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.

Protocells: Modular Mesoporous Silica Nanoparticle-Supported Lipid Bilayers for Drug Delivery.

Mesoporous silica nanoparticle-supported lipid bilayers, termed 'protocells,' represent a potentially transformative class of therapeutic and theranostic delivery vehicle. The field of targeted drug delivery poses considerable challenges that cannot be addressed with a single 'magic bullet'. Consequently, the protocell has been designed as a modular platform composed of interchangeable biocompatible components. The mesoporous silica core has variable size and shape to direct biodistribution and a controlled pore size and surface chemistry to accommodate diverse cargo. The encapsulating supported lipid bilayer can be modified with targeting and trafficking ligands as well as polyethylene glycol (PEG) to effect selective binding, endosomal escape of cargo, drug efflux prevention, and potent therapeutic delivery, while maintaining in vivo colloidal stability. This review describes the individual components of the platform, including the mesoporous silica nanoparticle core and supported lipid bilayer, their assembly (by multiple techniques) into a protocell, and the combined, often synergistic, performance of the protocell based on in vitro and in vivo studies, including the assessment of biocompatibility and toxicity. In closing, the many emerging variations of the protocell theme and the future directions for protocell research are commented on.

Mechanistic modeling identifies drug-uptake history as predictor of tumor drug resistance and nano-carrier-mediated response.

A quantitative understanding of the advantages of nanoparticle-based drug delivery vis-à-vis conventional free drug chemotherapy has yet to be established for cancer or other diseases despite numerous investigations. Here, we employ first-principles cell biophysics, drug pharmaco-kinetics, and drug pharmaco-dynamics to model the delivery of doxorubicin (DOX) to hepatocellular carcinoma (HCC) tumor cells and predict the resultant experimental cytotoxicity data. The fundamental, mechanistic hypothesis of our mathematical model is that the integrated history of drug uptake by the cells over time of exposure, which sets the cell death rate parameter, and the uptake rate are the sole determinants of the dose response relationship. A universal solution of the model equations is capable of predicting the entire, nonlinear dose response of the cells to any drug concentration based on just two separate measurements of these cellular parameters. This analysis reveals that nanocarrier-mediated delivery overcomes resistance to the free drug because of improved cellular uptake rates, and that dose response curves to nanocarrier mediated drug delivery are equivalent to those for free-drug, but "shifted to the left;" that is, lower amounts of drug achieve the same cell kill. We then demonstrate the model's general applicability to different tumor and drug types, and cell-exposure time courses by investigating HCC cells exposed to cisplatin and 5-fluorouracil, breast cancer MCF-7 cells exposed to DOX, and pancreatic adenocarcinoma PANC-1 cells exposed to gemcitabine. The model will help in the optimal design of nanocarriers for clinical applications and improve the current, largely empirical understanding of in vivo drug transport and tumor response.

Mesoporous silica nanoparticle nanocarriers: biofunctionality and biocompatibility.

The study of ordered mesoporous silica materials has exploded since their discovery by Mobil researchers 20 years ago. The ability to make uniformly sized, porous, and dispersible nanoparticles using colloidal chemistry and evaporation-induced self-assembly has led to many applications of mesoporous silica nanoparticles (MSNPs) as "nanocarriers" for delivery of drugs and other cargos to cells. The exceptionally high surface area of MSNPs, often exceeding 1000 m²/g, and the ability to independently modify pore size and surface chemistry, enables the loading of diverse cargos and cargo combinations at levels exceeding those of other common drug delivery carriers such as liposomes or polymer conjugates. This is because noncovalent electrostatic, hydrogen-bonding, and van der Waals interactions of the cargo with the MSNP internal surface cause preferential adsorption of cargo to the MSNP, allowing loading capacities to surpass the solubility limit of a solution or that achievable by osmotic gradient loading. The ability to independently modify the MSNP surface and interior makes possible engineered biofunctionality and biocompatibility. In this Account, we detail our recent efforts to develop MSNPs as biocompatible nanocarriers (Figure 1 ) that simultaneously display multiple functions including (1) high visibility/contrast in multiple imaging modalities, (2) dispersibility, (3) binding specificity to a particular target tissue or cell type, (4) ability to load and deliver large concentrations of diverse cargos, and (5) triggered or controlled release of cargo. Toward function 1, we chemically conjugated fluorescent dyes or incorporated magnetic nanoparticles to enable in vivo optical or magnetic resonance imaging. For function 2, we have made MSNPs with polymer coatings, charged groups, or supported lipid bilayers, which decrease aggregation and improve stability in saline solutions. For functions 3 and 4, we have enhanced passive bioaccumulation via the enhanced permeability and retention effect by modifying the MSNP surfaces with positively charged polymers. We have also chemically attached ligands to MSNPs that selectively bind to receptors overexpressed in cancer cells. We have used encapsulation of MSNPs within reconfigurable supported lipid bilayers to develop new classes of responsive nanocarriers that actively interact with the target cell. Toward function 4, we exploit the high surface area and tailorable surface chemistry of MSNPs to retain hydrophobic drugs. Finally, for function 5, we have engineered dynamic behaviors by incorporating molecular machines within or at the entrances of MSNP pores and by using ligands, polymers, or lipid bilayers. These provide a means to seal-in and retain cargo and to direct MSNP interactions with and internalization by target cells. Application of MSNPs as nanocarriers requires biocompatibility and low toxicity. Here the intrinsic porosity of the MSNP surface reduces the extent of hydrogen bonding or electrostatic interactions with cell membranes as does surface coating with polymers or lipid bilayers. Furthermore, the high surface area and low extent of condensation of the MSNP siloxane framework promote a high rate of dissolution into soluble silicic acid species, which are found to be nontoxic. Potential toxicity is further mitigated by the high drug capacity of MSNPs, which greatly reduces needed dosages compared with other nanocarriers. We anticipate that future generations of MSNPs incorporating molecular machines and encapsulated by membrane-like lipid bilayers will achieve a new level of controlled cellular interactions.

Delivery of ricin toxin a-chain by peptide-targeted mesoporous silica nanoparticle-supported lipid bilayers.

Mesoporous silica nanoparticle-supported lipid bilayers, or "protocells", exhibit a high loading capacity, enhanced colloidal stability, and peptide-directed, cell-specific uptake, making them especially well-suited for targeted delivery of protein toxins to cancer. Protocells loaded with ricin toxin A-chain (RTA) and targeted to hepatocellular carcinoma cause complete cell death at 30 pM of RTA without affecting the viability of control hepatocytes.

Delivery of small interfering RNA by peptide-targeted mesoporous silica nanoparticle-supported lipid bilayers.

The therapeutic potential of small interfering RNAs (siRNAs) is severely limited by the availability of delivery platforms that protect siRNA from degradation, deliver it to the target cell with high specificity and efficiency, and promote its endosomal escape and cytosolic dispersion. Here we report that mesoporous silica nanoparticle-supported lipid bilayers (or "protocells") exhibit multiple properties that overcome many of the limitations of existing delivery platforms. Protocells have a 10- to 100-fold greater capacity for siRNA than corresponding lipid nanoparticles and are markedly more stable when incubated under physiological conditions. Protocells loaded with a cocktail of siRNAs bind to cells in a manner dependent on the presence of an appropriate targeting peptide and, through an endocytic pathway followed by endosomal disruption, promote delivery of the silencing nucleotides to the cytoplasm. The expression of each of the genes targeted by the siRNAs was shown to be repressed at the protein level, resulting in a potent induction of growth arrest and apoptosis. Incubation of control cells that lack expression of the antigen recognized by the targeting peptide with siRNA-loaded protocells induced neither repression of protein expression nor apoptosis, indicating the precise specificity of cytotoxic activity. In terms of loading capacity, targeting capabilities, and potency of action, protocells provide unique attributes as a delivery platform for therapeutic oligonucleotides.

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.

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 exhibit a 10,000-fold greater affinity for human hepatocellular carcinoma than for hepatocytes, endothelial cells or immune cells. Furthermore, protocells can be loaded with combinations of therapeutic (drugs, small interfering RNA 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 enable a single protocell loaded with a drug cocktail to kill a drug-resistant human hepatocellular carcinoma cell, representing a 10(6)-fold improvement over comparable liposomes.

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 Qß show that this assembly process is not fully described by a simple Derjaguin-Landau-Verwey-Overbeek model alone.

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 integration into three-dimensional lipid-silica nanostructured matrices.

We report a unique approach in which living cells direct their integration into 3D solid-state nanostructures. Yeast cells deposited on a weakly condensed lipid/silica thin film mesophase actively reconstruct the surface to create a fully 3D bio/nano interface, composed of localized lipid bilayers enveloped by a lipid/silica mesophase, through a self-catalyzed silica condensation process. Remarkably, this integration process selects exclusively for living cells over the corresponding apoptotic cells (those undergoing programmed cell death), via the development of a pH gradient, which catalyzes silica deposition and the formation of a coherent interface between the cell and surrounding silica matrix. Added long-chain lipids or auxiliary nanocomponents are localized within the pH gradient, allowing the development of complex active and accessible bio/nano interfaces not achievable by other synthetic methods. Overall, this approach provides the first demonstration of active cell-directed integration into a nominally solid-state three-dimensional architecture. It promises a new means to integrate "bio" with "nano" into platforms useful to study and manipulate cellular behavior at the individual cell level and to interface living organisms with electronics, photonics, and fluidics.

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.

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-directed assembly of bio/nano interfaces-a new scheme for cell immobilization.

When lipid-directed assembly of silicic acid precursors is conducted in the presence of living cells, the cells intervene, surrounding themselves with a fluid, multilayered lipid vesicle that interfaces coherently with an ordered silica mesophase. This bio/nano interface is unique in that its uniform nanostructure prevents excessive drying of water, maintaining cell viability, yet provides accessibility of the cell surface to small molecules. In comparison to existing immobilization schemes, such as encapsulation within sol-gel matrices, we show this interface to form by an active interplay between the living cell and surrounding matrix, which we refer to as cell-directed assembly (CDA). Importantly and perhaps uniquely, CDA creates a localized nanostructured microenvironment within which three-dimensional chemical gradients are established and maintained.

Cell-directed assembly of lipid-silica nanostructures providing extended cell viability.

Amphiphilic phospholipids were used to direct the formation of biocompatible, uniform silica nanostructures in the presence of Saccharomyces cerevisiae and bacterial cell lines. The cell surfaces organize multilayered phospholipid vesicles that interface coherently with the silica host and help relieve drying stresses that develop with conventional templates. These host structures maintain cell accessibility, addressability, and viability in the absence of buffer or an external fluidic architecture. The cell surfaces are accessible and can be used to localize added proteins, plasmids, and nanocrystals. Prolonged cell viability combined with reporter protein expression enabled stand-alone cell-based sensing.