Generation of prototissues from functional and chemoselective protocells

Project Information

Smart Protocells

Grant agreement ID: 701876

DOI

10.3030/701876

Project closed

EC signature date 25 February 2016

Start date 1 February 2017

End date 31 January 2019

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 183 454,80

EU contribution

€ 183 454,80

Coordinated by

UNIVERSITY OF BRISTOL
United Kingdom

Periodic Reporting for period 1 - Smart Protocells (Generation of prototissues from functional and chemoselective protocells)

Reporting period: 2017-02-01 to 2019-01-31

Summary of the context and overall objectives of the project

Overall, the project achieved its objectives and milestones for the period. However, a few deviations from the original ideas were required. The main objective of the Smart Protocells project was the design and synthesis of novel protocell membranes capable of recognising each other, exchange materials, and self-assemble into prototissues. More specifically, the scientific objectives of the research project were the following:
1. The synthesis of novel block-copolymers containing dangling bioorthogonal functionalities.
2. The creation of functional and bioorthogonal protocells able to recognize partner protocells.
3. The development of techniques for the generation of macroscopic prototissues with high spatial and temporal control.
As for the ER training objectives, they were as follows:
1. Provide a supportive environment for knowledge-exchange in the multidisciplinary and intersectoral field of protocell research.
2. Establish a tailored mentoring and training plan for career development in both academia and industry.
A training plan for career development of the ER was established at the beginning of the fellowship. The ER improved his research skills by the completion of research activities, and attended workshops, conferences, and participated in outreach events to further hone his communication skills. A detailed description of these outreach activities is provided in the following section.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Deliverable: Bio-orthogonally reactive BSA/PNIPAM-co-MAA nanoconjugates
Bio-orthogonally reactive BSA/PNIPAM-co-MAA protein-polymer nanoconjugates were synthesised according to the following procedure. Initially, bovine serum albumin (BSA) was cationized to transform all the interfacial carboxylic residues into primary amines. Then a poly(N-isopropylacrylamide)-co-methacrylic acid (PNIPAM-co-MAA) random copolymer was grafted onto the cationized BSA to obtain a BSA/PNIPAM-co-MAA nanoconjugate. Finally, the desired bioorthogonally reactive BSA/PNIPAM-co-MAA nanoconjugates were obtained by conjugating either an azide-amine prosthetic group, or a bicyclo[6.1.0]nonyne (BCN)-amine prosthetic group.

Deliverable: Protocells capable of undergoing interfacial bio-orthogonal chemistry
Azide- or BCN-functionalised proteinosomes were prepared from the corresponding bio-orthogonally reactive BSA/PNIPAM-co-MAA nanoconjugates using the Pickering emulsion technique, crosslinked, and transferred into water using dialysis. They were characterised using brightfield and fluorescence microscopy, and their ability to chemoselectively bind to partner proteinosomes was investigated in water media.
Deliverable: Prototissue spheroids
Prototissue spheroids were generated using a stepwise process based on the encapsulation of 1:1 binary populations of the bio-orthogonal proteinosomes (mean diameter, ~30μm; volume, 10±3pl) within water-in-oil-in-water Pickering emulsion droplets stabilized by a non-bio-orthogonal membrane of BSA/PNIPAM-co-MAA nanoconjugates that was stabilized by a crosslinker. Fluorescence microscopy images of the emulsion droplets showed a nested arrangement of water-filled bio-orthogonally reactive proteinosomes that were closely packed, dispersed in an encapsulated oil phase, and housed within a bio-orthogonally non-reactive host proteinosome (see figure). Significantly, removal of the encapsulated oil triggered bio-orthogonal ligation of the guest proteinosomes to produce membrane-bounded spheroids (mean size, ~70 μm; volume, 180 ± 80 pl) with a spatially integrated tissue-like structure.

Deliverable: Rudimentary forms of mechano-chemical transduction within prototissue spheroids
The prototissue spheroids were capable of housing proteinosome-encapsulated enzyme cascade reactions, and show enhanced volume contractions compared with caged assemblies of unbound non-bio-orthogonal proteinosomes or individual proteinosomes when heated above 35°C. We exploit the collective contractibility to produce tissue-like constructs capable of reversible and sustainable contractions that could be enzymatically modulated and exploited for the mechanochemical transduction of a proteinosome-coordinated enzyme cascade. The emergence of this higher-order behaviour arose from collective interactions and coordinated responses that were facilitated by the spatial integration of the synthetic protocells within the prototissue spheroids.

Communication, dissemination and exploitation of results: The key results of the project were disseminated using different channels depending on the target audience.
For the scientific audience, a scientific journal article was published in Nature Materials (volume 17, pages 1145–1153, year 2018). In accordance with the regulations of the Horizon 2020 scheme, the publication was deposited in the repository of the University of Bristol, PURE, and made open access.
In addition the results were presented at the following scientific conferences:
• Multifunctional Hybrid and Nanomaterials Conference, Lisbon (Portugal), 6-10 Mar 2017.
• Canadian Chemistry Conference and Exhibition, Toronto (Canada), 28 May - 1 Jun. 2017.
• Royal Society Meeting on The artificial cell: biology-inspired compartmentalisation of chemical function, Kavli Royal Society Centre at Newport Pagnell (UK), 26-27 Feb 2018.
When the work was published in Nature Materials, we issued a press release where we disseminated our results and scientific advancements to a more general audience, see:
https://www.bristol.ac.uk/news/2018/october/synthetic-prototissue.html
The press release was then echoed through different online social media, such as Facebook, Twitter, and educational scientific blogs and forums, such as phys.org sci-news.com flipboard.com research-gate.net news-edical.net innovationtoronto.com and many others.

Training activities: The learning-through research activities carried out during the past two years enabled the ER to acquire knowledge in the multi-disciplinary and intersectoral field of protocell design and engineering.
In addition to receiving a high-quality scientific training, the ER attended conferences and career development courses. The ER attended three international conferences and was able to extend his network of international collaborations. The ER attended workshops to further hone his skills on grant writing and leadership. Finally, the ER also attended training sessions organised by the Centre of Public Engagement of the University of Bristol to improve his communication skills.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

In terms of scientific impact, the ER generated the first chemical approach to the generation of protocell-protocell adhesions. In general, the rational design and fabrication of prototissues bridges an important gap in bottom-up synthetic biology strategies, and contributes to the development of new bioinspired materials for potential use in areas such as tissue engineering, cell–protocell interactions (drug delivery, signalling, gene regulation) and micro-bioreactor technology.
In terms of impact on the experienced researcher’s career, through this research project the ER had the possibility to grow considerably as a scientist and young academic. In fact, during the course of the project the ER further honed his skills in writing grants, public engagement, and in writing papers for top ranked journals such as Nature Materials, where it is important to package the whole work in terms of wider impact on various research fields.

Prototissue spheroids as water-in-oil-in-water microdroplets