PURPOSE. The purpose of this study is to address the challenge of limited donor retinal ganglion cell (RGC) migration into the retina after transplantation, which is largely due to donor neuron accumulation at the inner limiting membrane (ILM). We present a minimally invasive technique, ILM photodisruption, to locally ablate the ILM and thereby promote RGC engraftment. METHODS. ILM photodisruption uses indocyanine green as a photosensitizer, which is delivered to the ILM and activated with ultra-short laser pulses. This process generates vapor nanobubbles (VNBs) that, upon collapse, create localized disruptions in the ILM. In this study, we finetuned this technology in bovine and postmortem human organotypic retinal explants to generate patterned ILM pores. To assess the impact of these photodisruption patterns on RGC transplantation, we applied induced pluripotent stem cell (iPSC)-derived RGCs to the ILM surface and co-cultured them for 7 days. Using advanced microscopy and spatial metric tools, we assessed donor RGC survival, spreading, and neurite localization. We compared ILM photodisruption to a current standard method of enzymatically digesting the ILM. RESULTS. ILM photodisruption was highly effective in creating pores in both the bovine ILM and the thicker, more complex human ILM. In contrast, collagenase treatment had no effect on the human ILM. Both collagenase and ILM photodisruption significantly promoted donor RGC survival, enhanced cell spreading, and resulted in more neurites that extended deeper into the retina. CONCLUSIONS. Our findings demonstrate that ILM photodisruption can overcome a key barrier in RGC replacement therapy and, as such, may help advance vision restoration strategies for glaucoma.

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. ### Competing Interest Statement The authors have declared no competing interest. China Scholarship Council, 202208320061 European Research Council, 101075873 (Project: DYE-LIGHT) European Innovation Council, 101071105 (Project: NOVISTEM) Kom op Tegen Kanker (KOTK), STI.VLK.2022.0003.01 Villa Joep, STI.DIV.2020.0019.01

Visual impairments affect over 2.2 billion people worldwide, yet delivering drugs to the posterior segment of the eye, including the retina, remains a major challenge. Intravitreal injection, the standard administration route, often results in suboptimal drug diffusion through the vitreous, limiting drug access to the retina. While various strategies have been explored to enhance the mobility of drug molecules and nanomedicines (drugs encapsulated in nanoparticles) in the vitreous, no method has demonstrated guided transport. Here, we investigate photothermal transport of nanoparticles in the vitreous using a pulsed laser and indocyanine green, both clinically approved modalities. We show that photothermal transport guides nanoparticles from one location in the vitreous toward the laser-illuminated area, away from the injection spot. Multiple-particle tracking and numerical simulations reveal that this motion is predominantly driven by thermal convection, with thermophoresis contributing to a lesser extent. We identified parameters for optimization, including dye concentration, particle size, distance from the laser focus, and laser fluence. These findings establish a novel and clinically relevant paradigm for light-guided drug delivery in the eye. To our knowledge, this is the first demonstration of guided light-controlled particle transport in the vitreous using ocular dyes and pulsed lasers routinely applied in ophthalmology.

Despite the natural capacity of extracellular vesicles (EVs) to encapsulate intracellular compounds and transfer these to nearby or distant recipient cells, the intentional loading of EVs with cargo molecules remains a challenging endeavor. Pre-formation EV loading (i.e., during EV biogenesis), offers advantages compared to post-formation loading (i.e., after EV isolation), as EV integrity and composition are minimally perturbed. Pre-formation EV loading is primarily achieved through the genetic engineering of the producer cell, which is time consuming and not very flexible regarding the types of molecules that can be incorporated into EVs. In this work, we investigated the possibility of loading cargo molecules into EVs by delivering the cargo directly into the cytosol of the producer cells, which can subsequently be encapsulated into EVs as they are formed. For the cytosolic delivery of cargo molecules, we evaluated the use of photoporation. This membrane disruption technology has been demonstrated to successfully deliver a broad range of cargo molecules into virtually any cell type, while minimally impacting the cell's normal functioning and homeostasis. As a proof-of-concept, we delivered fluorescently labeled dextran macromolecules and anti-EGFP nanobodies into HEK293T cells genetically engineered with gag-EGFP fusion proteins, which are shuttled into EVs. Colocalization of cargo and EGFP fluorescence in secreted EVs can then serve as a convenient readout for successful EV loading. We established that photoporation had minimal impact on EV characteristics such as concentration, size, zeta potential and the enrichment of EV tetraspanin membrane surface molecules. We found that using EGFP-targeted nanobodies resulted in up to 53% loaded EVs (relative to the amount of EGFP EVs), while non-targeted dextran molecules produced on average 12% loaded EVs (relative to the amount of EGFP EVs). These results highlight the promise of photoporation for pre-formation loading of EVs.

Electrokinetic phenomena offer promising tools for the manipulation of micro- and nanoparticles in liquid media. However, most existing techniques rely on complex configurations and are often limited to particle separation based on large size differences or distinct material properties. Here, we present a simple and tunable method for spatial control and separation of nanoparticles using interdigitated electrodes under AC electric fields. Our approach exploits subtle differences in the electroosmotic and dielectrophoretic responses of particles with small size variations but identical material compositions. By adjusting the frequency and amplitude of the applied voltage, particles can be selectively directed and accumulated at designated regions of the device, enabling precise control over their positioning and segregation. We demonstrate the effectiveness of our method using micro- and nanoparticles composed of the same material, achieving accurate spatial separation based solely on their electrokinetic behavior. This technique offers a low-cost, easily integrable platform for diverse applications, including cell manipulation, water purification, and targeted drug delivery.

The most lethal form of skin cancer is cutaneous melanoma, a tumor that develops in the melanocytes, which are found in the epidermis. The treatment strategy of melanoma is dependent on the stage of the disease and often requires combined local and systemic treatment. Over the years, systemic treatment of melanoma has been revolutionized and shifted toward immunotherapeutic approaches. Phototherapies like photothermal therapy (PTT) have gained considerable attention in the field, mainly because of their straightforward applicability in melanoma skin cancer, combined with the fact that these strategies are able to induce immunogenic cell death (ICD), linked with a specific antitumor immune response. However, PTT comes with the risk of uncontrolled heating of the surrounding healthy tissue due to heat dissipation. Here, we used pulsed laser irradiation of endogenous melanin-containing melanosomes to induce cell killing of B16-F10 murine melanoma cells in a nonthermal manner. Pulsed laser irradiation of the B16-F10 cells resulted in the formation of water vapor nanobubbles (VNBs) around endogenous melanin-containing melanosomes, causing mechanical cell damage. We demonstrated that laser-induced VNBs are able to kill B16-F10 cells with high spatial resolution. When looking more deeply into the cell death mechanism, we found that a large part of the B16-F10 cells succumbed rapidly after pulsed laser irradiation, reaching maximum cell death already after 4 h. Practically all necrotic cells demonstrated exposure of phosphatidylserine on the plasma membrane and caspase-3/7 activity, indicative of regulated cell death. Furthermore, calreticulin, adenosine triphosphate (ATP) and high-mobility group box 1 (HMGB1), three key damage-associated molecular patterns (DAMPs) in ICD, were found to be exposed from B16F10 cells upon pulsed laser irradiation to an extent that exceeded or was comparable to the bona fide ICD-inducer, doxorubicin. Finally, we could demonstrate that VNB formation from melanosomes induced plasma membrane permeabilization. This allowed for enhanced intracellular delivery of bleomycin, an ICD-inducing chemotherapeutic, which further boosted cell death with the potential to improve the systemic antitumor immune response.

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.

Cell therapies such as adoptive T cell transfer require ex vivo modification of cells with exogenous cargo to modulate their phenotype (e.g., to express a synthetic antigen receptor) for optimal therapeutic efficacy upon reinfusion in a patient. Several studies have shown superior anti-tumor activity of minimally differentiated T cell subsets over their activated counterparts. Therefore, developing techniques for safe and efficient manipulation of these quiescent cells is important for both clinical applications and fundamental studies of T cell biology. Photoporation with photothermal electrospun nanofibers (PEN) is an efficient and minimally perturbing nonviral intracellular delivery technique for activated and expanded T cells. However, the technique has not yet been applied to unstimulated T cells. Here, we investigated the potential of PEN photoporation for delivery of macromolecules into these cells. First, we confirmed with inductively coupled plasma tandem mass spectrometry that there was no significant iron release from fibers after laser activation of PEN substrates for laser fluences up to 0.36 J/cm2. Next, we demonstrated successful intracellular delivery of 150 kDa FITC-dextran as model macromolecule in resting and pre-activated lymphocytes with 55-60 % delivery efficiency. By analyzing metabolic activity, activation surface marker presentation and extracellular cytokine release, we found that PEN treatment had no effect on cell proliferation and limited impact on T cell activation propensity for all tested irradiation energies. Thus, our findings show that PEN photoporation holds promise as a safe and efficient delivery strategy, paving the way for its use in genetic modification of minimally differentiated T cells.

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.

End-to-end gold nanorod dimers provide unique plasmonic hotspots with extremely large near-field enhancements in the gaps. Thereby they are beneficial in a wide range of applications, such as enhancing the emissions from ultra-weak emitters. For practical purposes, synthesis of gold nanorod dimers with high yield, especially on the substrates, is essential. Here, we demonstrate two controllable strategies to synthesize gold nanorod dimers based on the self-assembly of gold nanorods, either in bulk solution or on the surface of a glass substrate directly. Both methods can give a high yield of gold nanorod dimers, yet, assembling them directly on the substrate provides more flexibility in controlling the shape and size of each nanorod within the dimer. We also show that these gold nanorod dimers can be used to enhance two-photon-excited fluorescence signals at the single-molecule level.

Optoplasmonic methods capable of single protein detection so far rely on analyte immobilization in order to facilitate detection [1-6]. These detection schemes, even if they facilitate transient single-molecule detection [7,8] via consequent formation and cleavage of chemical bonds, typically exhibit time resolutions on the order of milliseconds. The need for analyte immobilisation is a direct consequence of the minuscule dimensions of plasmonic near fields typically providing sub-attolitre-sized detection volumes which in turn demand sub-microsecond temporal resolution for the direct detection of proteins in motion. Here we show that such temporal resolution can indeed be achieved. We demonstrate the observation of single proteins as small as Hemoglobin (molecular weight: 64 kDa) as they traverse plasmonic near fields of gold nanorods and interact with their surface, all while maintaining signal-to-noise ratios larger than 5 and an unprecedented temporal resolution well below microseconds. This method enables the label-free observation of single-molecule dynamics on previously unaccessible timescales.

Optoplasmonic bio-detection assays commonly probe the response of plasmonic nanostructures to changes in their dielectric environment. The accurate detection of nanoscale entities such as virus particles, micelles and proteins requires optimization of multiple experimental parameters. Performing such optimization directly via analyte recognition is often not desirable or feasible, especially if the nanostructures exhibit limited numbers of analyte binding sites and if binding is irreversible. Here we introduce photothermal spectro-microscopy as a benchmarking tool for the characterization and optimization of optoplasmonic detection assays.

Fluorescence-based single-molecule optical detection techniques are widely chosen over other methods, owing to the ease of background screening and better signal-to-noise throughput. Nonetheless, the methodology still suffers from limitations imposed by weak emitting properties of most molecules. Plasmonic nanostructures, such as gold nanorods, can significantly enhance the fluorescence signal of a weak emitter, extending the application of these techniques to a wider range of species. In this work, we explore the lower limit of fluorescence quantum yield for single-molecule detection, using a single gold nanorod to enhance molecular fluorescence. We specifically designed an infrared dye with the extremely low quantum yield of 10(4) and a comparatively large Stokes shift of 3000 cm(1) to demonstrate single-molecule detection by fluorescence enhancement. This example allows us to discuss more general cases. We estimate theoretically the optimal excitation wavelength and the plasmon resonance of the rod that maximize the fluorescence signals. We then confirm experimentally the detection of single-molecule fluorescence with an enhancement factor of 3 orders of magnitude for the quantum yield 10(4). Theoretical simulations indicate that single-molecule signals should be detectable for molecules with quantum yield as low as 10(6), provided the dwell time of the molecules in the plasmonic hot spot is long enough.

Plasmonic antennas offer extremely promising strategies to enhance single molecule fluorescence sensing and breach the limitations set by diffraction. However, the technical difficulty and limited availability of top-down nanofabrication techniques enabling nanometer gap sizes are limiting the impact of plasmonic antennas for biochemical and biophysical applications. Here we demonstrate the effectiveness of self-assembled nanoparticle gap antennas to enhance single molecule fluorescence detection at high concentrations. For a dimer of 80 nm gold nanoparticles with 6 nm gap, we isolate detection volumes down to 70 zL (equivalent to lambda(3)/3600) and achieve 600-fold fluorescence enhancement, microsecond transit time, and operation of fluorescence correlation spectroscopy at concentrations exceeding 10 mu M. We quantify the near-field detection volume and the fluorescence enhancement for different self-assembled nanoantenna designs using fluorescence correlation spectroscopy. The combination of the fabrication simplicity with the large fluorescence enhancement makes the self-assembled colloidal nanoparticle gap antennas optimal to extend a wide variety of single-molecule applications toward the biologically relevant micromolar concentration regime.

Single-molecule approaches to biology offer a powerful new vision to elucidate the mechanisms that underpin the functioning of living cells. However, conventional optical single molecule spectroscopy techniques such as Förster fluorescence resonance energy transfer (FRET) or fluorescence correlation spectroscopy (FCS) are limited by diffraction to the nanomolar concentration range, far below the physiological micromolar concentration range where most biological reaction occur. To breach the diffraction limit, zero-mode waveguides (ZMW) and plasmonic antennas exploit the surface plasmon resonances to confine and enhance light down to the nanometer scale. The ability of plasmonics to achieve extreme light concentration unlocks an enormous potential to enhance fluorescence detection, FRET, and FCS. Single molecule spectroscopy techniques greatly benefit from ZMW and plasmonic antennas to enter a new dimension of molecular concentration reaching physiological conditions. The application of nano-optics to biological problems with FRET and FCS is an emerging and exciting field, and is promising to reveal new insights on biological functions and dynamics.

Single nanoparticles made of noble metals are strongly appealing to develop practical applications to detect fluorescent molecules in solution. Here, we detail the use of a single gold nanoparticle of 100 nm diameter to enhance the detection of single Alex Fluor 647 fluorescent molecules at high concentrations of several micromolar. We discuss the implementation of fluorescence correlation spectroscopy, and provide a new method to reliably extract the enhanced fluorescence signal stemming from the nanoparticle near-field from the background generated in the confocal volume. The applicability of our method is checked by reporting the invariance of the single molecule results as function of the molecular concentration, and the experimental data is found in good agreement with numerical simulations.

Plasmonics and photonic crystals are two complementary approaches to tailor single-emitter fluorescence, using strong local field enhancements near metals on one hand and spatially extended photonic band structure effects on the other hand. Here, we explore the emergence of spontaneous emission control by finite-sized hexagonal arrays of nanoapertures milled in gold film. We demonstrate that already small lattices enable highly directional and enhanced emission from single fluorescent molecules in the central aperture. Even for clusters just four unit cells across, the directionality is set by the plasmonic crystal band structure, as confirmed by full-wave numerical simulations. This realization of plasmonic phase array antennas driven by single quantum emitters opens a flexible toolbox to engineer fluorescence and its detection.

Single-molecule fluorescence techniques1,2,3 are key for a number of applications, including DNA sequencing4,5, molecular and cell biology6,7 and early diagnosis8. Unfortunately, observation of single molecules by diffraction-limited optics is restricted to detection volumes in the femtolitre range and requires pico- or nanomolar concentrations, far below the micromolar range where most biological reactions occur2. This limitation can be overcome using plasmonic nanostructures, which enable the confinement of light down to nanoscale volumes9,10,11,12,13. Although these nanoantennas enhance fluorescence brightness14,15,16,17,18,19,20, large background signals20,21,22 and/or unspecific binding to the metallic surface23,24,25 have hampered the detection of individual fluorescent molecules in solution at high concentrations. Here we introduce a novel ‘antenna-in-box’ platform that is based on a gap-antenna inside a nanoaperture. This design combines fluorescent signal enhancement and background screening, offering high single-molecule sensitivity (fluorescence enhancement up to 1,100-fold and microsecond transit times) at micromolar sample concentrations and zeptolitre-range detection volumes. The antenna-in-box device can be optimized for single-molecule fluorescence studies at physiologically relevant concentrations, as we demonstrate using various biomolecules. A plasmonic nanoantenna enables a thousand fold-enhanced fluorescence brightness allowing single-molecule analysis to be carried out in a zeptolitre volume at physiological concentrations.