In-depth analysis of vapor nanobubblemediated permeabilization at the level of the plasma membrane and nuclear envelope

Vapor nanobubble (VNB)-photoporation allows to deliver various compounds into a broad range of cell types with minimal toxicity. It relies on laser irradiation of plasma membrane (PM)-adhered gold nanoparticles (AuNPs), which results in the formation of VNBs. The subsequent collapse of VNBs near the PM facilitates its transient disruption, whereby cargo can enter the cytosol. Intracellular delivery is desirable in various biomedical applications, such as the delivery of nucleic acids for genetic engineering of cells for therapy. Despite its promise, however, to date little is known about the effects of VNB photoporation on cell physiology. Elucidation of the cellular response to VNB-induced PM disruption could aid in the development of strategies to diminish side effects and thereby increase its efficiency. To uncover the cellular response to VNB photoporation, we performed a longitudinal RNA sequencing experiment. We found a marked upregulation of the LMNA gene 6h after photoporation, which had disappeared at later timepoints. This was mirrored at the protein level (A-type lamins), and was accompanied by a temporary increase in chromatin condensation. We found this nuclear stiffening to be required for the cell to cope with the effects of VNB photoporation. Correspondingly, experimental increase of A-type lamin levels was able to increase the fraction of successfully transfected cells. The versatile nature of VNB photoporation motivated us to explore its potential for use at the nuclear envelope (NE). Transient ruptures of the NE (NERs) have recently been found to occur in laminopathy patient cells and cancer cells. Although cells are able to repair these NERs, the uncontrolled exchange of nuclear and cytoplasmic factors will undoubtedly have consequences on cell physiology. However, detailed investigation of the effects of NERs is currently hampered by their stochastic nature and variable frequency. Therefore, we set out to adapt VNB photoporation for the transient disruption of the NE. We demonstrated that perinuclear localization of AuNPs can be achieved after endocytic uptake or electroporation-facilitated delivery, and that both strategies result in NER upon laser irradiation. We found that the induced NERs mimicked spontaneous NERs to a high degree. We also demonstrated that NE photoporation is able to allow the intranuclear delivery of compounds that are otherwise not able to cross the NE. Once fully optimized, NE photoporation will not only be a substantial asset for research on NERs, it will become a valuable tool for a wide range of applications that require the transient and controlled disruption of the NE.

Jaar van publicatie:2021

Trefwoorden:Doctoral thesis

Toegankelijkheid:Open