Periodic Reporting for period 1 - SAPTiMeC (Semi-Autonomous ProtoTissues capable of photo-Mechano-Chemical transduction)
Periodo di rendicontazione: 2021-09-01 al 2023-08-31
The main objective of SAPTiMeC was to create unprecedented tissue-like materials capable of self-regulating the amount of luminous energy received from the environment to tune their endogenous photocatalytic reactivity.
The development of innovative and programmable tissue-like materials starting from synthetic components will provide key innovations that contribute to breakthrough knowledge in out-of-equilibrium materials, helping to bridge the gap between non-living and living matter. This work aimed to establish new areas in bottom-up synthetic biology by creating the first prototissues capable of higher-order functions. This research is also crucial for the development of emerging applications with impact in in tissue engineering, cell–protocell interactions (drug delivery, signalling, gene regulation), environmental remediation, soft robotics, and micro-bioreactor technology.
Objectives of the research project:
- Generation of large-scale prototissues with complex 3D architectures;
- Development of phototropic prototissues;
- Generation of prototissues capable of photo-mechano-chemical transduction.
By applying the methods developed during this action, it was possible to build large-scale prototissues (also called protocellular materials, PCMs) starting from adhesive protocell building blocks. The PCMs were developed to encapsulate a synthetic polymer network within the protocell building blocks to achieve robust PCMs which could reversibly change their volume and mechanical properties upon a temperature variation. It was also possible to include catalysts, such as enzymes, within the PCMs and achieve a modulation of the rate of the enzymatic reaction following a temperature variation, achieving a mechano-chemical transduction within these materials. Furthermore, it was possible to introduce an external control of the PCM movements using an light stimulus. We can conclude that the project achieved most its objectives and milestones for the period.
The development of innovative and programmable tissue-like materials starting from synthetic components will provide key innovations that contribute to breakthrough knowledge in out-of-equilibrium materials, helping to bridge the gap between non-living and living matter. This work aimed to establish new areas in bottom-up synthetic biology by creating the first prototissues capable of higher-order functions. This research is also crucial for the development of emerging applications with impact in in tissue engineering, cell–protocell interactions (drug delivery, signalling, gene regulation), environmental remediation, soft robotics, and micro-bioreactor technology.
Objectives of the research project:
- Generation of large-scale prototissues with complex 3D architectures;
- Development of phototropic prototissues;
- Generation of prototissues capable of photo-mechano-chemical transduction.
By applying the methods developed during this action, it was possible to build large-scale prototissues (also called protocellular materials, PCMs) starting from adhesive protocell building blocks. The PCMs were developed to encapsulate a synthetic polymer network within the protocell building blocks to achieve robust PCMs which could reversibly change their volume and mechanical properties upon a temperature variation. It was also possible to include catalysts, such as enzymes, within the PCMs and achieve a modulation of the rate of the enzymatic reaction following a temperature variation, achieving a mechano-chemical transduction within these materials. Furthermore, it was possible to introduce an external control of the PCM movements using an light stimulus. We can conclude that the project achieved most its objectives and milestones for the period.
Scientific part:
We developed a strategy to prepare prototissues (or protocellular materials – PCMs) starting from binary populations of bio-orthogonally reactive protocells (proteinosomes) in oil. We developed the so-called "floating mould technique" allowing the bio-orthogonal ligation of the binary proteinosome population. With this technique it was possible to obtain PCM sheets that retain their compartmentalised structure and have a large (cm2) size. It was also possible to prepare PCMs patterned with different binary populations of proteinosomes within one layer of PCM by means of manual pipetting, and also stratified PCMs. Moreover, by encapsulating an established enzyme pair within the binary proteinosome population, we demonstrated and explored the protocell-protocell and protocell-environment communication within the same PCM and also in arrays of PCMs.
Subsequently, we developed a strategy to fabricate PCMs enhanced with a thermo- and photoresponsive polymer network enabling them to perform remotely controlled movements. The method consisted in the synthesis of two reactive copolymers capable to crosslink and form a network when mixed together. The two polymers were incapsulated and crosslinked in-situ in the aqueous lumen of the proteinosomes. The presence of the thermoresponsive polymer network significantly improved the mechanical properties of the PCM. These PCMs show thermoresponsive properties, reversibly shrinking to about 30 % of their initial area upon heating above 30 °C. Moreover, we also managed to encapsulate a light nano-absorber capable to convert visible light to heat, thus achieving a photo-contractile behaviour in the PCMs, observing a fast (within seconds) and reversible contraction upon shining light on them. Additionally, we developed non-thermoresponsive polymers allowing to make PCMs with patterns of photoresponsive and non-responsive protocells, thus achieving PCMs that could perform light-triggered anisotropic movements, such as bending.
We worked towards the implementation of emergent light-gated communication behaviours in PCMs. To do this, we encapsulate different enzymes within the thermo- and photoresponsive PCMs developed in the project. We demonstrated the possibility to obtain a thermally induced mechano-chemical transduction within the PCMs, following the observation of a reversible inhibition of the enzyme cascade reaction rate upon environmental temperature variation. Unfortunately, with the above-mentioned system it was not possible to successfully demonstrate the emergence of a photo-mechano-chemical transduction behaviour following a number of technical issues related to the light used to induce PCM movements. Nevertheless, research focussed at solving these issues is still ongoing within the Supervisor’s group and will hopefully lead to the expected results in the foreseeable future.
Communication, dissemination and exploitation of the results:
The key results of the project were disseminated using different channels. For the scientific audience, five scientific journal articles were published during the course of the fellowship. The publications were made open access. In addition to the above-mentioned publications, we expect to submit two more articles to top-tier scientific journals within the next few months, in order to disseminate the yet-unpublished findings obtained during the course of this fellowship. In addition to the publications, the results have been presented at two international scientific conferences. The results obtained from the research performed during this fellowship were also echoed and disseminated online through social media such as Facebook, Twitter and Instagram, or through the research group and the hosting institution’s websites.
We developed a strategy to prepare prototissues (or protocellular materials – PCMs) starting from binary populations of bio-orthogonally reactive protocells (proteinosomes) in oil. We developed the so-called "floating mould technique" allowing the bio-orthogonal ligation of the binary proteinosome population. With this technique it was possible to obtain PCM sheets that retain their compartmentalised structure and have a large (cm2) size. It was also possible to prepare PCMs patterned with different binary populations of proteinosomes within one layer of PCM by means of manual pipetting, and also stratified PCMs. Moreover, by encapsulating an established enzyme pair within the binary proteinosome population, we demonstrated and explored the protocell-protocell and protocell-environment communication within the same PCM and also in arrays of PCMs.
Subsequently, we developed a strategy to fabricate PCMs enhanced with a thermo- and photoresponsive polymer network enabling them to perform remotely controlled movements. The method consisted in the synthesis of two reactive copolymers capable to crosslink and form a network when mixed together. The two polymers were incapsulated and crosslinked in-situ in the aqueous lumen of the proteinosomes. The presence of the thermoresponsive polymer network significantly improved the mechanical properties of the PCM. These PCMs show thermoresponsive properties, reversibly shrinking to about 30 % of their initial area upon heating above 30 °C. Moreover, we also managed to encapsulate a light nano-absorber capable to convert visible light to heat, thus achieving a photo-contractile behaviour in the PCMs, observing a fast (within seconds) and reversible contraction upon shining light on them. Additionally, we developed non-thermoresponsive polymers allowing to make PCMs with patterns of photoresponsive and non-responsive protocells, thus achieving PCMs that could perform light-triggered anisotropic movements, such as bending.
We worked towards the implementation of emergent light-gated communication behaviours in PCMs. To do this, we encapsulate different enzymes within the thermo- and photoresponsive PCMs developed in the project. We demonstrated the possibility to obtain a thermally induced mechano-chemical transduction within the PCMs, following the observation of a reversible inhibition of the enzyme cascade reaction rate upon environmental temperature variation. Unfortunately, with the above-mentioned system it was not possible to successfully demonstrate the emergence of a photo-mechano-chemical transduction behaviour following a number of technical issues related to the light used to induce PCM movements. Nevertheless, research focussed at solving these issues is still ongoing within the Supervisor’s group and will hopefully lead to the expected results in the foreseeable future.
Communication, dissemination and exploitation of the results:
The key results of the project were disseminated using different channels. For the scientific audience, five scientific journal articles were published during the course of the fellowship. The publications were made open access. In addition to the above-mentioned publications, we expect to submit two more articles to top-tier scientific journals within the next few months, in order to disseminate the yet-unpublished findings obtained during the course of this fellowship. In addition to the publications, the results have been presented at two international scientific conferences. The results obtained from the research performed during this fellowship were also echoed and disseminated online through social media such as Facebook, Twitter and Instagram, or through the research group and the hosting institution’s websites.
In terms of societal impact, this research was aimed to establish new areas in bottom-up synthetic biology and effectively moved the first steps towards the creation of the first generation of prototissues capable of higher-order functions. The application of well-known thermo- and photoresponsive functional materials to bottom-up synthetic biology to construct the first out-of-equilibrium tissue-like materials endowed with remotely controllable properties will lead to an important jump forward in the technology readiness level of prototissues. In general, the rational design and fabrication of prototissues bridges an important gap in bottom-up synthetic biology strategies, 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. Since the technology developed through this research project is still at TRL 1-2, it is difficult to estimate its socio-economic impact or the societal implications. However, the scientific methodologies that were developed during the course of this action open up a route to the fabrication of artificial tissue-like materials capable of collective behaviours, address important emerging challenges in bottom-up synthetic biology and bioinspired tissue engineering. Moreover, the materials chemistry approach outlined here has a much broader scope and will open up a route to the design and synthetic construction of different forms of tissue-like materials capable of emergent bio-inspired functions such as haptic sensing, muscle memory, and self-sustained contractility.