Elastic deformation of a fluid membrane upon colloid binding

When a colloidal particle adheres to a fluid membrane, it induces elastic deformations in the membrane which oppose its own binding. The structural and energetic aspects of this balance are theoretically studied within the framework of a Helfrich Hamiltonian. Based on the full nonlinear shape equations for the membrane profile, a line of continuous binding transitions and a second line of discontinuous envelopment transitions are found, which meet at an unusual triple point. The regime of low tension is studied analytically using a small gradient expansion, while in the limit of large tension scaling arguments are derived which quantify the asymptotic behavior of phase boundary, degree of wrapping, and energy barrier. The maturation of animal viruses by budding is discussed as a biological example of such colloid-membrane interaction events.Comment: 14 pages, 9 figures, REVTeX style, follow-up on cond-mat/021242

Small poly-L-lysines improve cationic lipid-mediated gene transfer in vascular cells in vitro and in vivo

The potential of two small poly-L-lysines ( sPLLs), low molecular weight sPLL ( LMW-L) containing 7 - 30 lysine residues and L18 with 18 lysine repeats, to enhance the efficiency of liposome-mediated gene transfer ( GT) with cationic lipid DOCSPER {[}1,3- dioleoyloxy- 2-( N-5-carbamoyl-spermine)-propane] in vascular smooth muscle cells ( SMCs) was investigated. Dynamic light scattering was used for determination of particle size. Confocal microscopy was applied for colocalization studies of sPLLs and plasmid DNA inside cells. GT was performed in proliferating and quiescent primary porcine SMCs in vitro and in vivo in porcine femoral arteries. At low ionic strength, sPLLs formed small complexes with DNA ( 50 100 nm). At high ionic strength, large complexes ( 11 mu m) were observed without any significant differences in particle size between lipoplexes ( DOCSPER/ DNA) and lipopolyplexes ( DOCSPER/ sPLL/ DNA). Both sPLLs were colocalized with DNA inside cells 24 h after transfection, protecting DNA against degradation. DOCSPER/ sPLL/ DNA formulations enhanced GT in vitro up to 5- fold, in a porcine model using local periadventitial application up to 1.5- fold. Both sPLLs significantly increased liposome- mediated GT. Poly-L-lysine L18 was superior to LMW-L since it enabled maximal GT at a 10-fold lower concentration. Thus, sPLLs may serve as enhancers for GT applications in SMCs in vitro and in vivo using local delivery. Copyright (c) 2007 S. Karger AG, Basel

Epidermal Growth Factor–PEG Functionalized PAMAM-Pentaethylenehexamine Dendron for Targeted Gene Delivery Produced by Click Chemistry

Aim of this study was the site-specific conjugation of an epidermal growth factor (EGF)-polyethylene glycol (PEG) chain by click chemistry onto a poly(amido amine) (PAMAM) dendron, as a key step toward defined multifunctional carriers for targeted gene delivery. For this purpose, at first propargyl amine cored PAMAM dendrons with ester ends were synthesized. The chain terminal ester groups were then modified by oligoamines with different secondary amino densities. The oligoamine-modified PAMAM dendrons were well biocompatible, as demonstrated in cytotoxicity assays. Among the different oligoamine-modified dendrons, PAMAM-pentaethylenehexamine (PEHA) dendron polyplexes displayed the best gene transfer ability. Conjugation of PAMAM-PEHA dendron with PEG spacer was conducted via click reaction, which was performed before amidation with PEHA. The resultant PEG-PAMAM-PEHA copolymer was then coupled with EGF ligand. pDNA transfections in HuH-7 hepatocellular carcinoma cells showed a 10-fold higher efficiency with the polyplexes containing conjugated EGF as compared to the ligand-free ones, demonstrating the concept of ligand targeting. Overall gene transfer efficiencies, however, were moderate, suggesting that additional measures for overcoming subsequent intracellular bottlenecks in delivery have to be taken

A strategy to discover new organizers identifies a putative heart organizer

Organizers are regions of the embryo that can both induce new fates and impart pattern on other regions. So far, surprisingly few organizers have been discovered, considering the number of patterned tissue types generated during development. This may be because their discovery has relied on transplantation and ablation experiments. Here we describe a new approach, using chick embryos, to discover organizers based on a common gene expression signature, and use it to uncover the anterior intestinal portal (AIP) endoderm as a putative heart organizer. We show that the AIP can induce cardiac identity from non-cardiac mesoderm and that it can pattern this by specifying ventricular and suppressing atrial regional identity. We also uncover some of the signals responsible. The method holds promise as a tool to discover other novel organizers acting during development

In vitro and in vivo mRNA delivery using lipid-enveloped pHresponsive polymer nanoparticles

Biodegradable core−shell structured nanoparticles with a poly(β-amino ester) (PBAE) core enveloped by a phospholipid bilayer shell were developed for in vivo mRNA delivery with a view toward delivery of mRNA-based vaccines. The pH-responsive PBAE component was chosen to promote endosome disruption, while the lipid surface layer was selected to minimize toxicity of the polycation core. Messenger RNA was efficiently adsorbed via electrostatic interactions onto the surface of these net positively charged nanoparticles. In vitro, mRNA-loaded particle uptake by dendritic cells led to mRNA delivery into the cytosol with low cytotoxicity, followed by translation of the encoded protein in these difficult-to-transfect cells at a frequency of 30%. Particles loaded with mRNA administered intranasally (i.n.) in mice led to the expression of the reporter protein luciferase in vivo as soon as 6 h after administration, a time point when naked mRNA given i.n. showed no expression. At later time points, luciferase expression was detected in naked mRNA-treated mice, but this group showed a wide variation in levels of transfection, compared to particle-treated mice. This system may thus be promising for noninvasive delivery of mRNA-based vaccines.United States. Dept. of Defense (Institute for Soldier Nanotechnology, contract W911NF-07-D-0004)Ragon Institute of MGH, MIT and HarvardSingapore. Agency for Science, Technology and ResearchHoward Hughes Medical Institute (Investigator

Phosphorothioate antisense oligonucleotides induce the formation of nuclear bodies

Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20-30 bright spherical foci designated phosphorothioate bodies (PS bodies), which were set against a diffuse nucleoplasmic population excluding nucleoli. PS bodies are nuclear structures that formed in cells after PS-ON delivery by transfection agents or microinjection but were observed irrespectively of antisense activity or sequence. Ultrastructurally, PS bodies corresponded to electron-dense structures of 150-300 nm diameter and resembled nuclear bodies that were found with lower frequency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entities that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-ON pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our data provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides

Layer-by-Layer Assembled Antisense DNA Microsponge Particles for Efficient Delivery of Cancer Therapeutics

Antisense oligonucleotides can be employed as a potential approach to effectively treat cancer. However, the inherent instability and inefficient systemic delivery methods for antisense therapeutics remain major challenges to their clinical application. Here, we present a polymerized oligonucleotides (ODNs) that self-assemble during their formation through an enzymatic elongation method (rolling circle replication) to generate a composite nucleic acid/magnesium pyrophosphate sponge-like microstructure, or DNA microsponge, yielding high molecular weight nucleic acid product. In addition, this densely packed ODN microsponge structure can be further condensed to generate polyelectrolyte complexes with a favorable size for cellular uptake by displacing magnesium pyrophosphate crystals from the microsponge structure. Additional layers are applied to generate a blood-stable and multifunctional nanoparticle via the layer-by-layer (LbL) assembly technique. By taking advantage of DNA nanotechnology and LbL assembly, functionalized DNA nanostructures were utilized to provide extremely high numbers of repeated ODN copies for efficient antisense therapy. Moreover, we show that this formulation significantly improves nucleic acid drug/carrier stability during in vivo biodistribution. These polymeric ODN systems can be designed to serve as a potent means of delivering stable and large quantities of ODN therapeutics systemically for cancer treatment to tumor cells at significantly lower toxicity than traditional synthetic vectors, thus enabling a therapeutic window suitable for clinical translation.United States. Dept. of Defense. Ovarian Cancer Research Program (Teal Innovator Award Grant OC120504)Natural Sciences and Engineering Research Council of Canada (Postdoctoral Fellowship)National Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award 1F32EB017614-01)National Science Foundation (U.S.). Graduate Research Fellowshi

Collision of millimetre droplets induces DNA and protein transfection into cells

Nonperturbing and simple transfection methods are important for modern techniques used in biotechnology. Recently, we reported that electrospraying can be applied to DNA transfection in cell lines, bacteria, and chicken embryos. However, the transfection efficiency was only about 2%. To improve the transfection rate, physical properties of the sprayed droplets were studied in different variations of the method. We describe a highly efficient technique (30–93%) for introduction of materials such as DNA and protein into living cells by electrospraying droplets of a high conductivity liquid onto cells incubated with the material for transfection. Electric conductivity has a sizable influence on the success of transfection. In contrast, molecular weight of the transfected material, types of ions in the electrospray solution, and the osmotic pressure do not influence transfection efficiency. The physical analysis revealed that collision of cells with millimetre-sized droplets activates intracellular uptake

Characterization of Ku702–NLS as Bipartite Nuclear Localization Sequence for Non-Viral Gene Delivery

Several barriers have to be overcome in order to achieve gene expression in target cells, e.g. cellular uptake, endosomal release and translocation to the nucleus. Nuclear localization sequences (NLS) enhance gene delivery by increasing the uptake of plasmid DNA (pDNA) to the nucleus. So far, only monopartite NLS were analysed for non-viral gene delivery. In this study, we examined the characteristics of a novel bipartite NLS like construct, namely NLS Ku70. We synthesized a dimeric structure of a modified NLS from the Ku70 protein (Ku702-NLS), a nuclear transport active mutant of Ku702-NLS (s1Ku702-NLS) and a nuclear transport deficient mutant of Ku702-NLS (s2Ku702). We examined the transfection efficiency of binary Ku702-NLS/DNA and ternary Ku702-NLS/PEI/DNA gene vector complexes in vitro by using standard transfection protocols as well as the magnetofection method. The application of Ku702-NLS and s1Ku702-NLS increased gene transfer efficiency in vitro and in vivo. This study shows for the first time that the use of bipartite NLS compounds alone or in combination with cationic polymers is a promising strategy to enhance the efficiency of non-viral gene transfer

Comparison of distribution and activity of nanoparticles with short interfering DNA (Dbait) in various living systems

Introducing small DNA molecules (Dbait) impairs the repair of damaged chromosomes and provides a new method for enhancing the efficiency of radiotherapy in radio-resistant tumors. The radiosensitizing activity is dependent upon the efficient delivery of Dbait molecules into the tumor cells. Different strategies have been compared, to improve this key step. We developed a pipeline of assays to select the most efficient nanoparticles and administration protocols before preclinical assays: (i) molecular analyses of complexes formed with Dbait molecules, (ii) cellular tests for Dbait uptake and activity, (iii) live zebrafish embryo confocal microscopy monitoring for in vivo distribution and biological activity of the nanoparticles and (iv) tumor growth and survival measurement on mice with xenografted tumors. Two classes of nanoparticles were compared, polycationic polymers with linear or branched polyethylenimine (PEI) and covalently attached cholesterol (coDbait). The most efficient Dbait transfection was observed with linear PEI complexes, in vitro and in vivo. Doses of coDbait ten-fold higher than PEI/Dbait nanoparticles, and pretreatment with chloroquine, were required to obtain the same antitumoral effect on xenografted melanoma. However, with a 22-fold lower ‘efficacy dose/toxicity dose' ratio as compared with Dbait/PEI, coDbait was selected for clinical trials