Cell Membranes Open “Doors” for Cationic Nanoparticles/Biomolecules: Insights into Uptake Kinetics

Cationic nanoparticles (NPs) and cell-penetrating peptides (CPPs) can enter cells in an energy-independent fashion, escaping the traditional endocytosis route, which is known as direct translocation. This unconventional entry, usually complementary to endocytosis, features rapid uptake and thus makes both cationic NPs and CPPs fascinating intracellular delivery agents. However, the mechanisms of the direct translocation of both cationic NPs and CPPs across cell membranes into the cytosol are not understood. Moreover, the relationship between direct translocation and endocytosis is also unclear. Here, using coarse-grained molecular dynamics simulations we show that a model cell membrane generates a nanoscale hole to assist the spontaneous translocation of cationic gold nanoparticles (AuNPs) as well as HIV-1 Tat peptides to the cytoplasm side under a transmembrane (TM) potential. After translocation, the AuNPs/Tat peptides move freely in the “cytoplasm” region and the membrane reseals itself within a microsecond, while the TM potential is strongly diminished. Furthermore, we show that the shape of the cationic object is crucial in determining if it can translocate or not across. The results provide insights into the uptake kinetics of cationic NPs/CPPs, which features the relationship between direction translocation and endocytosis. The mechanism put forward here establishes fundamental principles of the intracellular delivery of cationic nanocarriers

Cell Membranes Open “Doors” for Cationic Nanoparticles/Biomolecules: Insights into Uptake Kinetics

Cationic nanoparticles (NPs) and cell-penetrating peptides (CPPs) can enter cells in an energy-independent fashion, escaping the traditional endocytosis route, which is known as direct translocation. This unconventional entry, usually complementary to endocytosis, features rapid uptake and thus makes both cationic NPs and CPPs fascinating intracellular delivery agents. However, the mechanisms of the direct translocation of both cationic NPs and CPPs across cell membranes into the cytosol are not understood. Moreover, the relationship between direct translocation and endocytosis is also unclear. Here, using coarse-grained molecular dynamics simulations we show that a model cell membrane generates a nanoscale hole to assist the spontaneous translocation of cationic gold nanoparticles (AuNPs) as well as HIV-1 Tat peptides to the cytoplasm side under a transmembrane (TM) potential. After translocation, the AuNPs/Tat peptides move freely in the “cytoplasm” region and the membrane reseals itself within a microsecond, while the TM potential is strongly diminished. Furthermore, we show that the shape of the cationic object is crucial in determining if it can translocate or not across. The results provide insights into the uptake kinetics of cationic NPs/CPPs, which features the relationship between direction translocation and endocytosis. The mechanism put forward here establishes fundamental principles of the intracellular delivery of cationic nanocarriers

Structure of Mixed-Monolayer-Protected Nanoparticles in Aqueous Salt Solution from Atomistic Molecular Dynamics Simulations

Gold nanoparticles (AuNPs) protected by a grafted ligand monolayer are commonly used for applications in biosensing, bioimaging, and drug delivery, in part because of the ability to tune surface properties by modifying the composition of the protecting ligands. If the surface monolayer contains multiple distinct ligand species, the AuNPs are referred to as mixed-monolayer-protected particles. A typical mixed monolayer consists of two linear alkanethiol ligands, with one ligand species end-functionalized to confer aqueous solubility. However, the inclusion of multiple ligand species raises questions of how the nanoscale morphology and the relative lengths of the two ligands can affect properties, considerations that are unnecessary for single-component monolayers. In this work, we use atomistic molecular dynamics simulations to model the structure of mixed-monolayer-protected AuNPs in aqueous salt solution under typical biological conditions. We focus on identifying changes in the monolayer structure as a function of the diameter of the AuNP core, the morphology of the protecting ligands, and the relative ligand length, complementing existing studies of homogeneous monolayers. Our results show that increasing the particle diameter strongly inhibits ligand fluctuations, consistent with a reduction in free volume associated with higher-curvature substrates. We also show that, in aqueous solution, particles with striped, mixed, and random morphologies exhibit similar behaviors, as ligand fluctuations mask any influence of the grafting positions. Finally, our simulations indicate that long hydrophobic ligands always deform to allow shorter hydrophilic ligands to access water, leading to a significant distortion of the interface if the hydrophobic ligands are much longer than the hydrophilic ones. Our results thus provide new physical insight into the structure of mixed-monolayer-protected particles under typical biological conditions and can be used to guide the experimental design of new classes of AuNPs for biological applications

Membrane-Embedded Nanoparticles Induce Lipid Rearrangements Similar to Those Exhibited by Biological Membrane Proteins

Amphiphilic monolayer-protected gold nanoparticles (NPs) have recently been shown to spontaneously fuse with lipid bilayers under typical physiological conditions. The final configuration of these NPs after fusion is proposed to be a bilayer-spanning configuration resembling transmembrane proteins. In this work, we use atomistic molecular dynamics simulations to explore the rearrangement of the surrounding lipid bilayer after NP insertion as a function of particle size and monolayer composition. All NPs studied induce local bilayer thinning and a commensurate decrease in local lipid tail order. Bilayer thickness changes of similar magnitude have been shown to drive protein aggregation, implying that NPs may also experience a membrane-mediated attraction. Unlike most membrane proteins, the exposed surface of the NP has a high charge density that causes electrostatic interactions to condense and reorient nearby lipid head groups. The decrease in tail order also leads to an increased likelihood of lipid tails spontaneously protruding toward solvent, a behavior related to the kinetic pathway for both NP insertion and vesicle–vesicle fusion. Finally, our results show that NPs can even extract lipids from the surrounding bilayer to preferentially intercalate within the exposed monolayer. These drastic lipid rearrangements are similar to the lipid mixing encouraged by fusion peptides, potentially allowing these NPs to be tuned to perform a similar biological function. This work complements previous studies on the NP–bilayer fusion mechanism by detailing the response of the bilayer to an embedded NP and suggests guidelines for the design of nanoparticles that induce controllable lipid rearrangements

Ligand-Mediated Short-Range Attraction Drives Aggregation of Charged Monolayer-Protected Gold Nanoparticles

Monolayer-protected gold nanoparticles (AuNPs) are a promising new class of nanomaterials with applications in drug delivery, self-assembly, and biosensing. The versatility of the AuNP platform is conferred by the properties of the protecting monolayer which can be engineered to tune the surface functionality of the nanoparticles. However, many applications are hampered by AuNP aggregation, which can inhibit functionality or induce particles to precipitate out of solution, even for water-soluble AuNPs. It is critical to understand the mechanisms of aggregation in order to optimally engineer protecting monolayers that both inhibit aggregation and maintain functionality. In this work, we use implicit solvent simulations to calculate the free energy change associated with the aggregation of two small, charged, alkanethiol monolayer-protected AuNPs under typical biological conditions. We show that aggregation is driven by the hydrophobic effect related to the amphiphilic nature of the alkanethiol ligands. The critical factor that enables aggregation is the deformation of ligands in the monolayer to shield hydrophobic surface area from water upon close association of the two particles. Our results further show that ligand deformation, and thus aggregation, is highly dependent on the size of the AuNPs, choice of ligands, and environmental conditions. This work provides insight into the key role that ligand–ligand interactions play in stabilizing AuNP aggregates and suggests guidelines for the design of protecting monolayers that inhibit aggregation under typical biological conditions

Grafting Charged Species to Membrane-Embedded Scaffolds Dramatically Increases the Rate of Bilayer Flipping

The cell membrane is a barrier to the passive diffusion of charged molecules due to the chemical properties of the lipid bilayer. Surprisingly, recent experiments have identified processes in which synthetic and biological charged species directly transfer across lipid bilayers on biologically relevant time scales. In particular, amphiphilic nanoparticles have been shown to insert into lipid bilayers, requiring the transport of charged species across the bilayer. The molecular factors facilitating this rapid insertion process remain unknown. In this work, we use atomistic molecular dynamics simulations to calculate the free energy barrier associated with “flipping” charged species across a lipid bilayer for species that are grafted to a membrane-embedded scaffold, such as a membrane-embedded nanoparticle. We find that the free energy barrier for flipping a grafted ligand can be over 7 kcal/mol lower than the barrier for translocating an isolated, equivalent ion, yielding a 5 order of magnitude decrease in the corresponding flipping time scale. Similar results are found for flipping charged species grafted to either nanoparticle or protein scaffolds. These results reveal new mechanistic insight into the flipping of charged macromolecular components that might play an important, yet overlooked, role in signaling and charge transport in biological settings. Furthermore, our results suggest guidelines for the design of synthetic materials capable of rapidly flipping charged moieties across the cell membrane

Fusion of Ligand-Coated Nanoparticles with Lipid Bilayers: Effect of Ligand Flexibility

Amphiphilic, monolayer-protected gold nanoparticles (AuNPs) have recently been shown to insert into and fuse with lipid bilayers, driven by the hydrophobic effect. The inserted transmembrane state is stabilized by the “snorkeling” of charged ligand end groups out of the bilayer interior. This snorkeling process is facilitated by the backbone flexibility of the alkanethiol ligands that comprise the monolayer. In this work, we show that fusion is favorable even in the absence of backbone flexibility by modeling the ligands as rigid rods. For rigid ligands, snorkeling is still accommodated by rotations of the ligand with respect to the grafting point, but the process incurs a more significant free energy penalty than if the backbone were fully flexible. We show that the rigid rod model predicts similar trends in the free energy change for insertion as the previous flexible model when the size of the AuNPs is varied. However, the rigidity of the ligand backbone reduces the overall magnitude of the free energy change compared to that of the flexible model. These results thus generalize previous findings to systems with hindered backbone flexibility due to either structural constraints or low temperature

Diffusion of Entangled Rod–Coil Block Copolymers

The diffusion of entangled rod–coil block copolymers is investigated by molecular dynamics (MD) simulations, and theories are introduced that describe the observed features and underlying physics. The reptation of rod–coil block copolymers is dominated by the mismatch between the curvature of the rod and coil entanglement tubes, which results in dramatically slower diffusion of rod–coils compared to the rod and coil homopolymers. For small rods, a local curvature-dependent free energy penalty results in a rough energy surface inside the entanglement tube, causing diffusivity to decrease with rod length. For large rods, rotational hindrances on the rod dominate, causing the coil block to relax by an arm retraction mechanism and diffusivity to decrease exponentially with coil size

Oxepine-Based π‑Conjugated Ladder/Step-Ladder Polymers with Excited -State Aromaticity

Ladder polymers with backbones of uninterrupted ring-fused units have attracted academic and industrial attention for decades because of their extended π-conjugation and intrinsic microporosity for possible potential applications in organic optoelectronics and membrane gas separations. We report herein the synthesis and characterization of a new series of oxepine-based ladder/step-ladder polymers prepared by acid-promoted intramolecular aromatic electrophilic cyclization reactions on alkyne-containing poly(arylene ether) precursors. In contrast to the more common annulations that produce five- and six-membered rings, we report that a seven-membered ring can be regioselectively and quantitatively generated postpolymerization. Model compounds with repeating units of polymers have also been synthesized, and X-ray crystallographic analysis reveals a nonplanar contorted structure that is also present in the polymers. More interestingly, the oxepine-based ladder/step-ladder polymers appear to display a photoinduced planarization of the 8 π electron oxepine ring driven by the excited-state aromatic stabilization energy, as indicated by the large Stokes shift

Oxepine-Based π‑Conjugated Ladder/Step-Ladder Polymers with Excited -State Aromaticity

Ladder polymers with backbones of uninterrupted ring-fused units have attracted academic and industrial attention for decades because of their extended π-conjugation and intrinsic microporosity for possible potential applications in organic optoelectronics and membrane gas separations. We report herein the synthesis and characterization of a new series of oxepine-based ladder/step-ladder polymers prepared by acid-promoted intramolecular aromatic electrophilic cyclization reactions on alkyne-containing poly(arylene ether) precursors. In contrast to the more common annulations that produce five- and six-membered rings, we report that a seven-membered ring can be regioselectively and quantitatively generated postpolymerization. Model compounds with repeating units of polymers have also been synthesized, and X-ray crystallographic analysis reveals a nonplanar contorted structure that is also present in the polymers. More interestingly, the oxepine-based ladder/step-ladder polymers appear to display a photoinduced planarization of the 8 π electron oxepine ring driven by the excited-state aromatic stabilization energy, as indicated by the large Stokes shift