Nonviral In Vivo Delivery of CRISPR-Cas9 Using Protein-Agnostic, High-Loading Porous Silicon and Polymer Nanoparticles.

The complexity of CRISPR machinery is a challenge to its application for nonviral in vivo therapeutic gene editing. Here, we demonstrate that proteins, regardless of size or charge, efficiently load into porous silicon nanoparticles (PSiNPs). Optimizing the loading strategy yields formulations that are ultrahigh loading─>40% cargo by volume─and highly active. Further tuning of a polymeric coating on the loaded PSiNPs yields nanocomposites that achieve colloidal stability under cryopreservation, endosome escape, and gene editing efficiencies twice that of the commercial standard Lipofectamine CRISPRMAX. In a mouse model of arthritis, PSiNPs edit cells in both the cartilage and synovium of knee joints, and achieve 60% reduction in expression of the therapeutically relevant MMP13 gene. Administered intramuscularly, they are active over a broad dose range, with the highest tested dose yielding nearly 100% muscle fiber editing at the injection site. The nanocomposite PSiNPs are also amenable to systemic delivery. Administered intravenously in a model that mimics muscular dystrophy, they edit sites of inflamed muscle. Collectively, the results demonstrate that the PSiNP nanocomposites are a versatile system that can achieve high loading of diverse cargoes and can be applied for gene editing in both local and systemic delivery applications.

Impact of the Electric Polarizability on the Transport and Collective Dynamics of Metallodielectric Janus Particles.

The objective of this article is to investigate how the electric polarizability manifests on the propulsion and collective dynamics of metallodielectric Janus particles by comparing the velocity spectra under rotating and nonrotating AC fields. Janus particles were fabricated by depositing sequential layers of titanium and SiO2 on spherical cores. Model systems of known polarizability were created by varying the thickness of titanium or by adjusting the concentration of electrolyte. We found that the spectra for propulsion velocity displayed features (amplitude and transition frequencies) that were closely matched in the electrorotation spectra. That is, the transition frequency from dielectric- to metal-side forward matched closely the peak in counterfield rotation, while the minima in propulsion velocity matched the transition frequency from counterfield to cofield rotation. Furthermore, based on electroorientation measurements for prolate Janus ellipsoids, we conclude that the propulsion velocity of spherical Janus particles reflects the real part of their polarizability. Solutions of the Poisson-Nernst-Planck equations confirm the thickness of the metal cap facilitates adjusting the behavior from metal- to dielectric-like. These traits translate into different collective behaviors, such as the ability to traverse or become part of a lattice of nonpatchy silica particles. Overall, these results provide experimental evidence to either challenge or refine existing electrokinetic models of propulsion.

Long-range transport and directed assembly of charged colloids under aperiodic electrodiffusiophoresis.

Faradaic reactions often lead to undesirable side effects during the application of electric fields. Therefore, experimental designs often avoid faradaic reactions by working at low voltages or at high frequencies, where the electrodes behave as ideally polarizable. In this work, we show how faradaic processes under ac fields can be used advantageously to effect long-range transport, focusing and assembly of charged colloids. Herein, we use confocal microscopy and ratiometric analysis to confirm that ac fields applied in media of low conductivity induce significant pH gradients below and above the electrode charging frequency of the system. At voltages above 1 Vpp, and frequencies below 1.7 kHz, the pH profile becomes highly nonlinear. Charged particles respond to such conditions by migrating towards the point of highest pH, thereby focusing tens of microns away from both electrodes. Under the combination of oscillating electric fields and concentration gradients of electroactive species, particles experience aperiodic electrodiffusiophoresis (EDP). The theory of EDP, along with a mass transport model, describes the dynamics of particles. Furthermore, the high local concentration of particles near the focusing point leads to disorder-order transitions, whereby particles form crystals. The position and order within the levitating crystalline sheet can be readily tuned by adjusting the voltage and frequency. These results not only have significant implications for the fundamental understanding of ac colloidal electrokinetics, but also provide new possibilities for the manipulation and directed assembly of charged colloids.

Visualization of Concentration Gradients and Colloidal Dynamics under Electrodiffusiophoresis.

In this work, we present an experimental study of the dynamics of charged colloids under direct currents and gradients of chemical species (electrodiffusiophoresis). In our approach, we simultaneously visualize the development of concentration polarization and the ensuing dynamics of charged colloids near electrodes. With the aid of confocal microscopy and fluorescent probes, we show that the passage of current through water confined between electrodes, separated about a hundred microns, results in significant pH gradients. Depending on the current density and initial conditions, steep pH gradients develop, thus becoming a significant factor in the behavior of charged colloids. Furthermore, we show that steep pH gradients induce the focusing of charged colloids away from both electrodes. Our results provide the experimental basis for further development of models of electrodiffusiophoresis and the design of non-equilibrium strategies for the fabrication of advanced materials.

Electric polarizability of metallodielectric Janus particles in electrolyte solutions.

Metallodielectric Janus particles (JPs) and electric fields have been a useful combination for the development of innovative concepts on AC electrokinetics, directed transport and collective dynamics. The polarizability, and its frequency dependence, underlie the rich behavior exhibited by JPs. Nonetheless, direct measurements of polarizability are few and the interplay of different mechanisms remains unclear. This paper discusses measurements and strategies to tailor the magnitude of the polarizability of JPs. Our approach uses electrorotation to measure the polarizability of particles with different thicknesses of metal in electrolyte solutions. On the other hand, we gain further insight into the basic polarization mechanisms through modeling based on the fundamental transport equations. JPs exhibit rich polarization spectra that depend strongly on the thickness of the metal layer, the conductivity of the medium and the surface charge. At low frequencies-around 10 kHz-the results indicate that counter-field rotation stems from the charging of the double layer at the particle-electrolyte interface, while the transition to co-field rotation at high frequencies (above 100 kHz) stems from the Maxwell-Wagner relaxation. The latter polarization mechanism is significantly affected by the conductivity within the electrical double layer. The insights from this study provide helpful quantitative information for the design of colloidal machines with desirable features such as targeted propulsion, and tunable collective dynamics.

Magnetite-OmpA Nanobioconjugates as Cell-Penetrating Vehicles with Endosomal Escape Abilities.

Outer membrane protein A (OmpA) has been extensively studied in Gram-negative bacteria due to its relevance in the adhesion of pathogens to host cells and its surfactant capabilities. It consists of a hydrophobic ß-barrel domain and a hydrophilic periplasmic domain, that confers OmpA an amphiphilic structure. This study aims to elucidate the capacity of Escherichia coli OmpA to translocate liposomal membranes and serve as a potential cell-penetrating vehicle. We immobilized OmpA on magnetite nanoparticles and investigated the possible functional changes exhibited by OmpA after immobilization. Liposomal intake was addressed using egg lecithin liposomes as a model, where magnetite-OmpA nanobioconjugates were able to translocate the liposomal membrane and caused a disruptive effect when subjected to a magnetic field. Nanobioconjugates showed both low cytotoxicity and hemolytic tendency. Additional interactions within the intracellular space led to altered viability results via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Confocal microscopy images revealed that immobilized nanoparticles effectively enter the cytoplasm of THP-1 and Vero cells by different routes, and, subsequently, some escape endosomes, lysosomes, and other intracellular compartments with relatively high efficiencies. This was demonstrated by co-localization analyses with LysoTracker green that showed Pearson correlations of about 80 and 28%.

Controlled Levitation of Colloids through Direct Current Electric Fields.

We report the controlled levitation of surface-modified colloids in direct current (dc) electric fields at distances as far as 75 µm from an electrode surface. Instead of experiencing electrophoretic deposition, colloids modified through metallic deposition or the covalent bonding of poly(ethylene glycol) (PEG) undergo migration and focusing that results in levitation at these large distances. The levitation is a sensitive function of the surface chemistry and magnitude of the field, thus providing the means to achieve control over the levitation height. Experiments with particles of different surface charge show that levitation occurs only when the absolute zeta potential is below a threshold value. An electrodiffusiophoretic mechanism is proposed to explain the observed large-scale levitation.

Unique toxicological behavior from single-wall carbon nanotubes separated via selective adsorption on hydrogels.

Over the past decade, extensive research has been completed on the potential threats of single-wall carbon nanotubes (SWCNTs) to living organisms upon release to aquatic systems. However, these studies have focused primarily on the link between adverse biological effects in exposed test organisms on the length, diameter, and metallic impurity content of SWCNTs. In contrast, few studies have focused on the bioeffects of the different SWCNTs in the as-produced mixture, which contain both metallic (m-SWCNT) and semiconducting (s-SWCNT) species. Using selective adsorption onto hydrogels, high purity m-SWCNT and s-SWCNT fractions were produced and their biological impacts determined in dose-response studies with Pseudokirchneriella subcapitata as test organism. The results show significant differences in the biological responses of P. subcapitata exposed to high purity m- and s-SWCNT fractions. Contrary to the biological response observed using SWCNTs separated by density gradient ultracentrifugation, it is found that the high-pressure CO conversion (HiPco) s-SWCNT fraction separated by selective adsorption causes increased biological impact. These findings suggest that s-SWCNTs are the primary factor driving the adverse biological responses observed from P. subcapitata cells exposed to our as-produced suspensions. Finally, the toxicity of the s-SWCNT fraction is mitigated by increasing the concentration of biocompatible surfactant in the suspensions, likely altering the nature of surfactant coverage along SWCNT sidewalls, thereby reducing potential physical interaction with algal cells. These findings highlight the need to couple sample processing and toxicity response studies.

Isolation of specific small-diameter single-wall carbon nanotube species via aqueous two-phase extraction.

Aqueous two-phase extraction is demonstrated to enable isolation of single semiconducting and metallic single-wall carbon nanotube species from a synthetic mixture. The separation is rapid and robust, with remarkable tunability via modification of the surfactant environment set for the separation.

Interactive forces between sodium dodecyl sulfate-suspended single-walled carbon nanotubes and agarose gels.

Selective adsorption onto agarose gels has become a powerful method to separate single-walled carbon nanotubes (SWCNTs). A better understanding of the nature of the interactive forces and specific sites responsible for adsorption should lead to significant improvements in the selectivity and yield of these separations. A combination of nonequilibrium and equilibrium studies are conducted to explore the potential role that van der Waals, ionic, hydrophobic, π-π, and ion-dipole interactions have on the selective adsorption between agarose and SWCNTs suspended with sodium dodecyl sulfate (SDS). The results demonstrate that any modification to the agarose gel surface and, consequently, the permanent dipole moments of agarose drastically reduces the retention of SWCNTs. Because these permanent dipoles are critical to retention and the fact that SDS-SWCNTs function as macro-ions, it is proposed that ion-dipole forces are the primary interaction responsible for adsorption. The selectivity of adsorption may be attributed to variations in polarizability between nanotube types, which create differences in both the structure and mobility of surfactant. These differences affect the enthalpy and entropy of adsorption, and both play an integral part in the selectivity of adsorption. The overall adsorption process shows a complex behavior that is not well represented by the Langmuir model; therefore, calorimetric data should be used to extract thermodynamic information.

Analyzing surfactant structures on length and chirality resolved (6,5) single-wall carbon nanotubes by analytical ultracentrifugation.

The structure and density of the bound interfacial surfactant layer and associated hydration shell were investigated using analytical ultracentrifugation for length and chirality purified (6,5) single-wall carbon nanotubes (SWCNTs) in three different bile salt surfactant solutions. The differences in the chemical structures of the surfactants significantly affect the size and density of the bound surfactant layers. As probed by exchange of a common parent nanotube population into sodium deoxycholate, sodium cholate, or sodium taurodeoxycholate solutions, the anhydrous density of the nanotubes was least for the sodium taurodeoxycholate surfactant, and the absolute sedimentation velocities greatest for the sodium cholate and sodium taurodeoxycholate surfactants. These results suggest that the thickest interfacial layer is formed by the deoxycholate, and that the taurodeoxycholate packs more densely than either sodium cholate or deoxycholate. These structural differences correlate well to an observed 25% increase in fluorescence intensity relative to the cholate surfactant for deoxycholate and taurodeoxycholate dispersed SWCNTs displaying equivalent absorbance spectra. Separate sedimentation velocity experiments including the density modifying agent iodixanol were used to establish the buoyant density of the (6,5) SWCNT in each of the bile salt surfactants; from the difference in the buoyant and anhydrous densities, the largest hydrated diameter is observed for sodium deoxycholate. Understanding the effects of dispersant choice and the methodology for measurement of the interfacial density and hydrated diameter is critical for rationally advancing separation strategies and applications of nanotubes.

Aqueous suspension methods of carbon-based nanomaterials and biological effects on model aquatic organisms.

The preparation of aqueous suspensions of carbon-based nanomaterials (NMs) requires the use of dispersing agents to overcome their hydrophobic character. Although studies on the toxicity of NMs have focused primarily on linking the characteristics of particles to biological responses, the role of dispersing agents has been overlooked. This study assessed the biological effects of a number of commonly used dispersing agents on Pseudokirchneriella subcapitata and Ceriodaphnia dubia as model test organisms. The results show that for a given organism, NM toxicity can be mitigated by use of nontoxic surfactants, and that a multispecies approach is necessary to account for the sensitivity of different organisms. In addition to the intrinsic physicochemical properties of NMs, exposure studies should take into account the effects of used dispersing fluids.

Swelling the hydrophobic core of surfactant-suspended single-walled carbon nanotubes: a SANS study.

Localized solvent environments form around single-wall carbon nanotubes (SWCNTs) because of the ability of surfactant molecules to solubilize immiscible organic solvents. Although these microenvironments around SWCNTs have already been used for fundamental and applied studies, small-angle neutron scattering (SANS) was used here to assess the size and shape of the solvent domains, their uniformity and distribution on the sidewalls, and the effect of solvent swelling on the aggregation state of the suspension. SANS measurements confirm both the formation of local solvent environments and that no irreversible aggregation of the nanotube suspension occurs after the SDS molecules are swollen in solvent. The results also corroborate prior conclusions based on photoluminescence that the structure formed is dependent of the nature of the solvent-surfactant combination; SWCNTs suspended with SDS and swelled with benzene have a more uniform coating on the sidewall than those swelled with o-dichlorobenzene. These differences can be important to understanding the effect of the local environment on the photoluminescence properties and the interaction of SWCNTs with interfaces.

Solvatochromic shifts of single-walled carbon nanotubes in nonpolar microenvironments.

Single-walled carbon nanotubes (SWNTs) are encapsulated with microenvironments of nonpolar solvent, providing a new method to measure the photophysical properties of nanotubes in environments with known properties. Photoluminescence (PL) and absorbance spectra of SWNTs show solvatochromic shifts in 16 nonpolar solvents, which are proportional to the solvent induction polarization. The shifts in the emission energies (DeltaE(11)) range from approximately 25 to 100 meV and the smallest diameter SWNTs have the largest shifts. The PL intensity of SWNTs is very sensitive to changes in polarity. For example, SWNTs encapsulated with chloroform (epsilon approximately 5) show substantial reductions in intensity. The solvatochromic shifts of SWNTs were used to determine the relationship between the longitudinal polarizability, band gap and radius, alpha(11,||) proportional to 1/(R(2)E(11)(3)).

Long-term improvements to photoluminescence and dispersion stability by flowing SDS-SWNT suspensions through microfluidic channels.

Shearing single-walled carbon nanotubes (SWNTs) coated with sodium dodecyl sulfate in microfluidic channels significantly increases the photoluminescence (PL) intensity and dispersion stability of SWNTs. The PL quantum yield (QY) of SWNTs improves by a factor of 3 for initially bright suspensions; on the other hand, SWNT QYs in a "poor" suspension improve by 2 orders of magnitude. In both cases, the QYs of the sheared suspensions are approximately 1%. The increases in PL intensity persist for months and are most prominent in larger diameter SWNTs. These improvements are attributed to surfactant reorganization rather than disaggregation of SWNTs bundles or shear-induced alignment. The results also highlight potential opportunities to eliminate discrepancies in the PL intensity of different suspensions and further improve the PL of SWNTs by tailoring the surfactant structure around SWNTs.