Carlee E. Ashley

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.

Electrostatically mediated liposome fusion and lipid exchange with a nanoparticle-supported bilayer for control of surface charge, drug containment, and delivery.

The loading and containment of cargo within nanoparticles and their efficient delivery to cells represent a primary challenge in nanomedicine. We report lipid exchange between free and mesoporous silica nanoparticle-supported lipid bilayers as an effective means of containing cargo, controlling charge, and directing delivery to mammalian cells. The delivery of a membrane-impermeable dye (calcein) and a chemotherapeutic drug (doxorubicin) are demonstrated. Exchanged lipid bilayers minimized premature drug release, and an overall positive charge on the supported lipid bilayer effected enhanced delivery.

Cell-Specific Delivery of Diverse Cargos by Bacteriophage MS2 Virus-Like Particles

Virus-like particles (VLPs) of bacteriophage MS2 possess numerous features that make them well-suited for use in targeted delivery of therapeutic and imaging agents. MS2 VLPs can be rapidly produced in large quantities using in vivo or in vitro synthesis techniques. Their capsids can be modified in precise locations via genetic insertion or chemical conjugation, facilitating the multivalent display of targeting ligands. MS2 VLPs also self-assemble in the presence of nucleic acids to specifically encapsidate siRNA and RNA-modified cargos. Here we report the use of MS2 VLPs to selectively deliver nanoparticles, chemotherapeutic drugs, siRNA cocktails, and protein toxins to human hepatocellular carcinoma (HCC). MS2 VLPs modified with a peptide (SP94) that binds HCC exhibit a 10(4)-fold higher avidity for HCC than for hepatocytes, endothelial cells, monocytes, or lymphocytes and can deliver high concentrations of encapsidated cargo to the cytosol of HCC cells. SP94-targeted VLPs loaded with doxorubicin, cisplatin, and 5-fluorouracil selectively kill the HCC cell line, Hep3B, at drug concentrations <1 nM, while SP94-targeted VLPs that encapsidate a siRNA cocktail, which silences expression of cyclin family members, induce growth arrest and apoptosis of Hep3B at siRNA concentrations <150 pM. Impressively, MS2 VLPs, when loaded with ricin toxin A-chain (RTA) and modified to codisplay the SP94 targeting peptide and a histidine-rich fusogenic peptide (H5WYG) that promotes endosomal escape, kill virtually the entire population of Hep3B cells at an RTA concentration of 100 fM without affecting the viability of control cells. Our results demonstrate that MS2 VLPs, because of their tolerance of multivalent peptide display and their ability to specifically encapsidate a variety of chemically disparate cargos, induce selective cytotoxicity of cancer in vitro and represent a significant improvement in the characteristics of VLP-based delivery systems.

Cell-directed-assembly: Directing the formation of nano/bio interfaces and architectures with living cells ☆

BACKGROUND The desire to immobilize, encapsulate, or entrap viable cells for use in a variety of applications has been explored for decades. Traditionally, the approach is to immobilize cells to utilize a specific functionality of the cell in the system. SCOPE OF REVIEW This review describes our recent discovery that living cells can organize extended nanostructures and nano-objects to create a highly biocompatible nano//bio interface [1]. MAJOR CONCLUSIONS We find that short chain phospholipids direct the formation of thin film silica mesophases during evaporation-induced self-assembly (EISA) [2], and that the introduction of cells alter the self-assembly pathway. Cells organize an ordered lipid-membrane that forms a coherent interface with the silica mesophase that is unique in that it withstands drying-yet it maintains accessibility to molecules introduced into the 3D silica host. Cell viability is preserved in the absence of buffer, making these constructs useful as standalone cell-based sensors. In response to hyperosmotic stress, the cells release water, creating a pH gradient which is maintained within the nanostructured host and serves to localize lipids, proteins, plasmids, lipidized nanocrystals, and other components at the cellular surface. This active organization of the bio/nano interface can be accomplished during ink-jet printing or selective wetting-processes allowing patterning of cellular arrays-and even spatially-defined genetic modification. GENERAL SIGNIFICANCE Recent advances in the understanding of nanotechnology and cell biology encourage the pursuit of more complex endeavors where the dynamic interactions of the cell and host material act symbiotically to obtain new, useful functions. This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.

Cell-Directed Localization and Orientation of a Functional Foreign Transmembrane Protein within a Silica Nanostructure

A simple procedure for introducing functional exogenous membrane-bound proteins to viable cells encapsulated within a lipid templated silica nanostructure is described. In one method, bacteriorhodopsin (bR) was added directly to a Saccharomyces cerevisiae solution along with short zwitterionic diacylphosphatidylcholines (diC(6) PC) and mixed with equal volumes of a sol precursor solution. Alternatively, bR was first incorporated into liposomes (bR-proteoliposomes) and then added to an S. cerevisiae solution with diC(6) PC, and this was followed by mixing with sol precursor solution. Films prepared from bR added directly to diC(6) PC resulted in bR localization near S. cerevisiae cells in a disordered and diffuse fashion, while films prepared from bR-proteoliposomes added to the diC(6) PC/yeast solution resulted in preferential localization of bR near yeast cell surfaces, forming bR-containing multilayer vesicles. Importantly, bR introduced via proteoliposomes was observed to modulate pH gradients developed at the cell surface, demonstrating both retained functionality and preferential orientation. Localization of liposome lipid or bR did not occur around neutrally charged latex beads acting as cell surrogates, demonstrating that living cells actively organize the multilayered lipid during evaporation-induced self-assembly. We expect this simple procedure for introducing functional and oriented membrane-bound proteins to the surface of cells to be general and adaptable to other membrane-bound proteins. This advance may prove useful in fundamental studies of membrane protein function and cell-cell signaling and in imparting non-native characteristics to arbitrary cells.

Convective Assembly of 2D Lattices of Virus‐like Particles Visualized by In‐Situ Grazing‐Incidence Small‐Angle X‐Ray Scattering

The rapid assembly of icosohedral virus-like particles (VLPs) into highly ordered (domain size > 600 nm), oriented 2D superlattices directly onto a solid substrate using convective coating is demonstrated. In-situ grazing-incidence small-angle X-ray scattering (GISAXS) is used to follow the self-assembly process in real time to characterize the mechanism of superlattice formation, with the ultimate goal of tailoring film deposition conditions to optimize long-range order. From water, GISAXS data are consistent with a transport-limited assembly process where convective flow directs assembly of VLPs into a lattice oriented with respect to the water drying line. Addition of a nonvolatile solvent (glycerol) modified this assembly pathway, resulting in non-oriented superlattices with improved long-range order. Modification of electrostatic conditions (solution ionic strength, substrate charge) also alters assembly behavior; however, a comparison of in-situ assembly data between VLPs derived from the bacteriophages MS2 and Qβ show that this assembly process is not fully described by a simple Derjaguin-Landau-Verwey-Overbeek model alone.

Versatile Synthesis and Fluorescent Labeling of ZIF-90 Nanoparticles for Biomedical Applications.

We describe a versatile method for the synthesis and fluorescent labeling of ZIF-90 nanoparticles (NPs). Gram-scale quantities of NPs can be produced under mild conditions, circumventing the need for high temperatures and extended reaction periods required by existing procedures. Monitoring the reaction in situ using UV-vis spectroscopy reveals that ZIF-90 NP nucleation in solution starts within seconds. In addition to reporting a method to reproducibly form sub-100 nm ZIF-90 particles, we show that particles of various sizes can be produced, ranging from 30 to 1000 nm, by altering amine chemistry or reaction temperature. The presence of linker aldehyde groups on the NP surface allows for postsynthetic labeling with amine-functionalized fluorescent dyes, providing utility for imaging within biological systems. In vitro cell studies show that ZIF-90 NPs have a high rate of cellular internalization, provide finite degradation periods of the order of several weeks, and are biocompatible with six different cell lines (>90% viable when incubated with NPs for up to 7 days). These features highlight the potential for use of ZIF-90 nanostructures in bioimaging and targeted drug delivery applications.

Nature versus nurture in cellular behavior and disease.

This project pursued two complementary, interrelated goals: 1) the incorporation of individual or groups of bacterial, fungal, or mammalian cells within novel three-dimensional (3D) cell-built or lithographically defined matrices that provide an engineered chemical and physical background to inform cells and direct their behavior; and 2) the development of two classes of targeted nanoparticle delivery platforms, protocells (porous nanoparticle supported lipid bilayers) and virus-like particles (VLPs), which could be selected against dormant/drug resistant/metastatic cells and selectively deliver multicomponent cargos (cocktails) to this recalcitrant population. This project provided a unique means to understand environmental influences on cellular behavior, in particular, dormancy, drug resistance, metastasis and

Biocompatible self-assembly of nano-materials for Bio-MEMS and insect reconnaissance.

This report summarizes the development of new biocompatible self-assembly procedures enabling the immobilization of genetically engineered cells in a compact, self-sustaining, remotely addressable sensor platform. We used evaporation induced self-assembly (EISA) to immobilize cells within periodic silica nanostructures, characterized by unimodal pore sizes and pore connectivity, that can be patterned using ink-jet printing or photo patterning. We constructed cell lines for the expression of fluorescent proteins and induced reporter protein expression in immobilized cells. We investigated the role of the abiotic/biotic interface during cell-mediated self-assembly of synthetic materials.