MoleculAr harvesTing with electroporation, microfluidics and nanoparticles for dIagnoSticS and thErapy of heterogeneous solid tumours

A major challenge in cancer treatment is that a cancerous tumour often consists of areas with very different properties. Each responds differently when the same treatment is delivered. The diagnosis and treatment of choice is often based on the biopsy results, usually done by taking small pieces of tissue from the tumour. If a tumour has areas with properties that differ far from the tissue sample on which the diagnosis is based on, it is most probable that the treatment will not eradicate the tumour completely. The MATISSE project aims to combine several nanotechnology elements into a new, diagnostic tool of multiple areas in the same tumour, thereby facilitating a more informed selection of a treatment regime that is more likely to eradicate the entire tumour. This will be achieved by the development of a needle inserted into tumours for harvesting the molecular content from the tumour cells. Such approach may potentially eliminate the need for complex procedures to collect the tumour samples and to allow analysis of many areas in the same tumour. Inside the needle, the molecular content will be harvested using a technique called electroporation. It is based on forming pores in the cell membranes by applying an electric field to subsequently release the molecular content. The molecular content is then extracted from the tissue using the needle and transferred to a centrifugal microfluidic device that separates the relevant biomolecules, namely proteins and RNA, from the rest of the sample. These biomolecules are then analysed promptly to provide the diagnosis. The centrifugal microfluidic device can analyse several samples in parallel and thereby speeds up the process of diagnosis. Based on the diagnosis, the surface of therapeutic nanoparticles will be tailored to bind specifically to biomolecules discovered in the analysis. Thereby, therapeutics may hopefully reach all parts of the tumour and significantly increase the chances of successful therapy. Status: In the initial phase of the project, we worked with choice of isolation procedure for protein and RNA from the electroporated sample to be implemented on the centrifugal microfluidic device. We concluded to focus on liquid-liquid extraction protocols for protein isolation to start with. We narrowed our options down to two potential protocols. One is a commercial protein extraction kit from Biological Industries which is well known to the project coordinator in Israel. The other is a "green chemistry" procedure for protein extraction published in 2015: Xu K, Wang Y, Huang Y, Li N, Wen Q. A green deep eutectic solvent-based aqueous two-phase system for protein extracting. Anal Chim Acta. 2015 Mar 15;864:9-20. doi: 10.1016/j.aca.2015.01.026. Epub 2015 Jan 23. PMID: 25732422. The protocol from Biological Industries utilizes hazardous chemicals, and hence complicates experimental testing in the centrifugal set-up and is less practical in a clinical setting. The green protocol utilizes non-hazardous ("green") chemicals and is as such more suitable for the application but is not an established procedure in the consortium. In collaboration with the project partners in Israel, we have investigated the field of deep eutectic solvents and aqueous two-phase systems which the green protocol relies on, and the potential implementation with centrifugal microfluidics. The project partners in Israel have performed experiments to try to replicate the green protocol described in the paper by Xu et al. but have so far been unsuccessful. Viscosity and contact angles of the liquids used in the green protocol with relevant polymers for microfluidic device fabrication (PC, PMMA, COP and Flexdym) have been tested. Due to the uncertainty of which protein extraction protocol to be utilized in the end, we have chosen to focus the development of the centrifugal microfluidic design on unit operations which are common for all liquid-liquid extraction protocols and where we see the most potential for improvement and innovation; the mixing and subsequent separation of the two immiscible liquids to obtain efficient mass transfer between the phases. A detailed literature review has been performed to identify relevant approaches, and the applicability of already-existing approaches has been considered and used as a basis for development of new, innovative solutions to the problem. Theoretical considerations are currently being done to arrive at design suggestions for the new mixing principle. We will most likely utilize non-hazardous model liquids in the initial phase of experimental testing of the centrifugal microfluidic devices. We have investigated potential consumer-market-type laser ablation instruments and inspected test structures in PMMA to evaluate the dimensional results compared to design. However, the results did not meet our criteria for fabrication of microfluidic structures. We will therefore focus on other prototyping methods in this project.

Intratumor heterogeneity is a major challenge preventing the wide-spread adaptation of a personalized medicine. The objective of this proposal is to develop a complementary set of beyond state-of-the-art, nanotechnology-driven tools for effective diagnostics of intratumor heterogeneity and subsequent therapy with tailored nanoparticles. The methods include (i) collection of molecular biopsies at improved spatial resolution (~100 µm) by tissue permeabilisation with electroporation, (ii) automation of sample preparation on electroporated extracts (RNA and proteins extraction) using centrifugal microfluidics and (iii) application of therapeutic gold and silica nanoparticles addressing the tumor subclonality. The hypothesis behind the MATISSE project is that the combination of the proposed technologies will for the first time enable dissecting molecular cartography and heterogeneity of solid tumours addressable by nanoparticle drugs. To test the hypothesis, international consortium of experts in engineering, nanotechnology, medicine, biochemistry, and machine learning will jointly tackle the following specific challenges: 1) development of a novel e-biopsy device for molecular harvesting in vivo; 2) determination of the electric pulse parameters for RNA and protein extraction from tumour model in mice and excised human skin and internal tumours; 3) development of a microfluidic technology for interfacing e-biopsy devices and laboratory instrumentation that ensures integrity of data collection during biopsies analysis; 4) development of nanoparticles combinations to address the heterogeneity of the 4T1 model tested in vivo. The project involves partners from 4 countries (Project leader: Tel Aviv University). SINTEF will focus on the development of a centrifugal microfluidic device for automation of sample preparation (RNA and protein extraction), interfacing of e-biopsy devices with other instrumentation to ensure integrity of data collection during biopsies analysis.