Multifaceted Characterization for the Hepatic Clearance of Graphene Oxide and Size-Related Hepatic Toxicity.

Understanding the final fate of nanomaterials (NMs) in the liver is crucial for their safer application. As a representative two-dimensional (2D) soft nanomaterial, graphene oxide (GO) has shown to have high potential for applications in the biomedical field, including in biosensing, drug delivery, tissue engineering, therapeutics, etc. GO has been shown to accumulate in the liver after entering the body, and thus, understanding the GO-liver interaction will facilitate the development of safer bio-applications. In this study, the hepatic clearance of two types of PEGylated GOs with different lateral sizes (s-GOs: ~70 nm and l-GOs: ~300 nm) was carefully investigated. We found that GO sheets across the hepatic sinusoidal endothelium, which then may be taken up by the hepatocytes via the Disse space. The hepatocytes may degrade GO into dot-like particles, which may be excreted via the hepatobiliary route. In combination with ICP-MS, LA-ICP-MS, and synchrotron radiation FTIR techniques, we found that more s-GO sheets in the liver were prone to be cleared via hepatobiliary excretion than l-GO sheets. A Raman imaging analysis of ID/IG ratios further indicated that both s-GO and l-GO generated more defects in the liver. The liver microsomes may contribute to GO biotransformation into O-containing functional groups, which plays an important role in GO degradation and excretion. In particular, more small-sized GO sheets in the liver were more likely to be cleared via hepatobiliary excretion than l-GO sheets, and a greater clearance of s-GO will mitigate their hepatotoxicity. These results provide a better understanding of the hepatic clearance of soft NMs, which is important in the safer-by-design of GO.

Clusterbody Enables Flow Sorting-Assisted Single-Cell Mass Spectrometry Analysis for Identifying Reversal Agent of Chemoresistance.

Identifying effective reversal agents overcoming multidrug resistance with causal mechanisms from an efflux pump protein is of vital importance for enhanced tumor chemotherapy in clinic. To achieve this end, we construct a metal cluster-based probe, named clusterbody, to develop flow sorting-assisted single-cell mass spectrometry analysis. This clusterbody synthesized by biomimetic mineralization possesses an antibody-like property to selectively recognize an efflux pump protein. The intrinsic red fluorescence emission of the clusterbody facilitates fluorescence-activated high-throughput cell sorting of subpopulations with different multidrug resistance levels. Furthermore, based on the accurate formula of the clusterbody, the corresponding protein abundance at the single-cell level is determined through detecting gold content via precise signal amplification by laser ablation inductively coupled plasma mass spectrometry. Therefore, the effect of reversal agent treatment overcoming multidrug resistance is evaluated in a quantitative manner. This work opens a new avenue to identify reversal agents, shedding light on developing combined or synergetic tumor therapy.

Renal clearance of graphene oxide: glomerular filtration or tubular secretion and selective kidney injury association with its lateral dimension.

BACKGROUND: Renal excretion is one of the major routes of nanomaterial elimination from the body. Many previous studies have found that graphene oxide nanosheets are excreted in bulk through the kidneys. However, how the lateral size affects GO disposition in the kidneys including glomerular filtration, active tubular secretion and tubular reabsorption is still unknown. RESULTS: The thin, two-dimensional graphene oxide nanosheets (GOs) was observed to excrete in urine through the kidneys, but the lateral dimension of GOs affects their renal clearance pathway and renal injury. The s-GOs could be renal excreted via the glomerular filtration, while the l-GOs were predominately excreted via proximal tubular secretion at a much faster renal clearance rate than the s-GOs. For the tubular secretion of l-GOs, the mRNA level of basolateral organic anion transporters Oat1 and Oat2 in the kidney presented dose dependent increase, while no obvious alterations of the efflux transporters such as Mdr1 and Mrp4 mRNA expression levels were observed, suggesting the accumulation of l-GOs. During the GO renal elimination, mostly the high dose of 15 mg/kg s-GO and l-GO treatment showed obvious kidney injuries but at different renal compartment, i.e., the s-GOs induced obvious glomerular changes in podocytes, while the l-GOs induced more obvious tubular injuries including necrosis of renal tubular epithelial cells, loss of brush border, cast formation and tubular dilatation. The specifically tubular injury biomarkers KIM1 and NGAL were shown slight increase with mRNA levels in l-GO administrated mice. CONCLUSIONS: This study shows that the lateral size of GOs affected their interactions with different renal compartments, renal excretion pathways and potential kidney injuries.

Understanding the Role of the Lateral Dimensional Property of Graphene Oxide on Its Interactions with Renal Cells.

Renal excretion is expected to be the major route for the elimination of biomedically applied nanoparticles from the body. Hence, understanding the nanomedicine-kidney interaction is crucially required, but it is still far from being understood. Herein, we explored the lateral dimension- (~70 nm and ~300 nm), dose- (1, 5, and 15 mg/kg in vivo and 0.1~250 µg/mL in vitro), and time-dependent (48 h and 7 d in vivo) deposition and injury of PEGylated graphene oxide sheets (GOs) in the kidney after i.v. injection in mice. We specially investigated the cytotoxic effects on three typical kidney cell types with which GO renal excretion is related: human renal glomerular endothelial cells (HRGECs) and human podocytes, and human proximal tubular epithelial cells (HK-2). By using in vivo fluorescence imaging and in situ Raman imaging and spectroscopic analysis, we revealed that GOs could gradually be eliminated from the kidneys, where the glomeruli and renal tubules are their target deposition sites, but only the high dose of GO injection induced obvious renal histological and ultrastructural changes. We showed that the high-dose GO-induced cytotoxicity included a cell viability decrease and cellular apoptosis increase. GO uptake by renal cells triggered cellular membrane damage (intracellular LDH release) and increased levels of oxidative stress (ROS level elevation and a decrease in the balance of the GSH/GSSG ratio) accompanied by a mitochondrial membrane potential decrease and up-regulation of the expression of pro-inflammatory cytokines TNF-α and IL-18, resulting in cellular apoptosis. GO treatments activated Keap1/Nrf2 signaling; however, the antioxidant function of Nrf2 could be inhibited by apoptotic engagement. GO-induced cytotoxicity was demonstrated to be associated with oxidative stress and an inflammation reaction. Generally, the l-GOs presented more pronounced cytotoxicity and more severe cellular injury than s-GOs did, demonstrating lateral size-dependent toxicity to the renal cells. More importantly, GO-induced cytotoxicity was independent of renal cell type. The results suggest that the dosage of GOs in biomedical applications should be considered and that more attention should be paid to the ability of a high dose of GO to cause renal deposition and potential nephrotoxicity.

Single-Cell Isotope Dilution Analysis with LA-ICP-MS: A New Approach for Quantification of Nanoparticles in Single Cells.

Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) is an emerging method for the analysis of metal nanoparticles (NPs) in single cells. However, two main obstacles, low analytical throughput and lack of commercial reference materials, need to be overcome. In this work, we demonstrated the principles of a new approach termed "single-cell isotope dilution analysis" (SCIDA) to remove the two obstacles. For a proof of concept, macrophage cells were chosen as a model to study the uptake of silver NPs (AgNPs) at a single-cell level. Single cells exposed to AgNPs were placed in an array by a microfluidic technique; each cell in the array was precisely dispensed with a known picoliter droplet of an enriched isotope solution with a commercial inkjet printer; accurate quantification of AgNPs in single cells was done by using isotope dilution LA-ICP-MS. The average Ag mass of 1100 single cells, 396 ± 219 fg Ag per cell, was in good accord with the average of the population of cells determined by solution ICP-MS analysis. The detection limit was 0.2 fg Ag per cell. The SCIDA approach is expected to be widely applied for the study of cell-NP interactions and biological effects of NPs at the single-cell level.

Adsorption and oxidation of SO2 on the surface of TiO2 nanoparticles: the role of terminal hydroxyl and oxygen vacancy-Ti3+ states.

Herein, the absorption and oxidation reactions of SO2 on TiO2 nanoparticles (TiO2 NPs) at 296 K under various environmental conditions (humidity, UV irradiation, and ozone copresence) were investigated by using a flow chamber reaction system, synchrotron X-ray absorption near-edge structure (XANES) and high resolution synchrotron X-ray photoelectron spectroscopy (XPS) measurements. The results showed that oxidation of SO2 to sulfate via TiO2 NP catalysis happened at a very rapid rate. The appropriate relative humidity, UV irradiation and co-presence of ozone all markedly promoted SO2 oxidation on TiO2 NPs. High resolution XPS unraveled that the terminal hydroxyl (OHt) and oxygen vacancy (VO)-Ti3+ states on TiO2 NPs were the active sites for SO2 adsorption and oxidation. The data of XPS measurements suggest that SO2 was adsorbed on a OHt next to a Ti3+ VO and reacted to form HSO3-. HSO3- can then transform into SO32-via transfer of a proton. The resulting adsorbed SO32- could bind to a surface bridging O (Ob) atom and transform into SO42-. A H2O molecule could dissociate on VO-Ti3+ into two bridging hydroxyl (OHb) groups, subsequently forming new Ob, which provides an active O site for the adsorbed HSO3-/SO32- and oxidizes them into HSO4-/SO42- on the surface of the TiO2 NPs. The copresence of O3 could promote H2O dissociation into OHb, promoting the formation of Ob. The copresence of O3 may also promote the dissociation of adsorbed H2O into TiO2-O2- and hydroxyl radicals (˙OH) on VOs, facilitating the oxidation of adsorbed HSO3-/SO32-. Under UV irradiation, new VOs were created via oxidation of lattice O by photo-generated holes, resulting in increased Ob and subsequently enhanced oxidation of adsorbed HSO3-/SO32- on TiO2 NPs.

Surface chemistry governs the sub-organ transfer, clearance and toxicity of functional gold nanoparticles in the liver and kidney.

BACKGROUND: To effectively applied nanomaterials (NMs) in medicine, one of the top priorities is to address a better understanding of the possible sub-organ transfer, clearance routes, and potential toxicity of the NMs in the liver and kidney. RESULTS: Here we explored how the surface chemistry of polyethylene glycol (PEG), chitosan (CS), and polyethylenimine (PEI) capped gold nanoparticles (GNPs) governs their sub-organ biodistribution, transfer, and clearance profiles in the liver and kidney after intravenous injection in mice. The PEG-GNPs maintained dispersion properties in vivo, facilitating passage through the liver sinusoidal endothelium and Disse space, and were captured by hepatocytes and eliminated via the hepatobiliary route. While, the agglomeration/aggregation of CS-GNPs and PEI-GNPs in hepatic Kupffer and endothelial cells led to their long-term accumulation, impeding their elimination. The gene microarray analysis shows that the accumulation of CS-GNPs and PEI-GNPs in the liver induced obvious down-regulation of Cyp4a or Cyp2b related genes, suggesting CS-GNP and PEI-GNP treatment impacted metabolic processes, while the PEI-GNP treatment is related with immune responses. CONCLUSIONS: This study demonstrates that manipulation of nanoparticle surface chemistry can help NPs selectively access distinct cell types and elimination pathways, which help to clinical potential of non-biodegradable NPs.

Elemental analysis and imaging of sunscreen fingermarks by X-ray fluorescence.

Chemical composition in fingermarks could provide useful information for forensic studies and applications. Here, we evaluate the feasibility of analysis and imaging of fingermarks via elements by synchrotron radiation X-ray fluorescence (SRXRF) and commercial X-ray fluorescence (XRF). As a proof of concept, we chose four brands of sunscreens to make fingermarks on different substrates, including plastic film, glass, paper, and silicon wafer. We obtained an evident image of fingermarks via zinc and titanium by XRF methods. In addition, the ratios of element concentrations in sunscreen fingermarks were obtained, which were in accordance with the results obtained by acid digestion and ICP-OES analysis. In comparison, commercial XRF offers the most advantages in terms of non-destructive detection, easy accessibility, fast element images, and broad applicability. The possibility to acquire fingermark images simultaneously with element information opens up new avenues for forensic science. Graphical abstract.

Thermal Unfolding Process of Lysozyme on PEGylated Gold Nanoparticles Reveals Length-Dependent Effects of PEG Layer.

Characterization of bio-nano interface is crucial for developing safer and more efficient nanoparticles in nanomedical application. PEGylation is commonly used in nanocarrier for drug delivery, as it confers nanoparticles good stability, stealth effect and better targeting specificity compared with those without PEGylation. However, the protein binding state on PEGylated AuNP is still limited known. In present work, we prepared 13 nm AuNPs and then PEGylated them with thiol PEG methoxy. Lysozyme is selected as a model protein and to investigate the interactions on protein-PEGylated/AuNP interface. The thermal unfolding processes of lysozyme in absence and presence of PEGylated AuNP were measured by synchrotron radiation based circular dichroism (SRCD), dynamic light scattering (DLS) and infrared spectroscopy (IR). The results suggest that in terms of secondary structural changes, α helix content is decreased, while ß sheet content is increased, and thus the adsorbed lysozyme may be present in PEG layer.

Transferrin Adsorbed on PEGylated Gold Nanoparticles and Its Relevance to Targeting Specificity.

Polyethylene glycol (PEG) has been frequently used for surface modification of nanoparticles (NPs) to reduce non-specific binding of proteins on NPs. The investigation of protein absorption on PEGylated nanoparticles is necessary. In the work, the conjugation of transferrin (Tf) to PEGylated AuNPs via adsorption or bonding was studied. The 13 nm AuNPs were coated with various molecular weight (300, 2000, 5000) carboxyl and methoxy PEG thiol. The presence of Tf on PEGylated AuNP was characterized by dynamic light scattering (DLS) and infrared spectroscopy (IR). The data of IR confirmed the presence of Tf on PEGylated AuNPs. The diameter decrease of PEGylated AuNPs after Tf adsorption was observed by DLS measurement, which is attributed to competitive adsorption between Tf and PEG molecules. These phenomena may be important to the preservation of Tf targeting specificity on PEGylated AuNPs.

Inhibition of Lysozyme Fibrillation by Gold Nanorods and Nanoparticles.

Amyloid fibrillation has been implicated in many neurodegenerations, dialysis-related amyloidosis, type II diabetes and more than 30 other amyloid-related diseases. Nanomaterials as potential inhibitors of amyloid fibrillation have attracted increasing interests. In the present study, the effects of gold nanorods (AuNRs) and nanoparticles (AuNPs) on amyloid fibrillation were investigated using hen egg white lysozyme (HEWL) as a model system. Our results indicated that AuNRs and AuNPs, especially AuNRs, present significant inhibitory effects on HEWL amyloid fibril formation during all the kinetic processes, from nucleation to elongation and equilibration stages. The stronger adsorption capacity of HEWL on AuNRs surface is the key mechanism of inhibition of HEWL amyloid fibrillation. Furthermore, AuNRs lead to more stable α-helix conformation and hydrophobic microenvironment of aromatic side groups in HEWL molecules, which facilitate the system to form small amorphous aggregates rather than oligomer, profibril or mature fibril.

Interrogating the variation of element masses and distribution patterns in single cells using ICP-MS with a high efficiency cell introduction system.

Cellular heterogeneity is an inherent condition of cell populations, which results from stochastic expression of genes, proteins, and metabolites. The heterogeneity of individual cells can dramatically influence cellular decision-making and cell fate. So far, our knowledge about how the variation of endogenous metals and non-metals in individual eukaryotic cells is limited. In this study, ICP-MS equipped with a high efficiency cell introduction system (HECIS) was developed as a method of single-cell ICP-MS (SC-ICP-MS). The method was applied to the single-cell analysis of Mn, Fe, Co, Cu, Zn, P, and S in human cancer cell lines (HeLa and A549) and normal human bronchial epithelial cell line (16HBE). The analysis showed obvious variation of the masses of Cu, Fe, Zn, and P in individual HeLa cells, and variation of Fe, Zn, and P in individual A549 cells. On the basis of the single-cell data, a multimodal distribution of the elements in the cell population was fitted, which showed marked differences among the various cell lines. Importantly, subpopulations of the elements were found in the cell populations, especially in the HeLa cancer cells. This study demonstrates that SC-ICP-MS is able to unravel the extent of variation of endogenous elements in individual cells, which will help to improve our fundamental understanding of cellular biology and reveal novel insights into human biology and medicine. Graphical abstract The variations of masses and distribution patterns of elements Mn, Fe, Co, Cu, Zn, P, and S in single cells were successfully detected by ICP-MS coupled with a high efficiency cell introduction system (HECIS).

Magnetic Fe3O4 nanoparticle catalyzed chemiluminescence for detection of nitric oxide in living cells.

Direct and real-time measurement of nitric oxide (NO) in biological media is very difficult due to its transient nature. Fe3O4 nanoparticles (nanoFe3O4) because of their unique catalytic activities have attracted much attention as catalysts in a variety of organic and inorganic reactions. In this work, we have developed a magnetic Fe3O4 nanoparticle-based rapid-capture system for real-time detection of cellular NO. The basic principle is that the nanoFe3O4 can catalyze the decomposition of H2O2 in the system to generate superoxide anion (O2 (·-)) and the O2 (·-) can serve as an effective NO(·) trapping agent yielding peroxynitrite oxide anion, ONOO(-). Then the concentration of NO in cells can be facilely determined via peroxynitrite-induced luminol chemiluminescence. The linear range of the method is from 10(-4) to 10(-8) mol/L, and the detection of limit (3σ, n = 11) is as low as 3.16 × 10(-9) mol/L. By using this method, the NO concentration in 0.1 and 0.5 mg/L LPS-stimulated BV2 cells was measured as 4.9 and 11.3 µM, respectively. Surface measurements by synchrotron X-ray photoelectron spectroscopy (SRXPS) and scanning transmission X-ray microscopy (STXM) demonstrate the catalytic mechanism of the nanoFe3O4-based system is that the significantly excess Fe(II) exists on the surface of nanoFe3O4 and mediates the rapid heterogeneous electron transfer, thus presenting a new Fe2O3 phase on the surface.

Size-Dependent Translocation Pattern, Chemical and Biological Transformation of Nano- and Submicron-Sized Ferric Oxide Particles in the Central Nervous System.

The present study investigated the size-dependent translocation pattern and biological fate of intranasally instilled nano- and submicron-sized Fe2O3 particles (40 nm and 280 nm) in the CNS. The particle translocation in different parts of brain at 4 h, 12 h, 24 h, 3 d, 7 d, and 30 d after intranasal instillation were quantified using ICP-MS method. A biexponential model (correlation coefficient r = 0.98-0.99) was satisfactory to describe the particokinetic translocation behavior of Fe2O3 nanoparticles in brain. We found a size-dependent translocation pattern and a time-dependent translocation mode for nano- and submicron-sized Fe2O3 nanoparticles in the olfactory bulb, which are most significant in toxic concerns of nanoparticles in the CNS. The TEM images showed particle-like substances of approximately 35-50 nm were located in the axons of olfactory neurons and in the mitochondria and lysosomes of hippocampus cells in the 40 nm-Fe2O3 exposed mice. The synchrotron-based near-edge X-ray absorption spectroscopy (XANES) was used to identify the chemical forms of the nanoparticles in brain. The XANES results indicate that the presence of chemical speciation of the Fe2O3 nanoparticle (-17%) and protein-complex like apotransferrin-Fe2O3 (-16%) in the olfactory bulb, implying that self-coating of Fe2O3 nanoparticles with transferrin occurred in brain. All the findings suggest size-sensitive manners of nano- and submicron-sized Fe2O3 particles in the brain; the smaller one possesses evident detention properties in the CNS versus the larger one.

Facile approach to observe and quantify the α(IIb)ß3 integrin on a single-cell.

We report for the first time seeing and counting integrin α(IIb)ß3 on a single-cell level. The proposed method is based on the using of the Au cluster probe. With the fluorescent property of Au24 cluster and the specific targeting ability of peptide, our probe can directly visualize integrin α(IIb)ß3 on the membrane of human erythroleukemia cells (HEL) via confocal microscopy. On the basis of the accurate formula of our probe (Au24Peptide8), the number of integrin α(IIb)ß3 can be precisely counted by quantifying the gold content on a single HEL cell via laser ablation inductively coupled plasma mass spectrometry. Our results reveal that the number of integrin α(IIb)ß3 on a single cell varies from 5.75 × 10(-17) to 9.11 × 10(-17) mol, because of the heteroexpression levels of α(IIb)ß3 on individual cells.

Coculture with Low-Dose SWCNT Attenuates Bacterial Invasion and Inflammation in Human Enterocyte-like Caco-2 Cells.

Single walled carbon nanotubes (SWCNTs) have been shown to be highly effective against a wide range of bacteria. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) infection is a well-known mediator to prolong hospitalization and initiate chronic inflammation, yet the biological effects of SWCNTs on the pathogen-infected enterocytes remain unclear. Herein, it is shown that the low-dose SWCNT treatment attenuates the human enterocyte-like Caco-2 cells from the damage of E. coli and S. aureus infection by suppressing NLRP3 inflammasome activation. The relatively low-dose (1 and 10 µg mL(-1) ) SWCNT treatments reduce the adhesion and invasion of E. coli and S. aureus to Caco-2 cells, increase the cell viability and proliferation, reduce the tight junction permeability, and restitute the integrity of cell surface microvilli structure, meanwhile has low cytotoxicity to the host cells. The low-dose SWCNT treatment further reduces the NLRP3-mediated IL-1ß secretion in the infected cells. The results identify that a low-dose SWCNT treatment serves a protective function for the E. coli- and S. aureus-infected Caco-2 cells by negatively regulating mitochondrial reactive oxygen species-mediated NLRP3 inflammasome activation.

Time-resolved ICP-MS analysis of mineral element contents and distribution patterns in single cells.

Novel single cell techniques are attracting growing interest for clinical applications, because they can elucidate the cellular diversity and heterogeneity instead of the average masked by bulk measurements. Herein, time-resolved ICP-MS for the determination of essential mineral elements in single cells has been developed and is used to analyze the contents and distribution patterns of Fe, Cu, Zn, Mn, P and S in two types of cancer cells (HeLa and A549) and one type of normal cells (16HBE). The results show that there are obvious differences in contents and distribution patterns of the elements among the three types of cells. The mass of Fe, Zn, Cu, Mn, P, and S in individual HeLa cells is significantly higher and span a broader range of values than in the single 16HBE and A549 cells. The contents of Fe, Zn, and Cu follow log-normal distributions, and Mn, P, and S follow Poisson distributions with high λ values in single HeLa cells, indicating a large cell-to-cell variance. Comparatively, the contents of Cu, Zn, P, and S in 16HBE cells show the narrowest distribution range among the three tested cells, demonstrating the homogenous distribution of the elements in the cells. The method of single cell ICP-MS (SC-ICP-MS) provides potential applications for the monitoring of the variation of mineral elements at a single cell level.

Quantitative analysis of Gd@C82(OH)22 and cisplatin uptake in single cells by inductively coupled plasma mass spectrometry.

Cisplatin is a commonly used chemotherapeutic drug in cancer treatment, whereas Gd@C82(OH)22 is a new nanomaterial anti-tumor agent. In this study, we determined intracellular Gd@C82(OH)22 and cisplatin after treatment of Hela and 16HBE cells by single cell inductively coupled plasma-mass spectrometry (SC-ICP-MS), which could provide quantitative information at a single-cell level. The cell digestion method validated the accuracy of the SC-ICP-MS. The concentrations of Gd@C82(OH)22 and cisplatin in cells at different exposure times and doses were studied. The SC-ICP-MS is a promising complement to available methods for single cell analysis and is anticipated to be applied further to biomedical research.

Quantitative analysis of gold nanoparticles in single cells by laser ablation inductively coupled plasma-mass spectrometry.

Single cell analysis has become an important field of research in recent years reflecting the heterogeneity of cellular responses in biological systems. Here, we demonstrate a new method, based on laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS), which can quantify in situ gold nanoparticles (Au NPs) in single cells. Dried residues of picoliter droplets ejected by a commercial inkjet printer were used to simulate matrix-matched calibration standards. The gold mass in single cells exposed to 100 nM NIST Au NPs (Reference material 8012, 30 nm) for 4 h showed a log-normal distribution, ranging from 1.7 to 72 fg Au per cell, which approximately corresponds to 9 to 370 Au NPs per cell. The average result from 70 single cells (15 ± 13 fg Au per cell) was in good agreement with the result from an aqua regia digest solution of 1.2 × 10(6) cells (18 ± 1 fg Au per cell). The limit of quantification was 1.7 fg Au. This paper demonstrates the great potential of LA-ICPMS for single cell analysis and the beneficial study of biological responses to metal drugs or NPs at the single cell level.

Metabolism of nanomaterials in vivo: blood circulation and organ clearance.

Before researchers apply nanomaterials (NMs) in biomedicine, they need to understand the blood circulation and clearance profile of these materials in vivo. These qualities determine the balance between nanomaterial-induced activity and unwanted toxicity. NMs have heterogeneous characteristics: they combine the bulk properties of solids with the mobility of molecules, and their highly active contact interfaces exhibit diverse functionalities. Any new and unexpected circulation features and clearance patterns are of great concern in toxicological studies and pharmaceutical screens. A number of studies have reported that NMs can enter the bloodstream directly during their application or indirectly via inhalation, ingestion, and dermal exposure. Due to the small size of NMs, the blood can then transport them throughout the circulation and to many organs where they can be stored. In this Account, we discuss the blood circulation and organ clearance patterns of NMs in the lung, liver, and kidney. The circulation of NMs in bloodstream is critical for delivery of inhalable NMs to extrapulmonary organs, the delivery of injectable NMs, the dynamics of tissue redistribution, and the overall targeting of drug carriers to specific cells and organs. The lung, liver, and kidney are the major distribution sites and target organs for NMs exposure, and the clearance patterns of NMs in these organs are critical for understanding the in vivo fate of NMs. Current studies suggest that multiple factors control the circulation and organ clearance of NMs. The size, shape, surface charge, surface functional groups, and aspect ratio of NMs as well as tissue microstructures strongly influence the circulation of NMs in bloodstream, their site-specific extravasation, and their clearance profiles within organs. Therefore structure design and surface modification can improve biocompatibility, regulate the in vivo metabolism, and reduce the toxicity of NMs. The biophysicochemical interactions occurring between NMs and between NMs and the biological milieu after the introduction of NMs into living systems may further influence the blood circulation and clearance profiles of NMs. These interactions can alter properties such as agglomeration, phase transformations, dissolution, degradation, protein adsorption, and surface reactivity. The physicochemical properties of NMs change dynamically in vivo thereby making the metabolism of NMs complex and difficult to predict. The development of in situ, real-time, and quantitative techniques, in vitro assays, and the adaptation of physiologically-based pharmacokinetic (PBPK) and quantitative structure-activity relationship (QNSAR) modeling for NMs will streamline future in vivo studies.