Lela Vuković

Diffusion and filtration properties of self-assembled gold nanocrystal membranes.

Close-packed nanoparticle monolayers have recently been shown to form mechanically robust, free-standing membranes. We report the first measurements of molecular transport through such ultrathin sheets, self-assembled from dodecanethiol-ligated gold nanocrystals. For aqueous solutions we find filtration coefficients 2 orders of magnitude larger than those observed in polymer-based filters, sieving of large solutes, and for smaller solutes a pronounced dependence of rejection on being charged. These results open up new possibilities for controlled delivery and separation of nano-objects.

Structure and Dynamics of Highly PEG-ylated Sterically Stabilized Micelles in Aqueous Media

Molecular assemblies of highly PEG-ylated phospholipids are important in many biomedical applications. We have studied sterically stabilized micelles (SSMs) of self-assembled DSPE–PEG2000 in pure water and isotonic HEPES-buffered saline solution. The observed SSM sizes of 2–15 nm largely depend on the solvent and the lipid concentration used. The critical micelle concentration of DSPE–PEG2000 is 10 times higher in water than in buffer, and the viscosity of the dispersion dramatically increases with the lipid concentration. To explain the experimentally observed results, we performed atomistic molecular dynamics simulations of solvated SSMs. Our modeling revealed that the observed assemblies have very different aggregation numbers (N(agg) ≈ 90 in saline solution and N(agg) < 8 in water) because of very different screening of their charged PO4(–) groups. We also demonstrate that the micelle cores can inflate and their coronas can fluctuate strongly, thus allowing storage and delivery of molecules with different chemistries.

Dendron-mediated self-assembly of highly PEGylated block copolymers: a modular nanocarrier platform

PEGylated dendron coils (PDCs) were investigated as a novel potential nanocarrier platform. PDCs self-assembled into micelles at lower CMCs than linear copolymer counterparts by 1-2 orders of magnitude, due to the unique architecture of dendrons. MD simulations also supported thermodynamically favourable self-assembly mediated by dendrons.

Colloidal nanocube supercrystals stabilized by multipolar Coulombic coupling

We explore microscopic principles governing the self-assembly of colloidal octylamine-coated platinum nanocubes solvated in toluene. Our experiments show that regular nanocubes with an edge length of l(RC) = 5.5 nm form supercrystals with simple cubic packing, while slightly truncated nanocubes with an edge length of l(TC) = 4.7 nm tend to arrange in fcc packing. We model by averaged force fields and atomistic molecular dynamics simulations the coupling forces between these nanocrystals. Our detailed analysis shows that the fcc packing, which for cubes has a lower density than simple cubic packing, is favored by the truncated nanocubes due to their Coulombic coupling by multipolar electrostatic fields, formed during charge transfer between the octylamine ligands and the Pt cores.

Dynamic profiling of double-stranded RNA binding proteins

Double-stranded (ds) RNA is a key player in numerous biological activities in cells, including RNA interference, anti-viral immunity and mRNA transport. The class of proteins responsible for recognizing dsRNA is termed double-stranded RNA binding proteins (dsRBP). However, little is known about the molecular mechanisms underlying the interaction between dsRBPs and dsRNA. Here we examined four human dsRBPs, ADAD2, TRBP, Staufen 1 and ADAR1 on six dsRNA substrates that vary in length and secondary structure. We combined single molecule pull-down (SiMPull), single molecule protein-induced fluorescence enhancement (smPIFE) and molecular dynamics (MD) simulations to investigate the dsRNA-dsRBP interactions. Our results demonstrate that despite the highly conserved dsRNA binding domains, the dsRBPs exhibit diverse substrate specificities and dynamic properties when in contact with different RNA substrates. While TRBP and ADAR1 have a preference for binding simple duplex RNA, ADAD2 and Staufen1 display higher affinity to highly structured RNA substrates. Upon interaction with RNA substrates, TRBP and Staufen1 exhibit dynamic sliding whereas two deaminases ADAR1 and ADAD2 mostly remain immobile when bound. MD simulations provide a detailed atomic interaction map that is largely consistent with the affinity differences observed experimentally. Collectively, our study highlights the diverse nature of substrate specificity and mobility exhibited by dsRBPs that may be critical for their cellular function.

High-resolution imaging of cellular dopamine efflux using a fluorescent nanosensor array

Significance Cells communicate between themselves using waves of chemical concentration that change in both direction and time. Existing methods are not able to capture this spatial and temporal dependence at the necessary resolution to study the influence of cellular morphology. This study utilizes arrays of fluorescent nanosensors based on single-walled carbon nanotubes placed under and around neuroprogenitor cells to label-free image the efflux of the neurotransmitter dopamine. We use the spatiotemporal resolution of this approach to resolve where on the cell surface dopamine is released and how cell morphology affects the location of release sites. Intercellular communication via chemical signaling proceeds with both spatial and temporal components, but analytical tools, such as microfabricated electrodes, have been limited to just a few probes per cell. In this work, we use a nonphotobleaching fluorescent nanosensor array based on single-walled carbon nanotubes (SWCNTs) rendered selective to dopamine to study its release from PC12 neuroprogenitor cells at a resolution exceeding 20,000 sensors per cell. This allows the spatial and temporal dynamics of dopamine release, following K+ stimulation, to be measured at exceedingly high resolution. We observe localized, unlabeled release sites of dopamine spanning 100 ms to seconds that correlate with protrusions but not predominately the positive curvature associated with the tips of cellular protrusions as intuitively expected. The results illustrate how directionality of chemical signaling is shaped by membrane morphology, and highlight the advantages of nanosensor arrays that can provide high spatial and temporal resolution of chemical signaling.

Broad-spectrum non-toxic antiviral nanoparticles with a virucidal inhibition mechanism

Viral infections kill millions yearly. Available antiviral drugs are virus-specific and active against a limited panel of human pathogens. There are broad-spectrum substances that prevent the first step of virus-cell interaction by mimicking heparan sulfate proteoglycans (HSPG), the highly conserved target of viral attachment ligands (VALs). The reversible binding mechanism prevents their use as a drug, because, upon dilution, the inhibition is lost. Known VALs are made of closely packed repeating units, but the aforementioned substances are able to bind only a few of them. We designed antiviral nanoparticles with long and flexible linkers mimicking HSPG, allowing for effective viral association with a binding that we simulate to be strong and multivalent to the VAL repeating units, generating forces (∼190 pN) that eventually lead to irreversible viral deformation. Virucidal assays, electron microscopy images, and molecular dynamics simulations support the proposed mechanism. These particles show no cytotoxicity, and in vitro nanomolar irreversible activity against herpes simplex virus (HSV), human papilloma virus, respiratory syncytial virus (RSV), dengue and lenti virus. They are active ex vivo in human cervicovaginal histocultures infected by HSV-2 and in vivo in mice infected with RSV.

Molecular Friction-Induced Electroosmotic Phenomena in Thin Neutral Nanotubes.

We reveal by classical molecular dynamics simulations electroosmotic flows in thin neutral carbon (CNT) and boron nitride (BNT) nanotubes filled with ionic solutions of hydrated monovalent atomic ions. We observe that in (12,12) BNTs filled with single ions in an electric field, the net water velocity increases in the order of Na(+) < K(+) < Cl(-), showing that different ions have different power to drag water in thin nanotubes. However, the effect gradually disappears in wider nanotubes. In (12,12) BNTs containing neutral ionic solutions in electric fields, we observe net water velocities going in the direction of Na(+) for (Na(+), Cl(-)) and in the direction of Cl(-) for (K(+), Cl(-)). We hypothesize that the electroosmotic flows are caused by different strengths of friction between ions with different hydration shells and the nanotube walls.

Substrate recognition and specificity of double-stranded RNA binding proteins.

Recognition of double-stranded (ds) RNA is an important part of many cellular pathways, including RNA silencing, viral recognition, RNA editing, processing, and transport. dsRNA recognition is often achieved by dsRNA binding domains (dsRBDs). We use atomistic molecular dynamics simulations to examine the binding interface of the transactivation response RNA binding protein (TRBP) dsRBDs to dsRNA substrates. Our results explain the exclusive selectivity of dsRBDs toward dsRNA and against DNA–RNA hybrid and dsDNA duplexes. We also provide corresponding experimental evidence. The dsRNA duplex is recognized by dsRBDs through the A-form of three duplex grooves and by the chemical properties of RNA bases, which have 2′-hydroxyl groups on their sugar rings. Our simulations show that TRBP dsRBD discriminates dsRNA- from DNA-containing duplexes primarily through interactions at two duplex grooves. The simulations also reveal that the conformation of the DNA–RNA duplex can be altered by dsRBD proteins, resulting in a weak binding of dsRBDs to DNA–RNA hybrids. Our study reveals the structural and molecular basis of protein–RNA interaction that gives rise to the observed substrate specificity of dsRNA binding proteins.

Solubilization of therapeutic agents in micellar nanomedicines

We use atomistic molecular dynamics simulations to reveal the binding mechanisms of therapeutic agents in PEG-ylated micellar nanocarriers (SSM). In our experiments, SSM in buffer solutions can solubilize either ≈11 small bexarotene molecules or ≈6 (2 in low ionic strength buffer) human vasoactive intestinal peptide (VIP) molecules. Free energy calculations reveal that molecules of the poorly water-soluble drug bexarotene can reside at the micellar ionic interface of the PEG corona, with their polar ends pointing out. Alternatively, they can reside in the alkane core center, where several bexarotene molecules can self-stabilize by forming a cluster held together by a network of hydrogen bonds. We also show that highly charged molecules, such as VIP, can be stabilized at the SSM ionic interface by Coulombic coupling between their positively charged residues and the negatively charged phosphate headgroups of the lipids. The obtained results illustrate that atomistic simulations can reveal drug solubilization character in nanocarriers and be used in efficient optimization of novel nanomedicines.