Growing gold nanostructures for shape-selective cellular uptake.

With development in the synthesis of shape- and size-dependent gold (Au) nanostructures (NSs) and their applications in nanomedicine, one of the biggest challenges is to understand the interaction of these shapes with cancer cells. Herein, we study the interaction of Au NSs of five different shapes with glioblastoma-astrocytoma cells. Three different shapes (nanorods, tetrahexahedra, and bipyramids), possessing tunable optical properties, have been synthesized by a single-step seed-mediated growth approach employing binary surfactant mixtures of CTAB and a secondary surfactant. By the use of two-step seed-mediated approach, we obtained new NSs, named nanomakura (Makura is a Japanese word used for pillow) which is reported for the first time here. Spherical Au nanoparticles were prepared by the Turkevich method. To study NS-cell interactions, we functionalized the NSs using thiolated PEG followed by 11-Mercaptoundecanoic acid. The influence of shape and concentration of NSs on the cytotoxicity were assessed with a LIVE/DEAD assay in glioblastoma-astrocytoma cells. Furthermore, the time-dependent uptake of nanomakura was studied with TEM. Our results indicate that unlike the other shapes studied here, the nanomakura were taken up both via receptor-mediated endocytosis and macropinocytosis. Thus, from our library of different NSs with similar surface functionality, the shape is found to be an important parameter for cellular uptake.

Synthesis of gadolinium oxide nanodisks and gadolinium doped iron oxide nanoparticles for MR contrast agents.

Herein, we report the synthesis of differently sized gadolinium oxide nanodisks and gadolinium doped iron oxide spherical and cubic nanoparticles through the thermal decomposition of an oleate precursor. We also demonstrate that these nanoparticles are promising candidates for MR contrast agents.

Nanoparticle-stabilized microbubbles for multimodal imaging and drug delivery.

Microbubbles (MBs) are routinely used as contrast agents for ultrasound imaging. The use of ultrasound in combination with MBs has also attracted attention as a method to enhance drug delivery. We have developed a technology platform incorporating multiple functionalities, including imaging and therapy in a single system consisting of MBs stabilized by polyethylene glycol (PEG)-coated polymeric nanoparticles (NPs). The NPs, containing lipophilic drugs and/or contrast agents, are composed of the widely used poly(butyl cyanoacrylate) (PBCA) polymer and prepared in a single step. MBs stabilized by these NPs are subsequently prepared by self-assembly of NPs at the MB air-liquid interface. Here we show that these MBs can act as contrast agents for conventional ultrasound imaging. Successful encapsulation of iron oxide NPs inside the PBCA NPs is demonstrated, potentially enabling the NP-MBs to be used as magnetic resonance imaging (MRI) and/or molecular ultrasound imaging contrast agents. By precise tuning of the applied ultrasound pulse, the MBs burst and the NPs constituting the shell are released. This could result in increased local deposit of NPs into target tissue, providing improved therapy and imaging contrast compared with freely distributed NPs.

Self-assembly and characterization of transferrin-gold nanoconstructs and their interaction with bio-interfaces.

Transferrin (Tf) conjugated to gold nanoparticles and clusters combine the protein's site-specific receptor targeting capabilities with the optical properties imparted by the nano-sized gold. We have described two different synthesis protocols, one yielding fluorescent Tf-stabilized gold nanoclusters (AuNCs) and one yielding Tf-stabilized gold nanoparticles that exhibit localized surface plasmon resonance. We demonstrate that the synthetic route employed has a large influence both on the gold nanostructure formed, and also on the structural integrity of the protein. A slight protein unfolding allows stronger interaction with lipids, and was found to significantly perturb lipid monolayers. Interactions between the protein-gold nanostructures and three different cell types were also assessed, indicating that the enhanced membrane affinity may be attributed to intercellular membrane differences.

Synthesis, characterization, and cellular uptake of magnetic nanocarriers for cancer drug delivery.

HYPOTHESIS: The absence of targetability is the primary inadequacy of conventional chemotherapy. Targeted drug delivery systems are conceptualized to overcome this challenge. We have designed a targetable magnetic nanocarrier consisting of a superparamagnetic iron oxide (SPIO) core and biocompatible and biodegradable poly(sebacic anhydride)-block-methyl ether poly(ethylene glycol) (PSA-mPEG) polymer shell. The idea is that this type of carriers should facilitate the targeting of cancer cells. EXPERIMENTS: PSA-mPEG was synthesized with poly-condensation and the in vitro degradation rate of the polymer was monitored by gel permeation chromatography (GPC). The magnetic nanocarriers were fabricated devoid of any surfactants and were capable of carrying high payload of hydrophobic dye. The successful encapsulation of SPIO within the polymer shell was confirmed by TEM. The results we obtained from measuring the size of SPIO loaded in polymeric NPs (SPIO-PNP) by dynamic light scattering (DLS) and iron content measurement of these particles by ICP-MS, indicate that SPIO is the most suitable carrier for cancer drug delivery applications. FINDINGS: Measuring the hydrodynamic radii of SPIO-PNPs by DLS over one month revealed the high stability of these particles at both body and room temperature. We further investigated the cell viability and cellular uptake of SPIO-PNPs in vitro with MDA-MB-231 breast cancer cells. We found that SPIO-PNPs induce negligible toxicity within a concentration range of 1-2µg/ml. The TEM micrographs of thin cross-sectioned MDA-MBA-231 cells showed internalization of SPIO-PNPs within size range of 150-200nm after 24h. This study has provided a foundation for eventually loading these nanoparticles with anti-cancer drugs for targeted cancer therapy using an external magnetic field.

Cytotoxicity of bovine α-lactalbumin: oleic acid complexes correlates with the disruption of lipid membranes.

HAMLET/BAMLET (Human/Bovine α-Lactalbumin Made Lethal to Tumors) is a tumoricidal substance composed of partially unfolded human/bovine α-lactalbumin (HLA/BLA) and several oleic acid (OA) molecules. The HAMLET mechanism of interaction involves an insufficiently understood effect on the membrane or its embedded components. We examined the effect of BLAOA (bovine α-lactalbumin complexed with oleic acid, a HAMLET-like substance) and its individual components on cells and artificial lipid membranes using viability staining and metabolic dyes, fluorescence spectroscopy, leakage integrity assays and microscopy. Our results show a dose-dependency of OA used to prepare BLAOA on its ability to induce tumor cell death, and a correlation between leakage and cell death. BLAOA incorporates into the membrane, tightens the lipid packing and lowers their solvent accessibility. Fluorescence imaging reveals that giant unilamellar vesicles (GUVs) develop blebs and eventually collapse upon exposure to BLAOA, indicating that the lipid packing reorganization can translate into observable morphological effects. These effects are observed to be local in GUVs, and a tightly packed and solvent-shielded lipid environment is associated with leakage and GUV disruption. Furthermore, the effects of BLAOA on membrane are pH dependent, with an optimum of activity on artificial membranes near neutral pHs. While BLA alone is effective at membrane disruption at acidic pHs, OA is ineffective in a pH range of 4.5 to 9.1. Taken together, this supports a model where the lipid, fatty acid and protein components enhance each other's ability to affect the overall integrity of the membrane.

Interactions between bovine serum albumin and Langmuir films composed of charged and uncharged poly(N-isopropylacrylamide) block copolymers.

The thermoresponsive poly(N-isopropylacrylamide) (PNIPAAM) and NIPAAM block copolymer derivatives are attractive for drug delivery applications as they contract reversibly at lower critical solution temperatures (LCST) close to physiological conditions. In order to investigate biomaterial-protein compatibility, we have studied the interaction between PNIPAAM copolymer films spread at the air-water surface and bovine serum albumin (BSA) injected below the precompressed polymer films, using the Langmuir technique coupled with Brewster angle microscopy (BAM). A PNIPAAM homopolymer was applied together with a number of PNIPAAM-based di- and triblock copolymers, to assess effects of e.g., charge and hydrophobicity on protein-polymer interactions. The nature and strength of protein-polymer interaction was found to be tunable, ranging from complex formation (PNIPAAM homopolymer) to mixed monolayers and electrostatic cross-linking, according to the nature of the co-monomer. Results show that intercalation versus adsorption can be controlled through polymer composition.

HAMLET forms annular oligomers when deposited with phospholipid monolayers.

Recently, the anticancer activity of human α-lactalbumin made lethal to tumor cells (HAMLET) has been linked to its increased membrane affinity in vitro, at neutral pH, and ability to cause leakage relative to the inactive native bovine α-lactalbumin (BLA) protein. In this study, atomic force microscopy resolved membrane distortions and annular oligomers (AOs) produced by HAMLET when deposited at neutral pH on mica together with a negatively charged lipid monolayer. BLA, BAMLET (HAMLET's bovine counterpart) and membrane-binding Peptide C, corresponding to BLA residues 75-100, also form AO-like structures under these conditions but at higher subphase concentrations than HAMLET. The N-terminal Peptide A, which binds to membranes at acidic but not at neutral pH, did not form AOs. This suggests a correlation between the capacity of the proteins/peptides to integrate into the membrane at neutral pH-as observed by liposome content leakage and circular dichroism experiments-and the formation of AOs, albeit at higher concentrations. Formation of AOs, which might be important to HAMLET's tumor toxic action, appears related to the increased tendency of the protein to populate intermediately folded states compared to the native protein, the formation of which is promoted by, but not uniquely dependent on, the oleic acid molecules associated with HAMLET.

Emergent membrane-affecting properties of BSA-gold nanoparticle constructs.

By adsorbing bovine serum albumin (BSA) on gold nanoparticles (Aunps) with diameters 30 nm and 80 nm, different degrees of protein unfolding were obtained. Adsorption and adlayer conformation were characterized by UV-vis spectroscopy, ζ-potential measurements, steady-state and time-resolved fluorescence. The unfolding was also studied using 1-anilino-8-naphthalene sulfonate (ANS) as an extrinsic probe, showing that BSA unfolds more on 80 nm Aunp than on 30 nm Aunp. Langmuir monolayer studies using two distinct methods of introducing the BSA and BSA-Aunp constructs accompanied with Brewster Angle Microscopy (BAM) and Digital Video Microscope (DVM) imaging demonstrated that BSA-Aunp constructs induce film miscibility with L-α-phosphatidylethanolamine not seen for BSA or Aunp alone. The changes induced by partial unfolding clearly give better film-penetration ability, as well as disruption of liquid crystalline domains in the film, thereby inducing film miscibility. Gold or protein only does not possess the nanoscale film-affecting properties of the protein-gold constructs, and as such the surface-active and miscibility-affecting characteristics of the BSA-Aunp represent emergent qualities.

High-efficiency NO(x) absorption in water using equipment packed with a glass fiber filter.

NO(X) absorption in water is quite difficult by comparison with other exhausted gas, such as SO(2), CO(2), and NH(3) because of low solubility of NO(X) in water. We have been developed a NO(X) absorption equipment with a glass fiber filter having high porosity and surface area. When feed NO(X) gas concentration was high, high NO(X) removal efficiency was obtained. This was because the surface area per glass fiber filter volume was about 40 to 600 times higher than for common packing materials. For verification test and industrial application, a high concentration of NO(X) gas (206,000 ppm) produced by a metal dissolution process was treated with a series of two absorption experiments. We can attain 97.6% of NO(X) removal efficiency, and HNO(3) concentration in water was concentrated up to 56.3 wt %. Furthermore, ozone addition to gas and usage of ozone saturated water as an absorbent resulted in complete removal of NO(X) in the gas (up to 120 ppm). This result indicated the importance of aqueous phase oxidation of HNO(2), which produces NO in the gas phase.

Adsorption of cationic hydroxyethylcellulose derivatives onto planar and curved gold surfaces.

The adsorption of two positively charged hydroxyethylcellulose derivatives with 7 and 60 mol % positively charged groups and a cationic, hydrophobically modified hydroxyethylcellulose containing 1 mol % hydrophobic groups and 7 mol % charged groups onto flat and spherical citrate-coated gold surfaces of different sizes has been investigated. The planar surfaces were studied by means of the quartz crystal microbalance with dissipation monitoring, whereas nanoparticle suspensions were examined using dynamic light scattering and UV-vis spectroscopy. Two different driving forces for adsorption have been evaluated: the electrostatic interaction between the positive charges on the polymers and the negatively charged gold surfaces and the affinity of the polymers for gold due to hydrophobic interactions. The comparison between the data obtained from curved and planar surfaces suggests a strong correlation between surface curvature and adlayer conformation in the formation of the hybrid polymer-gold nanoparticles. The influence of particle size on the amount of adsorbed polymer has been evaluated for the different polymers. The impact of the ionic strength on polymer adsorption has been explored, and the adsorbed polymer layer has been found to protect the gold nanoparticles from aggregation when salt is added to the solution. The addition of salt to a mixture of gold particles and a charged polymer can induce a thicker adsorbed layer at low salinity, and desorption was found at high levels of salt addition.

Adsorption and bioactivity of tyrosine hydroxylase on gold surfaces and nanoparticles.

Tyrosine hydroxylase is studied in terms of adsorption behaviour on gold surfaces and various passivating layers. Results reveal differences in layer formation, where mercaptoundecanoic acid-coated gold shows the best potential in terms of adsorbed mass. Nanoparticles with this coating are subsequently tested for enzymatic activity, which remains at attenuated levels.

Temperature-dependent optical properties of gold nanoparticles coated with a charged diblock copolymer and an uncharged triblock copolymer.

We demonstrate that the optical properties of gold nanoparticles can be used to detect and follow stimuli-induced changes in adsorbed macromolecules. Specifically, we investigate thermal response of anionic diblock and uncharged triblock copolymers based on poly(N-isopropylacrylamide) (PNIPAAM) blocks adsorbed onto gold nanoparticles and planar gold surfaces in a temperature range between 25 and 60 degrees C. By employing a palette of analytical probes, including UV-visible spectroscopy, dynamic light scattering, fluorescence, and quartz crystal microbalance with dissipation monitoring, we establish that while the anionic copolymer forms monolayers at both low and high temperature, the neutral copolymer adsorbs as a monolayer at low temperatures and forms multilayers above the cloud point (T(C)). Raising the temperature above T(C) severely affects the optical properties of the gold particle/polymer composites, expelling associated water and altering the immediate surroundings of the gold nanoparticles. This effect, stronger for the uncharged polymer, is related to the amount of polymer adsorbed on the surface, where a denser shell influences the surface plasmon band to a greater degree. This is corroborated with light scattering experiments, which reveal that flocculation of the neutral polymer-coated particles occurs at high temperatures. The flocculation behavior of the neutral copolymer on planar gold surfaces results in multilayer formation. The observed effects are discussed within the framework of the Mie-Drude theory.

Temperature-induced adsorption and optical properties of an amphiphilic diblock copolymer adsorbed onto flat and curved silver surfaces.

Temperature-induced adsorption of a thermoresponsive amphiphilic copolymer, containing poly(N-isopropylacrylamide), on citrate-capped silver nanoparticles and planar silver surfaces has been studied with the aid of quartz crystal microbalance with dissipation monitoring (QCM-D) and dynamic light scattering (DLS). The results clearly show that both the amount of adsorbed copolymer and thickness of the adsorbed layer increase strongly at temperatures above the lower critical solution temperature (LCST). These findings are ascribed to enhanced hydrophobicity of the polymer and higher affinity for polymer adsorption at elevated temperatures and formation of intermicellar structures of the copolymer. The values of the layer thickness calculated from QCM-D data are practically identical to the values for the hydrodynamic thickness from DLS. This result suggests that the adsorbed layer is compact with few tails protruding out into the bulk. The surface plasmon peak for silver is observed at all temperatures, and the maximum is red-shifted with increasing temperature, which is attributed to an increase of the localized refractive index as more polymer chains are adsorbed onto the silver particles.

Use of cellulose derivatives on gold surfaces for reduced nonspecific adsorption of immunoglobulin G.

This study addresses the design of protein-repellent gold surfaces using hydroxyethyl- and ethyl(hydroxyethyl) cellulose (HEC and EHEC) and hydrophobically modified analogues of these polymers (HM-HEC and HM-EHEC). Adsorption behavior of the protein immunoglobulin G (IgG) onto pure gold and gold surfaces coated with cellulose polymers was investigated and described by quartz crystal microbalance with dissipation monitoring (QCM-D), atomic force microscopy (AFM) and contact angle measurements (CAM). Surfaces coated with the hydrophobically modified cellulose derivatives were found to significantly outperform a reference poly(ethylene glycol) (PEG) coating, which in turn prevented 90% of non-specific protein adsorption as compared to adsorption onto pure gold. HEC and EHEC prevented around 30% and 60% of the IgG adsorption observed on pure gold, while HM-HEC and HM-EHEC were both found to completely hinder biofouling when deposited on the gold substrates. Adsorption behavior of IgG has been discussed in terms of polymer surface coverage and roughness of the applied surfaces, together with hydrophobic interactions between protein and gold, and also polymer-protein interactions.

Same system-different results: the importance of protein-introduction protocols in Langmuir-monolayer studies of lipid-protein interactions.

For studies of protein-lipid interactions, thin films at the air-water surface are often employed as model systems for cell membranes. A convenient manner in which to study these interactions is the Langmuir technique, which allows for formation of monolayer phospholipid films together with a choice of where and how to introduce proteins, according to the desired response variable. Here, a distinction has been made between different interaction protocols and it is also commented upon to what extent introduction of protein to a solution prior to spreading of a lipid film affects the results. This paper describes commonly used methods when working with Langmuir monolayers as membrane mimics and compares the results of four different experimental protocols: formation of a lipid film on top of a protein-containing subphase, injection of protein under an existing, semicompressed phospholipid film (surface pressure 5 mN/m), and deposition of a protein solution on top of a lipid film contained at either surface pressure 0 mN/m or at surface pressure 5 mN/m. Results obtained from Langmuir isotherms and Brewster angle microscope clearly differentiate between these methods and give insight into under which conditions and at which interfaces the protein interactions are predominant (protein-air or protein-lipid).

Adsorption of cellulose derivatives on flat gold surfaces and on spherical gold particles.

The adsorption of hydroxyethylcellulose (HEC), ethyl(hydroxyethyl)cellulose (EHEC), and their hydrophobically modified counterparts HM-HEC and HM-EHEC has been studied on planar gold and citrate-covered gold surfaces by means of quartz crystal microbalance with dissipation monitoring (QCM-D), and on citrate-covered gold particles with the aid of dynamic light scattering (DLS). The QCM-D results indicate that larger amounts of polymer are adsorbed from aqueous solutions of HM-HEC and HM-EHEC on both substrates than from solutions of their unmodified analogues. The adsorption affinity for all the polymers, except EHEC, is higher on the citrate-covered surfaces than on the bare gold substrate. This indicates that more adsorption sites are activated in the presence of the citrate layer. The experimental adsorption data for all the polymers can be described fairly well by the Langmuir adsorption isotherm. However, at very low polymer concentrations significant deviations from the model are observed. The value of the hydrodynamic thickness of the adsorbed polymer layer (delta h), determined from DLS, rises with increasing polymer concentration for all the cellulose derivatives; a Langmuir type of isotherm can be used to roughly describe the adsorption behavior. Because of good solvent conditions for HEC the chains extend far out in the bulk at higher concentrations and the value of delta h is much higher than that of HM-HEC. The adsorption of EHEC and HM-EHEC onto gold particles discloses that the values of delta h are considerably higher for the hydrophobically modified cellulose derivative, and this finding is compatible with the trend in layer thickness estimated from the QCM-D measurements.

Adsorption behavior of acidic and basic proteins onto citrate-coated Au surfaces correlated to their native fold, stability, and pI.

The adsorption of eight different proteins (alpha-lactalbumin (types I and III), bovine serum albumin, hemoglobin, myoglobin, cytochrome c, alpha-casein, and lysozyme) onto a model anionic surface was performed at equivalent bulk (solvent, ionic strength, pH) and surface conditions. Adsorption was monitored on a quartz crystal microbalance with dissipation monitoring (QCM-D) with citrate-coated gold surfaces as adsorbents and has been correlated to native fold stability determined from near- and far-UV circular dichroism (CD) measurements. The proteins studied here were chosen based on their pI and documented knowledge about their structural stability and flexibility. Protein adsorption was found to be independent of global protein charge. Rather, binding occurs through oppositely charged patches on protein and surface. Moreover, data indicate that there is a correlation between secondary and tertiary structure stability and the adsorption characteristics at interfaces. Also, protein surface coverage, layer thickness, and flexibility can be tuned as a function of deposition method. This is discussed in terms of adsorption/spreading kinetics and intermolecular (protein-surface and protein-protein) interactions. Adsorption to surfaces can induce formation of supramolecular structures such as micelles (in the case of alpha-Cas) and multilayers (as for Hb). In the case of alpha-casein, this phenomenon depends on the deposition method and protein concentration. When ranking the surface coverage for proteins added in excess, the order is Lyz < Cyt c < Mb < BSA < alpha-La I < alpha-Cas < alpha-La III < Hb, which can be correlated to the proteins ability to form supramolecular structures (alpha-Cas, Hb), overall conformational flexibilities, and ability to form stable intermediates.

Synthesis, functionalisation and characterisation of mesoporous materials and sol-gel glasses for applications in catalysis, adsorption and photonics.

In this review, synthesis and functionalisation approaches for ordered mesoporous materials and sol-gel glasses are described. Catalytic and adsorption applications are emphasised for the ordered mesoporous materials, while optical applications are the focus for sol-gel glasses.

Spectroscopic characterization of cobalt-containing mesoporous materials.

Cobalt-containing mesoporous materials that have been prepared using different procedures have been comparatively characterized by transmission electron microscopy/energy-dispersive X-ray spectroscopy (TEM/EDS), extended X-ray absorption fine structure spectroscopy (EXAFS), X-ray absorption near edge spectroscopy (XANES), and ultraviolet-visible (UV-vis), near-infrared (NIR), and mid-infrared (mid-IR) spectroscopies, and the results provide new insights into the local environment and properties of cobalt in this type of material. TEM/EDS analyses have shown that tetraethyl orthosilicate (TEOS) may be less appropriate as a silicon source during the syntheses of cobalt-containing mesoporous materials, because the distribution of cobalt throughout the framework may become uneven. EXAFS has been determined to be the most suitable method for direct verification of framework incorporation, by identifying silicon as the backscatterer in the second shell. Such a direct verification may not be obtained using UV-vis spectroscopy. From EXAFS analyses, it is also possible to distinguish between surface-bound and framework-incorporated cobalt. There is a good agreement between the results obtained from XANES and UV-vis regarding the coordination symmetry of cobalt in the samples. The presence of cobalt in the silica framework has been determined to create Lewis acid sites, and these acid sites are suggested to be located at tetrahedral cobalt sites at the surface.