Biomedical subjects

Ayesha Ahmad

Publications and source records attributed to Ayesha Ahmad.

14 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

MAGfect: a novel liposome formulation for MRI labelling and visualization of cells.

Cellular entry of imaging probes, such as contrast agents for magnetic resonance imaging (MRI), is a key requirement for many molecular imaging studies, particularly imaging intracellular events and cell tracking. Here, we describe the successful development and in vitro analysis of MAGfect, a novel liposome formulation containing a lipidic gadolinium contrast agent for MRI, Gd-DOTA-Chol , designed to enter and label cells. Liposome formulation and cell incubation time were optimised for maximum cellular uptake of the imaging probe in a variety of cell lines. MRI analysis of cells incubated with MAGfect showed them to be highly MRI active. This formulation was examined further for cytotoxicity, cell viability and mechanism of cell labelling. One of the key advantages of using MAGfect as a labelling vehicle arises from its potential for additional functions, such as concomitant drug or gene delivery and fluorescent labelling. The gadolinium liposome was found to be an effective vehicle for transport of plasmid DNA (pDNA) into cells and expression levels were comparable to the commercial transfection agent Trojene.

Cells↗

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↗

PubMed2006

In vivo studies of dialkynoyl analogues of DOTAP demonstrate improved gene transfer efficiency of cationic liposomes in mouse lung.

A novel set of dialkynoyl analogues of the cationic, gene delivery lipid DOTAP (1) was synthesized. Structure-activity studies demonstrate that replacement of the cis-double bonds of DOTAP with triple bonds in varying positions alters both the physical properties of the resultant cationic liposome-DNA complexes and their biological functionalities, both in vitro and in vivo. Particularly, in vivo studies demonstrate that pDNA transfection of mouse lung endothelial cells with lead analogue DS(14-yne)TAP (4):cholesterol lipoplexes exhibits double the transfection level with less associated toxicity relative to the well-established DOTAP:cholesterol system. In fact, 4:cholesterol delivers up to 3 times the dose of pDNA in mice than can be tolerated by DOTAP, leading to nearly 3 times greater marker-gene expression. X-ray diffraction studies suggest that lipoplexes containing analogue 4 display increased stability at physiological temperatures. Our results thus suggest that analogue 4 is a potentially strong candidate for the gene therapy of lung tumors.

Animals↗

PubMed2005

A dialkynoyl analogue of DOPE improves gene transfer of lower-charged, cationic lipoplexes.

Positively-charged gene delivery agents, such as cationic liposomes, typically prepared by mixing a cationic lipid and a neutral lipid in a 1 : 1 molar ratio, exhibit a fundamental flaw: on the one hand, the charge encourages cell uptake; on the other hand, the charge leads to aggregation in vivo with anionic serum components. We herein report a more phase-stable analogue of the zwitterionic and fusogenic lipid DOPE that allows for the reduction of the cationic lipid component of the liposome from 50 to 9 mol% with almost no apparent loss in transfection activity. This reduction in charge may induce important in vivo stability whilst still imparting high cell uptake and transgene expression.

Cations↗

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

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

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↗

PubMedDate not supplied

Computerized stereotactic brain biopsies: an experience of 15 patients at Ayub Teaching Hospital.

BACKGROUND: Deep seated lesions of the brain cannot be approached by conventional neurosurgical approach stereotactic system offers minimally invasive and accurate approach to such lesions. This study was carried out with an objective to determine the safety, efficacy and diagnostic yield of stereotactic biopsies of brain lesions using Brown-Roberts-Wells (BRW) system. METHODS: This study was carried out in patients with intracranial lesions at Ayub teaching hospital Abbottabad from September 1999 to October 2003. Suitable patients with intra cranial lesions underwent computerized stereo tactic biopsy with BRW system. Tissue specimens were analyzed in histopathology department of the Ayub Medical College and results were obtained. Data was analyzed via computer software SPSS 8.0 version for windows. RESULTS: Fifteen patients were selected for stereotactic brain biopsy. Age ranged from 15 years to 54 years. Among them 09 (60%) were male and 06 (40%) were female patients. Out come of the procedure was highly promising in terms of safety and positive diagnostic yield in 14 patients (93.3%), and histopathalogical validity of results (93.3%). Only one patient suffered mild neurological deficit (6.7%), one patient had inconclusive tissue diagnosis and invalid result (6.7%). biopsy proven lesion was astrocytoma in 04 patients (26.7%), anaplastic astrocytoma in 04 (26.7%), gliomatosis in 02 (13.3%), tuberculomas in 03 (20.0%). metastatic adenocarcinomas in 01 (6.7%) and lymphoma in 01(6.7%). CONCLUSIONS: We conclude that computerized stereotactic brain biopsy is safe and effective procedure with a high diagnostic yield at our center.

Adolescent↗