Darren R. Dunphy

The targeted delivery of multicomponent cargos to cancer cells by nanoporous particle-supported lipid bilayers

Encapsulation of drugs within nanocarriers that selectively target malignant cells promises to mitigate side effects of conventional chemotherapy and to enable delivery of the unique drug combinations needed for personalized medicine. To realize this potential, however, targeted nanocarriers must simultaneously overcome multiple challenges, including specificity, stability, and a high capacity for disparate cargos. Here we report porous nanoparticle-supported lipid bilayers (protocells) that synergistically combine properties of liposomes and nanoporous particles. Protocells modified with a targeting peptide that binds to human hepatocellular carcinoma (HCC) exhibit a 10,000-fold greater affinity for HCC than for hepatocytes, endothelial cells, and immune cells. Furthermore, protocells can be loaded with combinations of therapeutic (drugs, siRNA, and toxins) and diagnostic (quantum dots) agents and modified to promote endosomal escape and nuclear accumulation of selected cargos. The enormous capacity of the high-surface-area nanoporous core combined with the enhanced targeting efficacy enabled by the fluid supported lipid bilayer allow a single protocell loaded with a drug cocktail to kill a drug-resistant HCC cell, representing a 106-fold improvement over comparable liposomes.

Surface Interactions with Compartmentalized Cellular Phosphates Explain Rare Earth Oxide Nanoparticle Hazard and Provide Opportunities for Safer Design

Growing international exploitation of rare earth oxides (REOs) for commercial and biological use has increased the possibility of human exposure and adverse health effects. Occupational exposure to rare earth materials in miners and polishers leads to a severe form of pneumoconiosis, while gadolinium-containing MRI contrast agents cause nephrogenic systemic fibrosis in patients with renal impairment. The mechanisms for inducing these adverse pro-fibrogenic effects are of considerable importance for the safety assessment of REO particles as well as presenting opportunities for safer design. In this study, using a well-prepared REO library, we obtained a mechanistic understanding of how REOs induce cellular and pulmonary damage by a compartmentalized intracellular biotransformation process in lysosomes that results in pro-fibrogenic growth factor production and lung fibrosis. We demonstrate that rare earth oxide ion shedding in acidifying macrophage lysosomes leads to biotic phosphate complexation that results in organelle damage due to stripping of phosphates from the surrounding lipid bilayer. This results in nanoparticle biotransformation into urchin shaped structures and setting in motion a series of events that trigger NLRP3 inflammasome activation, IL-1β release, TGF-β1 and PDGF-AA production. However, pretreatment of REO nanoparticles with phosphate in a neutral pH environment prevents biological transformation and pro-fibrogenic effects. This can be used as a safer design principle for producing rare earth nanoparticles for biological use.

The Electroactive Integrated Optical Waveguide: Ultrasensitive Spectroelectrochemistry of Submonolayer Adsorbates

Highly sensitive spectroelectrochemistry of adsorbed films on ITO is demonstrated with the electroactive integrated optical waveguide (EA-IOW). The EA-IOW, a single-mode planar waveguide coated with an ITO layer, is ∼10(4)-fold more sensitive to changes in absorbance occurring during electrochemical events versus a single-pass transmission spectroelectrochemical experiment, as demonstrated by reduction of surface-adsorbed methylene blue. Furthermore, the EA-IOW is selective to near-surface events, as it is relatively insensitive to absorbance by solutions of dissolved chromophores at <1 mM. The EA-IOW is also used to monitor the formation of Prussian Blue during the reduction of ferricyanide, an event that is not easily followed using current-detected cyclic voltammetry, due to interfering faradaic and non-faradaic electrochemical events. The optical background of the EA-IOW is potential-dependent and is explained by ion diffusion into the ITO and by voltage-dependent changes in optical constants for the material. Finally, the high sensitivity of the EA-IOW (relative to other evanescent-field-based spectroelectrochemical techniques) is discussed in terms of its design.

Free-Standing, Patternable Nanoparticle/Polymer Monolayer Arrays Formed by Evaporation Induced Self-Assembly at a Fluid Interface

We report a general and facile method to prepare free-standing, patternable nanoparticle/polymer monolayer arrays by interfacial NP assembly within a polymeric photoresist. The ultrathin monolayer nanoparticle/polymer arrays are sufficiently robust that they can be transferred to arbitrary substrates and suspended as free-standing membranes over cm-sized holeseven with free edges. More importantly, the polymethylmethacrylate (PMMA) in the system serves as a photoresist enabling two modes of electron beam (e-beam) patterning. Lower e-beam doses direct differential nanoparticle solubility and result in nanoparticle patterns with somewhat diffuse interfaces. At higher e-beam doses the PMMA serves as a negative resist resulting in submicrometer patterns with edge roughness comparable to that of the nanoparticle diameter. These ultrathin films of monolayer nanoparticle arrays are of fundamental interest as 2D artificial solids for electronic, magnetic, and optical properties and are also of technological inter...

Mesoporous Silica Nanoparticle-Supported Lipid Bilayers (Protocells) for Active Targeting and Delivery to Individual Leukemia Cells

Many nanocarrier cancer therapeutics currently under development, as well as those used in the clinical setting, rely upon the enhanced permeability and retention (EPR) effect to passively accumulate in the tumor microenvironment and kill cancer cells. In leukemia, where leukemogenic stem cells and their progeny circulate within the peripheral blood or bone marrow, the EPR effect may not be operative. Thus, for leukemia therapeutics, it is essential to target and bind individual circulating cells. Here, we investigate mesoporous silica nanoparticle (MSN)-supported lipid bilayers (protocells), an emerging class of nanocarriers, and establish the synthesis conditions and lipid bilayer composition needed to achieve highly monodisperse protocells that remain stable in complex media as assessed in vitro by dynamic light scattering and cryo-electron microscopy and ex ovo by direct imaging within a chick chorioallantoic membrane (CAM) model. We show that for vesicle fusion conditions where the lipid surface area exceeds the external surface area of the MSN and the ionic strength exceeds 20 mM, we form monosized protocells (polydispersity index <0.1) on MSN cores with varying size, shape, and pore size, whose conformal zwitterionic supported lipid bilayer confers excellent stability as judged by circulation in the CAM and minimal opsonization in vivo in a mouse model. Having established protocell formulations that are stable colloids, we further modified them with anti-EGFR antibodies as targeting agents and reverified their monodispersity and stability. Then, using intravital imaging in the CAM, we directly observed in real time the progression of selective targeting of individual leukemia cells (using the established REH leukemia cell line transduced with EGFR) and delivery of a model cargo. Overall, we have established the effectiveness of the protocell platform for individual cell targeting and delivery needed for leukemia and other disseminated disease.

Protein-Directed Assembly of Arbitrary Three-Dimensional Nanoporous Silica Architectures

Through precise control of nanoscale building blocks, such as proteins and polyamines, silica condensing microorganisms are able to create intricate mineral structures displaying hierarchical features from nano- to millimeter-length scales. The creation of artificial structures of similar characteristics is facilitated through biomimetic approaches, for instance, by first creating a bioscaffold comprised of silica condensing moieties which, in turn, govern silica deposition into three-dimensional (3D) structures. In this work, we demonstrate a protein-directed approach to template silica into true arbitrary 3D architectures by employing cross-linked protein hydrogels to controllably direct silica condensation. Protein hydrogels are fabricated using multiphoton lithography, which enables user-defined control over template features in three dimensions. Silica deposition, under acidic conditions, proceeds throughout protein hydrogel templates via flocculation of silica nanoparticles by protein molecules, as indicated by dynamic light scattering (DLS) and time-dependent measurements of elastic modulus. Following silica deposition, the protein template can be removed using mild thermal processing yielding high surface area (625 m(2)/g) porous silica replicas that do not undergo significant volume change compared to the starting template. We demonstrate the capabilities of this approach to create bioinspired silica microstructures displaying hierarchical features over broad length scales and the infiltration/functionalization capabilities of the nanoporous silica matrix by laser printing a 3D gold image within a 3D silica matrix. This work provides a foundation to potentially understand and mimic biogenic silica condensation under the constraints of user-defined biotemplates and further should enable a wide range of complex inorganic architectures to be explored using silica transformational chemistries, for instance silica to silicon, as demonstrated herein.

Cellular complexity captured in durable silica biocomposites

Tissue-derived cultured cells exhibit a remarkable range of morphological features in vitro, depending on phenotypic expression and environmental interactions. Translation of these cellular architectures into inorganic materials would provide routes to generate hierarchical nanomaterials with stabilized structures and functions. Here, we describe the fabrication of cell/silica composites (CSCs) and their conversion to silica replicas using mammalian cells as scaffolds to direct complex structure formation. Under mildly acidic solution conditions, silica deposition is restricted to the molecularly crowded cellular template. Inter- and intracellular heterogeneity from the nano- to macroscale is captured and dimensionally preserved in CSCs following drying and subjection to extreme temperatures allowing, for instance, size and shape preserving pyrolysis of cellular architectures to form conductive carbon replicas. The structural and behavioral malleability of the starting material (cultured cells) provides opportunities to develop robust and economical biocomposites with programmed structures and functions.

Measuring the Thermal Conductivity of Porous, Transparent SiO2 Films With Time Domain Thermoreflectance

Nanocomposites offer unique capabilities of controlling thermal transport through the manipulation of various structural aspects of the material. However, measurements of the thermal properties of these composites are often difficult, especially porous nanomaterials. Optical measurements of these properties, although ideal due to the noncontact nature, are challenging due to the large surface variability of nanoporous structures. In this work, we use a vector-based thermal algorithm to solve for the temperature change and heat transfer in which a thin film subjected to a modulated heat source is sandwiched between two thermally conductive pathways. We validate our solution with time domain thermoreflectance measurements on glass slides and extend the thermal conductivity measurements to SiO 2 -based nanostructured films.

Multiphoton Lithography of Nanocrystalline Platinum and Palladium for Site-Specific Catalysis in 3D Microenvironments.

Integration of catalytic nanostructured platinum and palladium within 3D microscale structures or fluidic environments is important for systems ranging from micropumps to microfluidic chemical reactors and energy converters. We report a straightforward procedure to fabricate microscale patterns of nanocrystalline platinum and palladium using multiphoton lithography. These materials display excellent catalytic, electrical, and electrochemical properties, and we demonstrate high-resolution integration of catalysts within 3D defined microenvironments to generate directed autonomous particle and fluid transport.

Dynamic Investigation of Gold Nanocrystal Assembly Using In Situ Grazing-Incidence Small-Angle X-ray Scattering

Here we investigate the dynamic self-assembly pathway of ordered gold nanocrystal arrays during the self-assembly of gold nanocrystal micelles, with and without the presence of colloidal silica precursors, using grazing-incidence X-ray scattering performed at a synchrotron source. With silica precursors present, a lattice with rhombohedral symmetry is formed from the partial collapse of a face-centered cubic structure. In the absence of silica, a transient body-centered orthorhombic phase appears, which rapidly collapses into a glassy nanocrystal film. The appearance of face-centered and body-centered structures is consistent with a phase diagram for charged colloidal particles with assembly modulated via Coulomb screening.