Xiue Jiang

New views on cellular uptake and trafficking of manufactured nanoparticles

Nanoparticles (NPs) are of similar size to typical cellular components and proteins, and can efficiently intrude living cells. A detailed understanding of the involved processes at the molecular level is important for developing NPs designed for selective uptake by specific cells, for example, for targeted drug delivery. In addition, this knowledge can greatly assist in the engineering of NPs that should not penetrate cells so as to avoid adverse health effects. In recent years, a wide variety of experiments have been performed to elucidate the mechanisms underlying cellular NP uptake. Here, we review some select recent studies, which are often based on fluorescence microscopy and sophisticated strategies for specific labelling of key cellular components. We address the role of the protein corona forming around NPs in biological environments, and describe recent work revealing active endocytosis mechanisms and pathways involved in their cellular uptake. Passive uptake is also discussed. The current state of knowledge is summarized, and we point to issues that still need to be addressed to further advance our understanding of cellular NP uptake.

Endo- and exocytosis of zwitterionic quantum dot nanoparticles by live HeLa cells.

Uptake and intracellular transport of D-penicillamine coated quantum dots (DPA-QDs) of 4 nm radius by live HeLa cells have been investigated systematically by spinning disk and 4Pi confocal microscopies. Unlike larger nanoparticles, these small DPA-QDs were observed to accumulate at the plasma membrane prior to internalization, and the uptake efficiency scaled nonlinearly with the nanoparticle concentration. Both observations indicate that a critical threshold density has to be exceeded for triggering the internalization process. By using specific inhibitors, we showed that DPA-QDs were predominantly internalized by clathrin-mediated endocytosis and to a smaller extent by macropinocytosis. Clusters of DPA-QDs were found in endosomes, which were actively transported along microtubules toward the perinuclear region. Later on, a significant fraction of endocytosed DPA-QDs were found in lysosomes, while others were actively transported to the cell periphery and exocytosed with a half-life of 21 min.

Quantitative analysis of the protein corona on FePt nanoparticles formed by transferrin binding

Nanoparticles are finding a rapidly expanding range of applications in research and technology, finally entering our daily life in medical, cosmetic or food products. Their ability to invade all regions of an organism including cells and cellular organelles offers new strategies for medical diagnosis and therapy (nanomedicine), but their safe use requires a deep knowledge about their interactions with biological systems at the molecular level. Upon incorporation, nanoparticles are exposed to biological fluids from which they adsorb proteins and other biomolecules to form a ‘protein corona’. These nanoparticle–protein interactions are still poorly understood and quantitative studies to characterize them remain scarce. Here we have quantitatively analysed the adsorption of human transferrin onto small (radius approx. 5 nm) polymer-coated FePt nanoparticles by using fluorescence correlation spectroscopy. Transferrin binds to the negatively charged nanoparticles with an affinity of approximately 26 µM in a cooperative fashion and forms a monolayer with a thickness of 7 nm. By using confocal fluorescence microscopy, we have observed that the uptake of FePt nanoparticles by HeLa cells is suppressed by the protein corona compared with the bare nanoparticles.

A facile one-pot synthesis of copper sulfide-decorated reduced graphene oxide composites for enhanced detecting of H2O2 in biological environments.

The high levels of H2O2 are closely associated with cancer and progressive neurodegenerative diseases, such as Parkinsons disease. In this study, we developed a novel CuS nanoparticle-decorated reduced graphene oxide-based electrochemical biosensor for the reliable detection of H2O2. The new electrocatalyst, CuS/RGO composites was successfully prepared by heating the mixture of CuCl2 and Na2S aqueous solutions in the presence of PVP-protected graphene oxide at 180 °C. A potential application of CuS/RGO composite-modified electrode as a biosensor to monitor H2O2 has been investigated. The steady-state current response increases linearly with H2O2 concentration from 5 to 1500 μM with a fast response time of less than 2 s. The detection limit (3σ) for determination of H2O2 has been estimated to be 0.27 μM, which was lower than certain enzymes and noble metal nanomaterial-based biosensors. In addition, the study of storage time on the amperometric response of the sensor indicates super stability. Due to these remarkable analytical advantages, the as-made sensor was applied to determine the H2O2 levels in human serum and urine samples and H2O2 released from human cervical cancer cells with satisfactory results. These results demonstrate that this new nanocomposite with the high surface area and electrocatalytic activity is a promising candidate for use as an enhanced electrochemical sensing platform in the design of nonenzymatic biosensors.

The effect of carboxydextran-coated superparamagnetic iron oxide nanoparticles on c-Jun N-terminal kinase-mediated apoptosis in human macrophages

Superparamagnetic iron oxide nanoparticles are frequently used for cell labeling or as diagnostic contrast media, yet studies analyzing their effects on immune cells remain scarce. Here we investigated how nanosized carboxydextran-coated superparamagnetic iron oxide (SPIO) and ultrasmall superparamagnetic iron oxide (USPIO) might affect human macrophages. Within 1 h, both SPIO and USPIO were rapidly taken up by macrophages. Confocal microscopy revealed that after 24 h the particles were almost exclusively localized within the lysosomal compartment. Continued cultivation of the macrophages for several days was associated with apoptosis induction caused by a long-lasting activation of the c-Jun N-terminal kinase (JNK) pathway. JNK activation was due to significantly elevated levels of reactive oxygen species, whereas no TNF-alpha was produced by the macrophages treated with nanoparticles. Compared to SPIO, USPIO induced more pronounced biochemical alterations and cytotoxicity, which could be antagonized by the JNK inhibitor V. Alternatively, treatment of macrophages with Trolox or N-acetyl-L-cysteine, two functionally different scavengers of reactive oxygen species, abolished both the JNK activation and the subsequent cytotoxic effects. These data indicate that nanosized superparamagnetic iron oxide-based contrast media exert cytotoxicity in human macrophages that can be functionally antagonized with radical scavengers.

High sensitivity hydrogen peroxide and hydrazine sensor based on silver nanocubes with rich {100} facets as an enhanced electrochemical sensing platform

We report a novel hydrogen peroxide (H2O2) and hydrazine sensor based on low-cost poly(vinylpyrrolidone)-protected silver nanocubes (PVP-AgNCs). The monodisperse silver nanocubes were prepared by adding a trace amount of sodium sulfide in the conventional polyol synthesis for fast reduction of silver nitrate under protection of argon. The sensor was fabricated by simple casting of PVP-AgNCs aqueous solution on a glassy carbon electrode and the performance was evaluated by cyclic voltammetry and amperometric techniques. It was found that the resulting sensor exhibited extremely good performance toward H2O2 detection with wide linear response ranging from 0.05 to 70mM (R=0.996) at -0.3V and low detection limit of 0.18μM estimated at a signal-to-noise ratio of 3. In addition, the fabricated sensor also exhibited high sensitivity toward the detection of hydrazine with a low detection limit of 1.1μM, wide linear range from 0.005 to 0.46mM (R=0.999) at 0.4V and rapid amperometric response time of less than 2s. For both analytes, the sensor exhibited good reproducibility, selectivity and stability. The excellent performance of the sensor might be attributed to the enhanced electrochemical sensing property of well-defined PVP-AgNCs with rich {100} facets.

Specific Effects of Surface Amines on Polystyrene Nanoparticles in their Interactions with Mesenchymal Stem Cells

We have investigated the uptake of cationic polystyrene nanoparticles by mesenchymal stem cells (MSCs) using confocal fluorescence microscopy and flow cytometry. Two types of nanoparticles of about 100 nm diameter with similar zeta potentials were employed in this study, plain polystyrene (PS) nanoparticles and amino-functionalized polystyrene (NPS) nanoparticles, each carrying about 6000 amino groups on the surface. To assess the relative importance of specific endocytosis mechanisms, uptake was observed in the presence of the drugs dynasore and chlorpromazine. NPS nanoparticles were rapidly internalized and accumulated to a much higher level in MSCs than PS nanoparticles, predominantly via the main clathrin-mediated pathway. PS nanoparticles were internalized mainly via clathrin-independent endocytosis. The pronounced difference in the internalization behavior of PS and NPS nanoparticles points to specific interactions of the amino groups on the nanoparticle surface with the endocytosis machinery of the cells.

Resolving voltage-dependent structural changes of a membrane photoreceptor by surface-enhanced IR difference spectroscopy

Membrane proteins are molecular machines that transport ions, solutes, or information across the cell membrane. Electrophysiological techniques have unraveled many functional aspects of ion channels but suffer from the lack of structural sensitivity. Here, we present spectroelectrochemical data on vibrational changes of membrane proteins derived from a single monolayer. For the seven-helical transmembrane protein sensory rhodopsin II, structural changes of the protein backbone and the retinal cofactor as well as single ion transfer events are resolved by surface-enhanced IR difference absorption spectroscopy (SEIDAS). Angular changes of bonds versus the membrane normal have been determined because SEIDAS monitors only those vibrations whose dipole moment are oriented perpendicular to the solid surface. The application of negative membrane potentials (ΔV = −0.3 V) leads to the selective halt of the light-induced proton transfer at the stage of D75, the counter ion of the retinal Schiff base. It is inferred that the voltage raises the energy barrier of this particular proton-transfer reaction, rendering the energy deposited in the retinal by light excitation insufficient for charge transfer to occur. The other structural rearrangements that accompany light-induced activity of the membrane protein, are essentially unaffected by the transmembrane electric field. Our results demonstrate that SEIDAS is a generic approach to study processes that depend on the membrane potential, like those in voltage-gated ion channels and transporters, to elucidate the mechanism of ion transfer with unprecedented spatial sensitivity and temporal resolution.

Impact of Shape and Pore Size of Mesoporous Silica Nanoparticles on Serum Protein Adsorption and RBCs Hemolysis

With the rapid development of nanotechnology, mesoporous silica nanoparticles (MSNs) with numerous forms and structures have been synthesized and extensively applied in biomedicine in the past decades. However, our knowledge about the biocompatibility of the developed MSNs has not matched their development. Therefore, in this work, we have synthesized sphere-shaped MSNs with different pore scales (s-SPs and l-SPs) and rod-shape (RPs-3) MSNs to evaluate the influence of the morphology and pore size on their interaction with serum proteins and red blood cells (RBCs). The adsorption of human albumin (HSA), globulin (HGG), and fibrinogen (HSF) onto different kinds of MSNs has been analyzed by pseudo second-order kinetic model, and the conformational changes of the adsorbed proteins have been studied by FTIR spectroscopy. We find that the conformation of absorbed HSA and HSF, while not HGG, will be affected by the pore size and morphology of the MSNs. The conformational changes of the adsorbed proteins will further affect their saturated adsorption capacity. However, the initial adsorption rate is only determined by the property of MSNs and proteins. Additional hemolysis assay shows that the pore size and morphology of the MSNs will also affect their hemolytic activity in RBCs which will be extremely depressed by the formation of protein corona. These systematic studies will provide an overall understanding in the blood compatibility of MSNs as well as useful guidelines for fabrication of blood-compatible nanomaterials.

[Ru(bpy)3]2+‐Doped Silica Nanoparticles within Layer‐by‐Layer Biomolecular Coatings and Their Application as a Biocompatible Electrochemiluminescent Tag Material

[Ru(bpy)3]2+-doped silica (RuSi) nanoparticles were synthesized by using a water/oil microemulsion method. Stable electrochemiluminescence (ECL) was obtained when the RuSi nanoparticles were immobilized on a glassy carbon electrode by using tripropylamine (TPA) as a coreactant. Furthermore, the ECL of the RuSi nanoparticles with layer-by-layer biomolecular coatings was investigated. Squential self-assembly of the polyelectrolytes and biomolecules on the RuSi nanoparticles gave nanocomposite suspensions, the ECL of which decreased on increasing the number of bilayers. Moreover, factors that affected the assembly and ECL signals were investigated. The decrease in ECL could be assigned to steric hindrance and limited diffusion of the coreactant molecules in the silica matrix after they were attached to the biomolecules. Since surface modification of the RuSi nanoparticles can improve their biocompatibility and prevent leaking of the [Ru(bpy)3]2+ ions, the RuSi nanoparticles can be readily used as efficient and stable ECL tag materials in immunoassay and DNA detection.