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Engineering the Biointerface of Nanowires to Direct Stem Cell Differentiation

Periodic Reporting for period 4 - EnBioN (Engineering the Biointerface of Nanowires to Direct Stem Cell Differentiation)

Reporting period: 2022-08-01 to 2023-07-31

The reduced functionality or failure of organs due to disease or old age is a major societal and healtcare burden in our aging society. Regenerative medicine and tissue engineering approaches aim to combine materials, cells and instructive signals to repair and restore organ functionality. In particular, Regenerative medicine aims to recover lost tissue/organ function by directing the behaviour and organization of cells. Stem cells are ideal candidates for regenerative approaches owing to their potential to orchestrate tissue reconstruction through self-renewal, differentiation and recruitment of specialized cells. Stem cells reside within a microenvironment (the niche) that provides the biochemical and biophysical cues to support and modulate their physiological functions throughout development, quiescence and regeneration. There is a need to engineer materials that can direct the regenerative potential of stem cells combining regulatory stimuli ranging from gene therapy to biochemical and biophysical niche engineering.
ENBION is a first-in-class approach to integrate three core regulatory strategies to direct stem cell differentiation. ENBION engineers the unique biointerface of nanowires to (i)regulate gene transfection, (ii) directly stimulate the nucleus to direct epigenetic remodelling, (iii) pattern and localize presentation and delivery of biochemical stimuli. Engineering these aspects will enable to spatiotemporally regulate the differentiation of stem cells by developing principles for the rational design of the nanowire biointerface.

Throughout this project, significant progress was made in comprehending the mechanisms underlying cell-nanoneedle interaction, pivotal for cell interrogation and manipulation. Moreover, advanced nanoneedle technologies were developed, enhancing their performance.In summary, this project has significantly advanced our understanding and utilization of nanoneedle technology as a tool for modelling microenvironments in vitro and as an approach to support regenerative medicine, paving the way for its widespread application in biomedical research and clinical practice.
Early investigations unveiled that nanoneedles exert biophysical stimuli on cells by engaging with multiple organelles, prompting dynamic cellular responses such as membrane remodeling and cytoskeletal rearrangements. Notably, the nucleus orchestrates a molecular cascade to safeguard its integrity, influencing the differentiation potential of stem cells. Furthermore, elucidation of nanoneedle access mechanisms highlighted their capacity to upregulate endocytic processes, facilitating enhanced cargo uptake, including nucleic acids.

Building upon these findings, a novel approach leveraging nanoneedles to package nucleic acidswas devised. This strategy achieved efficient payload loading and subsequent delivery into recipient cells, showcasing promise for targeted gene silencing. Additionally, we proposed a groundbreaking manufacturing process facilitated the integration of nanoneedles into diverse substrates, enabling their utilization in various clinical and biological applications.

Further applications emerged, including the restoration of corneal endothelial cells through siRNA transfection and the regeneration of soft-mineral tissue interfaces using lithiated porous silicon. Additionally, an innovative approach enabled intracellular sensing via CRISPR/Cas12 amplification, while another technique facilitated non-destructive -omics level analysis of tissue composition, offering valuable insights into spatial biology.
We have established new methods for cell transfection mediated by porous silicon nanostructured materials. We have identified new mechanisms for the delivery of payloads from nanowires and leveraged those for efficient delivery of nucleic acids. We have developed new methods and protocols to investigate the cell-material interface in three dimensions with nanoscale resolution combining both structural and functional information. We have determined the mechanisms of nanowire interaction with the cells, including their direct interaction with the nucleus, the cytoskeleton and the cell membrane, and observed the mechanosensory response of the cells to this stimuli. We have provided new approaches to control the delivery of soluble differentiation sitmuli using porous silicon nanostructures, and devised strategies to pattern both ECM cues and payload for intracellular delivery with single cell resolution.
Cells on nanoneedles imaged by SEM and false coloured