Cooperative Intelligence in Swarms of Enzyme-Nanobots

Project Information

i-NANOSWARMS

Grant agreement ID: 866348

DOI

10.3030/866348

EC signature date 28 April 2020

Start date 1 October 2020

End date 30 September 2026

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 999 819,00

EU contribution

€ 1 999 819,00

Coordinated by

FUNDACIO INSTITUT DE BIOENGINYERIA DE CATALUNYA
Spain

Periodic Reporting for period 3 - i-NANOSWARMS (Cooperative Intelligence in Swarms of Enzyme-Nanobots)

Reporting period: 2023-10-01 to 2025-03-31

Summary of the context and overall objectives of the project

Nature has been a source of inspiration to researchers to explore the possibility to mimic the rich multifunctionality of molecular motors, cells and microorganisms capable of move, interact, communicate and cooperate in complex biological environments and adjust their motion and behaviours in response to local stimuli and act collectively (schooling of fish, migration or sperms and bacteria in chemical gradients). Stigmergy, where the trace left in the environment by the first organism affects the motion of the following one, leading to a coherent self-organization, is observed even for very simple entities (ants, termites). Replicating this sophisticate intelligences from biology to active artificial systems could lead to emergent behaviours such as self-organization to accomplish complex tasks collectively, which single units cannot.
Engineering active nanosystems is a new and emerging area of research that comprises the design of large populations of nano- and microstructures that can harvest energy from their surroundings to move and self-organize to perform complex functions. Studying such out-of-the-equilibrium systems is intrinsically challenging and require a multidisciplinary expertise.
In this project, we study a paradigm shift from individual “passive” nanoparticles towards swarming intelligence of “active” nano-systems based on self-propelled nanobots. It will represent a step forward in systems nanotechnology, imaging, environmental applications, physics of active matter and nanomedicine (e.g. increasing the targeting efficiency from passive particles/active individual systems to swarms of different nanobots).
The main objective of this project is the production of intelligent self-powered nanosystems that cooperate, communicate and interact among themselves and with their environment presenting emergent behaviour at the nanoscale. The particular objectives can be divided in 3 aims:
1. e-Nanobots communication: To bioengineer enzyme-powered nanobots based on enzymes and nanoparticles containing different asymmetric architectures, to study their mechanism of motion and to assess their nanobot-nanobot communication via enzymatic cascades
2. Swarms of e-Nanobots: To investigate the cooperative intelligence and guidance of swarms of enanobots by chemotaxis and stigmergy phenomena
3. e-Nanobots as bio-nano-tools: To study the communication between living systems and e-nanobots and explore their use as novel tools for nanomedicine and molecular imaging

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

Design and fabrication of nanobots from a toolbox of nanoparticles and a library of enzymes
We explored different materials to produce nanobots (NB) beyond silica: among others, we incorporated Iron oxide into nanomotors (NMs) to provide them with photothermal properties upon irradiation, and we obtained thermoresponsive nanogels that change their size with temperature.
We have also explored the use of different enzymes. We found that purifying the hexameric form of urease, among other benefits, leads to faster motors than those loaded with unpurified urease. We incorporated catalase into the pores of metallic-organic frameworks for the generation of oxygen bubbles leading to propulsion. We expanded the chassis structure of enzyme-motors to liquid metals, which in combination with urease, can self-propel, engage chemotaxis, and change size and shape upon light illumination. Other than enzymes, we have explored the use of skeletal muscle as biological powering component as biohybrid robots.

Bot-Bot communication: signal propagation
We described that ions are important in the propulsion mechanism and studied the factors affecting the motion of single bots. Until now we have not implemented bot-to-bot communication, but we plan to do it in the following period.

Nanoswarms response to chemical gradients: chemotaxis
We have designed microfluidic chips for studying collective chemotaxis. The NM swarms showed collective chemotaxis behaviour towards higher concentration of fuel.

Nanobots swimming and modifying the environment for stigmergic cooperation
We have explored the interaction of NBs with different biological barriers, such as mucus, synovial fluid, and collagen-based extracellular matrix. We also developed peptide-functionalized urease NM capable of killing bacteria in vitro and swim larger distances than passive particles or even free peptides to kill biofilms in vivo.

Imaging nanobots in the bladder of a mice.
We reported the tracking of urease-nanobots inside the bladder of living mice using PET-CT imaging techniques.

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)

Progress beyond the state of the art:

We used microfabricated structures to guide collectively catalytic micromotors without external sources, connecting the two fields of active matter and solid state physics as the fabricated structures resemble topographical insulators. Moreover, by using artificial intelligence and neuronal networks we tracked mistake free large number of micromotors, something never achieved before.

The imaging of nanobots inside living organisms is of paramount importance to progress towards biomedical applications of nanomotors and nanobots. We collaborated with a group at CIC Biomagune using PET-CT to be the first to image swarms of enzyme-nanomotors inside the bladder of mice. The instravesical injection of the nanobots and the use of medical imaging techniques ensures its translational potential of nanobots.

We incorporated enzymes inside the pores of MOFs. Porous materials are important as they can capture large amounts of proteins, small molecules and other moieties. MOFs contain two different pore sizes asymmetrically, one for encapsulating catalase enzyme and the other for capturing contaminants.

We demonstrated the bactericidal capabilities of peptide-modified nano- and microbots. Those functional machines can kill bacteria and disrupt biofilms in skin infections in mice in vivo. We demonstrateted that motion provided by self-propelled nanobots is key to reach large areas of infected regions.

We found that purifying the commercial urease by size-exclusion chromatography and functionalizing hollow silica microcapsules leads to motors 2.5 times faster than those loaded with unpurified urease. Moreover, reusability and stability were enhanced when urease is purified. The results that we obtained by purifying the commercial enzyme are leading us to open a new line that is to produce our own enzyme.


Expected results until the end of the project:

We plan to develop a protocol for up-scale the synthesis of nanobots based on mesoporous silica, and the production of pure urease enzyme. Moreover, we plan to expand the library to organic-based nanobots which may enable biodegradable nanobots for different applications.
We plan to study the bot-bot communication and cell-bot communication in microfluidic channels but also expand this knowledge to in vivo settings. One important aim will be the study of biomaterial-nanobot interactions, namely how nanobot disrupt biological barriers and how to reach, penetrate and internalize into tumors.

Transmission electron microscopy image of urease-powered nanomotors (Hortelao et al.2021,SciRobotic)