Towards cell-inspired particle-based microrobots: Optimisation of the encapsulation of microparticles in lipid membranes Eugenia De Remigis1, Gaia Petrucci1, Elisa Roberti1, Dario Cecchi1, Hilda Gomez Bernal1, Elisa L. Petrocelli2, Jyoti Sharma1 , Francesco Bianciardi1 and Stefano Palagi1 1) The Biorobotics Institute, Scuola Superiore Sant’Anna, Pontedera (Pisa), Italy 2) Sapienza University of Rome (Rome), Italy Confinement of active matter can lead to the emergence of peculiar collective behaviour and interaction with the confining structure1,2. Encapsulating microscale active particles in flexible vesicles can be the basis for artificial active systems to study or emulate complex biological phenomena. ERC project CELLOIDS aims to employ this concept to fabricate soft, deformable and intelligent microrobots for medical applications, inspired by the amoeboid migration strategy of human leukocytes. Particle density has been shown to be crucial for the emergence of collective behaviour3,4. 1. Song, S. et al., J. Am. Chem. Soc. 144, 2022. 2. Vutukuri, H. R. et al., Nature 586, 2020. 3. Bricard, A. et al., Nature 503, 2013. 4. Buttinoni, I. et al., Phys. Rev. Lett. 110, 2013. 5. Pautot, S. et al., Langmuir 19, 2003. 6. Moga, A. et al., ChemBioChem 20, 2019. 7. Ghellab, S. E. et al., Biophys Chem 253, 2019. 8. NIST/SEMATECH e-Handbook of Statistical Methods, http://www.itl.nist.gov/div898/handbook/, accessed April 11, 2023. 9. Sakamoto, R. et al., Proceedings of the National Academy of Sciences 119, 2022. Icons from: Bioicons - high quality science illustrations Data analysis: ImageJ, Rstudio References Acknowledgements This work has been supported by the European Research Council under Grant Agreement No. 948590, project “CELLOIDS”. Further information Do not hesitate to contact us! eugenia.deremigis@santannapisa.it PI: stefano.palagi@santannapisa.it @MicroRobotLab @microrobot.lab Microscale Robotics Laboratory Optimization of microparticle encapsulation Objective 1: obtain controllable particle density Goal: design a bioinspired strategy to employ active microparticles as a propulsion system for ultra-deformable, autonomous microrobots. flexible membrane active core gives ultra- deformability and responsiveness allows for self- propulsion Fabrication of particle-loaded, phospholipid-based giant unilamellar vesicles (GUVs) by inverted emulsion method5,6 Critical process parameters • particle concentration in inner suspension • centrifugation acceleration and time Major issue: polydispersity in vesicle size and particle density Future directions Analysis: response surfaces7,8 Phase-contrast microscopy Motility of amoeboid model systems derives from the interaction with a confining environment9. Fabrication of GUVs encapsulating PS@Pt Janus microparticles A B Infiltration of empty/full GUVs in restrictive chambers Particle-loaded GUVs in confinement Objective 2: verify activity and stability GUVs + PS@Pt Janus microparticles in unconfined environment GUVs in hypertonic solution form protrusions, where active particles can move. Confinement of the GUV in an asymmetric restrictive environment Functionalisation of membrane phospholipids and Janus microparticles to induce contraction-based propulsion How to obtain directional movement of the GUV by exploiting the spontaneous behaviour of Janus microparticles? Activity Stability & Deformability contraction-based extension-based Response parameters • vesicle size • particle density combined Conclusions Objective 1: Results suggest that high particle concentration in the inner suspension helps obtain bigger and fuller GUVs. Long centrifugation time and high acceleration seem necessary to maximise GUV dimension; on the other hand, very high acceleration values could prove counterproductive for an efficient encapsulation. The optimal trade-off point is likely still outside the tested domain. hypertonic glucose solution 3% H2O2 in hypertonic glucose solution GUV suspension 1 2 3 no confineme