Stealth and Biocompatible Gold Nanoparticles through Surface Coating with a Zwitterionic Derivative of Glutathione.

Gold nanoparticles (AuNPs) hold promise in biomedicine, but challenges like aggregation, protein corona formation, and insufficient biocompatibility must be thoroughly addressed before advancing their clinical applications. Designing AuNPs with specific protein corona compositions is challenging, and strategies for corona elimination, such as coating with polyethylene glycol (PEG), have limitations. In this study, we introduce a commercially available zwitterionic derivative of glutathione, glutathione monoethyl ester (GSHzwt), for the surface coating of colloidal AuNPs. Particles coated with GSHzwt were investigated alongside four other AuNPs coated with various ligands, including citrate ions, tiopronin, glutathione, cysteine, and PEG. We then undertook a head-to-head comparison of these AuNPs to assess their behavior in biological fluid. GSHzwt-coated AuNPs exhibited exceptional resistance to aggregation and protein adsorption. The particles could also be readily functionalized with biotin and interact with streptavidin receptors in human plasma. Additionally, they exhibited significant blood compatibility and noncytotoxicity. In conclusion, GSHzwt provides a practical and easy method for the surface passivation of AuNPs, creating "stealth" particles for potential clinical applications.

Hurdles in translating science from lab to market in delivery systems for Cosmetics: An industrial perspective.

In recent decades, a sweeping technological wave has reshaped the global economic landscape. Fueled by the unceasing forces of digital innovation and venture capital investment, this transformative machine has left a significant mark across numerous economic sectors. More recently, the emergence of 'deep tech' start-ups, focusing on areas such as artificial intelligence, nanotechnology, and biotechnology, has infused a fresh wave of innovation into various sectors, including the pharmaceutical and cosmetic industry. This review explores the significance of innovation within the cosmetics sector, with a particular emphasis on delivery systems. It assesses the crucial process of bridging the gap between research and the market, particularly in the translation of nanotechnology into tangible real-world applications. With the rise of nanotechnology-based beauty ingredients, we can anticipate groundbreaking advancements that promise to surpass consumer expectations, ushering in a new era of unparalleled innovation in beauty products.

Time Evolution of Ultrasmall Gold Nanoparticle-Protein Interactions.

To date, much effort has been devoted toward the study of protein corona formation onto large gold nanoparticles (GNPs). However, the protein corona concept breaks down for GNPs in the ultrasmall size regime (<3 nm), and, as a result, our understanding of ultrasmall GNP (usGNP)-protein interactions remains incomplete. Herein, we used anionic usGNPs and six different proteins as model systems to systematically investigate usGNP-protein interactions, with particular focus on the time evolution and long-term behavior of complex formation. The different proteins comprised chymotrypsin (Cht), trypsin (Try), thrombin (Thr), serum albumin (HSA), cytochrome c (Cyt c), and factor XII (FXII). We used a range of biochemical and biophysical methods to estimate binding affinities, determine the effects of usGNPs on protein structure and function, assess the reversibility of any protein structural and functional changes, and evaluate usGNP-protein complex stability. Among the main findings, we observed that prolonged (24 h)─but not short-term (10 min)─interactions between proteins and usGNPs permanently altered protein function, including enzyme activities (Try, Thr, and FXIIa), peroxidase-like activity (Cyt c), and ligand-binding properties (HSA). Remarkably, this occurred without any large-scale loss of the native global conformation, implying time-dependent effects of usGNPs on local protein conformation or dynamics. We also found that both short-(10 min) and long-term (24 h) interactions between proteins and usGNPs yielded short-lived complexes, i.e., there was no time-dependent "hardening" of the interactions at the binding interface as usually seen with large GNPs. The present study increases our fundamental understanding of nano-bio interactions in the ultrasmall size regime, which may assist the safe and effective translation of usGNPs into the clinic.

Anionic Ultrasmall Gold Nanoparticles Bind to Coagulation Factors and Disturb Normal Hemostatic Balance.

Ultrasmall gold nanoparticles (usNPs) and nanoclusters are an emerging class of nanomaterials exhibiting distinctive physicochemical properties and in vivo behaviors. Although understanding the interactions of usNPs with blood components is of fundamental importance to advance their clinical translation, currently, little is known about the way that usNPs interact with the hemostatic system. This study describes the effects of a model anionic p-mercaptobenzoic acid-coated usNP on the coagulation cascade, with particular emphasis on the contact pathway. It is found that in a purified system, the anionic usNPs bind to and activate factor XII (FXII). The formed usNP-FXII complexes are short-lived (residence time of ∼10 s) and characterized by an affinity constant of ∼200 nM. In human plasma, the anionic usNPs activate the contact pathway and promote coagulation. The usNPs also exhibit anticoagulant activity in plasma by interfering with the thrombin-mediated cleavage of fibrinogen. Taken together, these findings establish that anionic usNPs can disturb the normal hemostatic balance, which in turn may hinder their clinical translation. Finally, it is shown that usNPs can be designed to be nearly inert in plasma by surface coating with the natural peptide glutathione.

Cell-surface glycosaminoglycans regulate the cellular uptake of charged polystyrene nanoparticles.

Engineered nanoparticles approaching the cell body will first encounter and interact with cell-surface glycosaminoglycans (GAGs) before reaching the plasma membrane and becoming internalized. However, how surface GAGs may regulate the cellular entry of nanoparticles remains poorly understood. Herein, it is shown that the surface GAGs of Chinese hamster ovary cells perform as a charge-based barrier against the cellular internalization of anionic polystyrene nanoparticles (PS NPs). In contrast, cationic PS NPs interact favorably with the surface GAGs and thereby are efficiently internalized. Anionic PS NPs eventually reaching the plasma membrane bind to scavenger receptors and are endocytosed by clathrin-mediated and lipid raft/cholesterol-dependent mechanisms, whereas cationic PS NPs are primarily internalized via clathrin-mediated endocytosis and macropinocytosis. Upon the enzymatic shedding of surface GAGs, the uptake of anionic PS NPs increases while that of cationic PS NPs is dramatically reduced. Interestingly, the diminished uptake of cationic PS NPs is observed only when heparan sulfate, but not chondroitin sulfate, is cleaved from the cell surface. Heparan sulfate therefore serves as anchors/first receptors to facilitate the cellular entry of cationic PS NPs. These findings contribute to advance the basic science of nanoparticle endocytosis while also having important implications for the use of engineered nanocarriers as intracellular drug-delivery systems.

Biomolecular interactions of ultrasmall metallic nanoparticles and nanoclusters.

The use of nanoparticles (NPs) in biomedicine has made a gradual transition from proof-of-concept to clinical applications, with several NP types meeting regulatory approval or undergoing clinical trials. A new type of metallic nanostructures called ultrasmall nanoparticles (usNPs) and nanoclusters (NCs), while retaining essential properties of the larger (classical) NPs, have features common to bioactive proteins. This combination expands the potential use of usNPs and NCs to areas of diagnosis and therapy traditionally reserved for small-molecule medicine. Their distinctive physicochemical properties can lead to unique in vivo behaviors, including improved renal clearance and tumor distribution. Both the beneficial and potentially deleterious outcomes (cytotoxicity, inflammation) can, in principle, be controlled through a judicious choice of the nanocore shape and size, as well as the chemical ligands attached to the surface. At present, the ability to control the behavior of usNPs is limited, partly because advances are still needed in nanoengineering and chemical synthesis to manufacture and characterize ultrasmall nanostructures and partly because our understanding of their interactions in biological environments is incomplete. This review addresses the second limitation. We review experimental and computational methods currently available to understand molecular mechanisms, with particular attention to usNP-protein complexation, and highlight areas where further progress is needed. We discuss approaches that we find most promising to provide relevant molecular-level insight for designing usNPs with specific behaviors and pave the way to translational applications.

Circadian Modulation of Neurons and Astrocytes Controls Synaptic Plasticity in Hippocampal Area CA1.

Most animal species operate according to a 24-h period set by the suprachiasmatic nucleus (SCN) of the hypothalamus. The rhythmic activity of the SCN modulates hippocampal-dependent memory, but the molecular and cellular mechanisms that account for this effect remain largely unknown. Here, we identify cell-type-specific structural and functional changes that occur with circadian rhythmicity in neurons and astrocytes in hippocampal area CA1. Pyramidal neurons change the surface expression of NMDA receptors. Astrocytes change their proximity to synapses. Together, these phenomena alter glutamate clearance, receptor activation, and integration of temporally clustered excitatory synaptic inputs, ultimately shaping hippocampal-dependent learning in vivo. We identify corticosterone as a key contributor to changes in synaptic strength. These findings highlight important mechanisms through which neurons and astrocytes modify the molecular composition and structure of the synaptic environment, contribute to the local storage of information in the hippocampus, and alter the temporal dynamics of cognitive processing.

Quantitative mechanistic model for ultrasmall nanoparticle-protein interactions.

To date, extensive effort has been devoted toward the characterization of protein interactions with synthetic nanostructures. However, much remains to be understood, particularly concerning microscopic mechanisms of interactions. Here, we have conducted a detailed investigation of the kinetics of nanoparticle-protein complexation to gain deeper insights into the elementary steps and molecular events along the pathway for complex formation. Toward that end, the binding kinetics between p-mercaptobenzoic acid-coated ultrasmall gold nanoparticles (AuMBA) and fluorescently-labeled ubiquitin was investigated at millisecond time resolution using stopped-flow spectroscopy. It was found that both the association and dissociation kinetics consisted of multiple exponential phases, hence suggesting a complex, multi-step reaction mechanism. The results fit into a picture where complexation proceeds through the formation of a weakly-bound first-encounter complex with an apparent binding affinity (KD) of ∼9 µM. Encounter complex formation is followed by unimolecular tightening steps of partial desolvation/ion removal and conformational rearrangement, which, collectively, achieve an almost 100-fold increase in affinity of the final bound state (apparent KD ∼0.1 µM). The final state is found to be weakly stabilized, displaying an average lifetime in the range of seconds. Screening of the electrostatic forces at high ionic strength weakens the AuMBA-ubiquitin interactions by destabilizing the encounter complex, whereas the average lifetime of the final bound state remains largely unchanged. Overall, our rapid kinetics investigation has revealed novel quantitative insights into the molecular-level mechanisms of ultrasmall nanoparticle-protein interactions.

Regulation of Thrombin Activity with Ultrasmall Nanoparticles: Effects of Surface Chemistry.

Nanomaterials displaying well-tailored sizes and surface chemistries can provide novel ways with which to modulate the structure and function of enzymes. Recently, we showed that gold nanoparticles (AuNPs) in the ultrasmall size regime could perform as allosteric effectors inducing partial inhibition of thrombin activity. We now find that the nature of the AuNP surface chemistry controls the interactions to the anion-binding exosites 1 and 2 on the surface of thrombin, the allosterically induced changes to the active-site conformation, and, by extension, the enzymatic activity. Ultrasmall AuNPs passivated with p-mercaptobenzoic acid ligands (AuMBA) and a peptide-based (Ac-ECYN) biomimetic coat (AuECYN) were utilized in our investigations. Remarkably, we found that while AuMBA binds to exosites 1 and 2, AuECYN interacts primarily with exosite 2. It was further established that AuMBA behaves as a "mild denaturant" of thrombin leading to catalytic dysfunction over time. Conversely, AuECYN resembles a proper allosteric effector leading to partial and reversible inhibition of the activity. Collectively, our findings reveal how the distinct binding modes of different AuNP types may uniquely influence thrombin structure and catalysis. The present study further contributes to our understanding of how synthetic nanomaterials could be exploited in the allosteric regulation of enzymes.

Ultrasmall Gold Nanoparticles Coated with Zwitterionic Glutathione Monoethyl Ester: A Model Platform for the Incorporation of Functional Peptides.

Ultrasmall gold nanoparticles (AuNPs) are an emerging class of nanomaterials exhibiting distinctive physicochemical, molecular, and in vivo properties. Recently, we showed that ultrasmall AuNPs encompassing a zwitterionic glutathione monoethyl ester surface coating (AuGSHzwt) were highly resistant to aggregation and serum protein interactions. Herein, we performed a new set of biointeraction studies to gain a more fundamental understanding into the behavior of both pristine and peptide-functionalized AuGSHzwt in complex media. Using the model Strep-tag peptide (WSHPQFEK) as an integrated functional group, we established that AuGSHzwt could be conjugated with increasing numbers of Strep-tags by simple ligand exchange, which provides a generic approach for AuGSHzwt functionalization. It was found that the strep-tagged AuGSHzwt particles were highly resistant to nonspecific protein interactions and retained their targeting capability in biological fluid, displaying efficient binding to Streptactin receptors in nearly undiluted serum. However, AuGSHzwt functionalized with multiple Strep-tags displayed somewhat lower resistance to protein interactions and lower levels of binding to Streptactin than monofunctionalized AuGSHzwt under given conditions. These results underscore the need for optimizing ligand density onto the surface of ultrasmall AuNPs for improved performance. Collectively, our findings support ultrasmall AuGSHzwt as an attractive platform for engineering functional, protein-mimetic nanostructures capable of specific protein recognition within the complex biological milieu.

Allosteric inhibition of α-thrombin enzymatic activity with ultrasmall gold nanoparticles.

The catalytic activity of enzymes can be regulated by interactions with synthetic nanoparticles (NPs) in a number of ways. To date, however, the potential use of NPs as allosteric effectors has not been investigated in detail. Importantly, targeting allosteric (distal) sites on the enzyme surface could afford unique ways to modulate the activity, allowing for either enzyme activation, partial or full inhibition. Using p-mercaptobenzoic acid-coated ultrasmall gold NPs (AuMBA) and human α-thrombin as a model system, here we experimentally tested the hypothesis that enzyme activity could be regulated through ultrasmall NP interactions at allosteric sites. We show that AuMBA interacted selectively and reversibly around two positively charged regions of the thrombin surface (exosites 1 and 2) and away from the active site. NP complexation at the exosites transmitted long-range structural changes over to the active site, altering both substrate binding affinity and catalysis. Significantly, thrombin activity was partially reduced - but not completely inhibited - by interactions with AuMBA. These findings indicate that interactions of proteins with ultrasmall NPs may mimic a typical biomolecular complexation event, and suggest the prospect of using ultrasmall particles as synthetic receptors to allosterically regulate protein function.

Impact of soft protein interactions on the excretion, extent of receptor occupancy and tumor accumulation of ultrasmall metal nanoparticles: a compartmental model simulation.

Ultrasmall metal nanoparticles (NPs) are next-generation nano-based platforms for in vivo disease diagnosis and treatment. Due to their small size below the kidney filtration threshold and marked resistance to nonspecific serum protein adsorption, ultrasmall NPs can be rapidly excreted through the kidneys and escape liver uptake. However, although ultrasmall particles may be deemed highly resistant to protein adsorption, the real extent of this resistance is not known. Here, a simple compartmental model simulation was therefore implemented to understand how NP behavior in vivo could be modulated by soft, transient NP-plasma protein interactions characterized by dissociation constants in the millimolar range. In Model 1, ultrasmall NPs functionalized with a targeting probe, plasma proteins and target receptors were assumed to co-exist within a single compartment. Simulations were performed to understand the synergistic effect of soft interactions, systemic clearance and NP size on receptor occupancy in the single compartment. The results revealed the existence of a narrow range of ultraweak affinities and optimal particle sizes leading to greater target occupancy. In Model 2, simulations were performed to understand the impact of soft interactions on NP accumulation into a peripheral (tumor) compartment. The results revealed that soft interactions - but not active targeting - enhanced tumor uptake levels when tumor accumulation was limited by 'fast' plasma clearance and 'slow' vascular extravasation. The simple model presented here provides a basic framework to quantitatively understand the blood and tumor pharmacokinetics of ultrasmall NPs under the influence of transient protein interactions.

Identification of PSD-95 in the Postsynaptic Density Using MiniSOG and EM Tomography.

Combining tomography with electron microscopy (EM) produces images at definition sufficient to visualize individual protein molecules or molecular complexes in intact neurons. When freeze-substituted hippocampal cultures in plastic sections are imaged by EM tomography, detailed structures emerging from 3D reconstructions reveal putative glutamate receptors and membrane-associated filaments containing scaffolding proteins such as postsynaptic density (PSD)-95 family proteins based on their size, shape, and known distributions. In limited instances, structures can be identified with enhanced immuno-Nanogold labeling after light fixation and subsequent freeze-substitution. Molecular identification of structure can be corroborated in their absence after acute protein knockdown or gene knockout. However, additional labeling methods linking EM level structure to molecules in tomograms are needed. A recent development for labeling structures for TEM employs expression of endogenous proteins carrying a green fluorescent tag, miniSOG, to photoconvert diaminobenzidine (DAB) into osmiophilic polymers. This approach requires initial mild chemical fixation but many of structural features in neurons can still be discerned in EM tomograms. The photoreaction product, which appears as electron-dense, fine precipitates decorating protein structures in neurons, may diffuse to fill cytoplasm of spines, thus obscuring specific localization of proteins tagged with miniSOG. Here we develop an approach to minimize molecular diffusion of the DAB photoreaction product in neurons, which allows miniSOG tagged molecule/complexes to be identified in tomograms. The examples reveal electron-dense clusters of reaction product labeling membrane-associated vertical filaments, corresponding to the site of miniSOG fused at the C-terminal end of PSD-95-miniSOG, allowing identification of PSD-95 vertical filaments at the PSD. This approach, which results in considerable improvement in the precision of labeling PSD-95 in tomograms without complications due to the presence of antibody complexes in immunogold labeling, may be applicable for identifying other synaptic proteins in intact neurons.

Binding kinetics of ultrasmall gold nanoparticles with proteins.

Synthetic ultrasmall nanoparticles (NPs) can be designed to interact with biologically active proteins in a controlled manner. However, the rational design of NPs requires a clear understanding of their interactions with proteins and the precise molecular mechanisms that lead to association/dissociation in biological media. Although much effort has been devoted to the study of the kinetics mechanism of protein corona formation on large NPs, the nature of NP-protein interactions in the ultrasmall regime is radically different and poorly understood. Using a combination of experimental and computational approaches, we studied the interactions of a model protein, CrataBL, with ultrasmall gold NPs passivated with p-mercaptobenzoic acid (AuMBA) and glutathione (AuGSH). We have identified this system as an ideal in vitro platform to understand the dependence of binding affinity and kinetics on NP surface chemistry. We found that the structural and chemical complexity of the passivating NP layer leads to quite different association kinetics, from slow and reaction-limited (AuGSH) to fast and diffusion-limited (AuMBA). We also found that the otherwise weak and slow AuGSH-protein interactions measured in buffer solution are enhanced in macromolecular crowded solutions. These findings advance our mechanistic understanding of biomimetic NP-protein interactions in the ultrasmall regime and have implications for the design and use of NPs in the crowded conditions common to all biological media.

PAR1 activation induces rapid changes in glutamate uptake and astrocyte morphology.

The G-protein coupled, protease-activated receptor 1 (PAR1) is a membrane protein expressed in astrocytes. Fine astrocytic processes are in tight contact with neurons and blood vessels and shape excitatory synaptic transmission due to their abundant expression of glutamate transporters. PAR1 is proteolytically-activated by bloodstream serine proteases also involved in the formation of blood clots. PAR1 activation has been suggested to play a key role in pathological states like thrombosis, hemostasis and inflammation. What remains unclear is whether PAR1 activation also regulates glutamate uptake in astrocytes and how this shapes excitatory synaptic transmission among neurons. Here we show that, in the mouse hippocampus, PAR1 activation induces a rapid structural re-organization of the neuropil surrounding glutamatergic synapses, which is associated with faster clearance of synaptically-released glutamate from the extracellular space. This effect can be recapitulated using realistic 3D Monte Carlo reaction-diffusion simulations, based on axial scanning transmission electron microscopy (STEM) tomography reconstructions of excitatory synapses. The faster glutamate clearance induced by PAR1 activation leads to short- and long-term changes in excitatory synaptic transmission. Together, these findings identify PAR1 as an important regulator of glutamatergic signaling in the hippocampus and a possible target molecule to limit brain damage during hemorrhagic stroke.

Physicochemical characterization of ferumoxytol, heparin and protamine nanocomplexes for improved magnetic labeling of stem cells.

Stem cell-based therapies have become a major focus in regenerative medicine and to treat diseases. A straightforward approach combining three drugs, heparin (H), protamine (P) with ferumoxytol (F) in the form of nanocomplexes (NCs) effectively labeled stem cells for cellular MRI. We report on the physicochemical characteristics for optimizing the H, P, and F components in different ratios, and mixing sequences, producing NCs that varied in hydrodynamic size. NC size depended on the order in which drugs were mixed in media. Electron microscopy of HPF or FHP showed that F was located on the surface of spheroidal shaped HP complexes. Human stem cells incubated with FHP NCs resulted in a significantly greater iron concentration per cell compared to that found in HPF NCs with the same concentration of F. These results indicate that FHP could be useful for labeling stem cells in translational studies in the clinic.

Biointeractions of ultrasmall glutathione-coated gold nanoparticles: effect of small size variations.

Recent in vivo studies have established ultrasmall (<3 nm) gold nanoparticles coated with glutathione (AuGSH) as a promising platform for applications in nanomedicine. However, systematic in vitro investigations to gain a more fundamental understanding of the particles' biointeractions are still lacking. Herein we examined the behavior of ultrasmall AuGSH in vitro, focusing on their ability to resist aggregation and adsorption from serum proteins. Despite having net negative charge, AuGSH particles were colloidally stable in biological media and able to resist binding from serum proteins, in agreement with the favorable bioresponses reported for AuGSH in vivo. However, our results revealed disparate behaviors depending on nanoparticle size: particles between 2 and 3 nm in core diameter were found to readily aggregate in biological media, whereas those strictly under 2 nm were exceptionally stable. Molecular dynamics simulations provided microscopic insight into interparticle interactions leading to aggregation and their sensitivity to the solution composition and particle size. These results have important implications, in that seemingly small variations in size can impact the biointeractions of ultrasmall AuGSH, and potentially of other ultrasmall nanoparticles as well.

PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density.

The postsynaptic density (PSD)-95 family of membrane-associated guanylate kinases (MAGUKs) are major scaffolding proteins at the PSD in glutamatergic excitatory synapses, where they maintain and modulate synaptic strength. How MAGUKs underlie synaptic strength at the molecular level is still not well understood. Here, we explore the structural and functional roles of MAGUKs at hippocampal excitatory synapses by simultaneous knocking down PSD-95, PSD-93, and synapse-associated protein (SAP)102 and combining electrophysiology and transmission electron microscopic (TEM) tomography imaging to analyze the resulting changes. Acute MAGUK knockdown greatly reduces synaptic transmission mediated by α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) and N-methyl-d-aspartate receptors (NMDARs). This knockdown leads to a significant rise in the number of silent synapses, diminishes the size of PSDs without changes in pre- or postsynaptic membrane, and depletes the number of membrane-associated PSD-95-like vertical filaments and transmembrane structures, identified as AMPARs and NMDARs by EM tomography. The differential distribution of these receptor-like structures and dependence of their abundance on PSD size matches that of AMPARs and NMDARs in the hippocampal synapses. The loss of these structures following MAGUK knockdown tracks the reduction in postsynaptic AMPAR and NMDAR transmission, confirming the structural identities of these two types of receptors. These results demonstrate that MAGUKs are required for anchoring both types of glutamate receptors at the PSD and are consistent with a structural model where MAGUKs, corresponding to membrane-associated vertical filaments, are the essential structural proteins that anchor and organize both types of glutamate receptors and govern the overall molecular organization of the PSD.

A Note on the use of Steady-State Fluorescence Quenching to Quantify Nanoparticle-Protein Interactions.

Steady-state fluorescence quenching is a commonly used technique to investigate the interactions between proteins and nanoparticles, providing quantitative information on binding affinity, stoichiometry and cooperativity. However, a failure to account for the limitations and pitfalls of the methodology can lead to significant errors in data analysis and interpretation. Thus, in this communication we first draw attention to a few common pitfalls in the use of fluorescence quenching to study nanoparticle-protein interactions. For example, we discuss a frequent mistake in the use of the Hill equation to determine cooperativity. We also test using both simulated and experimental data the application of a model-independent method of analysis to generate true thermodynamic nanoparticle-protein binding isotherms. This model-free approach allows for a quantitative description of the interactions independent of assumptions about the nature of the binding process [Bujalowski W, Lohman TM (1987) Biochemistry 26: 3099; Schwarz G (2000) Biophys. Chem. 86: 119].

Passive diffusion as a mechanism underlying ribbon synapse vesicle release and resupply.

Synaptic ribbons are presynaptic protein structures found at many synapses that convey graded, "analog" sensory signals in the visual, auditory, and vestibular pathways. Ribbons, typically anchored to the presynaptic membrane and surrounded by tethered synaptic vesicles, are thought to regulate or facilitate vesicle delivery to the presynaptic membrane. No direct evidence exists, however, to indicate how vesicles interact with the ribbon or, once attached, move along the ribbon's surface to reach the presynaptic release sites at its base. To address these questions, we have created, validated, and tested a passive vesicle diffusion model of retinal rod bipolar cell ribbon synapses. We used axial (bright-field) electron tomography in the scanning transmission electron microscopy to obtain 3D structures of rat rod bipolar cell terminals in 1-µm-thick sections of retinal tissue at an isotropic spatial resolution of ∼3 nm. The resulting structures were then incorporated with previously published estimates of vesicle diffusion dynamics into numerical simulations that accurately reproduced electrophysiologically measured vesicle release/replenishment rates and vesicle pool sizes. The simulations suggest that, under physiologically realistic conditions, diffusion of vesicles crowded on the ribbon surface gives rise to a flow field that enhances delivery of vesicles to the presynaptic membrane without requiring an active transport mechanism. Numerical simulations of ribbon-vesicle interactions predict that transient binding and unbinding of multiple tethers to each synaptic vesicle may achieve sufficiently tight association of vesicles to the ribbon while permitting the fast diffusion along the ribbon that is required to sustain high release rates.