The fellowship has obtained the following results so far:
1. Formulation of ‘SmartCubes’ under biologically relevant conditions. Studies of interactions with cells demonstrated uptake and cytotoxicity dependent on the formulation. Structural studies were also performed using small angle scattering (X ray and neutron) to quantify the effects of formulation and cargo loading on lipid nanoparticle structure. Studies showed that tailor made lipid nanoparticles could play a significant role in the delivery of protein therapeutics, particularly if formulations are optimized for specific applications.
2. Development of a fluorescent assay to quantify protein loading into ‘SmartCubes’ that was subsequently validated using small angle scattering (X ray and neutron). This functional assay enables benchtop quantification of protein loading into ‘SmartCubes’ and assisted a mechanistic understanding of cargo loading including the importance of electrostatics, coupling to the membrane via protein – protein interactions and the lipid formulation.
3. Proof of concept demonstration that large functional proteins could be encapsulated in ‘SmartCubes’. Fluorescence assays were used to demonstrate no loss of protein function upon encapsulation. Subsequent cytotoxicity studies demonstrated protein uptake only when the cargo was loaded into ‘SmartCubes’. These results are currently being prepared for publication and the optimized formulations developed for delivery of diverse biologically active cargo.
4. To understand the intracellular trafficking and payload delivery of ‘SmartCubes’, the interaction of ‘SmartCubes’ with lipid modifying proteins (lipases) was characterized using electron microscopy, small angle scattering and fluorescence assays. It was concluded that lipases would play a significant role in payload delivery from ‘SmartCubes’.
5. Proof of concept demonstration that the fluorescence assay developed could be optimized to quantify lipase activity on ‘SmartCubes’. This is currently under further development to define the limits of detection, particle stability and structural changes as a results of lipase activity.
As a Marie Skłodowska-Curie fellow at Karolinska Institute, I have gained international experience working in a highly multidisciplinary environment. I have disseminated the results from this project via participation in international conferences and seminars at Karolinska Institute and Imperial College London. Proof of concept data from this project was used to obtain experimental beamtime at both X ray and neutron facilities (Diamond Light Source, UK and ISIS Muon and Neutron Source, UK). I have already published a review on the applications of cubosome systems and am currently preparing a further two high quality publications. I have organised Outreach activities for Swedish school children as part of European Researchers Night which involved organising a team to demonstrate live experiments to school children and holding a question and answer session (in Swedish) with students. These publications and the planned future publications will all acknowledge the funding received from the Marie Skłodowska-Curie programme under Horizon 2020 and will comply with EU open access policies.
