicin, the parental drug from the metabolite, Doxorubicinol. These key-steps for Doxorubicin detection were implemented into an automatized point-of-care device in order to prevent the labour-intensive, time-consuming, and error-sensitive sample handling. Moreover, this step was translated into an automatized module. Also for SN-38, the strongly metabolism dependent pro-drug of Irinotecan, a system was developed to distinguish easily the drug from its pro-drug and quantify both. In combination with a fluorescence detection system allowing for the sensitivity required for clinically relevant quantities the two key modules for a POC device were developed and incorporated into a device.

Two prototypes, one for Irinotecan and one for doxorubicin, were developed and tested by both clinical partners (CRO Aviano in Italy and WWU in Germany) to evaluate handiness, time to result, and detection limit with the gold standard in clinical use. In summary, it can be said that the system integration of all devices was successful. Both clinical partners confirmed the handiness and ease to use the device and programs to manage it. Nevertheless, it will be necessary to optimize and improve the equipment. Especially with respect to the marketing of the devices.

In the future, the developed point-of-care devices will allow a tighter monitoring of actual therapeutic drug concentration in blood with a reduction of toxicity events and increase in efficiency for chemotherapeutic treatments. The reduction of overdosing will reduce costs for the health care system due to unnecessary drug use while the impact of under-dosing will be the avoidance of drug-resistance of the tumour to the drug as well as tumour growth because the necessary drug concentration was never reached.
First Prototype
Final Consortium Meeting