Lipid-coated mesoporous silica nanoparticles for anti-viral applications via delivery of CRISPR-Cas9 ribonucleoproteins.

Emerging and re-emerging viral pathogens present a unique challenge for anti-viral therapeutic development. Anti-viral approaches with high flexibility and rapid production times are essential for combating these high-pandemic risk viruses. CRISPR-Cas technologies have been extensively repurposed to treat a variety of diseases, with recent work expanding into potential applications against viral infections. However, delivery still presents a major challenge for these technologies. Lipid-coated mesoporous silica nanoparticles (LCMSNs) offer an attractive delivery vehicle for a variety of cargos due to their high biocompatibility, tractable synthesis, and amenability to chemical functionalization. Here, we report the use of LCMSNs to deliver CRISPR-Cas9 ribonucleoproteins (RNPs) that target the Niemann-Pick disease type C1 gene, an essential host factor required for entry of the high-pandemic risk pathogen Ebola virus, demonstrating an efficient reduction in viral infection. We further highlight successful in vivo delivery of the RNP-LCMSN platform to the mouse liver via systemic administration.

Characterizing and Tuning the Properties of Polydiacetylene Films for Sensing Applications.

Self-assembled, polymerized diacetylene (DA) nanostructures and two-dimensional films have been studied over the past two decades for sensor applications because of their straightforward visual readout. DA monomers, when exposed to UV light, polymerize to produce a visibly blue polymer. Blue phase polydiacetylenes (PDAs) when exposed to an external stimuli, such as temperature or UV light, undergo a chromatic phase transition to a fluorescent, visibly red phase. The tunability of the monomer to blue to red chromatic phase transitions by choice of diacetylene monomer in the presence of metal cations is systematically and comprehensively investigated to determine their effects on the properties of PDA Langmuir films. The polymerization kinetics and domain morphology of the PDA films were characterized using polarized fluorescent microscopy, UV-vis-fluorescent spectroscopy, and Fourier transform infrared spectroscopy (FTIR). Increasing the monomer alkyl tail length was found to strongly increase the UV dose necessary to produce optimally blue films and fully red films. A decrease in the polymer domain size was also correlated with longer-tailed DA molecules. Metal cations have a diverse effect on the film behavior. Alkaline-earth metals such as Mg, Ca, and Ba have a negligible effect on the phase transition kinetics but can be used to tune PDA polymer domain sizes. The Ni and Fe cations increase the UV dose necessary to produce red phase PDA films and significantly decrease the polymer domain sizes. The Zn, Cd, and Cu ions exhibit strong directed interactions with the PDA carboxylic acid headgroups, resulting in quenched fluorescence and a unique film morphology. FTIR analysis provides insight into the metal-PDA binding mechanisms and demonstrates that the coordination between the PDA film headgroups and the metal cations can be correlated with changes in the film morphology and kinetics. The findings from these studies will have broad utility for tuning PDA-based sensors for different applications and sensitivity ranges.

Mechanism of Acid-Triggered Cargo Release from Lipid Bilayer-Coated Mesoporous Silica Particles.

Lipid bilayer-coated mesoporous silica nanoparticles are unique core-shell nanomaterials currently being developed as drug delivery vehicles. To improve cargo loading and biocirculation, the pore structure and surface chemistry of the particle have been modified and well characterized. However, an understanding of cargo release mechanisms from cellular uptake pathways remains largely unexplored. Here, we present a study of the release mechanism of lipid bilayer-coated silica particles induced by endosomal-like pH change from 7.4 to 5.0. We found that this relatively small pH change produces rapid deformation of the supported lipid bilayer that ultimately results in holes in the membrane. Using a combination of dye release studies, wide-field and confocal fluorescence microscopies, and surface area modeling analysis, we determined that small blister-like structures are formed, which lead to lateral membrane displacement and hole formation. Possible mechanisms for the blister formation, which include curvature effects and interfacial interactions, are discussed.

Lipid-Coated Mesoporous Silica Nanoparticles for the Delivery of the ML336 Antiviral to Inhibit Encephalitic Alphavirus Infection.

Venezuelan equine encephalitis virus (VEEV) poses a major public health risk due to its amenability for use as a bioterrorism agent and its severe health consequences in humans. ML336 is a recently developed chemical inhibitor of VEEV, shown to effectively reduce VEEV infection in vitro and in vivo. However, its limited solubility and stability could hinder its clinical translation. To overcome these limitations, lipid-coated mesoporous silica nanoparticles (LC-MSNs) were employed. The large surface area of the MSN core promotes hydrophobic drug loading while the liposome coating retains the drug and enables enhanced circulation time and biocompatibility, providing an ideal ML336 delivery platform. LC-MSNs loaded 20 ± 3.4 µg ML336/mg LC-MSN and released 6.6 ± 1.3 µg/mg ML336 over 24 hours. ML336-loaded LC-MSNs significantly inhibited VEEV in vitro in a dose-dependent manner as compared to unloaded LC-MSNs controls. Moreover, cell-based studies suggested that additional release of ML336 occurs after endocytosis. In vivo safety studies were conducted in mice, and LC-MSNs were not toxic when dosed at 0.11 g LC-MSNs/kg/day for four days. ML336-loaded LC-MSNs showed significant reduction of brain viral titer in VEEV infected mice compared to PBS controls. Overall, these results highlight the utility of LC-MSNs as drug delivery vehicles to treat VEEV.

Dynamics of Crowding-Induced Mixing in Phase Separated Lipid Bilayers.

We use fluorescence microscopy to examine the dynamics of the crowding-induced mixing transition of liquid ordered (Lo)-liquid disordered (Ld) phase separated lipid bilayers when the following particles of increasing size bind to either the Lo or Ld phase: Ubiquitin, green fluorescent protein (GFP), and nanolipoprotein particles (NLPs) of two diameters. These proteinaceous particles contained histidine-tags, which were phase targeted by binding to iminodiacetic acid (IDA) head groups, via a Cu2+ chelating mechanism, of lipids that specifically partition into either the Lo phase or Ld phase. The degree of steric pressure was controlled by varying the size of the bound particle (10-240 kDa) and the amount of binding sites present (i.e., DPIDA concentrations of 9 and 12 mol%) in the supported lipid multibilayer platform used here. We develop a mass transfer-based diffusional model to analyze the observed Lo phase domain dissolution that, along with visual observations and activation energy calculations, provides insight into the sequence of events in crowding-induced mixing. Our results suggest that the degree of steric pressure and target phase influence not only the efficacy of steric-pressure induced mixing, but the rate and controlling mechanism for which it occurs.

Engineering Lipid Structure for Recognition of the Liquid Ordered Membrane Phase.

The selective partitioning of lipid components in phase-separated membranes is essential for domain formation involved in cellular processes. Identifying and tracking the movement of lipids in cellular systems would be improved if we understood how to achieve selective affinity between fluorophore-labeled lipids and membrane assemblies. Here, we investigated the structure and chemistry of membrane lipids to evaluate lipid designs that partition to the liquid ordered (Lo) phase. A range of fluorophores at the headgroup position and lengths of PEG spacer between the lipid backbone and fluorophore were examined. On a lipid body with saturated palmityl or palmitoyl tails, we found that although the lipid tails can direct selective partitioning to the Lo phase through favorable packing interactions, headgroup hydrophobicity can override the partitioning behavior and direct the lipid to the disordered membrane phase (Ld). The PEG spacer can serve as a buffer to mute headgroup-membrane interactions and thus improve Lo phase partitioning, but its effect is limited with strongly hydrophobic fluorophore headgroups. We present a series of lipid designs leading to the development of novel fluorescently labeled lipids with selective affinity for the Lo phase.

Forming Giant-sized Polymersomes Using Gel-assisted Rehydration.

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier properties, chemical versatility and tunable physical characteristics. Typical methods used to prepare giant-sized (> 4 µm) vesicles, however, are both time and labor intensive, yielding low numbers of intact polymersomes. Here, we present for the first time the use of gel-assisted rehydration for the rapid and high-yielding formation of giant (>4 µm) polymer vesicles (polymersomes). Using this method, polymersomes can be formed from a wide array of rehydration solutions including several different physiologically-compatible buffers and full cell culture media, making them readily useful for biomimicry studies. This technique is also capable of reliably producing polymersomes from different polymer compositions with far better yields and much less difficulty than traditional methods. Polymersome size is readily tunable by altering temperature during rehydration or adding membrane fluidizers to the polymer membrane, generating giant-sized polymersomes (>100 µm).

Crowding-Induced Mixing Behavior of Lipid Bilayers: Examination of Mixing Energy, Phase, Packing Geometry, and Reversibility.

In an effort to develop a general thermodynamic model from first-principles to describe the mixing behavior of lipid membranes, we examined lipid mixing induced by targeted binding of small (Green Fluorescent Protein (GFP)) and large (nanolipoprotein particles (NLPs)) structures to specific phases of phase-separated lipid bilayers. Phases were targeted by incorporation of phase-partitioning iminodiacetic acid (IDA)-functionalized lipids into ternary lipid mixtures consisting of DPPC, DOPC, and cholesterol. GFP and NLPs, containing histidine tags, bound the IDA portion of these lipids via a metal, Cu(2+), chelating mechanism. In giant unilamellar vesicles (GUVs), GFP and NLPs bound to the Lo domains of bilayers containing DPIDA, and bound to the Ld region of bilayers containing DOIDA. At sufficiently large concentrations of DPIDA or DOIDA, lipid mixing was induced by bound GFP and NLPs. The validity of the thermodynamic model was confirmed when it was found that the statistical mixing distribution as a function of crowding energy for smaller GFP and larger NLPs collapsed to the same trend line for each GUV composition. Moreover, results of this analysis show that the free energy of mixing for a ternary lipid bilayer consisting of DOPC, DPPC, and cholesterol varied from 7.9 × 10(-22) to 1.5 × 10(-20) J/lipid at the compositions observed, decreasing as the relative cholesterol concentration was increased. It was discovered that there appears to be a maximum packing density, and associated maximum crowding pressure, of the NLPs, suggestive of circular packing. A similarity in mixing induced by NLP1 and NLP3 despite large difference in projected areas was analytically consistent with monovalent (one histidine tag) versus divalent (two histidine tags) surface interactions, respectively. In addition to GUVs, binding and induced mixing behavior of NLPs was also observed on planar, supported lipid multibilayers. The mixing process was reversible, with Lo domains reappearing after addition of EDTA for NLP removal.

Cholesterol-Enriched Domain Formation Induced by Viral-Encoded, Membrane-Active Amphipathic Peptide.

The α-helical (AH) domain of the hepatitis C virus nonstructural protein NS5A, anchored at the cytoplasmic leaflet of the endoplasmic reticulum, plays a role in viral replication. However, the peptides derived from this domain also exhibit remarkably broad-spectrum virocidal activity, raising questions about their modes of membrane association. Here, using giant lipid vesicles, we show that the AH peptide discriminates between membrane compositions. In cholesterol-containing membranes, peptide binding induces microdomain formation. By contrast, cholesterol-depleted membranes undergo global softening at elevated peptide concentrations. Furthermore, in mixed populations, the presence of ∼100 nm vesicles of viral dimensions suppresses these peptide-induced perturbations in giant unilamellar vesicles, suggesting size-dependent membrane association. These synergistic composition- and size-dependent interactions explain, in part, how the AH domain might on the one hand segregate molecules needed for viral assembly and on the other hand furnish peptides that exhibit broad-spectrum virocidal activity.

Designing lipids for selective partitioning into liquid ordered membrane domains.

Self-organization of lipid molecules into specific membrane phases is key to the development of hierarchical molecular assemblies that mimic cellular structures. While the packing interaction of the lipid tails should provide the major driving force to direct lipid partitioning to ordered or disordered membrane domains, numerous examples show that the headgroup and spacer play important but undefined roles. We report here the development of several new biotinylated lipids that examine the role of spacer chemistry and structure on membrane phase partitioning. The new lipids were prepared with varying lengths of low molecular weight polyethylene glycol (EGn) spacers to examine how spacer hydrophilicity and length influence their partitioning behavior following binding with FITC-labeled streptavidin in liquid ordered (Lo) and liquid disordered (Ld) phase coexisting membranes. Partitioning coefficients (Kp Lo/Ld) of the biotinylated lipids were determined using fluorescence measurements in studies with giant unilamellar vesicles (GUVs). Compared against DPPE-biotin, DPPE-cap-biotin, and DSPE-PEG2000-biotin lipids, the new dipalmityl-EGn-biotin lipids exhibited markedly enhanced partitioning into liquid ordered domains, achieving Kp of up to 7.3 with a decaethylene glycol spacer (DP-EG10-biotin). We further demonstrated biological relevance of the lipids with selective partitioning to lipid raft-like domains observed in giant plasma membrane vesicles (GPMVs) derived from mammalian cells. Our results found that the spacer group not only plays a pivotal role for designing lipids with phase selectivity but may also influence the structural order of the domain assemblies.

Thermal annealing triggers collapse of biphasic supported lipid bilayers into multilayer islands.

The collapse of phase-separating single, supported lipid bilayers, consisting of mixtures of a zwitterionic phospholipid (POPC) and an anionic lipid (DPPA) upon thermal annealing in the presence of ions is examined using a combination of scanning probe, epifluorescence, and ellipsometric microscopies. We find that thermal annealing in the presence of ions in the bathing medium induces an irreversible transition from domain-textured, single supported bilayers to one comprising islands of multibilayer stacks, whose lateral area decays with lamellarity, producing pyramidal staircase "mesa" topography. The higher order lamellae are almost invariably localized above the anionic-lipid rich, gel-phase domains in the parent bilayer and depends on the ions in the bathing medium. The collapse mechanism appears to involve synergistic influences of two independent mechanisms: (1) stabilization of the incipient headgroup-headgroup interface in the emergent multibilayer configuration facilitated by ions in the bath and (2) domain-boundary templated folding. This collapse mechanism is consistent with previous theoretical predictions of topography-induced rippling instability in collapsing lipid monolayers and suggests the role of the mismatch in height and/or spontaneous curvature at domain boundaries in the collapse of phase-separated single supported bilayers.

Facile rearrangement of 3-oxoalkyl radicals is evident in low-temperature gas-phase oxidation of ketones.

The pulsed photolytic chlorine-initiated oxidation of methyl-tert-butyl ketone (MTbuK), di-tert-butyl ketone (DTbuK), and a series of partially deuterated diethyl ketones (DEK) is studied in the gas phase at 8 Torr and 550-650 K. Products are monitored as a function of reaction time, mass, and photoionization energy using multiplexed photoionization mass spectrometry with tunable synchrotron ionizing radiation. The results establish that the primary 3-oxoalkyl radicals of those ketones, formed by abstraction of a hydrogen atom from the carbon atom in γ-position relative to the carbonyl oxygen, undergo a rapid rearrangement resulting in an effective 1,2-acyl group migration, similar to that in a Dowd-Beckwith ring expansion. Without this rearrangement, peroxy radicals derived from MTbuK and DTbuK cannot undergo HO2 elimination to yield a closed-shell unsaturated hydrocarbon coproduct. However, not only are these coproducts observed, but they represent the dominant oxidation channels of these ketones under the conditions of this study. For MTbuK and DTbuK, the rearrangement yields a more stable tertiary radical, which provides the thermodynamic driving force for this reaction. Even in the absence of such a driving force in the oxidation of partially deuterated DEK, the 1,2-acyl group migration is observed. Quantum chemical (CBS-QB3) calculations show the barrier for gas-phase rearrangement to be on the order of 10 kcal mol(-1). The MTbuK oxidation experiments also show several minor channels, including ß-scission of the initial radicals and cyclic ether formation.

In-situ transmission electron microscopy of liposomes in an aqueous environment.

The characterization of liposomes was undertaken using in-situ microfluidic transmission electron microscopy. Liposomes were imaged without contrast enhancement staining or cryogenic treatment, allowing for the observation of functional liposomes in an aqueous environment. The stability and quality of the liposome structures observed were found to be highly dependent on the surface and liposome chemistries within the liquid cell. The successful imaging of liposomes suggests the potential for the extension of in-situ microfluidic TEM to a wide variety of other biological and soft matter systems and processes.

Nanoscale patterning of membrane-bound proteins formed through curvature-induced partitioning of phase-specific receptor lipids.

This work describes a technique for forming high-density arrays and patterns of membrane-bound proteins through binding to a curvature-organized compositional pattern of metal-chelating lipids (Cu(2+)-DOIDA or Cu(2+)-DSIDA). In this bottom-up approach, the underlying support is an e-beam formed, square lattice pattern of hemispheres. This curvature pattern sorts Cu(2+)-DOIDA to the 200 nm hemispherical lattice sites of a 600 nm × 600 nm unit cell in Ld - Lo phase separated lipid multibilayers. Binding of histidine-tagged green fluorescent protein (His-GFP) creates a high density array of His-GFP-bound pixels localized to the square lattice sites. In comparison, the negative pixel pattern is created by sorting Cu(2+)-DSIDA in Ld - Lß' phase separated lipid multibilayers to the flat grid between the lattice sites followed by binding to His-GFP. Lattice defects in the His-GFP pattern lead to interesting features such as pattern circularity. We also observe defect-free arrays of His-GFP that demonstrate perfect arrays can be formed by this method suggesting the possibility of using this approach for the localization of various active molecules to form protein, DNA, or optically active molecular arrays.

Lipid membrane domains for the selective adsorption and surface patterning of conjugated polyelectrolytes.

Conjugated polyelectrolytes (CPEs) are promising materials for generating optoelectronics devices under environmentally friendly processing conditions, but challenges remain to develop methods to define lateral features for improved junction interfaces and direct optoelectronic pathways. We describe here the potential to use a bottom-up approach that employs self-assembly in lipid membranes to form structures to template the selective adsorption of CPEs. Phase separation of gel phase anionic lipids and fluid phase phosphocholine lipids allowed the formation of negatively charged domain assemblies that selectively adsorb a cationic conjugated polyelectrolyte (P2). Spectroscopic studies found the adsorption of P2 to negatively charged membranes resulted in minimal structural change of the solution phase polymer but yielded an enhancement in fluorescence intensity (~50%) due to loss of quenching pathways. Fluorescence microscopy, dynamic light scattering, and AFM imaging were used to characterize the polymer-membrane interaction and the polymer-bound domain structures of the biphasic membranes. In addition to randomly formed circular gel phase domains, we also show that predefined features, such as straight lines, can be directed to form upon etched patterns on the substrate, thus providing potential routes toward the self-organization of optoelectronic architectures.

A continuous network of lipid nanotubes fabricated from the gliding motility of kinesin powered microtubule filaments.

Synthetic interconnected lipid nanotube networks were fabricated on the millimeter scale based on the simple, cooperative interaction between phospholipid vesicles and kinesin-microtubule (MT) transport systems. More specifically, taxol-stabilized MTs, in constant 2D motion via surface absorbed kinesin, extracted and extended lipid nanotube networks from large Lα phase multilamellar liposomes (5-25 µm). Based on the properties of the inverted motility geometry, the total size of these nanofluidic networks was limited by MT surface density, molecular motor energy source (ATP), and total amount and physical properties of lipid source material. Interactions between MTs and extended lipid nanotubes resulted in bifurcation of the nanotubes and ultimately the generation of highly branched networks of fluidically connected nanotubes. The network bifurcation was easily tuned by changing the density of microtubules on the surface to increase or decrease the frequency of branching. The ability of these networks to capture nanomaterials at the membrane surface with high fidelity was subsequently demonstrated using quantum dots as a model system. The diffusive transport of quantum dots was also characterized with respect to using these nanotube networks for mass transport applications.

Membrane bending by protein-protein crowding.

Curved membranes are an essential feature of dynamic cellular structures, including endocytic pits, filopodia protrusions and most organelles. It has been proposed that specialized proteins induce curvature by binding to membranes through two primary mechanisms: membrane scaffolding by curved proteins or complexes; and insertion of wedge-like amphipathic helices into the membrane. Recent computational studies have raised questions about the efficiency of the helix-insertion mechanism, predicting that proteins must cover nearly 100% of the membrane surface to generate high curvature, an improbable physiological situation. Thus, at present, we lack a sufficient physical explanation of how protein attachment bends membranes efficiently. On the basis of studies of epsin1 and AP180, proteins involved in clathrin-mediated endocytosis, we propose a third general mechanism for bending fluid cellular membranes: protein-protein crowding. By correlating membrane tubulation with measurements of protein densities on membrane surfaces, we demonstrate that lateral pressure generated by collisions between bound proteins drives bending. Whether proteins attach by inserting a helix or by binding lipid heads with an engineered tag, protein coverage above ~20% is sufficient to bend membranes. Consistent with this crowding mechanism, we find that even proteins unrelated to membrane curvature, such as green fluorescent protein (GFP), can bend membranes when sufficiently concentrated. These findings demonstrate a highly efficient mechanism by which the crowded protein environment on the surface of cellular membranes can contribute to membrane shape change.

Photonic gene circuits by optically addressable siRNA-Au nanoantennas.

The precise perturbation of gene circuits and the direct observation of signaling pathways in living cells are essential for both fundamental biology and translational medicine. Current optogenetic technology offers a new paradigm of optical control for cells; however, this technology relies on permanent genomic modifications with light-responsive genes, thus limiting dynamic reconfiguration of gene circuits. Here, we report precise control of perturbation and reconfiguration of gene circuits in living cells by optically addressable siRNA-Au nanoantennas. The siRNA-Au nanoantennas fulfill dual functions as selectively addressable optical receivers and biomolecular emitters of small interfering RNA (siRNA). Using siRNA-Au nanoantennas as optical inputs to existing circuit connections, photonic gene circuits are constructed in living cells. We show that photonic gene circuits are modular, enabling subcircuits to be combined on-demand. Photonic gene circuits open new avenues for engineering functional gene circuits useful for fundamental bioscience, bioengineering, and medical applications.

Orienting lipid domains in giant vesicles using an electric field.

Directing the orientation of molecular assemblies is a key step toward creating complex hierarchical structures that yield higher order functional materials. Here, we demonstrate the directed orientation of functionalized lipid domains and protein-membrane assemblies, using an electric field.

Targeting proteins to liquid-ordered domains in lipid membranes.

We demonstrate the construction of novel protein-lipid assemblies through the design of a lipid-like molecule, DPIDA, endowed with tail-driven affinity for specific lipid membrane phases and head-driven affinity for specific proteins. In studies performed on giant unilamellar vesicles (GUVs) with varying mole fractions of dipalymitoylphosphatidylcholine (DPPC), cholesterol, and diphytanoylphosphatidyl choline (DPhPC), DPIDA selectively partitioned into the more ordered phases, either solid or liquid-ordered (L(o)) depending on membrane composition. Fluorescence imaging established the phase behavior of the resulting quaternary lipid system. Fluorescence correlation spectroscopy confirmed the fluidity of the L(o) phase containing DPIDA. In the presence of CuCl(2), the iminodiacetic acid (IDA) headgroup of DPIDA forms the Cu(II)-IDA complex that exhibits a high affinity for histidine residues. His-tagged proteins were bound specifically to domains enriched in DPIDA, demonstrating the capacity to target protein binding selectively to both solid and L(o) phases. Steric pressure from the crowding of surface-bound proteins transformed the domains into tubules with persistence lengths that depended on the phase state of the lipid domains.