Tao Lu

Efficient and safe intracellular delivery of macromolecules, like nucleic acids, remains a major challenge in ex vivo cell engineering. While photoporation is a highly attractive method for cytosolic delivery of (macro)molecules, photosensitizers remaining in the photoporated cells may pose safety concerns, especially when the cells are engineered for therapeutic use. In this study we introduce the concept of “detachable photosensitizers” using gold nanoparticles (AuNPs) as photosensitizer. We demonstrate that phenylboronic acid (PBA)‐functionalized AuNPs can be anchored to membranes of HeLa cells and Jurkat cells, thereby enabling the efficient intracellular delivery of dextrans and mRNA upon pulsed laser irradiation (photoporation). Subsequently, the PBA‐AuNPs can be detached from the cells “on‐demand” by adding ATP to the cell medium, as cis‐ diol groups in ATP displace the boronate ester bonds between the PBA‐AuNPs and cell‐surface. ICP‐MS analysis demonstrated efficient detachment, with the average cell‐associated gold mass per viable cell falling below the theoretical mass of a single intact 80 nm AuNP. This “attach‐and‐detach” strategy offers a robust and effective approach to addressing safety concerns associated with the use of plasmonic nanomaterials as sensitizers for photoporation, and holds significant potential for the safe manufacturing of engineered cells in the context of cell therapy.

Photothermal nanomaterials enable precise tumor ablation but face limitations in biodistribution, tissue penetration, toxicity, and biodegradability. Here, we present a unique concept for nanoparticle-free photothermal therapy based on the lysosomal entrapment of cationic amphiphilic small molecular dyes for spatially controlled vapor bubble (VB)–mediated tumor cell ablation. This strategy, which exploits a universal biological and physical effect, uses intracellular pH gradients for extensive local dye enrichment in acidified organelles, transforming them into transient endogenous nanosized photothermal reactors for subsequent light activation. Using sunitinib, a clinically approved lysosomotropic anticancer drug, and the commercially available dye LysoTracker Deep Red, lacking intrinsic anticancer activity, we demonstrate pulsed laser-induced VB formation from dye-enriched lysosomal compartments, leading to selective photomechanical disruption of various ex vivo cancer cell models across two-dimensional (2D) cultures, 3D spheroids, patient-derived neuroblastoma tumoroids, and tumor fragments from a patient with ovarian carcinoma. This approach allows precise, low-fluence, and wavelength-tunable cancer tissue ablation without the need for synthetic photoresponsive nanoparticles.

Diseases of the ocular surface, such as dry eye disease (DED), can significantly impair vision and reduce the quality of life. Current treatments rely primarily on conventional approaches, including artificial tears, antibiotics, and anti‐inflammatory drugs. However, advances in nucleic acid (NA) therapeutics have opened new avenues for treating ocular surface disorders, with growing interest in small interfering RNA (siRNA) and messenger RNA (mRNA) as potential drugs. A key challenge remains the efficient delivery of these macromolecules into cells of the ocular surface, where both extracellular and intracellular barriers hinder their cellular uptake and activity. To address this limitation, we explore the use of photoporation, a laser‐based physical transfection technique, to enhance mRNA delivery into ocular surface cells. Photoporation employs pulsed‐laser irradiation of photosensitizers to generate vapor nanobubbles (VNBs), whose rapid expansion and implosion produce mechanical forces that transiently permeabilize cell membranes, thereby promoting intracellular delivery of co‐administered nucleic acids. Using polydopamine nanoparticles (PD NPs) as biocompatible photosensitizers for photoporation, we demonstrate mRNA transfection in the corneal epithelium with both an experimental and a clinically approved pulsed‐laser system. Our results highlight the potential of laser‐based approaches for noninvasive engineering of the ocular surface.

Having access to high‐quality cell populations is essential for biomedical research, diagnosis, prognosis, and therapeutic applications. Traditional enzymatic methods, such as trypsinization, remain widely used to detach cells from supporting material surfaces, though they often compromise cell surface integrity, viability and functionality. To address these limitations, diverse strategies have emerged that enable a much gentler release of cells from material surfaces. The use of external physical stimuli (like shear stresses, light, temperature, magnetic fields), chemical stimuli (based on changes in pH or ligand‐exchange based detachment) and biological stimuli (like aptamers and nucleic acid sequences) have been explored for the purpose of non‐invasive cell detachment. This review discusses the principles, mechanisms, advantages, and limitations of these most diverse cell detachment technologies. By providing this comprehensive perspective, we hope to guide researchers in selecting the most suitable techniques for their specific needs and to inspire further innovation in this vital biomedical area.

Photothermal nanomaterials enable precise tumor ablation but face limitations in biodistribution, tissue penetration, toxicity, and biodegradability. Here, we present a unique concept for nanoparticle-free photothermal therapy based on the lysosomal entrapment of cationic amphiphilic small molecular dyes for spatially controlled vapor bubble (VB)-mediated tumor cell ablation. This strategy, which exploits a universal biological and physical effect, employs intracellular pH gradients for extensive local dye enrichment in acidified organelles, transforming them into transient endogenous nanosized photothermal reactors for subsequent light activation. Using sunitinib, a clinically approved lysosomotropic anticancer drug, and the commercially available dye LysoTracker™ Deep Red, lacking intrinsic anticancer activity, we demonstrate pulsed laser-induced VB formation specifically from dye-loaded lysosomes, leading to selective photomechanical disruption of various cancer cell models across 2D cultures, 3D spheroids, patient-derived neuroblastoma tumoroids and tumor fragments from an ovarian carcinoma patient. This approach allows precise, low-fluence and wavelength-tunable cancer tissue ablation without the need for synthetic photoresponsive nanoparticles.