A Quantitative Study of the Intracellular Dynamics of Fluorescently Labelled Glyco‐Gold Nanoparticles via Fluorescence Correlation Spectroscopy

The dynamic behaviour of gold nanoparticles functionalised with glucose (Glc‐Au NPs) has been studied here by means of fluorescence correlation spectroscopy (FCS). Meaningful data on the state of aggregation and dynamics of Glc‐Au NPs fluorescently‐labelled with HiLyte Fluor647 (Glc‐Au‐Hi NPs) in the intracellular environment were obtained. Moreover, the work presented here shows that FCS can be used to visualise the presence of single NPs or NP aggregates following uptake and to estimate, locally, NP concentrations within the cell. FCS measurements become possible after applying a “prebleaching” methodology, when the immobile NP fraction has been effectively removed and thus significant FCS data has been recorded. In this study, Glc‐Au‐Hi NPs have been incubated with HepG2 cells and their diffusion time in the intracellular environment has been measured and compared with their diffusion value in water and cell media.     

Surface Functionality of Nanoparticles Determines Cellular Uptake Mechanisms in Mammalian Cells

Nanoparticles (NPs) are versatile scaffolds for numerous biomedical applications including drug delivery and bioimaging. The surface functionality of NPs essentially dictates intracellular NP uptake and controls their therapeutic action. Using several pharmacological inhibitors, it is demonstrated that the cellular uptake mechanisms of cationic gold NPs in both cancer (HeLa) and normal cells (MCF10A) strongly depend on the NP surface monolayer, and mostly involve caveolae and dynamin‐dependent pathways as well as specific cell surface receptors (scavenger receptors). Moreover, these NPs show different uptake mechanisms in cancer and normal cells, providing an opportunity to develop NPs with improved selectivity for delivery applications.     

Quantitative Evaluation of Cellular Uptake and Trafficking of Plain and Polyethylene Glycol‐Coated Gold Nanoparticles

This study addresses the cellular uptake and intracellular trafficking of 15‐nm gold nanoparticles (NPs), either plain (i.e., stabilized with citrate) or coated with polyethylene glycol (PEG), exposed to human alveolar epithelial cells (A549) at the air–liquid interface for 1, 4, and 24 h. Quantitative analysis by stereology on transmission electron microscopy images reveals a significant, nonrandom intracellular distribution for both NP types. No particles are observed in the nucleus, mitochondria, endoplasmic reticulum, or golgi. The cytosol is not a preferred cellular compartment for both NP types, although significantly more PEG‐coated than citrate‐stabilized NPs are present there. The preferred particle localizations are vesicles of different sizes (<150, 150–1000, >1000 nm). This is observed for both NP types and indicates a predominant uptake by endocytosis. Subsequent inhibition of caveolin‐ and clathrin‐mediated endocytosis by methyl‐β‐cyclodextrin (MβCD) results in a significant reduction of intracellular NPs. The inhibition, however, is more pronounced for PEG‐coated than citrate‐stabilized NPs. The latter are mostly found in larger vesicles; therefore, they are potentially taken up by macropinocytosis, which is not inhibited by MβCD. With prolonged exposure times, both NPs are preferentially localized in larger‐sized intracellular vesicles such as lysosomes, thus indicating intracellular particle trafficking. This quantitative evaluation reveals that NP surface coatings modulate endocytotic uptake pathways and cellular NP trafficking. Other nonendocytotic entry mechanisms are found to be involved as well, as indicated by localization of a minority of PEG‐coated NPs in the cytosol.     

Protein Corona Mediated Uptake and Cytotoxicity of Silver Nanoparticles in Mouse Embryonic Fibroblast

Medical applications of nanoparticles (NPs) require understanding of their interactions with living systems in order to control their physiological response, such as cellular uptake and cytotoxicity. When NPs are exposed to biological fluids, the adsorption of extracellular proteins on the surface of NPs, creating the so‐called protein corona, can critically affect their interactions with cells. Here, the effect of surface coating of silver nanoparticles (AgNPs) on the adsorption of serum proteins (SPs) and its consequence on cellular uptake and cytotoxicity in mouse embryonic fibroblasts are shown. In particular, citrate‐capped AgNPs are internalized by cells and show a time‐ and dose‐dependent toxicity, while the passivation of the NP surface with an oligo(ethylene glycol) (OEG)‐alkanethiol drastically reduces their uptake and cytotoxicity. The exposure to growth media containing SPs reveals that citrate‐capped AgNPs are promptly coated and stabilized by proteins, while the AgNPs resulting from capping with the OEG‐alkanethiol are more resistant to adsorption of proteins onto their surface. Using NIH‐3T3 cultured in serum‐free, the key role of the adsorption of SPs onto surface of NPs is shown as only AgNPs with a preformed protein corona can be internalized by the cells and, consequently, carry out their inherent cytotoxic activity.     

Near Infrared Light‐Triggered Drug Generation and Release from Gold Nanoparticle Carriers for Photodynamic Therapy

A photoprecursor Pc 227 is covalently bound onto gold nanoparticles (Au NPs) to produce the known photodynamic therapy (PDT) drug Pc 4 upon 660 nm photoirradiation. The photochemical formation of the photoproduct Pc 4 is identified by spectroscopy, chromatography, and mass spectrometry and its PDT efficacy is equal to Pc 4 when administered non‐covalently by Au NPs, with the added benefit of improved covalent delivery and targeted NIR‐triggered release from the covalent Pc 227‐Au NP conjugate, while during transport the attached Pc 227 is quenched by the Au NP and PDT inactivated.     

Elucidating the mechanism of cellular uptake and removal of protein-coated gold nanoparticles of different sizes and shapes

We investigated the mechanism by which transferrin-coated gold nanoparticles (Au NP) of different sizes and shapes entered mammalian cells. We determined that transferrin-coated Au NP entered the cells via clathrin-mediated endocytosis pathway. The NPs exocytosed out of the cells in a linear relationship to size. This was different than the relationship between uptake and size. Furthermore, we developed a mathematical equation to predict the relationship of size versus exocytosis for different cell lines. These studies will provide guidelines for developing NPs for imaging and drug delivery applications, which will require "controlling" NP accumulation rate. These studies will also have implications in determining nanotoxicity.     

Mammalian Cells Exhibit a Range of Sensitivities to Silver Nanoparticles that are Partially Explicable by Variations in Antioxidant Defense and Metallothionein Expression

While it is well known that there are interspecies differences in Ag sensitivity, differences in the cytotoxic responses of mammalian cells to silver nanoparticles (Ag NPs) are also observed. In order to explore these response outcomes, six cell lines, including epithelial cells (Caco‐2, NHBE, RLE‐6TN, and BEAS‐2B) and macrophages (RAW 264.7 and THP‐1) of human and rodent origin, are exposed to 20 nm citrate‐ and PVP‐coated Ag NPs with Au cores, as well as 20 nm citrate‐coated particles without cores. An MTS assay shows that while Caco‐2 and NHBE cells are resistant to particles over a 0.1–50 μg mL^?1 dose range, RAW 264.7, THP‐1, RLE‐6TN, and BEAS‐2B cells are more susceptible. While there are small differences in dissolution rates, there are no major differences in the cytotoxic potential of the different particles. However, differences in anti‐oxidant defense and metallothionein expression among different cell types are observed, which can partially explain differential Ag NP sensitivity. So, it is important to consider these differences in understanding the potential heterogeneous effects of nano Ag on mammalian biological systems.     

DNA Sensors and Aptasensors Based on the Hemin/G‐quadruplex‐Controlled Aggregation of Au NPs in the Presence of L‐Cysteine

L‐cysteine induces the aggregation of Au nanoparticles (NPs), resulting in a color transition from red to blue due to interparticle plasmonic coupling in the aggregated structure. The hemin/G‐quadruplex horseradish peroxidase‐mimicking DNAzyme catalyzes the aerobic oxidation of L‐cysteine to cystine, a process that inhibits the aggregation of the NPs. The degree of inhibition of the aggregation process is controlled by the concentration of the DNAzyme in the system. These functions are implemented to develop sensing platforms for the detection of a target DNA, for the analysis of aptamer‐substrate complexes, and for the analysis of L‐cysteine in human urine samples. A hairpin DNA structure that includes a recognition site for the DNA analyte and a caged G‐quadruplex sequence, is opened in the presence of the target DNA. The resulting self‐assembled hemin/G‐quadruplex acts as catalyst that controls the aggregation of the Au NPs. Also, the thrombin‐binding aptamer folds into a G‐quadruplex nanostructure upon binding to thrombin. The association of hemin to the resulting G‐quadruplex aptamer‐thrombin complex leads to a catalytic label that controls the L‐cysteine‐mediated aggregation of the Au NPs. The hemin/G‐qaudruplex‐controlled aggregation of Au NPs process is further implemented for visual and spectroscopic detection of L‐cysteine concentration in urine samples.     

Directed self-assembly of nanogold using a chemically modified nanopatterned surface

Electron-beam lithography (EBL) was used to define an aminosilane nanopatterned surface in order to electrostatically self-assemble gold nanoparticles (Au NPs). The chemically modified nanopatterned surfaces were immersed into a Au NP solution to allow the Au NPs to self-assemble. Equilibrium self-assembly was achieved in only 20 min. The number of Au NPs that self-assembled on an aminosilane dot was controlled by manipulating the diameters of both the Au NPs and the dots. Adding salt to the Au NP solution enabled the Au NPs to self-assemble in greater numbers on the same sized dot. However, the preparation of the Au NP solution containing salt was sensitive to spikes in the salt concentration. These spikes led to aggregation of the Au NPs and non-specific deposition of Au NPs on the substrate. The Au NP patterned surfaces were immersed in a sodium hydroxide solution in order to lift-off the patterned Au NPs, but no lift-off was observed without adequate physical agitation. The van der Waals forces are too strong to allow for lift-off despite the absence of electrostatic forces.     

Mechanotargeting: Mechanics‐Dependent Cellular Uptake of Nanoparticles

Targeted delivery of nanoparticle (NP)‐based diagnostic and therapeutic agents to malignant cells and tissues has exclusively relied on chemotargeting, wherein NPs are surface‐coated with ligands that specifically bind to overexpressed receptors on malignant cells. Here, it is demonstrated that cellular uptake of NPs can also be biased to malignant cells based on the differential mechanical states of cells, enabling mechanotargeting. Owing to mechanotransduction, cell lines (HeLa and HCT‐8) cultured on hydrogels of various stiffness are directed into different stress states, measured by cellular force microscopies. In vitro NP delivery reveals that increases in cell stress suppress cellular uptake, counteracting the enhanced uptake that occurs with increases in exposed surface area of spread cells. Upon prolonged culture on stiff hydrogels, cohesive HCT‐8 cell colonies undergo metastatic phenotypic change and disperse into individual malignant cells. The metastatic cells are of extremely low stress state and adopt an unspread, 3D morphology, resulting in several‐fold higher uptake than the nonmetastatic counterparts. This study opens a new paradigm of harnessing mechanics for the design of future strategies in nanomedicine.     

Surfactants-aided syntheses of different sizes and triangular shape of gold nanoparticles using trisodium citrate in environmentally friendly and photoinduced methods

We prepared gold nanoparticles (Au NPs) by only using trisodium citrate as the stabilizer. The detailed reaction mechanisms of S(N)1 and E1 reactions are examined and evidenced in this study by FTIR data. Citric acid is a kind of tertiary substrate. In aqueous solution, the substitution nucleophile path 1 (S(N)1) reaction and Elimination path 1 (E1) reaction usually occur simultaneously. Chloride ions, the substitution nucleophile, play a very important role to launch the mechanisms of S(N)1 and E1 reactions. Controlling the concentration of the chloride ions with the addition of HCl(aq) according to Le Chatelier theory, the average particle size of Au NPs (5.5 nm) was achieved to overcome the minimum limited size (approximately 10 nm). Two stages of the photoinduced method, aggregation into triangular conglomerates and growth into triangular particles, were determined form TEM observations. This preparation of Au NPs has potential in tuning the size, shape, and mechanism of Au NP formation by using only environmentally friendly trisodium citrate and the photoinduced method.     

Gold Nanoparticle–Protein Agglomerates as Versatile Nanocarriers for Drug Delivery

The fabrication of a versatile nanocarrier based on agglomerated structures of gold nanoparticle (Au NP)–lysozyme (Lyz) in aqueous medium is reported. The carriers exhibit efficient loading capacities for both hydrophilic (doxorubicin) and hydrophobic (pyrene) molecules. The nanocarriers are finally coated with an albumin layer to render them stable and also facilitate their uptake by cancer cells. The interaction between agglomerated structures and the payloads is non‐covalent. Cell viability assay in vitro showed that the nanocarriers by themselves are non‐cytotoxic, whereas the doxorubicin‐loaded ones are cytotoxic, with efficiencies higher than that of the free drug. Transmission electron microscopy and fluorescence microscopy along with flow cytometry analysis confirm the uptake of the drug‐loaded nanocarriers by a human cervical cancer HeLa cell line. Field‐emission scanning electron microscopy reveals the formation of apoptotic bodies leading to cell death, confirming the release of the payloads from the nanocarriers into the cell. Overall, the findings suggest the fabrication of novel Au NP–protein agglomerate‐based nanocarriers with efficient drug‐loading and ‐releasing capabilities, enabling them to act as multimodal drug‐delivery vehicles.     

Intracellular delivery of etoposide loaded biodegradable nanoparticles: cytotoxicity and cellular uptake studies

The preferred delivery systems for anticancer drugs would be the one which would have selective and effective destruction of cancer cells. In the present study etoposide (ETO) loaded nanoparticles (NP) were prepared using PLGA (ETO-PLGA NP), PLGA-MPEG block copolymer (ETO-PLGA-MPEG NP) and PLGA-Pluronic copolymer (ETO-PLGA-PLU NP) and they were evaluated for cytotoxicity and cellular uptake studies using two cancer cell lines, L1210 and DU145. The IC50 values for L1210 cells were 18.0, 6.2, 4.8 and 5.4 microM and for DU145 cells the IC50 values were 98.4, 75.1, 60.1 and 71.3 microM for ETO, ETO-PLGA NP, ETO-PLGA-MPEG NP and ETO-PLGA-PLU NP respectively. The increased cytotoxicities were attributed to increased uptake of the NPs by the cells. Moreover the ETO loaded PLGA-MPEG NP and PLGA-Pluronic NP showed a sustained cytotoxic effect till 5 days on both the cell lines. Results of the long term cytotoxicity study concluded that the drug loaded PLGA nanoparticulate formulations were efficient in decreasing the viability of the L1210 cells over a period of three days, whereas the pure drug exerted its maximum efficiency on the day one itself. Z-stack confocal images of NPs showed fluorescence activity in each section of DU 145 and L1210 cells indicating that the nanoparticles were internalized by the cells. The study concluded that ETO loaded PLGA NPs had higher cytotoxicity compared with that of the free drug and ETO-PLGA-MPEG NP and ETO-PLGA-PLU NP had higher cell uptake efficiency compared with that of ETO-PLGA NP. The developed PLGA based NPs shows promise to be used for cancer therapy.     

A 3D In Vitro Cancer Model as a Platform for Nanoparticle Uptake and Imaging Investigations

In order to maximize the potential of nanoparticles (NPs) in cancer imaging and therapy, their mechanisms of interaction with host tissue need to be fully understood. NP uptake is known to be dramatically influenced by the tumor microenvironment, and an imaging platform that could replicate in vivo cellular conditions would make big strides in NP uptake studies. Here, a novel NP uptake platform consisting of a tissue‐engineered 3D in vitro cancer model (tumoroid), which mimics the microarchitecture of a solid cancer mass and stroma, is presented. As the tumoroid exhibits fundamental characteristics of solid cancer tissue and its cellular and biochemical parameters are controllable, it provides a real alternative to animal models. Furthermore, an X‐ray fluorescence imaging system is developed to demonstrate 3D imaging of GNPs and to determine uptake efficiency within the tumoroid. This platform has implications for optimizing the targeted delivery of NPs to cells to benefit cancer diagnostics and therapy.     

Comparison of the abilities of ambient and manufactured nanoparticles to induce cellular toxicity according to an oxidative stress paradigm

Nanomaterial properties differ from those bulk materials of the same composition, allowing them to execute novel activities. A possible downside of these capabilities is harmful interactions with biological systems, with the potential to generate toxicity. An approach to assess the safety of nanomaterials is urgently required. We compared the cellular effects of ambient ultrafine particles with manufactured titanium dioxide (TiO2), carbon black, fullerol, and polystyrene (PS) nanoparticles (NPs). The study was conducted in a phagocytic cell line (RAW 264.7) that is representative of a lung target for NPs. Physicochemical characterization of the NPs showed a dramatic change in their state of aggregation, dispersibility, and charge during transfer from a buffered aqueous solution to cell culture medium. Particles differed with respect to cellular uptake, subcellular localization, and ability to catalyze the production of reactive oxygen species (ROS) under biotic and abiotic conditions. Spontaneous ROS production was compared by using an ROS quencher (furfuryl alcohol) as well as an NADPH peroxidase bioelectrode platform. Among the particles tested, ambient ultrafine particles (UFPs) and cationic PS nanospheres were capable of inducing cellular ROS production, GSH depletion, and toxic oxidative stress. This toxicity involves mitochondrial injury through increased calcium uptake and structural organellar damage. Although active under abiotic conditions, TiO2 and fullerol did not induce toxic oxidative stress. While increased TNF-alpha production could be seen to accompany UFP-induced oxidant injury, cationic PS nanospheres induced mitochondrial damage and cell death without inflammation. In summary, we demonstrate that ROS generation and oxidative stress are a valid test paradigm to compare NP toxicity. Although not all materials have electronic configurations or surface properties to allow spontaneous ROS generation, particle interactions with cellular components are capable of generating oxidative stress.     

Characterizing the Lateral Friction of Nanoparticles on On‐Chip Integrated Black Lipid Membranes

The use of nanoparticles (NPs) in biomedical applications creates a need for appropriate model systems to systematically investigate NP–membrane interactions under well‐defined conditions. Black lipid membranes (BLMs) are free‐floating membranes with defined composition that are ideally suited for characterizing NP–membrane interactions free of any potential perturbation through a supporting substrate. Herein, arrays of microfabricated BLMs are integrated into a chip‐based platform that is compatible with high‐speed optical NP tracking. This system is used to investigate the lateral diffusion of 40 nm gold spheres tethered to biotinylated lipids through antibody‐functionalized ligands (single‐stranded DNA or polyethylene glycol). Although the NPs show an almost free and ergodic diffusion, their lateral motion is subject to substantial drag at the membrane surface, which leads to systematically smaller diffusion coefficients than those obtained for lipids in the membrane through fluorescence recovery after photobleaching. The lateral mobility of the NPs is influenced by the chemical composition and salt concentration at the NP‐membrane interface, but is independent of the ligand density in the membrane. Together with the observation that nanoprisms, which have a larger relative contact area with the membrane than spherical NPs, show an even slower diffusion, these findings indicate that the lateral mobility of NPs tethered in close vicinity to a membrane is significantly reduced by the friction at the NP‐membrane interface.     

Controlling the aggregation behavior of gold nanoparticles

The aggregation of Au nanoparticles (NPs) in solution is influenced by cationic and oligocationic species. The polarization of the conduction electron oscillations in adjacent gold nanoparticles causes a new red-shifted plasmon absorbance attributed to the coupling of the plasmon absorbance of the particles. This appearance of an additional plasmon band is of particular interest to the field of SERS and has led to research works directed at the stabilization of small colloid aggregates in solution. The surface plasmon coupling can be tuned by controlling the aggregation of gold nanoparticles by the addition of some “cross-linking” agent. Here we develop a simple method to fabricate linear-chainlike aggregates of gold nanoparticles (so-called nanochains), tuning the linear optical properties in a wide wavelength range from visible to the near-infrared. The aggregation behavior and linear self-assembly mechanism of citrate-stabilized gold colloids as provoked by the addition of cetyltrimethylammonium bromide (CTAB) and 11-mercaptoundecanoic acid (MUA) are also analyzed. The line-assembly mechanism of gold nanochain is attributed to the preferential binding of CTAB molecules on a certain facet of gold NPs and the Au NP electrostatic interactions. We also found that the 11-mercaptoundecanoic acid was effective to prevent the further aggregation of CTAB-modified gold colloids.     

Uptake Kinetics and Nanotoxicity of Silica Nanoparticles Are Cell Type Dependent

In this study, it is shown that the cytotoxic response of cells as well as the uptake kinetics of nanoparticles (NPs) is cell type dependent. We use silica NPs with a diameter of 310 nm labeled with perylene dye and 304 nm unlabeled particles to evaluate cell type‐dependent uptake and cytotoxicity on human vascular endothelial cells (HUVEC) and cancer cells derived from the cervix carcinoma (HeLa). Besides their size, the particles are characterized concerning homogeneity of the labeling and their zeta potential. The cellular uptake of the labeled NPs is quantified by imaging the cells via confocal microscopy in a time‐dependent manner, with subsequent image analysis via a custom‐made and freely available digital method, Particle_in_Cell‐3D. We find that within the first 4 h of interaction, the uptake of silica NPs into the cytoplasm is up to 10 times more efficient in HUVEC than in HeLa cells. Interestingly, after 10 or 24 h of interaction, the number of intracellular particles for HeLa cells by far surpasses the one for HUVEC. Inhibitor studies show that these endothelial cells internalize 310 nm SiO₂ NPs via the clathrin‐dependent pathway. Remarkably, the differences in the amount of taken up NPs are not directly reflected by the metabolic activity and membrane integrity of the individual cell types. Interaction with NPs leads to a concentration‐dependent decrease in mitochondrial activity and an increase in membrane leakage for HUVEC, whereas HeLa cells show only a reduced mitochondrial activity and no membrane leakage. In addition, silica NPs lead to HUVEC cell death while HeLa cells survive. These findings indicate that HUVEC are more sensitive than HeLa cells upon silica NP exposure.