Complexes with low sigmaM remain trapped in the endosome. In the high sigmaM regime, accessible for the first time with the new MVLs, complexes escape by overcoming a kinetic barrier to fusion with the endosomal membrane (activated fusion), yet they exhibit a reduced level of efficiency, presumably due to the inability of the DNA to dissociate from the highly charged membranes in the cytosol. The intermediate, optimal regime reflects a compromise between the opposing demands on sigmaM for endosomal escape and dissociation in the cytosol.

Gene therapy holds great promise as a future approach to fighting disease and is explored in worldwide clinical trials. Cationic liposome (CL)-DNA complexes are a prevalent nonviral delivery vector, but their efficiency requires improvement and the understanding of their mechanism of action is incomplete. As part of our effort to investigate the structure-transfection efficiency relationships of self-assembled CL-DNA vectors, we have synthesized a new, highly charged (16+) multivalent cationic lipid, MVLBG2, with a dendritic headgroup. Our synthetic scheme allows facile variation of the headgroup charge and the spacer connecting hydrophobic and headgroup moieties as well as gram-scale synthesis. Complexes of DNA with mixtures of MVLBG2 and neutral 1,2-dioleoyl-sn-glycerophosphatidylcholine (DOPC) exhibit the well-known lamellar phase at 90 mol % DOPC. Starting at 20 mol % dendritic lipid, however, two novel nonlamellar phases are observed by synchrotron X-ray diffraction. The structure of one of these phases, present in a narrow range of composition around 25 mol % MVLBG2, has been solved. In this novel dual lattice structure, termed H(I)C, hexagonally arranged tubular lipid micelles are surrounded by DNA rods forming a three-dimensionally continuous substructure with honeycomb symmetry. Complexes in the H(I)C phase efficiently transfect mouse and human cells in culture. Their transfection efficiency, as well as that of the lamellar complexes containing only 10 mol% dendritic lipid, reaches and surpasses that of commercially available, optimized DOTAP-based complexes. In particular, complexes containing MVLBG2 are significantly more transfectant over the entire composition range in mouse embryonic fibroblasts, a cell line empirically known to be hard to transfect.

Cationic liposomes (CLs) are used as gene vectors (carriers) in worldwide human clinical trials of non-viral gene therapy. These lipid-gene complexes have the potential of transferring large pieces of DNA of up to 1 million base-pairs into cells. As our understanding of the mechanisms of action of CL-DNA complexes remains poor, transfection efficiencies are still low when compared to gene delivery with viral vectors. We describe recent studies with a combination of techniques (synchrotron x-ray diffraction for structure determination, laser-scanning confocal microscopy to probe the interactions of CL-DNA particles with cells, and luciferase reporter-gene expression assays to measure transfection efficiencies in mammalian cells), which collectively are beginning to unravel the relationship between the distinctly structured CL-DNA complexes and their transfection efficiency. The work described here is applicable to transfection optimization in ex vivo cell transfection, where cells are removed and returned to patients after transfection. CL-DNA complexes primarily form a multilayered sandwich structure with DNA layered between the cationic lipids (labeled L,~). On rare occasions, an inverted hexagonal structure with DNA encapsulated in lipid tubules (labeled H~~~) is observed. A major recent insight is that for L ,~ complexes the membrane charge density OM of the CL-vector, rather than the charge of the cationic lipid alone, is a key universal parameter that governs the transfection efficiency of L, ' complexes in cells. The parameter aM is a measure of the average charge per unit area of the membrane, thus taking into account the amount of neutral lipids. In contrast to LaC complexes, HITC complexes containing the lipid 1,2-dioleoyl-sn-glycerophosphatidylethanolamine (DOPE) exhibit no dependence on OM. The currenl limiting factor to transfection by cationic lipid vectors appears to be the tight association of a fraction of the delivered exogenous DNA with cationic cellular molecules, which may prevent optimal transcriptional activity. Future directions are outlined, which make use of surface-functionalized CL-DNA complexes suitable for transfection in vivo.

Despite widespread use of silk, it remains a significant challenge to fabricate fibers with properties similar to native silk. It has recently been recognized that the key to tuning silk fiber properties lies in controlling internal structure of assembled β-sheets. We report an advance in the precise control of silk fiber formation with control of properties via microfluidic solution spinning. We use an experimental approach combined with modeling to accurately predict and independently tune fiber properties including Young’s modulus and diameter to customize fibers. This is the first reported microfluidic approach capable of fabricating functional fibers with predictable properties and provides new insight into the structural transformations responsible for the unique properties of silk. Unlike bulk processes, our method facilitates the rapid and inexpensive fabrication of fibers from small volumes (50 μL) that can be characterized to investigate sequence-structure-property relationships to optimize recombinant silk technology to match and exceed natural silk properties.