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

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

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

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

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

Seedless Synthesis of Disulfide-Grafted Gold Nanoflowers with Size and Shape Control and Their Photothermally Mediated Cell Perforation

Thiol-based ligands have been widely used for passivating metal nanoparticles (NPs) to impart a variety of surface properties while maintaining high colloidal stability. However, their use in direct NP synthesis has been limited to small size (<15 nm) with spherical shape, and direct synthesis of anisotropic gold NPs using dithiol- or disulfide-based ligands has not been achieved. Here, we demonstrate a novel one-step method for the synthesis of multipod-shaped gold NPs (gold nanoflower; AuNF) with a wide size regime (20–500 nm) by controlling the reducing power, acidity, and reagent stoichiometry during NP synthesis using thioctic acid (TA). This strategy is also applied for derivatives of TA-based ligands (e.g., varied terminal groups and the length) to modulate the physicochemical function of AuNFs. To our knowledge, this is the first systematic study of seedless synthesis of anisotropic AuNPs using disulfide-based multifunctional ligands. The resulted morphological anisotropy, created by multiple lobes of “pods” of AuNFs, expands the surface plasmon resonance (SPR) absorption beyond 650 nm, which is longer than the SPR band of gold nanospheres with similar size. The significant red shift of a AuNF SPR band is evaluated by electromagnetic simulations using the finite-element method. We then demonstrate that cholesterol-modified 20 nm AuNFs efficiently convert the 640 nm excitation light to localized heat and perforate the cellular membrane to deliver cell-impermeable molecules (ethidium homodimer-1). Our approach provides a pathway to surfactantless and one-step formation of anisotropic nanostructures using disulfide-based ligands. Their improved photothermal effect under long-wavelength excitation can be used for cell perforation and direct cytosol delivery of various molecules

Determining the Cytosolic Stability of Small DNA Nanostructures <i>In Cellula</i>

DNA nanostructures have proven potential in biomedicine. However, their intracellular interactionsespecially cytosolic stabilityremain 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 structurescrosshairs and a tetrahedronwere 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