Photoporation-mediated spatial intracellular delivery of stem cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are promising candidates for disease modeling and therapeutic purposes, however, non-viral intracellular delivery in these cells remains challenging. Gold nanoparticle (AuNP)-sensitized photoporation creates transient pores in the cell membrane by vapor nanobubble formation, allowing diffusion of extracellular biomolecules. This non-viral technique was employed to test and optimize its distinct physical mode of action in iPSC-CMs. Photoporation optimization was aimed at achieving high delivery rates while minimizing cell death. Various AuNP concentrations, in conjunction with different laser fluences, were explored to facilitate the intracellular delivery of 10 kDa and 150 kDa FITC-labelled dextran as model macromolecules. Cardiomyocyte viability was assessed using the CellTiter-Glo® viability assay, while the delivery efficiency was quantified through flow cytometry. On 30 day-old cardiomyocytes, AuNP photoporation was able to yield ∼60 % delivery efficiency while maintaining a high cell viability (∼70 %). Overall, higher AuNP concentrations resulted in greater delivery efficiencies, albeit at the expense of lower cell viability. Finally, photoporation was capable of patterning a geometric shape, demonstrating its exceptional selective resolution in delivering molecules to spatially restricted regions of the cell culture. In conclusion, AuNP-photoporation exhibits considerable potential as an effective and gentle non-viral method for intracellular delivery in iPSC-CMs.•AuNP-photoporation is a non-viral intracellular delivery method suitable for iPSC-CMs with high efficiency and cell viability•This method is capable of spatially resolved intracellular delivery with excellent resolution.

A mini-review on gene delivery technique using nanoparticles-mediated photoporation induced by nanosecond pulsed laser.

Nanosecond pulsed laser induced photoporation has gained increasing attention from scholars as an effective method for delivering the membrane-impermeable extracellular materials into living cells. Compared with femtosecond laser, nanosecond laser has the advantage of high throughput and low costs. It also has a higher delivery efficiency than continuous wave laser. Here, we provide an extensive overview of current status of nanosecond pulsed laser induced photoporation, covering the photoporation mechanism as well as various factors that impact the delivery efficiency of photoporation. Additionally, we discuss various techniques for achieving photoporation, such as direct photoporation, nanoparticles-mediated photoporation and plasmonic substrates mediated photoporation. Among these techniques, nanoparticles-mediated photoporation is the most promising approach for potential clinical application. Studies have already been reported to safely destruct the vitreous opacities in vivo by nanosecond laser induced vapor nanobubble. Finally, we discuss the potential of nanosecond laser induced phototoporation for future clinical applications, particularly in the areas of skin and ophthalmic pathologies. We hope this review can inspire scientists to further improve nanosecond laser induced photoporation and facilitate its eventual clinical application.

Upconverting-photon quenching-mediated perforation influx as an intracellular delivery method using posAuNP@UCNPs nanocomposites for osteoarthritis treatment.

Photoporation techniques based on plasmonic nanoparticles such as gold nanoparticles have been extensively studied for the intracellular delivery of substances via cell membrane disruption. However, the clinical application of AuNP is challenging due to its absorption in the 500 nm region of the light spectrum. To overcome this challenge, upconversion nanoparticles were employed to stimulate AuNP at NIR wavelengths. posAuNP@UCNPs nanocomposites were produced by coating 30 nm UCNPs on 80 nm AuNPs using DOPA-PEI, which were then irradiated with 980 nm NIR light to facilitate their intracellular delivery. TEM and DLS confirmed that posAuNP and UCNP combine to form nanocomposites. Additionally, multiphysics simulation was used to analyze the distribution of the posAuNP electric field based on morphological differences that change as the UCNP ratio increases. Next, effective LED irradiation conditions were established by applying upconverting-photon quenching-mediated perforation influx to C28/I2 cells as suspensions or spheroids. posAuNP@UCNP nanocomposites were confirmed to be effective for the delivery of baricitinib as a treatment for osteoarthritis in a three-dimensional osteoarthritis model. Finally, chondrocyte differentiation was induced through intracellular delivery of baricitinib using posAuNP@UCNPs. The findings suggest that posAuNP@UCNPs have great potential as a tool for non-invasive drug delivery via UCPPin.

Response Surface Methodology to Efficiently Optimize Intracellular Delivery by Photoporation.

Photoporation is an up-and-coming technology for the gentle and efficient transfection of cells. Inherent to the application of photoporation is the optimization of several process parameters, such as laser fluence and sensitizing particle concentration, which is typically done one factor at a time (OFAT). However, this approach is tedious and runs the risk of missing a global optimum. Therefore, in this study, we explored whether response surface methodology (RSM) would allow for more efficient optimization of the photoporation procedure. As a case study, FITC-dextran molecules of 500 kDa were delivered to RAW264.7 mouse macrophage-like cells, making use of polydopamine nanoparticles (PDNPs) as photoporation sensitizers. Parameters that were varied to obtain an optimal delivery yield were PDNP size, PDNP concentration and laser fluence. Two established RSM designs were compared: the central composite design and the Box-Behnken design. Model fitting was followed by statistical assessment, validation, and response surface analysis. Both designs successfully identified a delivery yield optimum five- to eight-fold more efficiently than when using OFAT methodology while revealing a strong dependence on PDNP size within the design space. In conclusion, RSM proves to be a valuable approach to efficiently optimize photoporation conditions for a particular cell type.

Novel opto-fluidic drug delivery system for efficient cellular transfection.

Intracellular drug delivery is at the heart of many diagnosis procedures and a key step in gene therapy. Research has been conducted to bypass cell barriers for controlled intracellular drug release and made consistent progress. However, state-of-the-art techniques based on non-viral carriers or physical methods suffer several drawbacks, including limited delivery yield, low throughput or low viability, which are key parameters in therapeutics, diagnostics and drug delivery. Nevertheless, gold nanoparticle (AuNP) mediated photoporation has stood out as a promising approach to permeabilize cell membranes through laser induced Vapour NanoBubble (VNB) generation, allowing the influx of external cargo molecules into cells. However, its use as a transfection technology for the genetic manipulation of therapeutic cells is hindered by the presence of non-degradable gold nanoparticles. Here, we report a new optofluidic method bringing gold nanoparticles in close proximity to cells for photoporation, while avoiding direct contact with cells by taking advantage of hydrodynamic focusing in a multi-flow device. Cells were successfully photoporated with [Formula: see text] efficiency with no significant reduction in cell viability at a throughput ranging from [Formula: see text] to [Formula: see text]. This optofluidic approach provides prospects of translating photoporation from an R &D setting to clinical use for producing genetically engineered therapeutic cells.

Delivery of macromolecules in unstimulated T cells by photoporation with polydopamine nanoparticles.

Ex vivo modification of T cells with exogenous cargo is a common prerequisite for the development of T cell therapies, such as chimeric antigen receptor therapy. Despite the clinical success and FDA approval of several such products, T cell manufacturing presents unique challenges related to therapeutic efficacy after adoptive cell transfer and several drawbacks of viral transduction-based manufacturing, such as high cost and safety concerns. To generate cellular products with optimal potency, engraftment potential and persistence in vivo, recent studies have shown that minimally differentiated T cell phenotypes are preferred. However, genetic engineering of quiescent T cells remains challenging. Photoporation is an upcoming alternative non-viral transfection method which makes use of photothermal nanoparticles, such as polydopamine nanoparticles (PDNPs), to induce transient membrane permeabilization by distinct photothermal effects upon laser irradiation, allowing exogenous molecules to enter cells. In this study, we analyzed the capability of PDNP-photoporation to deliver large model macromolecules (FITC-dextran 500 kDa, FD500) in unstimulated and expanded human T cells. We compared different sizes of PDNPs (150, 250 and 400 nm), concentrations of PDNPs and laser fluences and found an optimal condition that generated high delivery yields of FD500 in both T cell phenotypes. A multiparametric analysis of cell proliferation, surface activation markers and cytokine production, revealed that unstimulated T cells photoporated with 150 nm and 250 nm PDNPs retained their propensity to become activated, whereas those photoporated with 400 nm PDNPs did less. Our findings show that PDNP-photoporation is a promising strategy for transfection of quiescent T cells, but that PDNPs should be small enough to avoid excessive cell damage.

Nanomaterial-mediated photoporation for intracellular delivery.

Translocation of extrinsic molecules into living cells is becoming increasingly crucial in biological studies ranging from cell engineering to biomedical applications. The concerns regarding biosafety and immunogenicity for conventional vectors and physical methods yet challenge effective intracellular delivery. Here, we begin with an overview of approaches for trans-membrane delivery up to now. These methods are featured with a relatively mature application but usually encounter low cell survival. Our review then proposes an advanced application for nanomaterial-sensitized photoporation triggered with a laser. We cover the mechanisms, procedures, and outcomes of photoporation-induced intracellular delivery with a highlight on its versatility to different living cells. We hope the review discussed here encourages researchers to further improvement and applications for photoporation-induced intracellular delivery. STATEMENT OF SIGNIFICANCE.

Transient nuclear lamin A/C accretion aids in recovery from vapor nanobubble-induced permeabilisation of the plasma membrane.

Vapor nanobubble (VNB) photoporation is a physical method for intracellular delivery that has gained significant interest in the past decade. It has successfully been used to introduce molecular cargo of diverse nature into different cell types with high throughput and minimal cytotoxicity. For translational purposes, it is important to understand whether and how photoporation affects cell homeostasis. To obtain a comprehensive view on the transcriptional rewiring that takes place after VNB photoporation, we performed a longitudinal shotgun RNA-sequencing experiment. Six hours after photoporation, we found a marked upregulation of LMNA transcripts as well as their protein products, the A-type lamins. At the same time point, we observed a significant increase in several heterochromatin marks, suggesting a global stiffening of the nucleus. These molecular features vanished 24 h after photoporation. Since VNB-induced chromatin condensation was prolonged in LMNA knockout cells, A-type lamins may be required for restoring the nucleus to its original state. Selective depletion of A-type lamins reduced cell viability after VNB photoporation, while pharmacological stimulation of LMNA transcription increased the percentage of successfully transfected cells that survived after photoporation. Therefore, our results suggest that cells respond to VNB photoporation by temporary upregulation of A-type lamins to facilitate their recovery.

Cas9 RNP transfection by vapor nanobubble photoporation for ex vivo cell engineering.

The CRISPR-Cas9 technology represents a powerful tool for genome engineering in eukaryotic cells, advancing both fundamental research and therapeutic strategies. Despite the enormous potential of the technology, efficient and direct intracellular delivery of Cas9 ribonucleoprotein (RNP) complexes in target cells poses a significant hurdle, especially in refractive primary cells. In the present work, vapor nanobubble (VNB) photoporation was explored for Cas9 RNP transfection in a variety of cell types. Proof of concept was first demonstrated in H1299-EGFP cells, before proceeding to hard-to-transfect stem cells and T cells. Gene knock-out levels over 80% and up to 60% were obtained for H1299 cells and mesenchymal stem cells, respectively. In these cell types, the unique possibility of VNB photoporation to knock out genes according to user-defined spatial patterns was demonstrated as well. Next, effective targeting of the programmed cell death 1 receptor and Wiskott-Aldrich syndrome gene in primary human T cells was demonstrated, reaching gene knock-out levels of 25% and 34%, respectively. With a throughput of >200,000 T cells per second, VNB photoporation is a scalable and versatile intracellular delivery method that holds great promise for CRISPR-Cas9-mediated ex vivo engineering of cell therapy products.

Delivery of Foreign Materials into Adherent Cells by Gold Nanoparticle-Mediated Photoporation.

Delivering extracellular materials into adherent cells presents several challenges. A homemade photoporation platform, mediated by gold nanoparticles (AuNPs), was constructed to find a suitable method for finding all adherent cells in this process with high delivery efficiency. The thermal dynamics of AuNPs could be monitored. Based on this system, 60 nm AuNPs were selected to be attached to cells for optimal photoporation. After irradiating the cells covered with AuNPs using a nanosecond pulse laser, fluorescein isothiocyanate-dextran in the medium were delivered into optoporated adherent HeLa (human cervical cell lines) cells. The delivery efficiency and cell viability of this process were evaluated using a fluorescence microscope and flow cytometry. The experimental results showed that targeting cells using antibodies, laser irradiation from the top of the cell culture well, and reducing the cell medium are important for improving the delivery efficiency. The optimal loading efficiency for adherent HeLa cells was 53.4%.

Electrochemical fabrication of TiO2 micro-flowers for an efficient intracellular delivery using nanosecond light pulse.

Introduction of foreign cargo into the targeted living cell with high transfection efficiency and high cell viability is an important mean for many biological and biomedical research purpose. Here, we have demonstrated a newly developed Titanium oxide micro-flower structure (TMS) for intracellular delivery. The TMS were formed on titanium (Ti) substrate using an electrochemical anodization process. The TMS consists of branches of titanium dioxide (TiO2) nanotubes, which play an important role in efficient cargo delivery. Due to nanosecond pulse laser exposure, Ti substrate heat-up, generating cavitation bubbles. These bubbles can rapidly grow, coalesce, and collapse to induce explosion resulting in very strong fluid flow through the TiO2 nanotubes and disrupt the cell plasma membrane promoting the delivery of biomolecules into cells. Using this platform, we successfully deliver dyes with 93% efficiency and nearly 98% cell viability into HCT cells, and this technique is potentially applicable for cellular therapy and diagnostics.

Optimization of intracellular macromolecule delivery by nanoparticle-mediated photoporation.

Nanoparticle-mediated photoporation is a novel delivery platform for intracellular molecule delivery. We studied the dependence of macromolecular delivery on molecular weight and sought to enhance delivery efficiency. DU145 prostate cancer cells were exposed to pulsed laser beam in the presence of carbon-black nanoparticles. Intracellular uptake of molecules decreased with increasing molecular weight. Attributing this dependence to molecular diffusivity, we hypothesized that macromolecular delivery efficiency could be enhanced by increasing either laser fluence or laser exposure duration at low fluence. We observed increased percentages of macromolecule uptake by cells in both cases. However, trade-off between cell uptake and viability loss was most favorable at low laser fluence (25-29 mJ/cm2) and longer exposure durations (4-5 min). We conclude that long exposure at low laser fluence optimizes intracellular macromolecule delivery by nanoparticle-mediated photoporation, which may be explained by longer time for macromolecules to diffuse into cells, during and between laser pulses.

Serum Protects Cells and Increases Intracellular Delivery of Molecules by Nanoparticle-Mediated Photoporation.

INTRODUCTION: Intracellular delivery of molecules is central to applications in biotechnology, medicine, and basic research. Nanoparticle-mediated photoporation using carbon black nanoparticles exposed to pulsed, near-infrared laser irradiation offers a physical route to create transient cell membrane pores, enabling intracellular delivery. However, nanoparticle-mediated photoporation, like other physical intracellular delivery technologies, necessitates a trade-off between achieving efficient uptake of exogenous molecules and maintaining high cell viability. METHODS: In this study, we sought to shift this balance by adding serum to cells during nanoparticle-mediated photoporation as a viability protectant. DU-145 prostate cancer cells and human dermal fibroblasts were exposed to laser irradiation in the presence of carbon black (CB) nanoparticles and other formulation additives, including fetal bovine serum (FBS) and polymers. RESULTS: Our studies showed that FBS can protect cells from viability loss, even at high-fluence laser irradiation conditions that lead to high levels of intracellular delivery in two different mammalian cell types. Further studies revealed that full FBS was not needed: viability protection was achieved with denatured FBS, with just the high molecular weight fraction of FBS (>30 kDa), or even with individual proteins like albumin or hemoglobin. Finally, we found that viability protection was also obtained using certain neutral water-soluble polymers, including Pluronic F127, polyvinylpyrrolidone, poly(2-ethyl-2-oxazoline), and polyethylene glycol, which were more effective at increased concentration, molecular weight, or hydrophobicity. CONCLUSION: Altogether, these findings suggest an interaction between amphiphilic domains of polymers with the cell membrane to help cells maintain viability, possibly by facilitating transmembrane pore closure. In this way, serum components or synthetic polymers can be used to increase intracellular delivery by nanoparticle-mediated photoporation while maintaining high cell viability.

Long-term live-cell microscopy with labeled nanobodies delivered by laser-induced photoporation.

Fluorescence microscopy is the method of choice for studying intracellular dynamics. However, its success depends on the availability of specific and stable markers. A prominent example of markers that are rapidly gaining interest are nanobodies (Nbs, ~ 15 kDa), which can be functionalized with bright and photostable organic fluorophores. Due to their relatively small size and high specificity, Nbs offer great potential for high-quality long-term subcellular imaging, but suffer from the fact that they cannot spontaneously cross the plasma membrane of live cells. We have recently discovered that laser-induced photoporation is well suited to deliver extrinsic labels to living cells without compromising their viability. Being a laser-based technology, it is readily compatible with light microscopy and the typical cell recipients used for that. Spurred by these promising initial results, we demonstrate here for the first time successful long-term imaging of specific subcellular structures with labeled nanobodies in living cells. We illustrate this using Nbs that target GFP/YFP-protein constructs accessible in the cytoplasm, actin-bundling protein Fascin, and the histone H2A/H2B heterodimers. With an efficiency of more than 80% labeled cells and minimal toxicity (~ 2%), photoporation proved to be an excellent intracellular delivery method for Nbs. Time-lapse microscopy revealed that cell division rate and migration remained unaffected, confirming excellent cell viability and functionality. We conclude that laser-induced photoporation labeled Nbs can be easily delivered into living cells, laying the foundation for further development of a broad range of Nbs with intracellular targets as a toolbox for long-term live-cell microscopy.

Nanobody click chemistry for convenient site-specific fluorescent labelling, single step immunocytochemistry and delivery into living cells by photoporation and live cell imaging.

While conventional antibodies have been an instrument of choice in immunocytochemistry for some time, their small counterparts known as nanobodies have been much less frequently used for this purpose. In this study we took advantage of the availability of nanobody cDNAs to site-specifically introduce a non-standard amino acid carrying an azide/alkyne moiety, allowing subsequent Cu(I)-catalyzed Azide-Alkyne Click Chemistry (CuAAC). This generated a fluorescently labelled nanobody that can be used in single step immunocytochemistry as compared to conventional two step immunocytochemistry. Two strategies were explored to label nanobodies with Alexa Fluor 488. The first involved enzymatic addition of an alkyne-containing peptide to nanobodies using sortase A, while the second consisted of incorporating para-azido phenylalanine at the nanobody C-terminus. Through these approaches, the fluorophore was covalently and site-specifically attached. It was demonstrated that cortactin and ß-catenin, cytoskeletal and adherens junction proteins respectively, can be imaged in cells in this manner through single step immunocytochemistry. However, fixation and permeabilization of cells can alter native protein structure and form a dense cross-linked protein network, encumbering antibody binding. It was shown that photoporation prior to fixation not only allowed delivery of nanobodies into living cells, but also facilitated ß-catenin nanobody Nb86 imaging of its target, which was not possible in fixed cells. Pharmacological inhibitors are lacking for many non-enzymatic proteins, and it is therefore expected that new biological information will be obtained through photoporation of fluorescent nanobodies, which allows the study of short term effects, independent of gene-dependent (intrabody) expression.

Surface Functionalization with Polyethylene Glycol and Polyethyleneimine Improves the Performance of Graphene-Based Materials for Safe and Efficient Intracellular Delivery by Laser-Induced Photoporation.

Nanoparticle mediated laser-induced photoporation is a physical cell membrane disruption approach to directly deliver extrinsic molecules into living cells, which is particularly promising in applications for both adherent and suspension cells. In this work, we explored surface modifications of graphene quantum dots (GQD) and reduced graphene oxide (rGO) with polyethylene glycol (PEG) and polyethyleneimine (PEI) to enhance colloidal stability while retaining photoporation functionality. After photoporation with FITC-dextran 10 kDa (FD10), the percentage of positive HeLa cells (81% for GQD-PEG, 74% for rGO-PEG and 90% for rGO-PEI) increased approximately two-fold compared to the bare nanomaterials. While for Jurkat suspension cells, the photoporation efficiency with polymer-modified graphene-based nanomaterial reached as high as 80%. Cell viability was >80% in all these cases. In addition, polymer functionalization proved to be beneficial for the delivery of larger macromolecules (FD70 and FD500) as well. Finally, we show that rGO is suitable for photoporation using a near-infrared laser to reach 80% FD10 positive HeLa cells at 80% cell viability. We conclude that modification of graphene-based nanoparticles with PEG and especially PEI provide better colloidal stability in cell medium, resulting in more uniform transfection and overall increased efficiency.

Vapor nanobubble is the more reliable photothermal mechanism for inducing endosomal escape of siRNA without disturbing cell homeostasis.

Strategies for controlled delivery of therapeutic siRNA into living cells are in high demand as endosomal escape remains the most prominent bottleneck at the intracellular level. Photothermal properties of gold nanoparticles (AuNP) can be used to overcome the endosomal membrane barrier upon laser irradiation by two mechanisms: endosomal rupture by mechanical energy from water vapor nanobubbles (VNBs), or permeabilization of the endosomal membrane by heat diffusion. Here we evaluated how both mechanisms influence cargo release, transfection efficiency, acute cytotoxicity and cell homeostasis. Using a siRNA/AuNP drug delivery system we found that the in vitro release of siRNA from the AuNP carrier occurs equally efficiently by VNB formation or heat generation. Heat-mediated endosomal escape happened more efficiently in cells that had more particles per endosome, resulting in variable siRNA-induced downregulation (20-50%). VNB-mediated endosomal escape did not dependent on the number of AuNP per endosome, yielding high downregulations (50-60%) independent of the cell type. Effects on cell homeostasis by whole transcriptome analysis, showed a quick recover after 24 h or 48 h for either of both photothermal mechanisms. We conclude that VNBs are more consistent to induce efficient endosomal escape and gene silencing independent of the cell type without long lasting effects on cell homeostasis.

Intracellular Delivery of mRNA in Adherent and Suspension Cells by Vapor Nanobubble Photoporation.

Efficient and safe cell engineering by transfection of nucleic acids remains one of the long-standing hurdles for fundamental biomedical research and many new therapeutic applications, such as CAR T cell-based therapies. mRNA has recently gained increasing attention as a more safe and versatile alternative tool over viral- or DNA transposon-based approaches for the generation of adoptive T cells. However, limitations associated with existing nonviral mRNA delivery approaches hamper progress on genetic engineering of these hard-to-transfect immune cells. In this study, we demonstrate that gold nanoparticle-mediated vapor nanobubble (VNB) photoporation is a promising upcoming physical transfection method capable of delivering mRNA in both adherent and suspension cells. Initial transfection experiments on HeLa cells showed the importance of transfection buffer and cargo concentration, while the technology was furthermore shown to be effective for mRNA delivery in Jurkat T cells with transfection efficiencies up to 45%. Importantly, compared to electroporation, which is the reference technology for nonviral transfection of T cells, a fivefold increase in the number of transfected viable Jurkat T cells was observed. Altogether, our results point toward the use of VNB photoporation as a more gentle and efficient technology for intracellular mRNA delivery in adherent and suspension cells, with promising potential for the future engineering of cells in therapeutic and fundamental research applications.

Delivery of Mixed-Lineage Kinase Domain-Like Protein by Vapor Nanobubble Photoporation Induces Necroptotic-Like Cell Death in Tumor Cells.

Modern molecular medicine demands techniques to efficiently deliver molecules directly into mammalian cells. As proteins are the final mediators of most cellular pathways, efficient intracellular protein delivery techniques are highly desired. In this respect, photoporation is a promising recent technique for the delivery of proteins directly into living cells. Here, we show the possibility to deliver a model saccharide (FD70) and a model protein (FITC-BSA) into murine B16 melanoma cells by using the vapor nanobubble photoporation technique with an efficiency of 62% and 38%, respectively. Next, we delivered the mixed-lineage kinase domain-like (MLKL) protein, the most terminal mediator of necroptosis currently known, and caspase-8 and -3 protein, which are important proteins in the initiation and execution of apoptosis. A significant drop in cell viability with 62%, 71% and 64% cell survival for MLKL, caspase-8 and caspase-3, respectively, was observed. Remarkably, maximal cell death induction was already observed within 1 h after protein delivery. Transduction of purified recombinant MLKL by photoporation resulted in rapid cell death characterized by cell swelling and cell membrane rupture, both hallmarks of necroptosis. As necroptosis has been identified as a type of cell death with immunogenic properties, this is of interest to anti-cancer immunotherapy. On the other hand, transduction of purified recombinant active caspase-3 or -8 into the tumor cells resulted in rapid cell death preceded by membrane blebbing, which is typical for apoptosis. Our results suggest that the type of cell death of tumor cells can be controlled by direct transduction of effector proteins that are involved in the executioner phase of apoptosis or necroptosis.

Targeted Perturbation of Nuclear Envelope Integrity with Vapor Nanobubble-Mediated Photoporation.

The nuclear envelope (NE) has long been considered to dismantle only during mitosis. However, recent observations in cancer cells and laminopathy patient cells have revealed that the NE can also transiently rupture during interphase, thereby perturbing cellular homeostasis. Although NE ruptures are promoted by mechanical force and the loss of lamins, their stochastic nature and variable frequency preclude the study of their direct downstream consequences. We have developed a method based on vapor nanobubble-mediated photoporation that allows for deliberately inducing NE ruptures in a spatiotemporally controlled manner. Our method relies on wide-field laser illumination of perinuclear gold nanoparticles, resulting in the formation of short-lived vapor nanobubbles that inflict minute mechanical damage to the NE, thus creating small pores. We demonstrate that perinuclear localization of gold nanoparticles can be achieved after endocytic uptake or electroporation-facilitated delivery and that both strategies result in NE rupture upon laser irradiation. Furthermore, we prove that photoporation-induced nuclear ruptures are transient and recapitulate hallmarks of spontaneous NE ruptures that occur in A-type lamin-depleted cells. Finally, we show that the same approach can be used to promote influx of macromolecules that are too large to passively migrate through the NE. Thus, by providing unprecedented control over nuclear compartmentalization, nuclear photoporation offers a powerful tool for both fundamental cell biology research and drug delivery applications.