Biomedical subjects

Cyrus R Safinya

Publications and source records attributed to Cyrus R Safinya.

At least 19 recordsLinked to original sources

PubMed2006

Cationic liposome-DNA complexes: from liquid crystal science to gene delivery applications.

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.

Cations↗

PubMed2006

Microtubule protofilament number is modulated in a stepwise fashion by the charge density of an enveloping layer.

Microtubules are able to adjust their protofilament (PF) number and, as a consequence, their dynamics and function, to the assembly conditions and presence of cofactors. However, the principle behind such variations is poorly understood. Using synchrotron x-ray scattering and transmission electron microscopy, we studied how charged membranes, which under certain conditions can envelop pre-assembled MTs, regulate the PF number of those MTs. We show that the mean PF number, , is modulated primarily by the charge density of the membranes. decreases in a stepwise fashion with increasing membrane charge density. does not depend on the membrane-protein stoichiometry or the solution ionic strength. We studied the effect of taxol and found that increases logarithmically with taxol/tubulin stoichiometry. We present a theoretical model, which by balancing the electrostatic and elastic interactions in the system accounts for the trends in our findings and reveals an effective MT bending stiffness of order 10-100 k(B)T/nm, associated with the observed changes in PF number.

Animals↗

PubMed2006

A columnar phase of dendritic lipid-based cationic liposome-DNA complexes for gene delivery: hexagonally ordered cylindrical micelles embedded in a DNA honeycomb lattice.

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.

Animals↗

PubMed2005

Hierarchical self-assembly of actin bundle networks: gels with surface protein skin layers.

The networklike structure of actin bundles formed with the cross-linking protein alpha-actinin has been investigated via x-ray scattering and confocal fluorescence microscopy over a wide range of alpha-actinin/F-actin ratios. We describe the hierarchical structure of bundle gels formed at high ratios. Isotropic actin bundle gels form via cluster-cluster aggregation in the diffusion-limited aggregation regime at high alpha-actinin/actin ratios. This process is clearly observed by confocal fluorescence microscopy. Polylysine is investigated as an alternative bundling agent in the high-ratio regime and the effects of F-actin length are also discussed. One particularly fascinating aspect of this system is the presence of a structured skin layer at the gel/water interface. Confocal microscopy has elucidated the full three-dimensional structure of this layer and revealed several interesting morphologies. The protein skin layer is a micron-scale structure composed of a directed network of bundles and exhibits flat, crumpled, and tubelike shapes. We show that crumpling of the skin layer results from stresses due to the underlying gel. These biologically based geometric structures may detach from the gel, demonstrating potential for the generation of biological scaffolds with defined shapes for applications in cell encapsulation and tissue engineering. We demonstrate manipulation of the skin layer, producing hemispherical structures in solution.

Actin Cytoskeleton↗

PubMed2005

Radial compression of microtubules and the mechanism of action of taxol and associated proteins.

Microtubules (MTs) are hollow cylindrical polymers composed of alphabeta-tubulin heterodimers that align head-to-tail in the MT wall, forming linear protofilaments that interact laterally. We introduce a probe of the interprotofilament interactions within MTs and show that this technique gives insight into the mechanisms by which MT-associated proteins (MAPs) and taxol stabilize MTs. In addition, we present further measurements of the mechanical properties of MT walls, MT-MT interactions, and the entry of polymers into the MT lumen. These results are obtained from a synchrotron small angle x-ray diffraction (SAXRD) study of MTs under osmotic stress. Above a critical osmotic pressure, P(cr), we observe rectangular bundles of MTs whose cross sections have buckled to a noncircular shape; further increases in pressure continue to distort MTs elastically. The P(cr) of approximately 600 Pa provides, for the first time, a measure of the bending modulus of the interprotofilament bond within an MT. The presence of neuronal MAPs greatly increases P(cr), whereas surprisingly, the cancer chemotherapeutic drug taxol, which suppresses MT dynamics and inhibits MT depolymerization, does not affect the interprotofilament interactions. This SAXRD-osmotic stress technique, which has enabled measurements of the mechanical properties of MTs, should find broad application for studying interactions between MTs and of MTs with MAPs and MT-associated drugs.

Animals↗

PubMed2005

Cationic liposome-microtubule complexes: pathways to the formation of two-state lipid-protein nanotubes with open or closed ends.

Intermolecular interactions between charged membranes and biological polyelectrolytes, tuned by physical parameters, which include the membrane charge density and bending rigidity, the membrane spontaneous curvature, the biopolymer curvature, and the overall charge of the complex, lead to distinct structures and morphologies. The self-assembly of cationic liposome-microtubule (MT) complexes was studied, using synchrotron x-ray scattering and electron microscopy. Vesicles were found to either adsorb onto MTs, forming a "beads on a rod" structure, or undergo a wetting transition and coating the MT. Tubulin oligomers then coat the external lipid layer, forming a tunable lipid-protein nanotube. The beads on a rod structure is a kinetically trapped state. The energy barrier between the states depends on the membrane bending rigidity and charge density. By controlling the cationic lipid/tubulin stoichiometry it is possible to switch between two states of nanotubes with either open ends or closed ends with lipid caps, a process that forms the basis for controlled chemical and drug encapsulation and release.

Cytoplasmic Vesicles↗

PubMed2005

New multivalent cationic lipids reveal bell curve for transfection efficiency versus membrane charge density: lipid-DNA complexes for gene delivery.

BACKGROUND: Gene carriers based on lipids or polymers-rather than on engineered viruses-constitute the latest technique for delivering genes into cells for gene therapy. Cationic liposome-DNA (CL-DNA) complexes have emerged as leading nonviral vectors in worldwide gene therapy clinical trials. To arrive at therapeutic dosages, however, their efficiency requires substantial further improvement. METHODS: Newly synthesized multivalent lipids (MVLs) enable control of headgroup charge and size. Complexes comprised of MVLs and DNA have been characterized by X-ray diffraction and ethidium bromide displacement assays. Their transfection efficiency (TE) in L-cells was measured with a luciferase assay. RESULTS: Plots of TE versus the membrane charge density (sigmaM, average charge/unit area of membrane) for the MVLs and monovalent 2,3-dioleyloxypropyltrimethylammonium chloride (DOTAP) merge onto a universal, bell-shaped curve. This bell curve leads to the identification of three distinct regimes, related to interactions between complexes and cells: at low sigmaM, TE increases with increasing sigmaM; at intermediate sigmaM, TE exhibits saturated behavior; and unexpectedly, at high sigmaM, TE decreases with increasing sigmaM. CONCLUSIONS: 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.

Animals↗

PubMed2005

Lipoplex Structures and Their Distinct Cellular Pathways.

Cationic liposomes (CLs) are used as non-viral vectors in worldwide clinical trials of gene therapy. Among other advantages, CL-DNA complexes have the ability to transfer very large genes into cells. However, since the understanding of their mechanisms of action is still incomplete, their transfection efficiencies remain low compared to those of viruses. We describe recent studies which have started to unravel the relationship between the distinct structures and physicochemical properties of CL-DNA complexes and their transfection efficiency by combining several 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. Most CL-DNA complexes form a multilayered structure with DNA sandwiched between the cationic lipids (lamellar complexes, L(alpha)(C)). Much more rarely, an inverted hexagonal structure (H(II)(C)) with single DNA strands encapsulated in lipid tubules is observed. An important recent insight is that the membrane charge density sigma(M) of the CL-vector, rather than, for example, the charge of the cationic lipid, is a universal parameter governing the transfection efficiency of L(alpha)(C) complexes. This has led to a new model of the intracellular release of L(alpha)(C) complexes, through activated fusion with endosomal membranes. In contrast to L(alpha)(C) complexes, H(II)(C) complexes exhibit no dependence on sigma(M), since their structure leads to a distinctly different mechanism of cell entry. Surface-functionalized complexes with poly(ethyleneglycol)-lipids (PEG-lipids), potentially suitable for transfection in vivo, have also been investigated, and the novel aspects of these complexes are discussed.

Journal Article↗

PubMed2005

Cationic lipid-DNA complexes for non-viral gene therapy: relating supramolecular structures to cellular pathways.

Cationic liposomes (CLs) are used as nonviral vectors in worldwide human clinical trials of gene therapy. Among other advantages, lipid-DNA complexes have the ability to transfer very large genes into cells, but their efficiency is much lower than that of viruses. Recent studies combining structural and biological techniques are beginning to unravel the relationship between the distinctly structured CL-DNA complexes and their transfection efficiency. Most CL-DNA complexes form a multilayered structure with DNA sandwiched between the cationic lipids (lamellar complexes, LalphaC). On rare occasions, an inverted hexagonal structure (HIIC) is observed. An important recent insight is that the membrane charge density (sigmaM) of the CL-vector is a universal parameter governing the transfection efficiency of LalphaC (but not HIIC) complexes. This has led to a new model of the cellular uptake of LalphaC complexes through activated fusion with endosomal membranes. Surface-functionalised complexes with poly(ethylene glycol)-lipids, potentially suitable for transfection invivo, have also been investigated, and the novel aspects of these complexes are discussed.

Cations↗

PubMed2005

Lipoplex structures and their distinct cellular pathways.

Cationic liposomes (CLs) are used as non-viral vectors in worldwide clinical trials of gene therapy. Among other advantages, CL-DNA complexes have the ability to transfer very large genes into cells. However, since the understanding of their mechanisms of action is still incomplete, their transfection efficiencies remain low compared to those of viruses. We describe recent studies which have started to unravel the relationship between the distinct structures and physicochemical properties of CL-DNA complexes and their transfection efficiency by combining several 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. Most CL-DNA complexes form a multilayered structure with DNA sandwiched between the cationic lipids (lamellar complexes, LalphaC). Much more rarely, an inverted hexagonal structure (HIIC) with single DNA strands encapsulated in lipid tubules is observed. An important recent insight is that the membrane charge density sigmaM of the CL-vector, rather than, for example, the charge of the cationic lipid, is a universal parameter governing the transfection efficiency of LalphaC complexes. This has led to a new model of the intracellular release of LalphaC complexes, through activated fusion with endosomal membranes. In contrast to LalphaC complexes, HIIC complexes exhibit no dependence on sigmaM, since their structure leads to a distinctly different mechanism of cell entry. Surface-functionalized complexes with poly(ethyleneglycol)-lipids (PEG-lipids), potentially suitable for transfection in vivo, have also been investigated, and the novel aspects of these complexes are discussed.

Biogenic Polyamines↗

PubMed2004

Ordered patterns of liquid crystal toroidal defects by microchannel confinement.

In this article we present experimental results demonstrating an approach to controlling the size and spatial patterning of defect domains in a smectic liquid crystal (LC) by geometric confinement in surface-modified microchannels. By confining the LC 4'-octyl-4-cyanobiphenyl in mum-sized rectangular channels with controlled surface polarity, we were able to generate defect domains that are not only nearly uniform in size but also arranged in quasi-2D ordered patterns. Atomic force microscopy measurements revealed that the defects have a toroidal topology, which we argue is dictated by the boundary conditions imposed by the walls of the microchannel. We show that the defects can be considered to be colloidal objects, which interact with each other to form ordered patterns. This method opens the possibility for exploiting the unique optical and rheological properties associated with LC defects to making new materials. For example, the control of the shape, size, and spatial arrangement of the defects at the mesoscale suggests applications in patterning, templating, and when extended to lyotropic LCs, a process leading to uniform-sized spherical particles for chemical encapsulation and delivery.

Biphenyl Compounds↗

PubMed2004

Higher-order assembly of microtubules by counterions: from hexagonal bundles to living necklaces.

Cellular factors tightly regulate the architecture of bundles of filamentous cytoskeletal proteins, giving rise to assemblies with distinct morphologies and physical properties, and a similar control of the supramolecular organization of nanotubes and nanorods in synthetic materials is highly desirable. However, it is unknown what principles determine how macromolecular interactions lead to assemblies with defined morphologies. In particular, electrostatic interactions between highly charged polyelectrolytes, which are ubiquitous in biological and synthetic self-assembled structures, are poorly understood. We have used a model system consisting of microtubules (MTs) and multivalent cations to examine how microscopic interactions can give rise to distinct bundle phases in biological polyelectrolytes. The structure of these supramolecular assemblies was elucidated on length scales from subnanometer to micrometer with synchrotron x-ray diffraction, transmission electron microscopy, and differential interference contrast microscopy. Tightly packed hexagonal bundles with controllable diameters were observed for large trivalent, tetravalent, and pentavalent counterions. Unexpectedly, in the presence of small divalent cations, we have discovered a living necklace bundle phase, comprised of 2D dynamic assemblies of MTs with linear, branched, and loop topologies. This new bundle phase is an experimental example of nematic membranes. The morphologically distinct MT assemblies give insight into general features of bundle formation and may be used as templates for miniaturized materials with applications in nanotechnology and biotechnology.

Animals↗

PubMed2004

Synchrotron X-ray diffraction study of microtubules buckling and bundling under osmotic stress: a probe of interprotofilament interactions.

Microtubules are hollow cylinders composed of tubulin heterodimers that stack into linear protofilaments that interact laterally to form the microtubule wall. Synchrotron x-ray diffraction of microtubules under increasing osmotic stress shows they transition to rectangular bundles with noncircular buckled cross sections, followed by hexagonally packed bundles. This new technique probes the strength of interprotofilamen bonds, yielding insight into the mechanism by which associated proteins and the chemotherapy drug taxol stabilize microtubules.

Guanosine Triphosphate↗

PubMed2004

Surface functionalized cationic lipid-DNA complexes for gene delivery: PEGylated lamellar complexes exhibit distinct DNA-DNA interaction regimes.

Cationic lipid-DNA (CL-DNA) complexes are abundantly used in nonviral gene therapy clinical applications. Surface functionality is the next step in developing these complexes as competent, target-specific gene carriers. Poly(ethylene glycol) (PEG) is the natural choice to serve as a protective coat or act as a tether for a specific ligand on the surface of these complexes due to its biocompatibility and ability to convey stealth-like properties. Understanding the effect of PEG on the internal structure and surface properties of CL-DNA complexes is essential in developing vectors with more complex derivatives of PEG, such as Arg-Gly-Asp (RGD)-based peptide-PEG-lipids. We report on x-ray diffraction studies to probe the internal structure of CL-DNA complexes consisting of a ternary mixture of cationic lipids, neutral lipids, and PEG-lipids. The PEG-coated complexes are found to exhibit a structure consistent with the lamellar phase. In addition, three distinct DNA interchain interaction regimes were found to exist, due to a), repulsive long-range electrostatic forces; b), short-range repulsive hydration forces; and c), novel polymer-induced depletion attraction forces in two dimensions. Optical microscopy and reporter gene assays further demonstrate the incorporation of the PEG-lipids into the lamellar CL-DNA complexes under biologically relevant conditions, revealing surface modification. Both techniques show that PEG-lipids with a polymer chain of molecular weight 400 do not provide adequate shielding of the PEGylated CL-DNA complexes, whereas PEG-lipids with a polymer chain of molecular weight 2000 confer stealth-like properties. This surface functionalization is a crucial initial step in the development of competent vectors for in vivo systemic gene delivery and suggests that a second type of surface functionality can be added specifically for targeting by the incorporation of peptide-PEG-lipids.

Animals↗

PubMed2004

Cationic lipid-DNA complexes for gene therapy: understanding the relationship between complex structure and gene delivery pathways at the molecular level.

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(alpha)(C)). On rare occasions, an inverted hexagonal structure with DNA encapsulated in lipid tubules (labeled H(II)(C)) is observed. A major recent insight is that for L(alpha)(C) complexes the membrane charge density sigma(M) 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(alpha)(C) complexes in cells. The parameter sigma(M) 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 L(alpha)(C) complexes, H(II)(C) complexes containing the lipid 1,2-dioleoyl-sn-glycerophosphatidylethanolamine (DOPE) exhibit no dependence on sigma(M). The current 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.

Animals↗

PubMed2003

Lamellar phase of stacked two-dimensional rafts of actin filaments.

We examined liquid crystalline phases of the cytoskeletal polyelectrolyte filamentous (F-)actin in the presence of multivalent counterions. As a function of increasing ion concentration, the F-actin rods in either an isotropic or a nematic phase will transform into a new and unexpected lamellar phase of cross-linked rafts (L(XR) phase), before condensing into a bundled phase of parallel, close-packed rods. This behavior is generic for alkali earth divalent ions Mg2+, Ca2+, Sr2+, and Ba2+, and the structural transitions are achieved without any architecture-specific actin-binding linker proteins.

Actins↗

PubMed2003

Three-dimensional imaging of lipid gene-carriers: membrane charge density controls universal transfection behavior in lamellar cationic liposome-DNA complexes.

Cationic liposomes (CLs) are used worldwide as gene vectors (carriers) in nonviral clinical applications of gene delivery, albeit with unacceptably low transfection efficiencies (TE). We present three-dimensional laser scanning confocal microscopy studies revealing distinct interactions between CL-DNA complexes, for both lamellar L(alpha)(C) and inverted hexagonal H(II)(C) nanostructures, and mouse fibroblast cells. Confocal images of L(alpha)(C) complexes in cells identified two regimes. For low membrane charge density (sigma(M)), DNA remained trapped in CL-vectors. By contrast, for high sigma(M), released DNA was observed in the cytoplasm, indicative of escape from endosomes through fusion. Remarkably, firefly luciferase reporter gene studies in the highly complex L(alpha)(C)-mammalian cell system revealed an unexpected simplicity where, at a constant cationic to anionic charge ratio, TE data for univalent and multivalent cationic lipids merged into a single curve as a function of sigma(M), identifying it as a key universal parameter. The universal curve for transfection by L(alpha)(C) complexes climbs exponentially over approximately four decades with increasing sigma(M) below an optimal charge density (sigma(M)(*)), and saturates for at a value rivaling the high transfection efficiency of H(II)(C) complexes. In contrast, the transfection efficiency of H(II)(C) complexes is independent of sigma(M). The exponential dependence of TE on sigma(M) for L(alpha)(C) complexes, suggests the existence of a kinetic barrier against endosomal fusion, where an increase in sigma(M) lowers the barrier. In the saturated TE regime, for both L(alpha)(C) complexes and H(II)(C), confocal microscopy reveals the dissociation of lipid and DNA. However, the lipid-released DNA is observed to be in a condensed state, most likely with oppositely charged macro-ion condensing agents from the cytoplasm, which remain to be identified. Much of the observed bulk of condensed DNA may be transcriptionally inactive and may determine the current limiting factor to transfection by cationic lipid gene vectors.

Animals↗

PubMed2002

Efficient synthesis and cell-transfection properties of a new multivalent cationic lipid for nonviral gene delivery.

Lipid-mediated delivery of DNA into cells holds great promise both for gene therapy and basic research applications. This paper describes the efficient and facile synthesis and the characterization of a new multivalent cationic lipid with a double-branched headgroup structure for gene delivery applications. The synthetic scheme can be extended to give cationic lipids of different charge, spacer, or lipid chain length. The chemical and physical properties of self-assembled complexes of the cationic liposomes (CLs) with DNA give indications of why multivalent cationic lipids possess superior transfection properties. The lipid bears a headgroup with five charges in the fully protonated state, which is attached to an unsaturated double-chain hydrophobic moiety based on 3,4-dihydroxybenzoic acid. Liposomes consisting of the new multivalent lipid and the neutral lipid 1,2-dioleoyl-sn-glycerophosphatidylcholine (DOPC) were used to prepare complexes with DNA. Investigations of the structures of these complexes by optical microscopy and small-angle X-ray scattering reveal a lamellar L(alpha)(C) phase of CL-DNA complexes with the DNA molecules sandwiched between bilayers of the lipids. Experiments using plasmid DNA containing the firefly luciferase reporter gene show that these complexes efficiently transfect mammalian cells. When compared to the monovalent cationic lipid 2,3-dioleyloxypropyltrimethylammonium chloride (DOTAP), the higher charge density of the membranes of CL-DNA complexes achievable with the new multivalent lipid greatly increases transfection efficiency in the regime of small molar ratios of cationic to neutral lipid. This is desired to minimize the known toxicity effects of cationic lipids.

Animals↗