In gene and siRNA based therapy, therapeutic benefits rely on the efficient transfer of foreign nucleic acids into cells. We investigate synthetic complexes capable of readily carrying nucleic acid across the cell membrane. The self-assembly, size and structure of lipid- and polycation-DNA complexes, known respectively as lipoplexes and polyplexes, are studied. We also endeavor to quantify and model transfection at the single cell level using GFP reporter and single cell time-lapse microscopy.
The delivery of mRNA through lipid-based transfection remains a key challenge for the development of RNA therapeutics. In order to elucidate the uptake-mechanism the expression of green fluorescence protein from a delivered reporter gene is monitored. We present the integration of scanning time-lapse microscopy with micro-structured single-cell arrays and mathematical modeling. Analysis of hundreds of individual time courses yields insight into the delivery kinetics. Surprisingly, faster delivery is not related to better efficiency at the single-cell level. However, we find that timing and efficiency of lipid-based gene carrier vary as function of the amount of serum in the cell culture medium. In conclusion, onset time distributions obtained from single-cell time courses provide a powerful tool to improve gene delivery systems. https://www.biorxiv.org/content/10.1101/607986v1 (published April 2019)
- A. Reiser, D. Woschée, N. Mehrotra, R. Krzysztoń, View ORCID ProfileH. H. Strey, J. O. Rädler
Multi-experiment nonlinear mixed effect modeling of single- cell translation kinetics after transfection
Single-cell time-lapse studies have advanced the quantitative understanding of cellular pathways and their inherent cell-to-cell variability. However, parameters retrieved from individual experiments are model dependent and their estimation is limited, if based on solely one kind of experiment. Hence, methods to integrate data collected under different conditions are expected to improve model validation and information content. Here we present a multi-experiment nonlinear mixed effect modeling approach for mechanistic pathway models, which allows the integration of multiple single- cell perturbation experiments. We apply this approach to the translation of green fluorescent protein after transfection using a massively parallel read-out of micropatterned single-cell arrays. We demonstrate that the integration of data from perturbation experiments allows the robust reconstruction of cell-to-cell variability, i.e., parameter densities, while each individual experiment provides insufficient information. Indeed, we show that the integration of the datasets on the population level also improves the estimates for individual cells by breaking symmetries, although each of them is only measured in one experiment. Moreover, we confirmed that the suggested approach is robust with respect to batch effects across experimental replicates and can provide mechanistic insights into the nature of batch effects. We anticipate that the proposed multi- experiment nonlinear mixed effect modeling approach will serve as a basis for the analysis of cellular heterogeneity in single-cell dynamics.
- Fabian Fröhlich, Anita Reiser, Laura Fink, Daniel Woschée, Thomas Ligon, Fabian Joachim Theis, Joachim Oskar Rädler & Jan Hasenauer
npj Systems Biology and Applicationsvolume 4, Article number: 42 (2018)
In non-viral gene delivery, the variance of transgenic expression stems from the low number of plas- mids successfully transferred. Here, we experimentally determine Lipofectamine- and PEI-mediated exogenous gene expression distributions from single cell time-lapse analysis. Broad Poisson-like distributions of steady state expression are observed for both transfection agents, when used with synchronized cell lines. At the same time, co- transfection analysis with YFP- and CFP-coding plasmids shows that multiple plasmids are simultaneously expressed, suggesting that plasmids are delivered in correlated units (complexes). We present a mathematical model of transfec- tion, where a stochastic, two-step process is assumed, with the first being the low-probability entry step of complexes into the nucleus, followed by the subsequent release and activation of a small number of plasmids from a delivered complex. This conceptually simple model consistently pre- dicts the observed fraction of transfected cells, the cotrans- fection ratio and the expression level distribution. It yields the number of efficient plasmids per complex and elucidates the origin of the associated noise, consequently providing a platform for evaluating and improving non-viral vectors.
- Schwake, G., Youssef, S., Kuhr, J.-T., Gude, S., David, M. P., Mendoza, E., et al. (2010, February 12). Predictive Modeling of Non-Viral Gene Transfer. arXiv.org. doi:10.1002/bit.22604
The design of efficient nucleic acid complexes is key to progress in genetic research and therapies based on RNA interference. For optimal transport within tissue andacross extracellular barriers, nucleic acid carriers need to be small and stable. In this Article, we prepare and characterize mono-nucleic acid lipid particles (mono-NALPs). The particles consist of single short double-stranded oligonucleotides or single siRNAmolecules each encapsulated within a closed shell of a cationic-zwitterionic lipid bilayer, furnished with an outer polyethylene glycol (PEG) shield. The particles self-assemble by solvent exchange from a solution containing nucleic acid mixed with the four lipid components DOTAP, DOPE, DOPC, and DSPE-PEG(2000). Using fluorescence correlation spectroscopy, we monitor the formation of mono-NALPs from short double-stranded oligonucleotides or siRNA and lipids into monodisperse particles of approximately 30 nm in diameter. Small angle neutron and X-ray scattering and transmission electron microscopy experiments substantiate a micelle-like core!shell structure of the particles. The PEGylated lipid shell protects the nucleic acid core against degradation by nucleases, sterically stabilizes the mono-NALPs against disassembly in collagen networks, and prevents nonspecific binding to cells. Hence, PEG-lipid shielded mono-NALPs are the smallest stable siRNA lipid system possible and may provide a structural design to be built upon for the development of novel nucleic acid delivery systems with enhanced biodistribution in vivo.
- Rudorf, S., & Rädler, J. O. (2012). "Self-Assembly of Stable Monomolecular Nucleic Acid Lipid Particles with a Size of 30 nm."
Journal of the American Chemical Society, 134(28), 11652–11658.
The complexation of linear DNA fragments with cationic diblock copolymers was studied as a model system for understanding “bottom-up” self-assembly of nanoscopic gene delivery systems. Fluorescence correlation spectroscopy (FCS) measurements were performed on monodisperse linear DNA fragments complexed with diblock copolymers consisting of a cationic charged moiety, branched polyethyleneimine (bPEI), of 2, 10 or 25kDa, and a neutral shielding moiety, poly(ethylene glycol) (PEG, 20kDa). For 10 and 25kDa bPEI-PEG diblocks, severe aggregation is observed despite the presence of the shielding PEG. By decreasing the bPEI length to 2 kDa, or conversely increasing the grafting density of PEG chains per DNA, controlled nanoparticle formation is observed. The resulting decorated particles are consistent with a “core-shell” particle consisting of a single DNA surrounded by a brush layer of densely packed PEG chains. Diffusion coefficients for both DNA and decorated DNA fragments were measured as a function of DNA length ranging from 75 to 1018 bp and are well described by a diffusing rod model. Decorated rod DNA nanoparticles showed high stability against both NaCl salt and bovine serum albumin and are of potential interest for gene delivery of short antisense DNA or siRNA.
• J. DeRouchey, G. Walker, E. Wagner, J.O. Rädler "Decorated rods: A “bottom-up” self-assembly of monomolecular DNA complexes" Journal of physical chemistry B, 110 (10): 4548-4554 (2006).
The internal ordering of DNA-polycation complexes were investigated by synchrotron small-angle x-ray scattering (SAXS). Hexagonal packing of DNA is observed for DNA complexes formed with poly-L-lysine (PL), poly-L-arginine (PA), spermine (Sp), and linear and branched polyethyleneimine (lPEI and bPEI, respectively). Variations in the internal spacings and degree of long-range ordering are dependent on both polycation type and concentration of added salt. With increasing concentration of monovalent salt, a continuous phase transition is observed from compact bundles to loose bundles and finally to an isotropic network phase. This salt-induced melting transition is universal for all polyplexes studied, but the critical ionic strength of melting was highly dependent on the polycation and scales approximately with the respective binding energies. Using the osmotic stress method, bulk modulus and compressibility (K and b) were measured for PL-DNA and PA-DNA polyplexes at different salt concentrations. Additionally, we show that the salt-induced melting transition can be reversibly crossed with increasing osmotic force.
• J. DeRouchey, R. R. Netz, J. O. Rädler, "Structural Investigations of DNA-Polycation complexes." Eur. Phys. J. E, (2005), 16 (1), 17-28.
- Artzner, F., Zantl, R., & Rädler, J. (2000). Lipid-DNA and lipid-polyelectrolyte mesophases: Structure and exchange kinetics. Cellular and Molecular Biology, 46(5), 967–978.
- Artzner, F., Zantl, R., Rapp, G., & Rädler, J. (1998). Observation of a rectangular columnar phase in condensed lamellar cationic lipid-DNA complexes. Physical Review Letters, 81(22), 5015–5018.
- Koltover, I., Rädler, J., & Safinya, C. (1999a). Membrane mediated attraction and ordered aggregation of colloidal particles bound to giant phospholipid vesicles. Physical Review Letters, 82(9), 1991–1994.
- Koltover, I., Rädler, J., Salditt, T., Rothschild, K., & Safinya, C. (1999b). Phase behavior and interactions of the membrane-protein bacteriorhodopsin. Physical Review Letters, 82(15), 3184–3187.
- Koltover, I., Salditt, T., Rädler, J., & Safinya, C. (1998). An inverted hexagonal phase of cationic liposome-DNA complexes related to DNA release and delivery. Science, 281(5373), 78–81.
- Pohle, W., Selle, C., Gauger, D., Zantl, R., Artzner, F., & Rädler, J. (2000). FTIR spectroscopic characterization of a cationic lipid-DNA complex and its components. Physical Chemistry Chemical Physics, 2(20), 4642–4650.
- Rädler, J., Koltover, I., Jamieson, A., Salditt, T., & Safinya, C. (1998). Structure and interfacial aspects of self-assembled cationic lipid-DNA gene carrier complexes. Langmuir, 14(15), 4272–4283.
- Rädler, J., Koltover, I., Salditt, T., & Safinya, C. (1997). Structure of DNA-cationic liposome complexes: DNA intercalation in multilamellar membranes in distinct interhelical packing regimes. Science, 275(5301), 810–814.
- Safinya, C., Koltover, I., & Rädler, J. (1998). DNA at membrane surfaces: an experimental overview. Current Opinion in Colloid & Interface Science, 3(1), 69–77.
- Salditt, T., Koltover, I., Rädler, J., & Safinya, C. (1997). Two-dimensional smectic ordering of linear DNA chains in self-assembled DNA-cationic liposome mixtures. Physical Review Letters, 79(13), 2582–2585.
- Salditt, T., Koltover, I., Rädler, O., & Safinya, C. (1998). Self-assembled DNA-cationic-lipid complexes: Two-dimensional smectic ordering, correlations, and interactions. Physical Review E, 58(1), 889–904.
- Zantl, R., Artzner, F., Rapp, G., & Rädler, J. (1999a). Thermotropic structural changes of saturated-cationic-lipid-DNA complexes. Europhysics Letters, 45(1), 90–96.
- Zantl, R., Baicu, L., Artzner, F., Sprenger, I., Rapp, G., & Rädler, J. (1999b). Thermotropic phase behavior of cationic lipid-DNA complexes compared to binary lipid mixtures. The Journal of Physical Chemistry B, 103(46), 10300–10310.