Iain E. Dunlop

The Stability of Silver Nanoparticles in a Model of Pulmonary Surfactant

The growing use of silver nanoparticles (AgNPs) in consumer products has raised concerns about their potential impact on the environment and human health. Whether AgNPs dissolve and release Ag(+) ions, or coarsen to form large aggregates, is critical in determining their potential toxicity. In this work, the stability of AgNPs in dipalmitoylphosphatidylcholine (DPPC), the major component of pulmonary surfactant, was investigated as a function of pH. Spherical, citrate-capped AgNPs with average diameters of 14 ± 1.6 nm (n = 200) were prepared by a chemical bath reduction. The kinetics of Ag(+) ion release was strongly pH-dependent. After 14 days of incubation in sodium perchlorate (NaClO4) or perchloric acid (HClO4) solutions, the total fraction of AgNPs dissolved varied from ∼10% at pH 3, to ∼2% at pH 5, with negligible dissolution at pH 7. A decrease in pH from 7 to 3 also promoted particle aggregation and coarsening. DPPC (100 mg·L(-1)) delayed the release of Ag(+) ions, but did not significantly alter the total amount of Ag(+) released after two weeks. In addition, DPPC improved the dispersion of the AgNPs and inhibited aggregation and coarsening. TEM images revealed that the AgNPs were coated with a DPPC layer serving as a semipermeable layer. Hence, lung lining fluid, particularly DPPC, can modify the aggregation state and kinetics of Ag(+) ion release of inhaled AgNPs in the lung. These observations have important implications for predicting the potential reactivity of AgNPs in the lung and the environment.

Direct Measurement of Normal and Shear Forces between Surface-Grown Polyelectrolyte Layers†

This paper presents measurements, using the surface force balance (SFB), of the normal and shear forces in aqueous solutions between polyelectrolyte layers grown directly on mica substrates (grafted-from). The grafting-from was via surface-initiated atom transfer radical polymerization (surface-initiated ATRP) using a positively charged methacrylate monomer. X-ray reflectometry measurements confirm the successful formation of polyelectrolyte layers by this method. Surface-inititated ATRP has the advantages that the polymer chains can be strongly grafted to the substrate, and that high grafting densities should be achievable. Measured normal forces in water showed a long-range repulsion arising from an electrical double layer that extended beyond the polyelectrolyte layers, and a stronger, shorter-range repulsion when the polyelectrolyte brushes were in contact. Swollen layer thicknesses were in the range 15-40 nm. Upon addition of approximately 10(-2)-10(-1) M sodium nitrate, screening effects reduced the electrical double layer force to an undetectable level. Shear force measurements in pure water were performed, and the measured friction may arise from polymer chains bridging between the surfaces.

Nanoscale ligand spacing influences receptor triggering in T cells and NK cells.

Bioactive nanoscale arrays were constructed to ligate activating cell surface receptors on T cells (the CD3 component of the TCR complex) and natural killer (NK) cells (CD16). These arrays are formed from biofunctionalized gold nanospheres with controlled interparticle spacing in the range 25-104 nm. Responses to these nanoarrays were assessed using the extent of membrane-localized phosphotyrosine in T cells stimulated with CD3-binding nanoarrays and the size of cell contact area for NK cells stimulated with CD16-binding nanoarrays. In both cases, the strength of response decreased with increasing spacing, falling to background levels by 69 nm in the T cell/anti-CD3 system and 104 nm for the NK cell/anti-CD16 system. These results demonstrate that immune receptor triggering can be influenced by the nanoscale spatial organization of receptor/ligand interactions.

Structure and Collapse of a Surface-Grown Strong Polyelectrolyte Brush on Sapphire

We have used neutron reflectometry to investigate the behavior of a strong polyelectrolyte brush on a sapphire substrate, grown by atom-transfer radical polymerization (ATRP) from a silane-anchored initiator layer. The initiator layer was deposited from vapor, following treatment of the substrate with an Ar/H(2)O plasma to improve surface reactivity. The deposition process was characterized using X-ray reflectometry, indicating the formation of a complete, cross-linked layer. The brush was grown from the monomer [2-(methacryloyloxy)ethyl]trimethylammonium chloride (METAC), which carries a strong positive charge. The neutron reflectivity profile of the swollen brush in pure water (D(2)O) showed that it adopted a two-region structure, consisting of a dense surface region ∼100 Å thick, in combination with a diffuse brush region extending to around 1000 Å from the surface. The existence of the diffuse brush region may be attributed to electrostatic repulsion from the positively charged surface region, while the surface region itself most probably forms due to polyelectrolyte adsorption to the hydrophobic initiator layer. The importance of electrostatic interactions in maintaining the brush region is confirmed by measurements at high (1 M) added 1:1 electrolyte, which show a substantial transfer of polymer from the brush to the surface region, together with a strong reduction in brush height. On addition of 10(-4) M oppositely charged surfactant (sodium dodecyl sulfate), the brush undergoes a dramatic collapse, forming a single dense layer about 200 Å in thickness, which may be attributed to the neutralization of the monomers by adsorbed dodecyl sulfate ions in combination with hydrophobic interactions between these dodecyl chains. Subsequent increases in surfactant concentration result in slow increases in brush height, which may be caused by stiffening of the polyelectrolyte chains due to further dodecyl sulfate adsorption.

Effect of end-group sticking energy on the properties of polymer brushes: comparing experiment and theory.

Using surface force balance measurements we have established that polystyrene chains bearing three zwitterionic groups have a higher end-group sticking energy than equivalent chains bearing a single zwitterionic group. In a good solvent, polystyrene chains end-functionalized with three zwitterionic groups form brushes of a higher surface coverage than those bearing a single zwitterion. The increase in surface coverage is slow compared with the initial formation of the brush. Measurements of the refractive index allow us to directly quantify the variation of surface coverage, permitting comparison with models for the kinetics of brush formation based on scaling theory and an analytical self-consistent field. We find qualitative support for associating the kinetic barrier with the energy required for an incoming chain to stretch as it penetrates the existing brush.

Modular and Versatile Spatial Functionalization of Tissue Engineering Scaffolds through Fiber-Initiated Controlled Radical Polymerization

Native tissues are typically heterogeneous and hierarchically organized, and generating scaffolds that can mimic these properties is critical for tissue engineering applications. By uniquely combining controlled radical polymerization (CRP), end‐functionalization of polymers, and advanced electrospinning techniques, a modular and versatile approach is introduced to generate scaffolds with spatially organized functionality. Poly‐ε‐caprolactone is end functionalized with either a polymerization‐initiating group or a cell‐binding peptide motif cyclic Arg‐Gly‐Asp‐Ser (cRGDS), and are each sequentially electrospun to produce zonally discrete bilayers within a continuous fiber scaffold. The polymerization‐initiating group is then used to graft an antifouling polymer bottlebrush based on poly(ethylene glycol) from the fiber surface using CRP exclusively within one bilayer of the scaffold. The ability to include additional multifunctionality during CRP is showcased by integrating a biotinylated monomer unit into the polymerization step allowing postmodification of the scaffold with streptavidin‐coupled moieties. These combined processing techniques result in an effective bilayered and dual‐functionality scaffold with a cell‐adhesive surface and an opposing antifouling non‐cell‐adhesive surface in zonally specific regions across the thickness of the scaffold, demonstrated through fluorescent labelling and cell adhesion studies. This modular and versatile approach combines strategies to produce scaffolds with tailorable properties for many applications in tissue engineering and regenerative medicine.

Cytotoxicity and intracellular dissolution of nickel nanowires

Abstract The assessment of cytotoxicity of nanostructures is a fundamental step for their development as biomedical tools. As widely used nanostructures, nickel nanowires (Ni NWs) seem promising candidates for such applications. In this work, Ni NWs were synthesized and then characterized using vibrating sample magnetometry, energy dispersive X-Ray analysis, and electron microscopy. After exposure to the NWs, cytotoxicity was evaluated in terms of cell viability, cell membrane damage, and induced apoptosis/necrosis on the model human cell line HCT 116. The influence of NW to cell ratio (10:1 to 1000:1) and exposure times up to 72 hours was analyzed for Ni NWs of 5.4 μm in length, as well as for Ni ions. The results show that cytotoxicity markedly increases past 24 hours of incubation. Cellular uptake of NWs takes place through the phagocytosis pathway, with a fraction of the dose of NWs dissolved inside the cells. Cell death results from a combination of apoptosis and necrosis, where the latter is the outcome of the secondary necrosis pathway. The cytotoxicity of Ni ions and Ni NWs dissolution studies suggest a synergistic toxicity between NW aspect ratio and dissolved Ni, with the cytotoxic effects markedly increasing after 24 hours of incubation.

Multibranched Gold Nanoparticles with Intrinsic LAT-1 Targeting Capabilities for Selective Photothermal Therapy of Breast Cancer

Because of the critical role of the large neutral amino acid transporter-1 (LAT-1) in promoting tumor growth and proliferation, it is fast emerging as a highly attractive biomarker for the imaging and treatment of human malignancies, including breast cancer. While multibranched gold nanoparticles (AuNPs) have emerged as a promising modality in the photothermal therapy (PTT) of cancers, some of the key challenges limiting their clinical translation lie in the need to develop reproducible and cost-effective synthetic methods as well as the selective accumulation of sufficient AuNPs at tumor sites. In this study, we report a simple and direct seed-mediated synthesis of monodispersed multibranched AuNPs using the catechol-containing LAT-1 ligands, L- and D-dopa, to confer active cancer targeting. This route obviates the need for additional conjugation with targeting moieties such as peptides or antibodies. Nanoflower-like AuNPs (AuNF) with diameters of approximately 46, 70, and 90 nm were obtained and were found to possess excellent colloidal stability and biocompatibility. A significantly higher intracellular accumulation of the L- and D-dopa functionalized AuNFs was observed in a panel of breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-468, and MDA-MB-453) when compared to the nontargeting control AuNFs synthesized with dopamine and 4-ethylcatechol. Importantly, no significant difference in uptake between the targeting and nontargeting AuNFs was observed in a non-tumorigenic MCF-10A breast epithelial cell line, hence demonstrating tumor selectivity. For PTT of breast cancer, Ag+ was introduced during synthesis to obtain L-dopa functionalized nanourchin-like AuNPs (AuNUs) with strong near-infrared (NIR) absorbance. The L-dopa functionalized AuNUs mediated selective photothermal ablation of the triple negative MDA-MB-231 breast cancer cell line and sensitized the cells to the anticancer drugs cisplatin and docetaxel. This work brings forward an effective strategy for the facile preparation of cancer targeting multibranched AuNPs with potential for the in vivo PTT of breast cancer.

Inhibition of Leptin-ObR Interaction Does not Prevent Leptin Translocation Across a Human Blood-Brain Barrier Model.

The adipocyte‐derived hormone leptin regulates appetite and energy homeostasis through the activation of leptin receptors (ObR) on hypothalamic neurones; hence, leptin must be transported through the blood–brain barrier (BBB) to reach its target sites in the central nervous system. During obesity, however, leptin BBB transport is decreased, in part precluding leptin as a viable clinical therapy against obesity. Although the short isoform of the ObR (ObRa) has been implicated in the transport of leptin across the BBB as a result of its elevated expression in cerebral microvessels, accumulating evidence indicates that leptin BBB transport is independent of ObRa. In the present study, we employed an ObR‐neutralising antibody (9F8) to directly examine the involvement of endothelial ObR in leptin transport across an in vitro human BBB model composed of the human endothelial cell line hCMEC/D3. Our results indicate that, although leptin transport across the endothelial monolayer was nonparacellular, and energy‐ and endocytosis‐dependent, it was not inhibited by pre‐treatment with 9F8, despite the ability of the latter to recognise hCMEC/D3‐expressed ObR, prevent leptin–ObR binding and inhibit leptin‐induced signal transducer and activator of transcription 3 (STAT‐3) phosphorylation in hCMEC/D3 cells. Furthermore, hCMEC/D3 cells expressed the transporter protein low‐density lipoprotein receptor‐related protein‐2 (LRP‐2), which is capable of binding and endocytosing leptin. In conclusion, our results demonstrate that leptin binding to and signalling through ObR is not required for efficient transport across human endothelial monolayers, indicating that ObR is not the primary leptin transporter at the human BBB, a role which may fall upon LRP‐2. A deeper understanding of leptin BBB transport will help clarify the exact causes for leptin resistance seen in obesity and aid in the development of more efficient BBB‐penetrating leptin analogues.

Scalable High-Affinity Stabilization of Magnetic Iron Oxide Nanostructures by a Biocompatible Antifouling Homopolymer

Iron oxide nanostructures have been widely developed for biomedical applications because of their magnetic properties and biocompatibility. In clinical applications, stabilization of these nanostructures against aggregation and nonspecific interactions is typically achieved using weakly anchored polysaccharides, with better-defined and more strongly anchored synthetic polymers not commercially adopted because of their complexity of synthesis and use. Here, we show for the first time stabilization and biocompatibilization of iron oxide nanoparticles by a synthetic homopolymer with strong surface anchoring and a history of clinical use in other applications, poly(2-methacryloyloxyethyl phosphorylcholine) [poly(MPC)]. For the commercially important case of spherical particles, binding of poly(MPC) to iron oxide surfaces and highly effective individualization of magnetite nanoparticles (20 nm) are demonstrated. Next-generation high-aspect-ratio nanowires (both magnetite/maghemite and core-shell iron/iron oxide) are, furthermore, stabilized by poly(MPC) coating, with the nanowire cytotoxicity at large concentrations significantly reduced. The synthesis approach exploited to incorporate functionality into the poly(MPC) chain is demonstrated by random copolymerization with an alkyne-containing monomer for click chemistry. Taking these results together, poly(MPC) homopolymers and random copolymers offer a significant improvement over current iron oxide nanoformulations, combining straightforward synthesis, strong surface anchoring, and well-defined molecular weight.