Wilhelm R. Glomm

Functionalized Gold Nanoparticles for Applications in Bionanotechnology

Gold nanoparticles have unique optical and chemical properties that make them ideally suited for a number of applications in bionanotechnology, including optical probes, targeted drug delivery, and programmed materials synthesis. The recurring theme is the versatility of gold nanoparticles and how this versatility can be exploited to adapt gold nanoparticles to fit into different chemical environments—from aqueous suspensions to the hostile conditions of a cell culture or the human bloodstream. Herein, the optical and chemical properties of gold nanoparticles are described, and a number of specific applications within bionanotechnology are reviewed.

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.

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).

L‐DOPA‐Coated Manganese Oxide Nanoparticles as Dual MRI Contrast Agents and Drug‐Delivery Vehicles

Manganese oxide nanoparticles (MONPs) are capable of time-dependent magnetic resonance imaging contrast switching as well as releasing a surface-bound drug. MONPs give T2/T2* contrast, but dissolve and release T1-active Mn(2+) and L-3,4-dihydroxyphenylalanine. Complementary images are acquired with a single contrast agent, and applications toward Parkinsons disease are suggested.

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.

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 (HAMLETs 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 HAMLETs 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.

Tunable photophysical properties, conformation and function of nanosized protein–gold constructs

Protein-stabilized gold nanoconstructs are widely studied due to their potential applications in biosensing, drug and gene delivery, and bioimaging. While a number of studies have focused on the novel properties of such materials emanating from the gold, there has been little focus on how the protein shell is affected by nanocluster formation with respect to conformation, stability and function. Herein, we show the synthesis of protein-stabilized gold nanoconstructs varying in size from small clusters (~8 Au atoms) dispersed within proteins to nanoparticles stabilized by multiple proteins by varying the concentration of gold precursor and reducing agent. Proteins used were bovine serum albumin (BSA), bovine α-lactalbumin (BLA) and lysozyme (LYZ). Photophysical properties of the gold nanostructures were monitored using UV-vis and fluorescence measurements, revealing that the gold constructs can be tuned from luminescent clusters to nanoparticles displaying localized surface plasmon resonance (LSPR). Conformational changes of the protein following conjugation to gold nanostructures were studied using steady-state and time-resolved Trp fluorescence measurements and circular dichroism. The degree of conformational perturbation varied greatly between the proteins used, with BLA being the most tunable in terms of gradual unfolding, whereas the conformational stability of LYZ was very sensitive to the reducing agent used. To assess the impact of the gold nanostructures as well as the reducing agent on protein function, the LYZ–gold nanoconstructs were subjected to an activity test by degradation of Micrococcus lysodeikticus cell walls, revealing that the activity of the LYZ–Au constructs was retained and tunable, albeit at attenuated levels.

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.

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.

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.