Nanoparticle Anisotropy Increases Targeting Interactions on Live-Cell Membranes.

This paper describes how branch lengths of anisotropic nanoparticles can affect interactions between grafted ligands and cell-membrane receptors. Using live-cell, single-particle tracking, we found that DNA aptamer-gold nanostar nanoconstructs with longer branches showed improved binding efficacy to human epidermal growth factor receptor 2 (HER2) on cancer cell membranes. Inhibiting nanoconstruct-HER2 binding promoted nonspecific interactions, which increased the rotational speed of long-branched nanoconstructs but did not affect that of short-branched constructs. Bivariate analysis of the rotational and translational dynamics showed that longer branch lengths increased the ratio of targeting to nontargeting interactions. We also found that longer branches increased the nanoconstruct-cell interaction times before internalization and decreased intracellular trafficking velocities. Differences in binding efficacy revealed by single-particle dynamics can be attributed to the distinct protein corona distributions on short- and long-branched nanoconstructs, as validated by transmission electron microscopy. Minimal protein adsorption at the high positive curvature tips of long-branched nanoconstructs facilitated binding of DNA aptamer ligands to HER2. Our study reveals the significance of nanoparticle branch length in regulating local chemical environment and interactions with live cells at the single-particle level.

Ligand Separation on Nanoconstructs Affects Targeting Selectivity to Protein Dimers on Cell Membranes.

This work demonstrates that targeting ligand density on nanoparticles can affect interactions between the nanoconstructs and cell membrane receptors. We discovered that when the separation between covalently grafted DNA aptamers on gold nanostars was comparable to the distance between binding sites on a receptor dimer (matched density; MD), nanoconstructs exhibited a higher selectivity for binding to the dimeric form of the protein. Single-particle dynamics of MD nanoconstructs showed slower rotational rates and larger translational footprints on cancer cells expressing more dimeric forms of receptors (dimer+) compared with cells having more monomeric forms (dimer-). In contrast, nanoconstructs with either increased (nonmatched density; NDlow) or decreased ligand spacing (NDhigh) had minimal changes in dynamics on either dimer+ or dimer- cells. Real-time, single-particle analyses can reveal the importance of nanoconstruct ligand density for the selective targeting of membrane receptors in live cells.

Nanoparticle-based delivery of nitric oxide for therapeutic applications.

Nitric oxide (NO), a low molecular weight signaling molecule, plays critical roles in both cellular health and disease. There is continued interest in new modalities for the controlled therapeutic delivery of NO to cells and tissues. The physicochemical properties of NO (including its short half-life and on-demand synthesis at the point of function), however, pose considerable challenges for its specific and efficient delivery. Recently, a number of nanoparticle (NP)-based systems are described that address some of these issues by taking advantage of the unique attributes of the NP carrier to effect efficient NO delivery. This review highlights the progress that has been made over the past 5 years in the use of various constructs for the therapeutic delivery of NO.

This review details progress made over the past 5 years in the implementation of various nanoparticle (NP) bioconjugates for the therapeutic delivery of nitric oxide. Various NP formulations including liposomes, polymeric NPs, and hard NPs such as AuNPs and upconversion NPs are covered and we discuss the inherent advantages and challenges in using these materials for the controlled delivery of nitric oxide to cells and tissues.

Determining the Cytosolic Stability of Small DNA Nanostructures In Cellula.

DNA nanostructures have proven potential in biomedicine. However, their intracellular interactions─especially cytosolic stability─remain mostly unknown and attempts to discern this are confounded by the complexities of endocytic uptake and entrapment. Here, we bypass the endocytic uptake and evaluate the DNA structural stability directly in live cells. Commonly used DNA structures─crosshairs and a tetrahedron─were labeled with a multistep Förster resonance energy transfer dye cascade and microinjected into the cytosol of transformed and primary cells. Energy transfer loss, as monitored by fluorescence microscopy, reported the structure's direct time-resolved breakdown in cellula. The results showed rapid degradation of the DNA crosshair within 20 min, while the tetrahedron remained consistently intact for at least 1 h postinjection. Nuclease assays in conjunction with a current understanding of the tetrahedron's torsional rigidity confirmed its higher stability. Such studies can inform design parameters for future DNA nanostructures where programmable degradation rates may be required.

Liquid Crystal Nanoparticle Conjugates for Scavenging Reactive Oxygen Species in Live Cells.

The elevated intracellular production of or extracellular exposure to reactive oxygen species (ROS) causes oxidative stress to cells, resulting in deleterious irreversible biomolecular reactions (e.g., lipid peroxidation) and disease progression. The use of low-molecular weight antioxidants, such as 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), as ROS scavengers fails to achieve the desired efficacy because of their poor or uncontrolled cellular uptake and off-target effects, such as dysfunction of essential redox homeostasis. In this study, we fabricated a liquid crystal nanoparticle (LCNP) conjugate system with the fluorescent dye perylene (PY) loaded in the interior and poly (ethylene glycol) (PEG) decorated on the surface along with multiple molecules of TEMPO (PY-LCNP-PEG/TEMPO). PY-LCNP-PEG/TEMPO exhibit enhanced cellular uptake, and efficient ROS-scavenging activity in live cells. On average, the 120 nm diameter PY-LCNPs were conjugated with >1800 molecules of TEMPO moieties on their surface. PY-LCNP-PEG/TEMPO showed significantly greater reduction in ROS activity and lipid peroxidation compared to free TEMPO when the cells were challenged with ROS generating agents, such as hydrogen peroxide (H2O2). We suggest that this is due to the increased local concentration of TEMPO molecules on the surface of the PY-LCNP-PEG/TEMPO NPs, which efficiently bind to the plasma membrane and enter cells. Overall, these results demonstrate the enhanced capability of TEMPO-conjugated LCNPs to protect live cells from oxidative stress by effectively scavenging ROS and reducing lipid peroxidation.

Delivery Order of Nanoconstructs Affects Intracellular Trafficking by Endosomes.

This paper reports how the endosomal pathways of nanoparticle (NP) constructs with different surface curvatures are affected by their order of delivery. Sequential incubation of cytosine-phosphate-guanine (CpG)-conjugated spiky and spherical gold NPs with macrophages resulted in different nanoconstruct ratios at the interior edges of endosomes. Application of spiky NPs after spherical NPs accelerated the formation of late-stage endosomes and resulted in larger endosomes, and the spherical NPs were enclosed by the spiky NPs. In contrast, the reverse incubation order produced an asymmetric distribution of the two nanoconstruct shapes in smaller endosomes. Macrophages with a higher proportion of the enclosed spherical NPs as well as a larger ratio of spiky to spherical NPs at the endosomal edge showed enhanced toll-like receptor 9 activation and secretion of proinflammatory cytokines and chemokines. Our results indicate that the subcellular trafficking of targeting nanoconstructs by vesicles is affected by both the delivery order and the endosomal distribution. Our study also establishes a new approach for nanoscale monitoring of intracellular therapeutics delivery with conventional electron microscopy.

Endosomal Organization of CpG Constructs Correlates with Enhanced Immune Activation.

This Letter describes how the endosomal organization of immunostimulatory nanoconstructs can tune the in vitro activation of macrophages. Nanoconstructs composed of gold nanoparticles conjugated with unmethylated cytosine-phosphate-guanine (CpG) oligonucleotides have distinct endosomal distributions depending on the surface curvature. Mixed-curvature constructs produce a relatively high percentage of hollow endosomes, where constructs accumulated primarily along the interior edges. These constructs achieved a higher level of toll-like receptor (TLR) 9 activation along with the enhanced secretion of proinflammatory cytokines and chemokines compared to constant-curvature constructs that aggregated mostly in the center of the endosomes. Our results underscore the importance of intraendosomal interactions in regulating immune responses and targeted delivery.

Lipid bilayer disruption induced by amphiphilic Janus nanoparticles: the non-monotonic effect of charged lipids.

In this study, we report the complex effects of charged lipids on the interaction between amphiphilic Janus nanoparticles and lipid bilayers. Janus nanoparticles are cationic on one hemisphere and hydrophobic on the other. We show that the nanoparticles, beyond threshold concentrations, induce holes in both cationic and anionic lipid bilayers mainly driven by hydrophobic interactions. However, the formation of these defects is non-monotonically dependent on ionic lipid composition. The electrostatic attraction between the particles and anionic lipid bilayers enhances particle adsorption and lowers the particle concentration threshold for defect initiation, but leads to more localized membrane disruption. Electrostatic repulsion leads to reduced particle adsorption on cationic bilayers and extensive defect formation that peaks at intermediate contents of cationic lipids. This study elucidates the significant role lipid composition plays in influencing how amphiphilic Janus nanoparticles interact with and perturb lipid membranes.

Lipid Bilayer Disruption by Amphiphilic Janus Nanoparticles: The Role of Janus Balance.

Amphiphilic nanoparticles are known to cause defects in lipid bilayers. However, we have shown recently that their disruptive effects are significantly enhanced when surface charges and hydrophobic groups are spatially segregated on opposite hemispheres of a single particle. Using the same amphiphilic cationic/hydrophobic Janus particle system, here we investigate the role of the hydrophilic-lipophilic balance of the particles (namely the Janus balance) in their interaction with zwitterionic lipid bilayers. We show that Janus nanoparticles induce holes in lipid bilayers only when the hydrophobic side of particles occupies 20% or more of their surfaces. Beyond this threshold, the larger the hydrophobic surface area, the more attractive the particles are to lipid bilayers, and a lower particle concentration is needed for causing defects in the bilayers. The results establish a quantitative relationship between the surface coverage of hydrophobicity on the Janus particles and the particle-induced disruption to the lipid membranes.

Rupture of Lipid Membranes Induced by Amphiphilic Janus Nanoparticles.

The surface coatings of nanoparticles determine their interaction with biomembranes, but studies have been limited almost exclusively to nanoparticles with a uniform surface chemistry. Although nanoparticles are increasingly made with complex surface chemistries to achieve multifunctionalities, our understanding of how a heterogeneous surface coating affects particle-biomembrane interaction has been lagging far behind. Here we report an investigation of this question in an experimental system consisting of amphiphilic "two-faced" Janus nanoparticles and supported lipid membranes. We show that amphiphilic Janus nanoparticles at picomolar concentrations induce defects in zwitterionic lipid bilayers. In addition to revealing the various effects of hydrophobicity and charge in particle-bilayer interactions, we demonstrate that the Janus geometry-the spatial segregation of hydrophobicity and charges on particle surface-causes nanoparticles to bind more strongly to bilayers and induce defects more effectively than particles with uniformly mixed surface functionalities. We combine experiments with computational simulation to further elucidate how amphiphilic Janus nanoparticles extract lipids to rupture intact lipid bilayers. This study provides direct evidence that the spatial arrangement of surface functionalities on a nanoparticle, rather than just its overall surface chemistry, plays a crucial role in determining how it interacts with biological membranes.

Janus Nanoparticles for T Cell Activation: Clustering Ligands to Enhance Stimulation.

The in vitro activation of T cells by synthetic particles is a promising technique for adoptive cancer immunotherapy. While it is known that cell-surface receptors form clusters during T cell activation, the use of clustered ligands on synthetic particles to modulate T cell response is a largely unexplored concept. Building upon our previous finding that T cells respond differently to various micro-sized patterns of ligands, we here investigate the effect of nano-sized ligand clusters on T cell activation. Two-faced Janus nanoparticles were fabricated to display ligands of different functions in spatially segregated clusters on single nanoparticles. Going beyond our earlier qualitative study, here we precisely quantified and controlled the surface density and the total amount of ligands on single nanoparticles. We show that nanoparticles with clustered ligands activate T cells to a greater level than ones uniformly coated with the same number of ligands. The enhanced effect is due to increased local surface density of ligands. The results demonstrate that the spatial arrangement of ligands on particles influences activation response of T cells and may be used as a new strategy to increase T cell stimulation in the presence of insufficient amount of stimuli. This fundamental study also represents an initial step in using nanoscale Janus particles for manipulating immune cell responses.

Interrogating Cellular Functions with Designer Janus Particles.

Janus particles have two distinct surfaces or compartments. This enables novel applications that are impossible with homogeneous particles, ranging from the engineering of active colloidal metastructures to creating multimodal therapeutic materials. Recent years have witnessed a rapid development of novel Janus structures and exploration of their applications, particularly in the biomedical arena. It, therefore, becomes crucial to understand how Janus particles with surface or structural anisotropy might interact with biological systems and how such interactions may be exploited to manipulate biological responses. This perspective highlights recent studies that have employed Janus particles as novel toolsets to manipulate, measure, and understand cellular functions. Janus particles have been shown to have biological interactions different from uniform particles. Their surface anisotropy has been used to control the cell entry of synthetic particles, to spatially organize stimuli for the activation of immune cells, and to enable direct visualization and measurement of rotational dynamics of particles in living systems. The work included in this perspective showcases the significance of understanding the biological interactions of Janus particles and the tremendous potential of harnessing such interactions to advance the development of Janus structure-based biomaterials.

Remote Control of T Cell Activation Using Magnetic Janus Particles.

We report a strategy for using magnetic Janus microparticles to control the stimulation of T cell signaling with single-cell precision. To achieve this, we designed Janus particles that are magnetically responsive on one hemisphere and stimulatory to T cells on the other side. By manipulating the rotation and locomotion of Janus particles under an external magnetic field, we could control the orientation of the particle-cell recognition and thereby the initiation of T cell activation. This study demonstrates a step towards employing anisotropic material properties of Janus particles to control single-cell activities without the need of complex magnetic manipulation devices.

Oxidant-resistant imaging and ratiometric luminescence detection by selective oxidation of silver nanodots.

Reactive oxygen species selectively accelerated transitions between various silver nanodots. The blue was developed as an oxidant-resistant imaging agent and analyte reporter. In addition to the spectral response of nanodots to ROS, silver nanodots were formulated to detect analytes with excellent selectivity and picomolar detection limit when coupled to glucose oxidase.

Autofluorescence generation and elimination: a lesson from glutaraldehyde.

Glutaraldehyde causes especially high autofluorescence. It reacted with proteins and peptides to generate visible to near-IR emitters. A model indicated that ethylenediamine and a secondary amine in the molecule were key components for the formation of emissive species. The mechanism enables us to control the generation and elimination of autofluorescence.