Nanoparticles In Living Cells Research Articles

A high-throughput lysosome trafficking assay guides ligand selection and elucidates differences in CD22-targeted nanodelivery

ABSTRACT Targeted nanoparticles offer potential to selectively deliver therapeutics to cells; however, their subcellular fate following endocytosis must be understood to properly design mechanisms of drug release. Here we describe a nanoparticle platform and associated cell-based assay to observe lysosome trafficking of targeted nanoparticles in live cells. The nanoparticle platform utilizes two fluorescent dyes loaded onto PEG-poly(glutamic acid) and PEG-poly(Lysine) block co-polymers that also comprise azide reactive handles on PEG termini to attach antibody-based targeting ligands. Fluorophores were selected to be pH-sensitive (pHrodo Red) or pH-insensitive (Alexafluor 488) to report when nanoparticles enter low pH lysosomes. Dye-labelled block co-polymers were further assembled into polyion complex micelle nanoparticles and crosslinked through amide bond formation to form stable nano-scaffolds for ligand attachment. Cell binding and lysosome trafficking was determined in live cells by fluorescence imaging in 96-well plates and quantification of red- and green-fluorescence signals over time. The platform and assay was validated for selection of optimal antibody-derived targeting ligands directed towards CD22 for nanoparticle delivery. Kinetic analysis of uptake and lysosome trafficking indicated differences between ligand types and the ligand with the highest lysosome trafficking efficiency translated into effective DNA delivery with nanoparticles bearing the optimal ligand.

Long-Term Intracellular Tracking of Label-Free Nanoparticles in Live Cells and Tissues with Confocal Microscopy.

The label-free imaging of inorganic nanoparticles (NPs) using confocal laser scanning microscopy (CLSM) provides a powerful and versatile tool for studying interactions between NPs and biological systems. Without the need for exogenous labels or markers, it simply benefits from the differential scattering of visible photons between biomaterials and inorganic NPs. Validation experiments conducted on fixed and living cells in real-time, as well as mouse tissue sections following parenteral administration of NPs. Additionally, by incorporating reporter fluorophores and utilizing both reflectance and fluorescence imaging modalities, the method enables high-resolution multiplex imaging of cellular structures and NPs. Different sizes and concentrations of Au NPs are tested as for Ag, Fe3O4, and CeO2 NPs, all with biological interest. Overall, the comprehensive study of NP imaging by confocal microscopy in reflectance mode provides valuable insights and tools for researchers interested in monitoring the nano-bio interactions.

Resonance Light-Scattering Correlation Spectroscopy and Its Application in Analytical Chemistry for Life Science.

Resonance light-scattering correlation spectroscopy (RLSCS) is a new single-particle detection method with its working principle being like fluorescence correlation spectroscopy (FCS). RLSCS is obtained by autocorrelation function analysis on the measured fluctuation of the resonance light scattering (RLS) intensity occurring within a subfemtoliter volume when a single nanoparticle (such as gold nanoparticles (NPs) or silver (SNPs)) freely diffuses through the volume. The RLSCS technique can detect such parameters as concentration, diffusion coefficient (translation and rotation), etc. Compared with the FCS technique, the correlated fluorescence intensity signal in RLSCS is replaced with the RLS signal of the nanoparticles, overcoming some limits of the fluorescent probes such as photobleaching under high-intensity or long-term illumination. In this Account, we showcase RLSCS methods, theoretical models at different optical configurations, and some key applications. First, the RLSCS optical detection system was constructed based on the confocal optics, its theoretical model was proposed, and the diffusion behaviors of the nanoparticles in the solution were studied including the rotational and translational diffusion. And, methods were developed to measure the concentration, size, aspect ratio, and size distribution of the NPs. Second, based on the RLSCS methods, some detection strategies were developed for homogeneous DNA detection, immunoassay, apoptosis assay, self-thermophoresis of the nanomotor, and quantitative assay in single living cells. Meanwhile, a new fluorescence/scattering cross-correlation spectroscopy (FSCCS) method was proposed for monitoring the molecule-particle interaction. This method enriched the conventional fluorescence/fluorescence cross-correlation spectroscopy (FCCS) method. Third, using the EMCCD with high sensitivity and rapid response as an optical detector, two temporospatially resolved scattering correlation spectroscopy methods and their theoretical models were developed: total internal reflection (TIR) configuration-based spatially resolved scattering correlation spectroscopy (SRSCS) and dark-field illumination-based scattering correlation spectroscopy (DFSCS). These methods extended single-spot confocal RLSCS to imaging RLSCS, which makes RLSCS have the ability for multiple channel detection with temporospatial resolution. The method was successfully used for investigating the dynamic behaviors of gold NPs in live cells and obtained its temporospatial concentration distribution and diffusion behaviors. The final section of this Account outlines future directions in the development of RLSCS.

Real-time six-dimensional spatiotemporal tracking of single anisotropic nanoparticles in live cells by integrated multifunctional light-sheet nanoscopy

An integrated multifunctional light-sheet nanoscopy (iMLSN) combined with differential interference contrast, total internal reflection, epifluorescence, a super-resolution radial fluctuation-stream module, and a wavelength-dependent light sheet was developed to simultaneously realize the six-dimensional (6D) vector-valued (three coordinates + rotational dynamics (azimuth and elevation angles) + transport speed) tracking of anisotropic nanoparticles in single living cells. The wavelength-dependent asymmetric scattering of light by gold nanorods was used to trigger signals depending on the polarizer angle, and real-time photo-switching was achieved by turning the polarizer, obtaining a series of super-resolution images, and tracking using different polarization directions and two channels. This technique was employed to directly observe native gold nanorods (AuNRs; 5 nm diameter × 15 nm length) and surface-functionalized AuNRs during their endocytosis and transport at the upper and attaching side membrane regions of single living cells, revealing that the AuNRs bound to the membrane receptors. The nanorods were subsequently internalized and transported away from the original entry spots. Detailed dynamic information regarding the rotation properties and endocytosis speed during the transmembrane process was also acquired for each region. The developed technique can be considered useful for the real-time monitoring of intracellular transport at various regions in single living cells, as well as for 6D vector-valued non-fluorescence super-resolution imaging and tracking.Graphical

Application of Rapid Fluorescence Lifetime Imaging Microscopy (RapidFLIM) to Examine Dynamics of Nanoparticle Uptake in Live Cells.

A key challenge in nanomedicine stems from the continued need for a systematic understanding of the delivery of nanoparticles in live cells. Complexities in delivery are often influenced by the biophysical characteristics of nanoparticles, where even subtle changes to nanoparticle designs can alter cellular uptake, transport and activity. Close examination of these processes, especially with imaging, offers important insights that can aid in future nanoparticle design or translation. Rapid fluorescence lifetime imaging microscopy (RapidFLIM) is a potentially valuable technology for examining intracellular mechanisms of nanoparticle delivery by directly correlating visual data with changes in the biological environment. To date, applications for this technology in nanoparticle research have not been explored. A PicoQuant RapidFLIM system was used together with commercial silica nanoparticles to follow particle uptake in glioblastoma cells. Importantly, RapidFLIM imaging showed significantly improved image acquisition speeds over traditional FLIM, which enabled the tracking of nanoparticle uptake into subcellular compartments. We determined mean lifetime changes and used this to delineate significant changes in nanoparticle lifetimes (>0.39 ns), which showed clustering of these tracks proximal to both extracellular and nuclear membrane boundaries. These findings demonstrate the ability of RapidFLIM to track, localize and quantify changes in single nanoparticle fluorescence lifetimes and highlight RapidFLIM as a valuable tool for multiparameter visualization and analysis of nanoparticle molecular dynamics in live cells.

Intracellular Transport Dynamics of Upconverting Nanoparticles in Living Cells

Intracellular Transport Dynamics of Upconverting Nanoparticles in Living Cells

Silk nanoparticles: proof of lysosomotropic anticancer drug delivery at single-cell resolution

Silk nanoparticles are expected to improve chemotherapeutic drug targeting to solid tumours by exploiting tumour pathophysiology, modifying the cellular pharmacokinetics of the payload and ultimately resulting in trafficking to lysosomes and triggering drug release. However, experimental proof for lysosomotropic drug delivery by silk nanoparticles in live cells is lacking and the importance of lysosomal pH and enzymes controlling drug release is currently unknown. Here, we demonstrate, in live single human breast cancer cells, the role of the lysosomal environment in determining silk nanoparticle-mediated drug release. MCF-7 human breast cancer cells endocytosed and trafficked drug-loaded native and PEGylated silk nanoparticles (∼100 nm in diameter) to lysosomes, with subsequent drug release from the respective carriers and nuclear translocation within 5 h of dosing. A combination of low pH and enzymatic degradation facilitated drug release from the silk nanoparticles; perturbation of the acidic lysosomal pH and inhibition of serine, cysteine and threonine proteases resulted in a 42% ± 2.2% and 33% ± 3% reduction in nuclear-associated drug accumulation for native and PEGylated silk nanoparticles, respectively. Overall, this study demonstrates the importance of lysosomal activity for anticancer drug release from silk nanoparticles, thereby providing direct evidence for lysosomotropic drug delivery in live cells.

In Situ Monitoring of the Intracellular Stability of Nanoparticles by Using Fluorescence Lifetime Imaging.

FLIMaging nanoparticle degradation: semiconductor and metal nanoparticle degradation has been observed in live cells over 3 d via the change of the characteristic luminescence lifetime using fluorescence lifetime imaging microscopy (FLIM). Thus, FLIM is a simple yet robust tool to examine the intracellular stability of photoluminescent nanoparticles in live cells, tissues, and organisms.

PEGylation density-modulated anticancer drug release on gold nanoparticles in live cells

Glutathione (GSH)-triggered drug releases of the anticancer drug mitoxantrone from a polyethylene glycol (PEG) (molecular weight: 5000) density-modulated drug delivery system up to 0.57nm−2 of gold nanoparticles (AuNPs) were examined using bimodal spectroscopic imaging techniques of live-cell fluorescence imaging and surface-enhanced resonance Raman scattering (SERRS). Prolonged delivery amounts and sustained drug releases after introducing a high PEG density on AuNPs was supported by a cell viability test in HeLa cells. A subcutaneous in vivo test also indicated that the SERRS spectra could be observed only for the PEGylated AuNPs after external GSH treatment in consistent with the fluorescence measurements.

Mapping the Diffusive Route of Nanoparticles in Live Cells Reveals Shape to Control Nuclear Accessibility

Viruses have developed mechanisms of cellular entry over millions of years and shape has evolved as a critical feature to overcoming cellular barriers which restrict access to a target destination. The efficiency with which pathogens overcome cellular barriers raises the question whether drug carriers can be designed to take an ‘optimal’ route and enhance the efficacy of chemotherapeutics. Mapping molecular mobility across cellular compartments, however, has been difficult since live cell imaging has insufficient spatiotemporal resolution to follow a population of molecules in real time. Here, we used pair correlation analysis, a form of correlation spectroscopy, to map the molecular mobility of polymeric nanoparticles with millisecond and sub-micron resolution across subcellular compartments. We measured differences in nuclear accessibility for polymeric nanoparticles of different shape that correlates with doxorubicin toxicity. The latter was caused by efficient drug release in the nucleus by pathogen shaped carriers.

Tempo-Spatially Resolved Scattering Correlation Spectroscopy under Dark-Field Illumination and Its Application to Investigate Dynamic Behaviors of Gold Nanoparticles in Live Cells

In this study, a new tempo-spatially resolved fluctuation spectroscopy under dark-field illumination is described, named dark-field illumination-based scattering correlation spectroscopy (DFSCS). DFSCS is a single-particle method, whose principle is similar to that of fluorescence correlation spectroscopy (FCS). DFSCS correlates the fluctuations of the scattered light from single nanoparticle under dark-field illumination. We developed a theoretical model for translational diffusion of nanoparticles in DFSCS system. The results of computer simulations documented that this model was able to well describe the diffusion behaviors of nanoparticles in uniformly illuminated field. The experimental setup of DFSCS was achieved by introducing a dark-field condenser to the frequently used bright-field microscope and an electron multiplying charge-coupled device (EMCCD) as the array detector. In the optimal condition, a stack of 500 000 frames were collected simultaneously on 64 detection channels for a single measurement with acquisition rate of 0.5 ms per frame. We systematically investigated the effect of certain factors such as particle concentration, viscosity of the solution, and heterogeneity of gold nanoparticles (GNPs) samples on DFSCS measurements. The experiment data confirmed theoretical model proposed. Furthermore, this new method was successfully used for investigating dynamic behaviors of GNPs in live cells. Our preliminary results demonstrate that DFSCS is a practical and affordable tool for ordinary laboratories to investigate the dynamic information of nanoparticles in vitro as well as in vivo.

Reducible polyamidoamine-magnetic iron oxide self-assembled nanoparticles for doxorubicin delivery

We report a reducible copolymer self-assembled with superparamagnetic iron oxide nanoparticles (SPIONs) to deliver doxorubicin (DOX) for cancer therapy. The copolymer of reducible polyamidoamine (rPAA) with poly(ethylene glycol)(PEG)/dodecyl amine graft was synthesized by Michael addition. rPAA@SPIONs were formed by the alkyl grafts of reducible copolymers intercalated with the oleic acid layer capped on the surface of magnetite nanocrystals. The intercalating area formed a reservoir for hydrophobic anti-cancer drug (DOX), whilst the PEG moiety in the copolymers helped the nanoparticle well-dispersible in aqueous solution. We employed two-photon excited fluorescence (TPEF) and coherent anti-Stokes Raman (CARS) to investigate drug delivery in intra-cellular structures of live cells, and used Vivaview® technique to show real-time inhibition efficacy of nanoparticles in live cells. rPAA@SPIONs present efficiently drug loading with reducible responsibility in vitro tests. Finally, rPAA@SPIONs were tested in mice with xenograft MDA-MB-231 breast tumor though i.v. injection and inhibited tumor growth efficiently. MRI was used to monitor nanoparticles aggregation in tumor site. Histology and Prussian blue on kidney, liver, and heart in mice indicated that DOX/rPAA@SPIONs showed no significant toxicity for mice organs after 24 days treatment.

A Programmable Sensor to Probe the Internalization of Proteins and Nanoparticles in Live Cells

Ein molekularer Sensor wurde entwickelt, der die Internalisierung von Proteinen und Nanopartikeln in lebende Zellen anzeigt. Diese einfache, hochdurchsatzfähige Technik ist mit Zellphänotypisierungsexperimenten kompatibel und ist unabhängig vom zellulären Schicksal des Materials. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

A Programmable Sensor to Probe the Internalization of Proteins and Nanoparticles in Live Cells

A Programmable Sensor to Probe the Internalization of Proteins and Nanoparticles in Live Cells

Temporal Techniques: Dynamic Tracking of Nanomaterials in Live Cells

Temporal analytical techniques to track nanoparticles in live cell would provide rich information to well understand the biologic properties of nanoparticles in molecular level. Significant advances in fluorescence microscopy techniques with high temporal and spatial resolution allow single nanoparticles to label biomolecules, ions, and microstructures in live cells, which will address many fundamental questions in cell biology. This review highlights the real time tracking techniques for monitoring the movement of nanomaterials such as carbon nanotubes (CNTs), quantum dots (QDs), metal clusters, upconver-sional nanomaterials, and polystyrene (PS) nanoparticles etc. in live cells. The biological properties of nanoparticles in live cells are also briefly summarized according to fluorescence microscopy studies.

Charge-driven selective localization of fluorescent nanoparticles in live cells

Covalent grafting of amino groups onto the carboxylic acid functionalities, naturally covering the surface of fluorescent nanoparticles produced from silicon carbide (SiC NPs), allowed tuning of their surface charge from negative to highly positive. Incubating 3T3-L1 fibroblast cells with differently charged SiC NPs demonstrates the crucial role of the charge in cell fluorescent targeting. Negatively charged SiC NPs concentrate inside the cell nuclei. Close to neutrally charged SiC NPs are present in both cytoplasm and nuclei while positively charged SiC NPs are present only in the cytoplasm and are not able to move inside the nuclei. This effect opens the door for the use of SiC NPs for easy and fast visualization of long-lasting biological processes taking place in the cell cytosol or nucleus as well as providing a new long-term cell imaging tool. Moreover, here we have shown that the interaction between charged NPs and nuclear pore complex plays an essential role in their penetration into the nuclei.

Photothermal release of small molecules from gold nanoparticles in live cells

The ability of gold (Au) nanoparticles (NPs) to generate heat efficiently by absorbing visible and near-infrared (NIR) light holds great promise as a means to trigger chemical and biochemical events near the NPs. Previous demonstrations show that pulsed laser irradiation can selectively elicit the release of a fluorescent dye covalently anchored to the NP surface through a heat-labile linker without measurably changing the temperature of the surroundings. This article reports that the authors demonstrate the biological efficacy of this approach to photodelivery by showing that the decorated Au NPs are rapidly internalized by cells, are stable under physiological conditions, are nontoxic, and exhibit nonlethal photorelease following exposure to pulsed laser radiation. These observations, further supported by the versatility of our delivery motif, reaffirm the potential for further development of nonlethal photothermal therapeutics and their future relevance to such fields as gene therapy and stem-cell differentiation. From the Clinical EditorThe authors further refine previous observations suggesting that Au NP-s may be useful in targeted drug or gene delivery systems. Due to a strong photothermal release effect and their generally low toxicity, Au NP-s may become an important subject of choice in targeted delivery systems.

Cellular uptake pathway and drug release characteristics of drug‐encapsulated glycol chitosan nanoparticles in live cells

Herein, we evaluated the cellular uptake pathways of hydrophobically modified glycol chitosan (HGC) nanoparticles as nano-sized drug carriers using cellular imaging technology. The endocytic pathway of nanocarriers for intracellular drug delivery is of great interest for the design of high efficacy delivery carriers for therapeutic agents. To evaluate the cellular uptake pathways of HGC nanoparticles, HGC was chemically labeled with near infrared (NIR) fluorescence dye, Cy5.5, to visualize the nanoparticle under confocal laser scanning microscopy. The internalization pathways of HGC nanoparticles were evaluated after treatment of specific endocytosis inhibitors. Importantly, HCG nanoparticles showed different cellular uptake efficiency and intracellular fate in cytoplasm according to the internalization pathways. Furthermore, drug distribution also evaluated according to the endocytic pathways after treatment of drug encapsulated HGC nanoparticles. As a model drug, fluorescent photosensitizer, Ce6, was encapsulated into HGC (Ce6-HGC) nanoparticles and the distribution of Ce6 in cytoplasm was evaluated using confocal laser scanning microscopy. The intracellular drug distribution showed different manner through specific endocytic pathways. The cellular imaging technology is highly useful for evaluation of endocytosis pathways and intracellular fate of drug delivery carrier which are closely related to drug distribution and therapeutic efficacy.

In Situ Monitoring of Bindings between Dasatinib and Its Target Protein Kinases Using Magnetic Nanoparticles in Live Cells

We report a novel technology, called InCell IT, for in situ monitoring of bindings between a small molecule kinase inhibitor and its target protein kinases in live cells using a kinase inhibitor, dasatinib, as a model compound. Streptavidin-attached MNPs were coated by biotinylated dasatinib, and then these dasatinib-MNPs were transferred into cells. In the cells, the MNPs were aligned to the same direction of the magnetic field and EGFP-tagged target protein kinases were bound to dasatinib-MNPs. Using this technology, we demonstrated the bindings between dasatinib and its target protein kinases including SRC, ABL1, and CSK in live cells. The specificity of these bindings was also confirmed by cold competition using unbiotinylated dasatinib in the same cells.