Heather M Evans

Max Planck Institute for Dynamics and Self-Organization, Göttingen, Lower Saxony, Germany

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Publications (42)125.42 Total impact

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    ABSTRACT: Collagen-based biomaterials are currently used as cell culture scaffolds in tissue engineering approaches. These materials are being developed with increased functional complexity, such as the incorporation of glycosaminoglycans. Our study shows the impact of heparin intercalation at specific binding sites in telopeptide-free collagen fibrils in terms of their structure, mechanics, and cell response. We demonstrate that heparin binds specifically and in a competitive manner along the tropocollagen helix at places that are occupied in vivo by telopeptides in fibrillar collagen type I. On the basis of this finding, we elucidate the reason for the in vivo dogma that heparin does not intercalate in fibrillar collagens. We further reveal the direct relationship among structure, mechanics, and function in terms of the effect of incorporation of intercalated heparin on the fibrillar structure, fibrillar bending modulus and flexural rigidity and the dynamic response of adherent cells to collagen scaffolds. This tight relationship is considered particularly important when designing xenogeneic scaffolds based on natural collagen type I to trigger cell proliferation and differentiation.
    Biomaterials 10/2011; 32(30):7444-53. · 8.31 Impact Factor
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    ABSTRACT: 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.
    Biomacromolecules 03/2011; 12(5):1504-11. · 5.37 Impact Factor
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    ABSTRACT: 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.
    Topics in current chemistry 01/2010; 296:191-226. · 8.46 Impact Factor
  • Biophysical Journal 01/2010; 98(3). · 3.67 Impact Factor
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    ABSTRACT: The protein fibrin plays a principal role in blood clotting and forms robust three dimensional networks. Here, microfluidic devices have been tailored to strategically generate and study these bionetworks by confinement in nanoliter volumes. The required protein components are initially encapsulated in separate droplets, which are subsequently merged by electrocoalescence. Next, distinct droplet microenvironments are created as the merged droplets experience one of two conditions: either they traverse a microfluidic pathway continuously, or they "park" to fully evolve an isotropic network before experiencing controlled deformations. High resolution fluorescence microscopy is used to image the fibrin networks in the microchannels. Aggregation (i.e."clotting") is significantly affected by the complicated flow fields in moving droplets. In stopped-flow conditions, an isotropic droplet-spanning network forms after a suitable ripening time. Subsequent network deformation, induced by the geometric structure of the microfluidic channel, is found to be elastic at low rates of deformation. A shape transition is identified for droplets experiencing rates of deformation higher than an identified threshold value. In this condition, significant densification of protein within the droplet due to hydrodynamic forces is observed. These results demonstrate that flow fields considerably affect fibrin in different circumstances exquisitely controlled using microfluidic tools.
    Lab on a Chip 08/2009; 9(13):1933-41. · 5.70 Impact Factor
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    ABSTRACT: Motivated by its important role in gene delivery, we have studied the effect of cholesterol and analogs on the transfection efficiency (TE) of lamellar cationic liposome-DNA (CL-DNA) complexes in vitro. Addition of cholesterol to low-transfecting DOTAP/DOPC-DNA complexes increases TE, with 15 mol % cholesterol already yielding 10-fold improvement. Steroids lacking the alkyl tail only modestly enhance TE, while molecules retaining it strongly enhance TE. All steroid-containing CL-DNA complexes exhibit the lamellar structure. The increase in experimentally determined membrane charge density (a universal parameter governing the TE of lamellar CL-DNA complexes) with cholesterol content alone cannot account for the rapid increase of TE. Instead, the reduction of the hydration repulsion layer of the membrane, caused by replacement of DOPC by cholesterol, promotes fusion between cationic membranes of CL-DNA complexes and anionic endosomal membranes, thus facilitating release of complexes and enhancing TE.
    The Journal of Physical Chemistry B 05/2009; 113(15):5208-16. · 3.61 Impact Factor
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    ABSTRACT: The prospects of gene therapy have generated unprecedented interest in the properties and structures of complexes of nucleic acids (NAs) with cationic liposomes (CLs), which are used as nonviral NA carriers in worldwide clinical trials. An improved understanding of the mechanisms of action of CL-NA complexes is required to enable their widespread therapeutic use. In prior studies of CL-mediated DNA delivery, membrane charge density (sigma(M)) was identified as a key parameter for transfection efficiency (TE) of lamellar (L(alpha)(C)) CL-DNA complexes. The TE of CL-DNA complexes containing cationic lipids with headgroup valencies from 1+ to 5+ follows a universal bell-shaped curve as a function of sigma(M). As we report here, the TE of CL-DNA complexes containing new multivalent lipids with dendritic headgroups (DLs) strongly deviates from this curve at high sigma(M). We have investigated four DLs, MVLG2 (4+), MVLG3 (8+), MVLBisG1 (8+), and MVLBisG2 (16+), in mixtures with neutral 1,2-dioleoyl-sn-glycerophosphatidyl-choline (DOPC). To understand the TE behavior, we have performed X-ray diffraction (XRD), optical microscopy, and cryo-TEM studies of the DL/DOPC mixtures and their DNA complexes. XRD reveals a complex phase behavior of DL-DNA complexes which strongly depends on the headgroup charge. MVLG2(4+)/DOPC-DNA complexes exhibit the lamellar phase at all molar fractions of DL, Phi(DL). In stark contrast, MVLBisG2(16+)/DOPC-DNA complexes remain lamellar only for Phi(DL) </= 0.2. In a narrow regime around Phi(DL) = 0.25, the hexagonal phase H(I)(C), consisting of a hexagonal lattice of cylindrical lipid micelles and a DNA honeycomb lattice, is formed. At Phi(DL) > 0.3, XRD suggests formation of a distorted H(I)(C) phase. For Phi(DL) >/= 0.5 under high salt conditions, this phase coexists with a bundle phase of DNA condensed by the depletion-attraction effect of DL micelles. The transitions at high sigma(M) from the lamellar phase to the new hexagonal phases of DL-DNA complexes coincide with the deviation from the universal TE behavior of lamellar complexes. The observed high TE, which is independent of sigma(M), strongly suggests a novel mechanism of action for these DL-DNA complex phases.
    The Journal of Physical Chemistry B 01/2009; · 3.61 Impact Factor
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    ABSTRACT: Cationic lipids (CLs) have found widespread use as nonviral gene carriers (vectors), including applications in clinical trials of gene therapy. However, their observed transfection efficiencies (TEs) are inferior to those of viral vectors, providing a strong incentive for a detailed understanding of CL-DNA complex behavior. In recent systematic studies employing monovalent as well as newly synthesized multivalent lipids (MVLs), the membrane cationic charge density has been identified as a key parameter governing the TE of lamellar CL-DNA complexes. In this work, we use x-ray scattering and molecular simulations to investigate the structural properties of complexes containing MVLs. At low mole fraction of neutral lipids (NLs), Phi(NL), the complexes show dramatic DNA compaction, down to essentially close-packed DNA arrays with a DNA interaxial spacing d(DNA) = 25 A. A gradual increase in Phi(NL) does not lead to a continuous increase in d(DNA) as observed for DNA complexes of monovalent CLs. Instead, distinct spacing regimes exist, with sharp transitions between the regimes. Three packing states have been identified: 1), close packed, 2), condensed, but not close packed, with d(DNA) = 27-28 A, and 3), an expanded state, where d(DNA) increases gradually with Phi(NL). Based on our experimental and computational results, we conclude that the DNA condensation is mediated by the multivalent cationic lipids, which assemble between the negatively charged DNA rods. Quite remarkably, the computational results show that the less tightly packed structure in regime 2 is thermodynamically more stable than the close packed structure in regime 1. Accordingly, the constant DNA spacing observed in regime 2 is attributed to lateral phase coexistence between this stable CL-DNA complex and neutral membranes. This finding may explain the reduced TE measured for such complexes: transfection involves endosomal escape and disassembly of the complex, and these processes are inhibited by the high thermodynamic stability. Our results, which demonstrate the existence of an inverse correlation between the stability and transfection activity of lamellar CL-DNA complexes are, therefore, consistent with a recently proposed model of cellular entry.
    Biophysical Journal 08/2008; 95(2):836-46. · 3.67 Impact Factor
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    ABSTRACT: Motivated by its important role in lipid-mediated gene delivery, we have studied the effect of cholesterol on the transfection efficiency (TE) of lamellar, cationic lipid-DNA (CL-DNA) complexes. A successful in vivo liposome mixture seems to require cholesterol. Recent work in our group has identified the membrane charge density (sigma) as a universal parameter for TE of lamellar, DOPC containing CL-DNA complexes (A.J. Lin et al, Biophys. J., 2003, K. Ewert et al, J. Med. Chem., 2002, A. Ahmad et al., J. Gene Med., 2005), with TE following a universal bell-shaped curve as a function of sigma. Theoretical calculations considering the headgroup area of cholesterol and thus necessarily counting for an increase in sigma, when DOPC is replaced by cholesterol, show that TE strongly deviates from the TE universal curve. However, experimental determination of sigma via X-ray diffraction shows full agreement with the TE universal curve demonstrating that the real sigma is higher as predicted, therefore the effective headgroup area of cholesterol is lower as expected by theory, suggesting that cholesterol is inserted deep into lipid bilayer partially hidden by the neighboring lipids. Funding provided by NIH GM-59288 and NSF DMR-0503347.
    03/2008;
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    ABSTRACT: We present in situ studies on the self-assembly and dynamic evolution of collagen gels from semidilute solutions in a microfluidic device. Collagen fibrils not only reinforce the mechanical properties of bone and tissues, but they also influence cellular motility and morphology. We access the initial steps of the hierarchical self-assembly of collagen fibrils and networks by using hydrodynamic focusing to form oriented fibers. The accurate description of the conditions within the microchannel requires a numerical expression for the pH in the device as well as a modified mathematical description of the viscosity, which increases nearly 300-fold as collagen fibrils form around neutral pH. Finite element modeling profiles overlay impressively with cross-polarized microscopy images of the birefringent fibrils in the channel. Real-time X-ray microdiffraction measurements in flow indicate an enhanced supramolecular packing having a unit spacing commensurate with that of a pentameric collagen subunit. These results have significant implications for the field of biomedicine, wherein new aligned, cellularly active, and mechanically strengthened materials continue to be in demand. However, this work is also remarkable from a more fundamental, biophysical point of view because the underlying concepts may be generalized to a large pool of systems.
    Biomacromolecules 02/2008; 9(1):199-207. · 5.37 Impact Factor
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    ABSTRACT: 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.
    Methods in molecular biology (Clifton, N.J.) 02/2008; 433:159-75. · 1.29 Impact Factor
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    ABSTRACT: DNA condensation in vivo usually requires proteins and/or multivalent salts. Here, we explore the in vitro compaction of DNA by cationic dendrimers having an intermediate size and charge. The dynamic assembly of DNA-dendrimer mesophases is discernible due to the laminar flow in a specially designed X-ray compatible microfluidic device. The setup ensures a nonequilibrium ascent of reactant concentration, and the resulting progression of DNA compaction was detected online using microfocused small-angle X-ray diffraction. The evolution of a DNA-dendrimer columnar square mesophase as a function of increasing dendrimer content is described. Additionally, in regions of maximum shear, an unexpected high-level perpendicular ordering of this phase is recorded. Furthermore, these assemblies are found to be in coexistence with a densely packed DNA-only mesophase in regions of excess DNA. The latter is reminiscent of dense packing found in bacteriophage and chromosomes, although surprisingly, it is not stabilized by direct dendrimer contact.
    Biomacromolecules 08/2007; 8(7):2167-72. · 5.37 Impact Factor
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    ABSTRACT: Spatially resolved X-ray microdiffraction in hydrodynamic focusing microdevices provides new opportunities to study time-resolved reactions of complex fluids. A demonstration of this technique as applied to the liquid crystal 8CB was recently reported [1]. Here, we discuss the dynamics of the compaction of DNA by polyimine dendrimers, as studied using microfluidic devices. Due to the laminar flow inside the channels a highly defined, diffusion controlled compaction of DNA occurs. Different snapshots in the time of the reaction are accessible at varying spatial positions along the interaction jet. We use newly developed X-ray compatible microflow foils made from PDMS and Kapton and having dimensions ranging from 30 to 150 micrometers. The real-time evolution of a DNA-dendrimer columnar mesophase with an in-plane square symmetry is reported. These studies are also extended to include a larger library of dendrimers whose size and charge approach those of the biological histone proteins. [1] Dootz, Evans, Koester, Pfohl, accepted to Small.
    03/2007;
  • Heather M. Evans, Sarah Koester, Thomas Pfohl
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    ABSTRACT: Microfluidic structures are particularly amenable to controlled investigations of protein bundle and network formation. Hydrodynamic focusing is utilized to create a diffusion-controlled gradient of reactants, enabling non-equilibrium investigations. We present studies of the blood clotting protein fibrin, a three-dimensional network formed from the enzymatic cleavage of fibrinogen monomers by the protein thrombin. Fibrin is a vital component of blood clots, and has been implicated in a variety of diseases. Real-time fluorescence microscopy and x-ray micro-diffraction are used to quantify supramolecular assembly and provide snapshots of the evolution of fibrin network formation. We also show that collagen, a ubiquitous extracellular protein, can be bundled in situ through the use of a pH gradient. An outlook toward artificial blood vessels arises from the insight that both fibrin and collagen can easily be used to coat microchannel structures. The resulting mesh forms an ideal environment for red blood cells and other cell types.
    03/2007;
  • Small 02/2007; 3(1):96-100. · 7.82 Impact Factor
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    ABSTRACT: Actin filaments, aside from their biological renown as providing the `skeleton' of cells, also proffer an ideal platform from which to study -- more generally -- the properties of semi-flexible polymers. Microfluidic devices made using soft-lithography are easily adapted in dimension and geometry to create well-defined flow environments. Actin filaments are visualized in continuous flow in a microfluidic channel by stroboscopic laser light illumination. A detailed analysis of filament orientation, center-of-mass distribution, and thermal fluctuations as a function of flow rate and channel geometry is reported. In addition, the non-equilibrium bundling behavior of actin in the presence of actin-binding proteins or multivalent ions is studied in microchannel devices using FRET microscopy.
    01/2007;
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    ABSTRACT: At present, there is an unprecedented level of interest in the properties and structures of complexes consisting of DNA mixed with oppositely charged cationic liposomes (CLs). The interest arises because the complexes mimic natural viruses as chemical carriers of DNA into cells in worldwide human gene therapy clinical trials. However, since our understanding of the mechanisms of action of CL-DNA complexes interacting with cells remains poor, significant additional insights and discoveries will be required before the development of efficient chemical carriers suitable for long-term therapeutic applications. Recent studies describe synchrotron X-ray diffraction, which has revealed the liquid crystalline nature of CL-DNA complexes, and three-dimensional laser-scanning confocal microscopy, which reveals CL-DNA pathways and interactions with cells. The importance of the liquid crystalline structures in biological function is revealed in the application of these modern techniques in combination with functional transfection efficiency measurements, which shows that the mechanism of gene release from complexes in the cell cytoplasm is dependent on their precise liquid crystalline nature and the physical and chemical parameters (for example, the membrane charge density) of the complexes. In [section sign] 5, we describe some recent new results aimed at developing bionanotube vectors for gene delivery.
    Philosophical Transactions of The Royal Society A Mathematical Physical and Engineering Sciences 11/2006; 364(1847):2573-96. · 2.89 Impact Factor
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    ABSTRACT: 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.
    Journal of the American Chemical Society 04/2006; 128(12):3998-4006. · 10.68 Impact Factor
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    ABSTRACT: Motivated by its important role in lipid-mediated gene delivery, we have studied the effect of cholesterol on membrane fusion. While recent work in our group has identified the membrane charge density as a critical parameter for transfection efficiency (TE) of lamellar, DOPC containing cationic lipid-DNA (CL-DNA) complexes [1-3], this model cannot fully explain the effect of cholesterol, suggesting that a different mechanism is responsible for the observed enhancement of TE. A model system using negatively charged giant vesicles has been developed to mimic the interaction of the cell membrane with CL-DNA complexes containing cholesterol. Differences in fusogenic properties have been observed as a function of the amount of cholesterol present in the CL-DNA complexes, and a fluorescence resonance energy transfer based assay was employed to quantify this effect. X-ray diffraction confirms that the lamellar structure seen with CL- DNA complexes is retained with the addition of cholesterol. Funding provided by NIH GM-59288 and NSF DMR-0503347. [1] A.J. Lin et al, Biophys. J., 2003, V84:3307-3316. [2] K. Ewert et al, J. Med. Chem., 2002, V45:5023-5029. [3] A. Ahmad et al., J. Gene Med., 2005, V7:739-748.
    03/2006;
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    ABSTRACT: Gene therapy is expected to lead to powerful new approaches for curing many diseases, a potential that is currently explored in worldwide clinical trials. Nonviral DNA delivery systems are desirable to overcome the inherent problems of viral vectors, but their current efficiency requires improvement and the understanding of their mechanism of action is incomplete. We have synthesized new multivalent cationic lipids with highly charged dendritic headgroups to probe the structure-transfection efficiency relationships of cationic liposome (CL)-DNA complexes, a prevalent nonviral vector. The lipid headgroups are constructed from ornithine cores and ornithine or carboxyspermine endgroups. The dendritic lipids were prepared on a gram scale, using a synthetic scheme that permits facile variation of the lipid building blocks headgroup, spacer, and hydrophobic moiety. They carry four to sixteen positive charges in their headgroups. Complexes of DNA with mixtures of the dendritic lipids and neutral 1,2-dioleoyl-sn-glycero phosphatidylcholine (DOPC) exhibit novel structures at high contents of the highly charged lipids, while the well-known lamellar phase is formed at high contents of DOPC. DNA complexes of the new dendritic lipids efficiently transfect mammalian cells in culture without cytotoxicity and, in contrast to lamellar complexes, maintain high transfection efficiency over a broad range of composition.
    Bioconjugate Chemistry 01/2006; 17(4):877-88. · 4.58 Impact Factor

Publication Stats

646 Citations
125.42 Total Impact Points

Institutions

  • 2007–2011
    • Max Planck Institute for Dynamics and Self-Organization
      • Department of Dynamics of Complex Fluids
      Göttingen, Lower Saxony, Germany
  • 2002–2010
    • University of California, Santa Barbara
      • • Department of Molecular, Cellular, and Developmental Biology
      • • Department of Biomolecular Science and Engineering
      Santa Barbara, CA, United States
  • 2008
    • Molecular and Cellular Biology Program
      Seattle, Washington, United States
    • Ben-Gurion University of the Negev
      • Department of Biomedical Engineering
      Beersheba, Southern District, Israel