Analysis of Silicon Quantum Dots and Serum Proteins Interactions Using Asymmetrical Flow Field-Flow Fractionation.

Semiconductor nanocrystals or quantum dots (QDs) have gained significant attention in biomedical research as versatile probes for imaging, sensing, and therapies. However, the interactions between proteins and QDs, which are crucial for their use in biological applications, are not yet fully understood. Asymmetric flow field-flow fractionation (AF4) is a promising method for analyzing the interactions of proteins with QDs. This technique uses a combination of hydrodynamic and centrifugal forces to separate and fractionate particles based on their size and shape. By coupling AF4 with other techniques, such as fluorescence spectroscopy and multi-angle light scattering, it is possible to determine the binding affinity and stoichiometry of protein-QD interactions. Herein, this approach has been utilized to determine the interaction between fetal bovine serum (FBS) and silicon quantum dots (SiQDs). Unlike metal-containing conventional QDs, SiQDs are highly biocompatible and photostable in nature, making them attractive for a wide range of biomedical applications. In this study, AF4 has provided crucial information on the size and shape of the FBS/SiQD complexes, their elution profile, and their interaction with serum components in real time. The differential scanning microcalorimetric technique has also been employed to monitor the thermodynamic behavior of proteins in the presence of SiQDs. We have investigated their binding mechanisms by incubating them at temperatures below and above the protein denaturation. This study yields various significant characteristics such as their hydrodynamic radius, size distribution, and conformational behavior. The compositions of SiQD and FBS influence the size distribution of their bioconjugates; the size increases by intensifying the concentration of FBS, with their hydrodynamic radii ranging between 150 and 300 nm. The results signify that in the alliance of SiQDs to the system, there is an augmentation of the denaturation point of the proteins and hence their thermal stability, providing a more comprehensive understanding of the interactions between FBS and QDs.

Comparative Thermodynamic Studies of the Micellization of Amphiphilic Block Copolymers before and after Cyclization.

The enthalpy and entropy of micellization in water, ΔHmic and ΔSmic, respectively, of three linear amphiphilic BAB block copolymers consisting of either poly(methyl acrylate) (Mn ∼ 1200 and 700 Da) or poly(ethyl acrylate) (Mn ∼ 800 Da) as hydrophobic (B) segments and poly(ethylene oxide) (PEO) as the hydrophilic (A, Mn ∼ 3000 Da) segment were determined by isothermal titration calorimetry (ITC). The ΔHmic and ΔSmic of the cyclic AB block copolymers obtained by cyclization of the linear triblock copolymers were determined under the same conditions. The ΔHmic value of the cyclic copolymers was smaller than that of their linear precursors. The ΔSmic value showed the same trend, but the relative difference between the cyclized and linear copolymers was less pronounced. The hydrodynamic diameter (Dh), critical micelle concentration (CMC), molecular weight (Mw-mic), and second virial coefficient (A2) of the micelles were determined. The Dh value of the cyclic copolymer micelles was smaller than the linear counterpart. On the other hand, the CMC value became larger, whereas the A2 value was comparable or increased by cyclization. Overall, the results suggest that, in the unimer state, the hydrophobic segments of the cyclized copolymers form a tightly coiled structure to minimize contact with water, resulting in the smaller ΔHmic value. Contrary to the linear copolymer micelles, the cyclic copolymer micelles have no "dangling chains", which may explain the topology-driven slight difference in the ΔSmic value.

Heat-Induced Flower Nanogels of Both Cholesterol End-Capped Poly(N-isopropylacrylamide)s in Water.

Thermoresponsive self-assembled nanogels were conveniently prepared by cholesterol end-capped poly(N-isopropylacrylamide) (PNIPAM) in water. Both cholesterol end-capped PNIPAMs (telelchelic cholesterol PNIPAM, tCH-PNIPAM) formed flower-like nanogels by the self-assembling of four to five polymer chains with multiple domains of cholesterol in water at 20 °C. Meanwhile, one end-group cholesterol-capped PNIPAM (semitelechelic cholesterol PNIPAM, stCH-PNIPAM) was also formed as a nanogel by the self-assembling of 15-20 polymer chains with 3 to 4 cholesterol domains. The hydrophobic cholesterol domains of tCH-PNIPAM nanogels were maintained above the lower critical solution temperature (LCST) of PNIPAM (>32 °C). Differently, the hydrophobic domains of stCH-PNIPAM were disrupted by cholesterol-free PNIPAM chain ends and formed large mesoglobules above the LCST. These transition controls of hydrophilic end-capped smart polymers may open new methodologies to design thermoresponsive nanosystems.

Synthesis of New Thermoresponsive Polymers Possessing the Dense 1,2,3-Triazole Backbone.

Thermoresponsive water-soluble polymers, aqueous solutions of which undergo lower critical solution temperature (LCST)-type phase separation, have been investigated in detail for several decades. To develop LCST-type thermoresponsive polymers with new polymer backbone, 4-azido-5-hexynamide (AHA) derivatives were designed as monomers for copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) polymerization. AHA derivatives carrying secondary amide side chains, that is, 4-azido-N-methyl-5-hexynamide (M), 4-azido-N-ethyl-5-hexynamide (E), and 4-azido-N-isopropyl-5-hexynamide (iP), were first synthesized and polymerized by CuAAC to obtain polymers (poly(M), poly(E), and poly(iP)). Contrary to our expectation, poly(M), poly(E), and poly(iP) were insoluble in water and many organic solvents presumably because of the formation of hydrogen bonding between the amide side chains or between the amide side chains and triazole residues in the backbone. Thus, AHA derivatives carrying tertiary amide side chains, that is, 4-azido-N,N-dimethyl-5-hexynamide (MM), 4-azido-N-ethyl-N-methyl-5-hexynamide (ME), 4-azido-N-isopropyl-N-methyl-5-hexynamide (MiP), and 4-azido-N,N-diethyl-5-hexynamide (EE), were also synthesized and polymerized to yield polymers (poly(MM), poly(ME), poly(MiP), and poly(EE)). These polymers were soluble in a number of common organic solvents. It is noteworthy that poly(MM) and poly(ME) were also soluble in water. The phase separation behavior of 1.0 wt % aqueous solutions of poly(MM) and poly(ME) was then investigated by transmittance measurements. These data indicated that poly(ME) was an LCST-type thermoresponsive polymer, whereas poly(MM) was not. A large hysteresis was observed in the transmittance measurements for the poly(ME) aqueous solution because of slow rehydration after phase separation. The phase separation behavior was investigated preliminarily by differential scanning calorimetry and 1H NMR.

Polymeric Nanoparticles Limit the Collective Migration of Cellular Aggregates.

Controlling the propagation of primary tumors is fundamental to avoiding the epithelial to mesenchymal transition process leading to the dissemination and seeding of tumor cells throughout the body. Here we demonstrate that nanoparticles (NPs) limit the propagation of cell aggregates of CT26 murine carcinoma cells used as tumor models. The spreading behavior of these aggregates incubated with NPs is studied on fibronectin-coated substrates. The cells spread with the formation of a cell monolayer, the precursor film, around the aggregate. We study the effect of NPs added either during or after the formation of aggregates. We demonstrate that, in both cases, the spreading of the cell monolayer is slowed down in the presence of NPs and occurs only above a threshold concentration that depends on the size and surface chemistry of the NPs. The density of cells in the precursor films, measured by confocal microscopy, shows that the NPs stick cells together. The mechanism of slowdown is explained by the increase in cell-cell interactions due to the NPs adsorbed on the membrane of the cells. The present results demonstrate that NPs can modulate the collective migration of cells; therefore, they may have important implications for cancer treatment.

Spreading of Cell Aggregates on Zwitterion-Modified Chitosan Films.

The sulfobetaine (SB) moiety, which comprises a quaternary ammonium group linked to a negatively charged sulfonate ester, is known to impart nonfouling properties to interfaces coated with polysulfobetaines or grafted with SB-polymeric brushes. Increasingly, evidence emerges that the SB group is, overall, a better antifouling group than the phosphorylcholine (PC) moiety extensively used in the past. We report here the synthesis of a series of SB-modified chitosans (CH-SB) carrying between 20 and 40 mol % SB per monosaccharide unit. Chitosan (CH) itself is a naturally derived copolymer of glucosamine and N-acetyl-glucosamine linked with a ß-1,4 bond. Analysis by quartz crystal microbalance with dissipation (QCM-D) indicates that CH-SB films (thickness ∼ 20 nm) resist adsorption of bovine serum albumin (BSA) with increasing efficiency as the SB content of the polymer augments (surface coverage ∼ 15 µg cm-2 for films of CH with 40 mol % SB). The cell adhesivity of CH-SB films coated on glass was assessed by determining the spreading dynamics of CT26 cell aggregates. When placed on chitosan films, known to be cell-adhesive, the CT26 cell aggregates spread by forming a cell monolayer around them. The spreading of CT26 cell aggregates on zwitterion-modified chitosans films is thwarted remarkably. In the cases of CH-SB30 and CH-SB40 films, only a few isolated cells escape from the aggregates. The extent of aggregate spreading, quantified based on the theory of liquid wetting, provides a simple in vitro assay of the nonfouling properties of substrates toward specific cell lines. This assay can be adopted to test and compare the fouling characteristics of substrates very different from the chemical viewpoint.

Thermoresponsive Pentablock Copolymer on Silica: Temperature Effects on Adsorption, Surface Forces, and Friction.

The adsorption of hydrophilic or amphiphilic multiblock copolymers provides a powerful means to produce well-defined "smart" surfaces, especially if one or several blocks are sensitive to external stimuli. We focus here on an A-B-A-B-A copolymer, where A is a cationic poly((3-acrylamido-propyl)-trimethylammonium chloride) (PAMPTMA) block containing 15 (end blocks) or 30 (middle block) repeat units and B is a neutral thermosensitive water-soluble poly(2-isopropyl-2-oxazoline) (PIPOZ) block with 50 repeat units. X-ray reflectivity and quartz crystal microbalance with dissipation monitoring were employed to study the adsorption of PAMPTMA15-PIPOZ50-PAMPTMA30-PIPOZ50-PAMPTMA15 on silica surfaces. The latter technique was employed at different temperatures up to 50 °C. Surface forces and friction between the two silica surfaces across aqueous pentablock copolymer solutions at different temperatures were determined with the atomic force microscopy colloidal probe force and friction measurements. The cationic pentablock copolymer was found to have a high affinity to the negatively charged silica surface, leading to a thin (2 nm) and rigid adsorbed layer. A steric force was encountered at a separation of around 3 nm from hard wall contact. A capillary condensation of a polymer-rich phase was observed at the cloud point of the solution. The friction forces were evaluated using Amontons' rule modified with an adhesion term.

Phototropic Multiresponsive Active Nanogels.

Aqueous dispersions of nanogels that respond to switches in environmental pH and/or temperature by changes in their hydrodynamic radius (Rh ) and/or ζ-potential are prepared by reversible addition-fragmentation chain-transfer (RAFT) polymerization-induced thermal (70 °C) self-assembly (PITSA) of N-isopropylacrylamide (NIPAM) in the presence of a poly(methacrylic acid) (PMAA)-substituted macromolecular chain transfer agent and a cross-linker. Photochromic spiropyran (SP) moieties are coupled to the carboxylic acid groups of the nanogels. Upon UV irradiation, the neutral SP isomerizes to the zwitterionic merocyanine (ME) form. Upon UV light irradiation, microgels formed by assembly of SP nanogels undergo a collective motion toward the UV-light source.

Transition-Metal-Doped NIR-Emitting Silicon Nanocrystals.

Impurity-doping in nanocrystals significantly affects their electronic properties and diversifies their applications. Herein, we report the synthesis of transition metal (Mn, Ni, Co, Cu)-doped oleophilic silicon nanocrystals (SiNCs) through hydrolysis/polymerization of triethoxysilane with acidic aqueous metal salt solutions, followed by thermal disproportionation of the resulting gel into a doped-Si/SiO2 composite that, upon HF etching and hydrosilylation with 1-n-octadecene, produces free-standing octadecyl-capped doped SiNCs (diameter≈3 to 8 nm; dopant <0.2 atom %). Metal-doping triggers a red-shift of the SiNC photoluminescence (PL) of up to 270 nm, while maintaining high PL quantum yield (26 % for Co doping).

Membrane Translocation and Organelle-Selective Delivery Steered by Polymeric Zwitterionic Nanospheres.

The majority of nanoparticles designed for cellular delivery of drugs and imaging agents enter the cell via endocytotic pathways leading to their entrapment in endosomes that present a robust barrier to further trafficking of the nanoparticles within the cells. A few materials, such as the cell penetrating peptides (CPPs), are known to enter cells not only via endocytosis, but also via translocation through the cell membrane into the cytoplasm, successfully bypassing the endosomes. We report here that random copolymers of 3-dimethyl(methacryloyloxyethyl)ammonium propanesulfonate and poly(ethylene glycol) methacrylate, p(DMAPS-ran-PEGMA), are internalized in cells primarily via translocation through the cell membrane rather than endocytosis. The properties of the polymers and their modes of uptake were investigated systematically by dynamic light scattering, confocal fluorescence microscopy, and flow cytometry. Using specific inhibitors of the cellular uptake machinery in a human cervical carcinoma cell line (HeLa), we show that these nontoxic synthetic polyzwitterions exist in cell media as self-assembled nanospheres that unravel as they adsorb on the plasma membrane and translocate through it. Conjugates of p(DMAPS-ran-PEGMA) with rhodamine B were delivered selectively to the mitochondria, whereas doxorubicin (Dox)-p(DMAPS-ran-PEGMA) conjugates were accumulated in both the nucleus and the mitochondria, effectively inducing apoptosis in HeLa cells. These findings suggest that the noncytotoxic and readily synthesized p(DMAPS-ran-PEGMA) can find applications as bioimaging tools and drug nanocarriers.

Nanostickers for cells: a model study using cell-nanoparticle hybrid aggregates.

We present direct evidence that nanoparticles (NPs) can stick together cells that are inherently non-adhesive. Using cadherin-depleted S180 murine cells lines, which exhibit very low cell-cell adhesion, we show that NPs can assemble dispersed single cells into large cohesive aggregates. The dynamics of aggregation, which is controlled by diffusion and collision, can be described as a second-order kinetic law characterized by a rate of collision that depends on the size, concentration, and surface chemistry of the NPs. We model the cell-cell adhesion induced by the "nanostickers" using a three-state dynamical model, where the NPs are free, adsorbed on the cell membrane or internalized by the cells. We define a "sticking efficiency parameter" to compare NPs and look for the most efficient type of NP. We find that 20 nm carboxylated polystyrene NPs are more efficient nanostickers than 20 nm silica NPs which were reported to induce fast wound healing and to glue soft tissues. Nanostickers, by increasing the cohesion of tissues and tumors, may have important applications for tissue engineering and cancer treatment.

Formation of Tethers from Spreading Cellular Aggregates.

Membrane tubes are commonly extruded from cells and vesicles when a point-like force is applied on the membrane. We report here the unexpected formation of membrane tubes from lymph node cancer prostate (LNCaP) cell aggregates in the absence of external applied forces. The spreading of LNCaP aggregates deposited on adhesive glass substrates coated with fibronectin is very limited because cell-cell adhesion is stronger than cell-substrate adhesion. Some cells on the aggregate periphery are very motile and try to escape from the aggregate, leading to the formation of membrane tubes. Tethered networks and exchange of cargos between cells were observed as well. Growth of the tubes is followed by either tube retraction or tube rupture. Hence, even very cohesive cells are successful in escaping aggregates, which may lead to epithelial mesenchymal transition and tumor metastasis. We interpret the dynamics of formation and retraction of tubes in the framework of membrane mechanics.

Tethered poly(2-isopropyl-2-oxazoline) chains: temperature effects on layer structure and interactions probed by AFM experiments and modeling.

Thermoresponsive polymer layers on silica surfaces have been obtained by utilizing electrostatically driven adsorption of a cationic-nonionic diblock copolymer. The cationic block provides strong anchoring to the surface for the nonionic block of poly(2-isopropyl-2-oxazoline), referred to as PIPOZ. The PIPOZ chain interacts favorably with water at low temperatures, but above 46 °C aqueous solutions of PIPOZ phase separate as water becomes a poor solvent for the polymer. We explore how a change in solvent condition affects interactions between such adsorbed layers and report temperature effects on both normal forces and friction forces. To gain further insight, we utilize self-consistent lattice mean-field theory to follow how changes in temperature affect the polymer segment density distributions and to calculate surface force curves. We find that with worsening of the solvent condition an attraction develops between the adsorbed PIPOZ layers, and this observation is in good agreement with predictions of the mean-field theory. The modeling also demonstrates that the segment density profile and the degree of chain interpenetration under a given load between two PIPOZ-coated surfaces rise significantly with increasing temperature.

Poly(N-isopropylacrylamide) Phase Diagrams: Fifty Years of Research.

In 1968, Heskins and Guillet published the first systematic study of the phase diagram of poly(N-isopropylacrylamide) (PNIPAM), at the time a "young polymer" first synthesized in 1956. Since then, PNIPAM became the leading member of the growing families of thermoresponsive polymers and of stimuli-responsive, "smart" polymers in general. Its thermal response is unanimously attributed to its phase behavior. Yet, in spite of 50 years of research, a coherent quantitative picture remains elusive. In this Review we survey the reported phase diagrams, discuss the differences and comment on theoretical ideas regarding their possible origins. We aim to alert the PNIPAM community to open questions in this reputably mature domain.

Quantum dot cytotoxicity and ways to reduce it.

The dramatic increase in the use of nanoparticles (NP) in industry and research has raised questions about the potential toxicity of such materials. Unfortunately, not enough is known about how the novel, technologically-attractive properties of NPs correlate with the interactions that may take place at the nano/bio interface. The academic, industrial, and regulatory communities are actively seeking answers to the growing concerns on the impact of nanotechnology on humans. In this Account we adopt quantum dots (QDs) as an illustrative example of the difficulties associated with the development of a rational science-based approach to nanotoxicology. The optical properties of QDs are far superior to those of organic dyes in terms of emission and absorption bandwidths, quantum yield, and resistance to photobleaching. Moreover, QDs may be decorated with targeting moieties or drugs and, therefore, are candidates for site-specific medical imaging and for drug delivery, for example in cancer treatment. Earlier this year researchers demonstrated that QD-based imaging using monkeys caused no adverse effects although QDs accumulated in lymph nodes, bone marrow, liver, and spleen for up to 3 months after injection. Such persistence of QDs in live animals does, however, raise concerns about the safety of using QDs both in the laboratory and in the clinic. Researchers anticipate that QDs will be increasingly used not only in clinical applications but also in various manufactured products. For example, QD-solar cells have emerged as viable contenders to complement or replace dye-sensitized solar cells; CdTe/CdS thin film cells have already captured approximately 10 percent of the global market, and in addition, QDs can serve as components of sensors and as emitting materials in LEDs. Given the clear indications that QDs will inevitably become components of a wide range of manufactured and consumer products, researchers and policy makers need to understand the possible health risks associated with exposure to QDs. In this Account, we initially review the known mechanisms by which QDs can damage cells, including oxidative stress elicited by reactive oxygen species (ROS). We discuss lesser-known impairments induced in cells by nanomolar to picomolar concentrations of QDs, which imply that cadmium-containing QDs can exert genotoxic, epigenetic, and metalloestrogenic effects. These observations strongly suggest that minute concentrations of QDs could be sufficient to cause long lasting, even transgenerational, effects. We also consider various modes by which humans could be exposed to QDs in their work or through the environment. Although considerable advances have been made in enhancing the stability and overall quality of QDs, over time they can partially degrade in the environment or in biological systems, and eventually cause small, but cumulative undesirable effects. A combination of toxicological, genetic, epigenetic and imaging approaches is required to create comprehensive guidelines for evaluating the nanotoxicity of nanomaterials, including QDs. Prior to biological investigations with these materials, an indispensible step must be the full characterization of NPs by complementary techniques. Specifically, the concentration, size, charge, and ligand stability of NPs in biological media must be known if we are to understand fully how the properties of nanoparticles and of their biological environment contribute to cytotoxicity.

Length fractionation of boron nitride nanotubes using creamed oil-in-water emulsions.

The fractionation by length of multiwalled boron nitride nanotubes (BNNTs) was achieved by emulsification and creaming of an oil/water/surfactant mixture. The length separation is based on the fact that nanoparticle-coated oil droplets polydisperse in size move toward the upper surface or the bottom of an emulsified mixture depending on the density of the droplets, such that droplets of different sizes are located at different heights. By sampling heightwise an unstable hexane/water/Tween 20/BNNT (1-20 µm long) emulsion, we observed that the lengths of the BNNTs adsorbed on the droplets display a strong correlation with the droplets sizes, thus leading to selective separation of the BNNT lengths, as confirmed by dark-field optical imaging and dynamic light scattering. This method may potentially be extended to other high aspect ratio nanomaterials exhibiting emulsification properties similar to those of BNNTs.

Temperature-dependent adsorption and adsorption hysteresis of a thermoresponsive diblock copolymer.

A nonionic-cationic diblock copolymer, poly(2-isopropyl-2-oxazoline)60-b-poly((3-acrylamidopropyl)trimethylammonium chloride)17, (PIPOZ60-b-PAMPTMA17), was utilized to electrostatically tether temperature-responsive PIPOZ chains to silica surfaces by physisorption. The effects of polymer concentration, pH, and temperature on adsorption were investigated using quartz crystal microbalance with dissipation monitoring and ellipsometry. The combination of these two techniques allows thorough characterization of the adsorbed layer in terms of surface excess, thickness, and water content. The high affinity of the cationic PAMPTMA17 block to the negatively charged silica surface gives rise to a high affinity adsorption isotherm, leading to (nearly) irreversible adsorption with respect to dilution. An increase in solution pH lowers the affinity of PIPOZ to silica but enhances the adsorption of the cationic block due to increasing silica surface charge density, which leads to higher adsorption of the cationic diblock copolymer. Higher surface excess is also achieved at higher temperatures due to the worsening of the solvent quality of water for the PIPOZ block. Interestingly, a large hysteresis in adsorbed mass and other layer properties was observed when the temperature was cycled from 25 to 45 °C and then back to 25 °C. Possible causes for this temperature hysteresis are discussed.

Prevention of thermally induced aggregation of IgG antibodies by noncovalent interaction with poly(acrylate) derivatives.

Prevention of thermal aggregation of antibodies in aqueous solutions was achieved by noncovalent association with hydrophobically modified poly(acrylate) copolymers. Using a polyclonal immunoglobin G (IgG) as a model system for antibodies, we have studied the mechanisms by which this multidomain protein interacts with polyanions when incubated at physiological pH and at temperatures below and above the protein unfolding/denaturation temperature, in salt-free solutions and in 0.1 M NaCl solutions. The polyanions selected were sodium poly(acrylates), random copolymers of sodium acrylate and N-n-octadecylacrylamide (3 mol %), and a random copolymer of sodium acrylate, N-n-octylacrylamide (25 mol %), and N-isopropylacrylamide (40 mol %). They were derived from two poly(acrylic acid) parent chains of Mw 5000 and 150000 g·mol(-1). The IgG/polyanion interactions were monitored by static and dynamic light scattering, fluorescence correlation spectroscopy, capillary zone electrophoresis, and high sensitivity differential scanning calorimetry. In salt-free solutions, the hydrophilic PAA chains form complexes with IgG upon thermal unfolding of the protein (1:1 w/w IgG/PAA), but they do not interact with native IgG. The complexes exhibit a remarkable protective effect against IgG aggregation and maintain low aggregation numbers (average degree of oligomerization <12 at a temperature up to 85 °C). These interactions are screened in 0.1 M NaCl and, consequently, PAAs lose their protective effect. Amphiphilic PAA derivatives (1:1 w/w IgG/polymer) are able to prevent thermal aggregation (preserving IgG monomers) or retard aggregation of IgG (formation of oligomers and slow growth), revealing the importance of both hydrophobic interactions and modulation of the Coulomb interactions with or without NaCl present. This study leads the way toward the design of new formulations of therapeutic proteins using noncovalent 1:1 polymer/protein association that are transient and require a markedly lower additive concentration compared to conventional osmolyte protecting agents. They do not modify IgG permanently, which is an asset for applications in therapeutic protein formulations since the in vivo efficacy of the protein should not be affected.