Nathan F. Bouxsein

Highly Efficient Gene Silencing Activity of siRNA Embedded in a Nanostructured Gyroid Cubic Lipid Matrix

RNA interference (RNAi) is an evolutionarily conserved sequence-specific post-transcriptional gene silencing pathway with wide-ranging applications in functional genomics, therapeutics, and biotechnology. Cationic liposome-small interfering RNA (CL-siRNA) complexes have emerged as vectors of choice for delivery of siRNA, which mediates RNAi. However, siRNA delivery by CL-siRNA complexes is often inefficient and accompanied by lipid toxicity. We report the development of CL-siRNA complexes with a novel cubic phase nanostructure, which exhibit efficient silencing at low toxicity. The inverse bicontinuous gyroid cubic nanostructure was unequivocally established from synchrotron X-ray scattering data, while fluorescence microscopy revealed colocalization of lipid and siRNA in complexes. We attribute the efficient silencing to enhanced fusion of complex and endosomal membranes, facilitated by the cubic phase membranes positive Gaussian modulus, which may enable spontaneous formation of transient pores. The findings underscore the importance of understanding membrane-mediated interactions between CL-siRNA complex nanostructure and cell components in developing CL-based gene silencing vectors.

Cationic liposome-nucleic acid complexes for gene delivery and silencing: pathways and mechanisms for plasmid DNA and siRNA.

Motivated by the promises of gene therapy, there is great interest in developing non-viral lipid-based vectors for therapeutic applications due to their low immunogenicity, low toxicity, ease of production, and the potential of transferring large pieces of DNA into cells. In fact, cationic liposome (CL) based vectors are among the prevalent synthetic carriers of nucleic acids (NAs) currently used in gene therapy clinical trials worldwide. These vectors are studied both for gene delivery with CL-DNA complexes and gene silencing with CL-siRNA (short interfering RNA) complexes. However, their transfection efficiencies and silencing efficiencies remain low compared to those of engineered viral vectors. This reflects the currently poor understanding of transfection-related mechanisms at the molecular and self-assembled levels, including a lack of knowledge about interactions between membranes and double stranded NAs and between CL-NA complexes and cellular components. In this review we describe our recent efforts to improve the mechanistic understanding of transfection by CL-NA complexes, which will help to design optimal lipid-based carriers of DNA and siRNA for therapeutic gene delivery and gene silencing.

Non-Viral Gene Delivery with Cationic Liposome–DNA Complexes

A large amount of research activity worldwide is currently directed towards developing lipid- or polymer-based, non-viral gene vectors for therapeutic applications. This strong interest is motivated by their low toxicity, ease of production, ability to transfer large pieces of DNA into cells, and lack of immunogenic protein components. Cationic liposomes (CLs) are one of the most powerful non-viral vectors. In fact, CL-based vectors are among the prevalent synthetic carriers of nucleic acids currently used in human clinical gene therapy trials as well as in cell transfection applications for biological research. Our understanding of the mechanisms of action of CL-DNA complexes is still in its infancy. However, the relevance of a few crucial parameters, such as the lipid/DNA charge ratio (rho(chg)) and the membrane charge density of lamellar complexes (sigma(M)), is well established. To arrive at true comparisons of lipid performance, one must optimize both these parameters using a reproducible, reliable transfection assay. In this chapter, we aim to provide the reader with detailed procedures for liposome formation and transfection. It is our hope that the use of such optimized protocols will improve the comparability of transfection data obtained with novel lipids.

Synthesis and Characterization of Degradable Multivalent Cationic Lipids with Disulfide-Bond Spacers for Gene Delivery

Gene therapy provides powerful new approaches to curing a large variety of diseases, which are being explored in ongoing worldwide clinical trials. To overcome the limitations of viral gene delivery systems, synthetic nonviral vectors such as cationic liposomes (CLs) are desirable. However, improvements of their efficiency at reduced toxicity and a better understanding of their mechanism of action are required. We present the efficient synthesis of a series of degradable multivalent cationic lipids (CMVLn, n=2 to 5) containing a disulfide bond spacer between headgroup and lipophilic tails. This spacer is designed to be cleaved in the reducing milieu of the cytoplasm and thus decrease lipid toxicity. Small angle X-ray scattering demonstrates that the initially formed lamellar phase of CMVLn-DNA complexes completely disappears when reducing agents such as DTT or the biologically relevant reducing peptide glutathione are added to mimic the intracellular milieu. The CMVLs (n=3 to 5) exhibit reduced cytotoxicity and transfect mammalian cells with efficiencies comparable to those of highly efficient non-degradable analogs and benchmark commercial reagents such as Lipofectamine 2000. Thus, our results demonstrate that degradable disulfide spacers may be used to reduce the cytotoxicity of synthetic nonviral gene delivery carriers without compromising their transfection efficiency.

Nanogyroids incorporating multivalent lipids: enhanced membrane charge density and pore forming ability for gene silencing.

The self-assembly of a custom-synthesized pentavalent cationic lipid (MVL5) and glycerol monooleate (GMO) with small interfering RNA (siRNA) results in the formation of a double-gyroid bicontinuous inverted cubic phase with colocalized lipid/siRNA domains as shown by synchrotron X-ray scattering and fluorescence microscopy. The high charge density (due to MVL5) and positive Gaussian modulus of the GMO-containing membranes confer optimal electrostatic and elastic properties for endosomal escape, enabling efficient siRNA delivery and effective, specific gene silencing.

Two-dimensional packing of short DNA with nonpairing overhangs in cationic liposome-DNA complexes: from Onsager nematics to columnar nematics with finite-length columns.

We report the formation of liquid crystalline (LC) phases of short double-stranded DNA with nonpairing (nonsticky) overhangs, confined between two-dimensional (2D) lipid bilayers of cationic liposome-DNA complexes. In a landmark study (Science2007, 318, 1276), Nakata et al. reported on the discovery of strong end-to-end stacking interactions between short DNAs (sDNAs) with blunt ends, leading to the formation of 3D nematic (N) and columnar LC phases. Employing synchrotron small-angle X-ray scattering, we have studied the interplay between shape anisotropy-induced and DNA end-to-end interaction-induced N ordering for 11, 24, and 48 bp sDNA rods with single-stranded oligo-thymine (T) overhangs modulating the end-to-end interactions. For suppressed stacking interactions with 10-T overhangs, the volume fraction of sDNA at which the 2D isotropic (I)-to-N transition occurs for 24 and 48 bp sDNA rods depended on their length-to-width (L/D) shape anisotropy, qualitatively consistent with Onsagers theory for the entropic alignment of rigid rods. As the overhang length is reduced from 10 to 5 and 2 T for 24 and 48 bp sDNA, the N-to-I transition occurs at lower volume fractions, indicating the onset of some degree of end-to-end stacking interactions. The 11 bp sDNA rods with 5- and 10-T overhangs remain in the I phase, consistent with their small shape anisotropy (L/D ≈ 1.9) below the limit for Onsager LC ordering. Unexpectedly, in contrast to the behavior of 24 and 48 bp sDNA, the end-to-end interactions between 11 bp sDNA rods with 2-T overhangs set in dramatically, and a novel 2D columnar N phase (N(C)) with finite-length columns formed. The building blocks of this phase are comprised of 1D stacks of (on average) four 11 bp DNA-2T rods with an effective L(stacked)/D ≈ 8.2. Our findings have implications for the DNA-directed assembly of nanoparticles on 2D platforms via end-to-end interactions and in designing optimally packed LC phases of short anisotropic biomolecules (such as peptides and short-interfering RNAs) on nanoparticle membranes, which are used in gene silencing and chemical delivery.

Structural responses of DNA-DDAB films to varying hydration and temperature

The structure of a DNA-dimethyldidodecylammonium bromide (DDAB) film was recently described to undergo a distinctive transition in response to the water content in the surrounding environment. The existence, preparation, and basic properties of DNA-surfactant films have been known in the literature for some time. Here, we describe the structural response of DNA-DDAB films to environmental changes, particularly temperature and humidity, in greater detail revealing new structural states. We can direct the lamellar structure of the film into three distinct states--double-stranded DNA (dsDNA) paired with an interdigitated bilayer of DDAB (bDDAB), single-stranded DNA (ssDNA) with monolayer of DDAB (mDDAB), and ssDNA with bDDAB. Both temperature and humidity cause the molecules composing the lamellar structure to change reversibly from ssDNA to dsDNA and/or from mDDAB to bDDAB. We found that the structural transition from dsDNA to ssDNA and bDDAB to mDDAB is concerted and follows apparent first-order kinetics. We also found that the double-stranded conformation of DNA in the film can be stabilized with the inclusion of cholesterol even while the DDAB in the film is able to form either a monolayer or bilayer depending on the environmental conditions. Films treated with ethidium bromide prompt switching of dsDNA to ssDNA before bDDAB transitions to mDDAB. Swelling experiments have determined that there is a direct proportionality between the macroscopic increase in volume and the nanoscopic increase in lamellar spacing when a film is allowed to swell in water. Finally, experiments with phosphate-buffered saline (PBS) indicate that the films can disassemble in a simulated biological environment due to screening of their charges by buffer salt. We conclude that the structure of DNA in the film depends on the water content (as measured by the relative humidity) and temperature of the environment, while the state of DDAB depends essentially only on the water content. The structure of the film is quite flexible and can be altered by changing environmental conditions as well as the chemical ingredients. These films will have useful, new applications as responsive materials, for example, in drug and gene delivery.

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.

Single Filament Behavior of Microtubules in the Presence of Added Divalent Counterions

Microtubules (MTs) are hollow biopolymeric filaments that function to define the shape of eukaryotic cells, serve as a platform for intracellular vesicular transport, and separate chromosomes during mitosis. One means of physiological regulation of MT mechanics and dynamics, critical to their adaptability in such processes, is through electrostatics due to the strong polyelectrolyte nature of MTs. Here, we show that in the presence of physiologically relevant amounts of divalent salts, MTs experience a dramatic increase in persistence length or stiffness, which is counter to theoretical expectations and experimental observations in similar systems (e.g., DNA). Divalent salt-dependent effects on MT dynamics were also observed with respect to suppressing depolymerization as well as reducing dispersion in kinesin-driven molecular motor transport assays. These results establish a novel mechanism by which MT dynamics, mechanics, and interaction with molecular motors may be regulated by physiologically relevant concentrations of divalent salts.

Alignment of filamentous proteins and associated molecules through confinement in microchannels

A technique has been developed to study the structure and interaction of aligned filamentous proteins by confining them in surface-treated silicon microchannels. The micron-size channels induce the semiflexible biopolymers with comparable or larger persistence lengths than the channel width to naturally align parallel to the channel in solution, which facilitates structural studies by x-ray diffraction and optical imaging techniques. As a model system, we investigated the cross-linking of filamentous actin (F-actin) with the bundling protein α-actinin in the microchannels. Synchrotron x-ray diffraction and fluorescence microscopy were used to confirm that F-actin, when bundled in the device, conforms to the alignment of the channel geometry.