RESUMEN
Nucleic acid biopharmaceuticals are being investigated as potential therapeutics. They need to be incorporated into a biocompatible carrier so as to overcome several biological barriers. Rational development of suitable nanocarriers requires high-quality characterization techniques. While size, concentration, and stability can be very well measured these days, even in complex biological fluids, a method to accurately quantify the number of nucleic acid therapeutics encapsulated in nanocarriers is still missing. Here we present a method, based on concentration measurements with single particle tracking microscopy, with which it is possible to directly measure the number of plasmid DNA molecules per nanoparticle, referred to as the plasmid/NP ratio. Using DOTAP/DOPE liposomes as a model carrier, we demonstrate the usefulness of the method by investigating the influence of various experimental factors on the plasmid/NP ratio. We find that the plasmid/NP ratio is inversely proportional with the size of the pDNA and that the plasmid/NP decreases when lipoplexes are prepared at lower concentrations of pDNA and nanocarrier, with values ranging from 6.5 to 3 plasmid/NP. Furthermore, the effect of pre- and post-PEGylation of lipoplexes was examined, finding that pre-PEGylation results in a decreased plasmid/NP ratio, while post-PEGylation did not alter the plasmid/NP ratio. These proof-of-concept experiments show that single particle tracking offers an extension of the nanoparticle characterization toolbox and is expected to aid in the efficient development of nanoformulations for nucleic acid-based therapies.
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Productos Biológicos/administración & dosificación , Portadores de Fármacos/química , Ácidos Nucleicos/administración & dosificación , Ácidos Grasos Monoinsaturados/química , Liposomas , Microscopía/métodos , Nanopartículas/química , Fosfatidiletanolaminas/química , Plásmidos/genética , Compuestos de Amonio Cuaternario/química , Transfección/métodosRESUMEN
Light sheet microscopy is a relatively new form of fluorescence microscopy that has been receiving a lot of attention recently. The strong points of the technique, such as high signal to noise ratio and its reduced photodamage of fluorescently labelled samples, come from its unique feature to illuminate only a thin plane in the sample that coincides with the focal plane of the detection lens. Typically this requires two closely positioned perpendicular objective lenses, one for detection and one for illumination. Apart from the fact that this special configuration of objective lenses is incompatible with standard microscope bodies, it is particularly problematic for high-resolution lenses which typically have a short working distance. To address these issues we developed sample holders with an integrated micromirror to perform single lens light sheet microscopy, also known as single objective single plane illumination microscopy (SoSPIM). The first design is based on a wet-etched silicon substrate, the second on a microfabricated polished polymer plug. We achieved an on-chip light sheet thickness of 2.3 µm (FWHM) at 638 nm with the polymer micromirror and of 1.7 µm (FWHM) at 638 nm with the silicon micromirror, comparable to reported light sheet thicknesses obtained on dedicated light sheet microscopes. A marked contrast improvement was obtained with both sample holders as compared to classic epi-fluorescence microscopy. In order to evaluate whether this technology could be made available on a larger scale, in a next step we evaluated the optical quality of inexpensive replicas from both types of master molds. We found that replicas from the polished polymer based mold have an optical quality close to that of the master component, while replicas from the silicon based mold were of slightly lower but still acceptable quality. The suitability of the replicated polymer based sample holder for single-lens light sheet microscopy was finally demonstrated by imaging breast cancer spheroids.
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BACKGROUND: Nanoparticle interactions with cellular membranes and the kinetics of their transport and localization are important determinants of their functionality and their biological consequences. Understanding these phenomena is fundamental for the translation of such NPs from in vitro to in vivo systems for bioimaging and medical applications. Two CdSe/ZnS quantum dots (QD) with differing surface functionality (NH2 or COOH moieties) were used here for investigating the intracellular uptake and transport kinetics of these QDs. RESULTS: In water, the COOH- and NH2-QDs were negatively and positively charged, respectively, while in serum-containing medium the NH2-QDs were agglomerated, whereas the COOH-QDs remained dispersed. Though intracellular levels of NH2- and COOH-QDs were very similar after 24 h exposure, COOH-QDs appeared to be continuously internalised and transported by endosomes and lysosomes, while NH2-QDs mainly remained in the lysosomes. The results of (intra)cellular QD trafficking were correlated to their toxicity profiles investigating levels of reactive oxygen species (ROS), mitochondrial ROS, autophagy, changes to cellular morphology and alterations in genes involved in cellular stress, toxicity and cytoskeletal integrity. The continuous flux of COOH-QDs perhaps explains their higher toxicity compared to the NH2-QDs, mainly resulting in mitochondrial ROS and cytoskeletal remodelling which are phenomena that occur early during cellular exposure. CONCLUSIONS: Together, these data reveal that although cellular QD levels were similar after 24 h, differences in the nature and extent of their cellular trafficking resulted in differences in consequent gene alterations and toxicological effects.
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Autofagia/efectos de los fármacos , Compuestos de Cadmio/toxicidad , Puntos Cuánticos/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Compuestos de Selenio/toxicidad , Sulfuros/toxicidad , Compuestos de Zinc/toxicidad , Compuestos de Cadmio/análisis , Compuestos de Cadmio/metabolismo , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Puntos Cuánticos/análisis , Puntos Cuánticos/metabolismo , Compuestos de Selenio/análisis , Compuestos de Selenio/metabolismo , Sulfuros/análisis , Sulfuros/metabolismo , Compuestos de Zinc/análisis , Compuestos de Zinc/metabolismoRESUMEN
Long-term in vivo imaging of cells is crucial for the understanding of cellular fate in biological processes in cancer research, immunology, or in cell-based therapies such as beta cell transplantation in type I diabetes or stem cell therapy. Traditionally, cell labeling with the desired contrast agent occurs ex vivo via spontaneous endocytosis, which is a variable and slow process that requires optimization for each particular label-cell type combination. Following endocytic uptake, the contrast agents mostly remain entrapped in the endolysosomal compartment, which leads to signal instability, cytotoxicity, and asymmetric inheritance of the labels upon cell division. Here, we demonstrate that these disadvantages can be circumvented by delivering contrast agents directly into the cytoplasm via vapor nanobubble photoporation. Compared to classic endocytic uptake, photoporation resulted in 50 and 3 times higher loading of fluorescent dextrans and quantum dots, respectively, with improved signal stability and reduced cytotoxicity. Most interestingly, cytosolic delivery by photoporation prevented asymmetric inheritance of labels by daughter cells over subsequent cell generations. Instead, unequal inheritance of endocytosed labels resulted in a dramatic increase in polydispersity of the amount of labels per cell with each cell division, hindering accurate quantification of cell numbers in vivo over time. The combined benefits of cell labeling by photoporation resulted in a marked improvement in long-term cell visibility in vivo where an insulin producing cell line (INS-1E cell line) labeled with fluorescent dextrans could be tracked for up to two months in Swiss nude mice compared to 2 weeks for cells labeled by endocytosis.
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The advent of nanotechnology has revolutionized drug delivery in terms of improving drug efficacy and safety. Both polymer-based and lipid-based drug-loaded nanocarriers have demonstrated clinical benefit to date. However, to address the multifaceted drug delivery challenges ahead and further expand the spectrum of therapeutic applications, hybrid lipid-polymer nanocomposites have been designed to merge the beneficial features of both polymeric drug delivery systems and liposomes in a single nanocarrier. This review focuses on different classes of nanohybrids characterized by a drug-loaded polymeric matrix core enclosed in a lipid shell. Various nanoengineering approaches to obtain lipid-polymer nanocomposites with a core-shell nanoarchitecture will be discussed as well as their predominant applications in drug delivery.
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Sistemas de Liberación de Medicamentos/métodos , Nanocompuestos/administración & dosificación , Nanocompuestos/química , Animales , Biomimética , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato , Lípidos/química , Nanocompuestos/toxicidad , Poliésteres/química , Dióxido de SilicioRESUMEN
The use of quantum dots (QDots) as bright and photostable probes for long-term fluorescence imaging is gaining more interest. Thus far, (pre)clinical use of QDots remains limited, which is primarily caused by the potential toxicity of QDots. Most QDots consist of Cd2+ ions, which are known to cause high levels of toxicity. In order to overcome this problem, several strategies have been tested, such as the generation of cadmium-free QDots. In the present study, two types of cadmium-free QDots, composed of ZnSe/ZnS (QDotZnSe) and InP/ZnS (QDotInP), were studied with respect to their cytotoxicity and cellular uptake in a variety of cell types. A multiparametric cytotoxicity approach is used, where the QDots are studied with respect to cell viability, oxidative stress, cell morphology, stem cell differentiation, and neurite outgrowth. The data reveal slight differences in uptake levels for both types of QDots (maximal for QDotZnSe), but clear differences in cytotoxicity and cell functionality effects exist, with highest toxicity for QDotZnSe. Differences between cell types and between both types of QDots can be explained by the intrinsic sensitivity of certain cell types and chemical composition of the QDots. At concentrations at which no toxic effects can be observed, the functionality of the QDots for fluorescence cell visualization is evaluated, revealing that the higher brightness of QDotZnSe overcomes most of the toxicity issues compared to that of QDotInP. Comparing the results obtained with common Cd2+-containing QDots tested under identical conditions, the importance of particle functionality is demonstrated, revealing that cadmium-free QDots tested in this study are not significantly better than Cd2+-containing QDots for long-term cell imaging and that more work needs to be performed in optimizing the brightness and surface chemistry of cadmium-free QDots for them to replace currently used Cd2+-containing QDots.
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Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Imagen Molecular/métodos , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Puntos Cuánticos/química , Puntos Cuánticos/toxicidad , Animales , Cadmio , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Indio/química , Indio/metabolismo , Indio/toxicidad , Ratones , Células-Madre Neurales/citología , Estrés Oxidativo/efectos de los fármacos , Células PC12 , Fosfinas/química , Fosfinas/metabolismo , Fosfinas/toxicidad , Puntos Cuánticos/metabolismo , Ratas , Compuestos de Selenio/química , Compuestos de Selenio/metabolismo , Compuestos de Selenio/toxicidad , Relación Estructura-Actividad , Sulfuros/química , Sulfuros/metabolismo , Sulfuros/toxicidad , Compuestos de Zinc/química , Compuestos de Zinc/metabolismo , Compuestos de Zinc/toxicidadRESUMEN
The development of biotechnological pharmaceutics, like macro- and nanocarriers, can benefit greatly from studying their characteristics in situ using advanced fluorescence microscopy methods. While choosing the optimal labeling method for visualizing the carrier or its cargo is crucial, it seldom receives attention. The possibility that high labeling densities alter the intracellular processing of the molecule is considered, but how and at which point this interference happens is not yet studied. The aim of this study was to elucidate the effect of labeling density on the cellular trafficking of labeled pDNA. Due to the drastic effect on expression levels for higher labeling densities, we tried to determine at which steps in the intracellular processing labeled pDNA behaves different than its nonlabeled counterpart. Therefore, different labeling densities, up to the manufacturer's recommended density, were tested. It was found that the cellular uptake remains unaffected, while the affinity for lipids is increased, which affects dissociation from the lipid-based complex and may affect endosomal escape. Also, nuclear injections clearly demonstrated that transcription is affected. The information and methodology, included in this work, could be helpful in determining if the labeling method and density used yields biological relevant results for the intended research question.
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Plásmidos/metabolismo , Transfección/métodos , Transporte Biológico/fisiología , Endosomas/metabolismo , Células HeLa , Humanos , Lípidos/química , Liposomas/química , Liposomas/metabolismo , Microscopía Fluorescente , Plásmidos/química , Plásmidos/genética , Reacción en Cadena de la PolimerasaRESUMEN
Fluorescence recovery after photobleaching (FRAP) is a fluorescence microscopy technique that has attracted a lot of interest in pharmaceutical research during the last decades. The main purpose of FRAP is to measure diffusion on a micrometer scale in a non-invasive and highly specific way, making it capable of measurements in complicated biomaterials, even in vivo. This has proven to be very useful in the investigation of drug diffusion inside different tissues of the body and in materials for controlled drug delivery. FRAP has even found applications for the improvement of several medical therapies and in the field of diagnostics. In this review, an overview is given of the different applications of FRAP in pharmaceutical research, together with essential guidelines on how to perform and analyse FRAP experiments.
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Sistemas de Liberación de Medicamentos/métodos , Recuperación de Fluorescencia tras Fotoblanqueo/métodos , Animales , Difusión , HumanosRESUMEN
Interactions between objects inside living cells are often investigated by looking for colocalization between fluorescence microscopy images that are recorded in separate colours corresponding to the fluorescent label of each object. The fundamental limitation of this approach in the case of dynamic objects is that coincidental colocalization cannot be distinguished from true interaction. Instead, correlation between motion trajectories obtained by dual colour single particle tracking provides a much stronger indication of interaction. However, frequently occurring phenomena in living cells, such as immobile phases or transient interactions, can limit the correlation to small parts of the trajectories. The method presented here, developed for the detection of interaction, is based on the correlation inside a window that is scanned along the trajectories, covering different subsets of the positions. This scanning window method was validated by simulations and, as an experimental proof of concept, it was applied to the investigation of the intracellular trafficking of polymeric gene complexes by endosomes in living retinal pigment epithelium cells, which is of interest to ocular gene therapy.
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Análisis de la Célula Individual/métodos , Algoritmos , Línea Celular , Simulación por Computador , Endosomas/metabolismo , Colorantes Fluorescentes/metabolismo , Humanos , Microscopía Fluorescente/métodos , Modelos Biológicos , Nanomedicina , Nanopartículas/metabolismoRESUMEN
Complexes between mRNA and GL67:DOPE:DMPE-PEG5000 (GL67) liposomes were formulated and characterized. Subsequently, the in vitro and in vivo expression characteristics of mRNA/GL67 complexes and pDNA/GL67 complexes, each produced at their optimal ratio, were compared in respiratory cells. Transfection of A549 cells with mRNA/GL67 complexes resulted in a much faster expression than after transfection with pDNA/GL67 complexes. The percentage of GFP-positive cells after mRNA and pDNA transfection peaked after 8 and 24 h, respectively. At these time points the percentage of GFP-positive cells was two times higher after mRNA transfection than after pDNA transfection. Furthermore, the efficacy of mRNA/GL67 complexes was independent of the cell cycle. This was in sharp contrast with pDNA/GL67 complexes that caused only a weak expression in nondividing cells. This confirms that the nuclear barrier is a crucial obstacle for pDNA but not for mRNA. Finally, mRNA/GL67 and pDNA/GL67 complexes encoding luciferase were administered intranasally to the lungs of mice. The mRNA/GL67 complexes did not give rise to a measurable luciferase expression in the murine lungs. In contrast, a detectable bioluminescent signal was present in the lungs of mice that received the pDNA/GL67 complexes. We showed that mRNA/GL67 complexes have a lower stability in biological fluids. Consequently, this may be an explanation for their lower performance in vivo.
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Liposomas/química , ARN Mensajero/química , ARN Mensajero/genética , Animales , Línea Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Técnicas de Transferencia de Gen , Humanos , Ratones , Ratones Endogámicos BALB C , Transfección/métodosRESUMEN
Because of their large surface area, the lungs appear an attractive route for noninvasive vaccine delivery, harboring the potential to induce local mucosal immune responses in addition to systemic immunity. To evoke adaptive immunity, Ags require the addition of adjuvants that not only enhance the strength of the immune response but also determine the type of response elicited. In this study, we evaluate the adjuvant characteristics of polyelectrolyte microcapsules (PEMs) consisting of the biopolymers dextran-sulfate and poly-L-arginine. PEMs form an entirely new class of microcapsules that are generated by the sequential adsorption of oppositely charged polymers (polyelectrolytes) onto a sacrificial colloidal template, which is subsequently dissolved leaving a hollow microcapsule surrounded by a thin shell. Following intratracheal instillation, PEMs were not only efficiently taken up by APCs but also enhanced their activation status. Pulmonary adaptive immune responses were characterized by the induction of a strongly Th17-polarized response. When compared with a mixture of soluble Ag with empty microcapsules, Ag encapsulation significantly enhanced the strength of this local mucosal response. Given their unique property to selectively generate Th17-polarized immune responses, PEMs may become of significant interest in the development of effective vaccines against fungal and bacterial species.
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Implantes Absorbibles , Adyuvantes Inmunológicos/administración & dosificación , Cápsulas/administración & dosificación , Interleucina-17/inmunología , Pulmón/inmunología , Vacunación/métodos , Adyuvantes Inmunológicos/síntesis química , Animales , Células Presentadoras de Antígenos/inmunología , Antígenos/administración & dosificación , Antígenos/inmunología , Lavado Broncoalveolar , Cápsulas/síntesis química , Sulfato de Dextran/inmunología , Electrólitos/inmunología , Femenino , Citometría de Flujo , Inmunidad Mucosa/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Péptidos/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Stimuli-responsive electrospun nanofibers are gaining considerable attention as highly versatile tools which offer great potential in the biomedical field. In this critical review, an overview is given on recent advances made in the development and application of stimuli-responsive fibers. The specific features of these electrospun fibers are highlighted and discussed in view of the properties required for the diverse applications. Furthermore, several novel biomedical applications are discussed and the respective advantages and shortcomings inherent to stimuli-responsive electrospun fibers are addressed (136 references).
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Nanofibras/química , Técnicas Electroquímicas , Nanofibras/ultraestructura , Polímeros/químicaRESUMEN
This study reports on the encapsulation efficiency of proteins in dextran sulfate/poly-L-arginine-based microcapsules, fabricated via layer-by-layer assembly (LbL). For this purpose, radiolabeled proteins are entrapped in CaCO(3) microparticles, followed by LbL coating of the CaCO(3) cores and subsequent dissolving of the CaCO(3) using EDTA. To allow to improve protein encapsulation in LbL microcapsules, we studied all steps in the preparation of the microcapsules where loss of protein load might occur. The encapsulation efficiency of proteins in LbL microcapsules turns out to be strongly dependent on both the charge and molecular weight of the protein as well as on the number of polyelectrolyte bilayers the microcapsules consist of.
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Carbonato de Calcio/química , Cápsulas , Proteínas/química , Microscopía Confocal , Microscopía Electrónica de RastreoRESUMEN
PURPOSE: To evaluate if lyophilization can be used to obtain a dry formulation of polyelectrolyte microcapsules, which have emerged as a new class of microparticles for the encapsulation and delivery of biomacromolecules. METHODS: Microcapsules composed of dextran sulfate and poly-L-arginine were obtained by coating CaCO(3) microparticles by means of the layer-by-layer technique. Microcapsules were lyophilized using different stabilizers; intactness was checked by CLSM and SEM. Horseradish peroxidase was encapsulated as model enzyme and retained activity after freeze-drying was determined using a fluorescence assay. Ovalbumin was encapsulated as model antigen; immunogenicity after lyophilization was evaluated in vitro by a T-cell proliferation assay and in vivo by measuring the antibody titer in mice. RESULTS: The results clearly demonstrate the necessity of using polyols in the formulation to prevent rupture of the microcapsules and to preserve the activity of encapsulated enzymes. Lyophilized microcapsules appeared as a promising adjuvant for antigen delivery, as both in vitro as in vivo assays showed higher immune activation compared to free antigen. CONCLUSIONS: Lyophilization is a promising strategy towards improved stability of protein-loaded microcapsules.
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Cápsulas/química , Ovalbúmina/química , Poliaminas/química , Animales , Ensayo de Inmunoadsorción Enzimática , Femenino , Liofilización , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Modelos Biológicos , PolielectrolitosAsunto(s)
Autofagia/efectos de los fármacos , Nanopartículas/toxicidad , Antineoplásicos/uso terapéutico , Sinergismo Farmacológico , Humanos , Nanopartículas del Metal/química , Nanopartículas del Metal/toxicidad , Nanomedicina , Nanopartículas/química , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patologíaRESUMEN
Accurate sizing of nanoparticles in biological media is important for drug delivery and biomedical imaging applications since size directly influences the nanoparticle processing and nanotoxicity in vivo. Using fluorescence single particle tracking we have succeeded for the first time in following the aggregation of drug delivery nanoparticles in real time in undiluted whole blood. We demonstrate that, by using a suitable surface functionalization, nanoparticle aggregation in the blood circulation is prevented to a large extent.
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Análisis Químico de la Sangre/métodos , Interpretación de Imagen Asistida por Computador/métodos , Microscopía Fluorescente/métodos , Nanoestructuras/análisis , Nanoestructuras/ultraestructura , Tamaño de la Partícula , HumanosRESUMEN
We present a new convenient method for quantitative three-dimensionally resolved diffusion measurements based on the photobleaching (FRAP) or photoactivation (FRAPa) of a disk-shaped area by the scanning laser beam of a multiphoton microscope. Contrary to previously reported spot-photobleaching protocols, this method has the advantage of full scalability of the size of the photobleached area and thus the range of diffusion coefficients, which can be measured conveniently. The method is compatible with low as well as high numerical aperture objective lenses, allowing us to perform quantitative diffusion measurements in three-dimensional extended samples as well as in very small volumes, such as cell nuclei. Furthermore, by photobleaching/photoactivating a large area, diffusion along the optical axis can be measured separately, which is convenient when studying anisotropic diffusion. First, we show the rigorous mathematical derivation of the model, leading to a closed-form formula describing the fluorescence recovery/redistribution phase. Next, the ability of the multiphoton FRAP method to correctly measure absolute diffusion coefficients is tested thoroughly on many test solutions of FITC-dextrans covering a wide range of diffusion coefficients. The same is done for the FRAPa method on a series of photoactivatable green fluorescent protein solutions with different viscosities. Finally, we apply the method to photoactivatable green fluorescent protein diffusing freely in the nucleus of living NIH-3T3 mouse embryo fibroblasts.
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Fotoblanqueo , Fotones , Animales , Bovinos , Núcleo Celular/metabolismo , Difusión , Proteínas Fluorescentes Verdes/metabolismo , Espacio Intracelular/metabolismo , Rayos Láser , Ratones , Microscopía Confocal , Células 3T3 NIH , Reproducibilidad de los ResultadosRESUMEN
In this communication we report on microcapsules which eject nanoparticles into the environment. The speed of the nanoparticles ejected in water is approximately 800-fold faster than their Brownian diffusion. Such microcarriers may allow nanosized species to travel long distances, in short times, through highly viscous environments and may find applications in e.g. drug delivery and tissue engineering.
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Cápsulas/química , Dextranos/química , Metacrilatos/química , Nanopartículas/química , Compuestos Azo/química , Difusión , Geles/química , Cinética , Microscopía de Fuerza Atómica , Modelos Moleculares , Poliestirenos/química , Agua/químicaRESUMEN
Dextrans modified with alkyne and azide groups through hydrolysable carbonate esters form degradable microcapsules after Cu(I) catalysed 'click' reaction between azides and alkynes yielding triazole cross-links.
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Cápsulas/síntesis química , Cápsulas/metabolismo , Cápsulas/química , Carbono/química , Dextranos/síntesis química , Dextranos/química , Dextranos/metabolismo , Dextranos/ultraestructura , Microscopía Electrónica de Rastreo , Estructura Molecular , Nitrógeno/química , Espectrofotometría InfrarrojaRESUMEN
Neurodevelopmental and neurodegenerative disorders are characterized by subtle alterations in synaptic connections and perturbed neuronal network functionality. A hallmark of neuronal connectivity is the presence of dendritic spines, micron-sized protrusions of the dendritic shaft that compartmentalize single synapses to fine-tune synaptic strength. However, accurate quantification of spine density and morphology in mature neuronal networks is hampered by the lack of targeted labeling strategies. To resolve this, we have optimized a method to deliver cell-impermeable compounds into selected cells based on Spatially resolved NAnoparticle-enhanced Photoporation (SNAP). We show that SNAP enables efficient labeling of selected individual neurons and their spines in dense cultured networks without affecting short-term viability. We compare SNAP with widely used spine labeling techniques such as the application of lipophilic dyes and genetically encoded fluorescent markers. Using SNAP, we demonstrate a time-dependent increase in spine density in healthy cultures as well as a reduction in spine density after chemical mimicry of hypoxia. Since the sparse labeling procedure can be automated using an intelligent acquisition scheme, SNAP holds promise for high-content screening campaigns of neuronal connectivity in the context of neurodevelopmental and neurodegenerative disorders.