Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - SmartCubes (An innovative class of nanoparticles (“SmartCubes”) for the targeted delivery of protein therapeutics)

Teaser

The objective of the ‘SmartCubes’ project was to develop a lipid nanoparticle that could be used to deliver protein therapeutics. Protein therapeutics offer exciting alternatives to common treatments as they are highly specific and commonly well tolerated. However, a...

Summary

The objective of the ‘SmartCubes’ project was to develop a lipid nanoparticle that could be used to deliver protein therapeutics. Protein therapeutics offer exciting alternatives to common treatments as they are highly specific and commonly well tolerated. However, a significant challenge with protein therapeutics is how to package the protein to promote uptake and prevent rapid degradation before reaching the site of interest. Lipid nanoparticles offer a potential solution to this problem. Previous attempts to use similar lipid nanoparticles in protein therapeutics had reached a bottleneck due to limited pore sizes inside the nanoparticle and hence ability for loading larger proteins. This project demonstrated that it was possible to load large proteins into lipid nanoparticles and maintain their function.
Novel pharmaceuticals are important for society to enable the continued development of medical treatments. Traditionally small drug molecules have been the cornerstone of treatment options, however scientific advances have led to new therapeutics such as protein therapeutics and therefore new challenges surrounding effective delivery.
The overall goals of the project were to demonstrate that ‘SmartCubes’ could be used as a novel carriers of protein cargo for therapeutic delivery of large proteins. This was divided into two key areas; cargo loading and delivery to cell lines. The Karolinska Institute was an ideal location for this ambitious multidisciplinary project enabling me to apply my skills in fundamental nanoparticle science to biological applications. Working in a medically focused environment has enabled me to gain new insights into the challenges of developing novel therapeutics, build collaborations to develop the project and increase my skill set. The image attached to this report schematically demonstrates the intelligent design of cubosomes for biomedical applications and has been reproduced with permission from Barriga et al. Angew. Chem. Int. Ed. Engl. 2019 Mar 4, 58(10), 2958 - 2978.

Work performed

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.

Final results

The ‘SmartCubes’ project has advanced the state of the art in the application of lipid nanoparticles for protein therapeutic delivery. Firstly, the fluorescence assay developed to quantify protein loading will accelerate the benchtop to clinic pipeline. Previously loading could only be validated via small angle scattering at large scale facilities to observe structural changes. Secondly the proof of concept work showing that functional proteins can be tethered to the lipid nanoparticle membrane via a protein – protein interaction will enable a variety of diverse cargo to be loaded and efficacy tested. Thirdly the consideration of lipase activity on payload delivery will make a significant contribution to understanding how novel lipid nanoparticles and therefore their cargo will be trafficked by cells.

Website & more info

More info: http://www.stevensgroup.org/index.php/people.