Biomedical subjects

Ana Martin-Herranz

Publications and source records attributed to Ana Martin-Herranz.

3 recordsLinked to original sources

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

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

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↗