Enzyme-triggered PEGylated pDNA-nanoparticles for controlled release of pDNA in tumors.

Nanoparticle mediated functional delivery of plasmid DNA (pDNA) in vivo typically requires the formulation of pDNA-nanoparticles with a surface layer of stealth/biocompatibility polymer (usually poly(ethylene glycol) [PEG]). This PEG layer ensures the colloidal stability of pDNA-nanoparticles in biological fluids and minimizes nanoparticle interactions with the reticulo-endothelical system. Unfortunately, the presence of the PEG layer appears to contribute to a reduction in efficiency of functional delivery of pDNA once target cells are reached. For this reason, we have focused recent research efforts on "triggerable" nanoparticle systems. These are designed to be stable from the point of administration until a target site of interest is reached, then triggered for the controlled release of therapeutic agent payload(s) at the target site by changes in local endogenous conditions or through the application of some exogenous stimulus. Here, we describe investigations into the potential use of enzymes to trigger pDNA-mediated therapy through a process of enzyme-assisted nanoparticle triggerability. Our approach is to use PEG(2000)-peptidyl lipids with peptidyl moieties sensitive to tumor-localized elastase or matrix metalloproteinase-2 digestion, and from these prepare putative enzyme-triggered PEGylated pDNA-nanoparticles. Our results provide initial proof of concept in vitro. From these data, we propose that this concept should be applicable for functional delivery of therapeutic nucleic acids to tumor cells in vivo, although the mechanism for enzyme-assisted nanoparticle triggerability remains to be fully characterized.

Treatment of the metabolic syndrome by siRNA targeting apolipoprotein CIII.

Apolipoprotein CIII (apoCIII) is increased in obesity-induced insulin resistance and type-2 diabetes. Emerging evidences support the advantages of small interfering RNAs (siRNAs) to target disease-causing genes. The aim of this study was to develop siRNAs for in vivo silencing of apoCIII and investigate if this results in metabolic improvements comparable to what we have seen using antisense oligonucelotides against apoCIII. Twenty-four siRNAs were synthesized and tested in a dual luciferase reporter assay. The eight best were selected, based on knockdown at 20 nM, and of these, two were selected based on IC50 values. In vivo experiments were performed in ob/ob mice, an obese animal model for diabetes. To determine the dose-dependency, efficacy, duration of effect and therapeutic dose we used a short protocol giving the apoCIII-siRNA mix for three days. To evaluate long-term metabolic effects mice were treated for three days, every second week for eight weeks. The siRNA mix effectively and selectively reduced expression of apoCIII in liver in vivo. Treatment had to be repeated every two weeks to maintain a suppression of apoCIII. The reduction of apoCIII resulted in increased LPL activity, lower triglycerides, reduced liver fat, ceased weight gain, enhanced insulin sensitivity, and improved glucose homeostasis. No off-target or side effects were observed during the eight-week treatment period. These results suggest that in vivo silencing of apoCIII with siRNA, is a promising approach with the potential to be used in the battle against obesity-induced metabolic disorders.

Synthesis, characterization, and in vitro transfection activity of charge-reversal amphiphiles for DNA delivery.

A series of charge-reversal lipids were synthesized that possess varying chain lengths and end functionalities. These lipids were designed to bind and then release DNA based on a change in electrostatic interaction with DNA. Specifically, a cleavable ester linkage is located at the ends of the hydrocarbon chains. The DNA release from the amphiphile was tuned by altering the length and position of the ester linkage in the hydrophobic chains of the lipids through the preparation of five new amphiphiles. The amphiphiles and corresponding lipoplexes were characterized by DSC, TEM, and X-ray, as well as evaluated for DNA binding and DNA transfection. For one specific charge-reversal lipid, stable lipoplexes of approximately 550 nm were formed, and with this amphiphile, effective in vitro DNA transfection activities was observed.

The effect of charge-reversal amphiphile spacer composition on DNA and siRNA delivery.

A series of charge-reversal amphiphiles with different spacers separating the headgroup from the hydrophobic chains are described for delivery of DNA and siRNA. Among them, the amphiphiles possessing a glycine spacer (e.g., B-GlyGly) showed effective DNA transfection in CHO and NIH 3T3 cells, as well as siRNA gene knockdown in HepG2 and UASMC cells. Ethidium bromide quenching assays revealed that DNA was released the fastest from the lipoplex of B-GlyGly in the presence of esterase. Also, X-ray diffraction results indicated that the DNA was located between the adjacent lipid bilayers in the lipoplex of B-GlyGly. These distinct features appear to be required for high transfection activity.

Cationic nucleoside lipids derived from universal bases: A rational approach for siRNA transfection.

Cationic nucleoside lipids (CNLs) derived from 5-nitroindole and 4-nitroimidazole bases were prepared from d-ribose by using a straightforward chemical synthesis. TEM experiments indicate that these amphiphilic molecules self-assemble to form supramolecular organizations in aqueous solutions. Electrophoresis and standard ethidium bromide (EB) fluorescence displacement assay shows that CNLs are able to bind siRNA. We demonstrated that both the nature of the universal bases and the stereochemistry of the anomeric position (alpha, beta) have an impact on the CNLs-siRNA complex formation. Correlations among chemical structure, stereochemistry, siRNA knockdown effect, and binding affinities for all the compounds were shown and analyzed with a simple molecular modeling study. The best binding affinities for siRNA were found for the beta anomer of the 5-nitroindole CNL which exhibits protein knockdown activity similar to the standard siPORT NeoFX positive control. It is noteworthy that no significant cytotoxicity at the tested concentration was observed for the novel CNLs.

Cationic nucleoside lipids based on a 3-nitropyrrole universal base for siRNA delivery.

Cationic nucleoside lipids based on a 3-nitropyrrole universal base were prepared from D-ribose using a straightforward chemical synthesis. Several studies including DLS, TEM, and ethidium bromide (EthBr) assay demonstrated that these amphiphilic molecules form supramolecular organizations of nanometer size in aqueous solutions and are able to bind nucleic acids. siRNA knockdown experiments were performed with these nucleolipids, and we observed protein knockdown activity similar to the siPORT NeoFX positive control. No significant cytotoxicity was found.

Ligand Conjugated Multimeric siRNAs Enable Enhanced Uptake and Multiplexed Gene Silencing.

Small interfering RNAs (siRNAs) conjugated to N-acetylgalactosamine (GalNAc) ligands have been used to treat disease in patients. However, conjugates with other ligands deliver siRNA less efficiently, limiting the development of new targeted therapies. Most approaches to enhancing the potency of such conjugates have concentrated on increasing ligand effectiveness and/or the chemical stability of the siRNA drug. One complementary and unexplored alternative is to increase the number of siRNAs delivered per ligand. An ideal system would be a single chemical entity capable of delivering multiple copies of an oligonucleotide drug and/or several such drugs simultaneously. Here we report that siRNAs can be stably linked together under neutral aqueous conditions to form chemically defined siRNA "multimers," and that these multimers can be delivered in vivo by a GalNAc ligand. Conjugates containing multiple copies of the same siRNA showed enhanced activity per unit of ligand, whereas siRNAs targeting different genes linked to a single ligand facilitated multigene silencing in vivo; this is the first demonstration of silencing several genes simultaneously in vivo using ligand-directed multimeric siRNA. Multimeric oligonucleotides represent a powerful and practical new approach to improve intracellular conjugate delivery.

A new helper phospholipid for gene delivery.

Enhanced gene transfection activity is observed when using a new helper lipid with DOTAP, compared to DOPE.

Peptide-PEG amphiphiles as cytophobic coatings for mammalian and bacterial cells.

Amphiphilic macromolecules containing a polystyrene-adherent peptide domain and a cell-repellent poly(ethylene glycol) domain were designed, synthesized, and evaluated as a cytophobic surface coating. Such cytophobic, or cell-repellent, coatings are of interest for varied medical and biotechnological applications. The composition of the polystyrene binding peptide domain was identified using an M13 phage display library. ELISA and atomic force spectroscopy were used to evaluate the binding affinity of the amphiphile peptide domain to polystyrene. When coated onto polystyrene, the amphiphile reduced cell adhesion of two distinct mammalian cell lines and pathogenic Staphylococcus aureus strains.

Dendritic supramolecular assemblies for drug delivery.

Dendritic supramolecular assemblies were formed in water with Reichardt's dye or the anticancer drug 10-hydroxycamptothecin and the dendritic macromolecule, ([G4]-PGLSA-OH)2-PEG3400.

Supramolecular assemblies of DNA with neutral nucleoside amphiphiles.

A neutral uridine-based amphiphile is described which condenses plasmid DNA. AFM studies show that the three distinct structural components of the amphiphile (i.e, nucleobase, alkyl chains, and poly(ethylene glycol)) are required for the formation of DNA-amphiphile supramolecular assemblies on a mica surface.

Nucleoside phosphocholine amphiphile for in vitro DNA transfection.

A new transfection reagent based on nucleoside phosphocholine amphiphile leading to high transfection efficacy and low cytotoxicity is described. TEM, ethidium bromide displacement assays, agarose gel electrophoresis and SAXS studies support the formation of lipoplexes for the transfection of CHO cells.

A new dihydroxysterol from the marine phytoplankton Diacronema sp.

Diacronema sp. was cultured and its sterols were separated by column chromatography on silica gel. The new sterol 24-ethyl-4alpha-methyl-cholestane-3,20-diol (1) was characterised by NMR and MS spectrometry, as well as (22E)-24-ethyl-4alpha-methyl-5alpha-cholest-22-en-3beta-ol (2) and beta-sitosterol, the major components of the sterol fractions. Neither the biosynthetic origin of the new dihydroxysterol nor its role in the biochemistry of Diacronema is known.

Enzyme-triggered PEGylated siRNA-nanoparticles for controlled release of siRNA.

A key goal of our recent research efforts has been to develop novel 'triggerable nanoparticle' systems with real potential utility in vivo. These are designed to be stable from the point of administration until a target site of interest is reached, then triggered for the controlled release of therapeutic agent payload(s) at the target site by changes in local endogenous conditions or through the application of some exogenous stimulus. Here we describe investigations into the use of enzymes to trigger RNAi-mediated therapy through a process of enzyme-assisted nanoparticle triggerability. Our approach is to use PEG(2000)-peptidyl lipids with peptidyl moieties sensitive to tumour-localized elastase or matrix metalloproteinase-2 digestion, and from these prepare putative enzyme-triggered PEGylated siRNA-nanoparticles. Our results provide initial proof of concept in vitro. From these data, we propose that this concept should be applicable for functional delivery of therapeutic nucleic acids to tumour cells in vivo, although the mechanism for enzyme-assisted nanoparticle triggerability remains to be fully characterized.

Lipophilic peptides for gene delivery.

DNA transfections are widely performed in research laboratories and in vivo gene delivery holds the promise for curing many diseases. The synthetic carriers or vectors for DNA are typically cationic lipids. However, in biology, the recognition of nucleic acids by proteins involves both electrostatic and stacking contributions. As such we have prepared a series of new lipophilic peptide vectors that possess lysine and tryptophan amino acids for evaluation. These lipophilic peptides show minimal cytotoxicity and enhanced in vitro gene transfection activity.

Non-viral charge reversal vectors for pDNA delivery.

A synthetic vector that transform from a cationic to an anionic lipid intracellularly is described. This charge-reversal lipid was synthesized and characterized, including the supramolecular complex it forms with DNA. Enhanced gene transfection was observed using this synthetic vector compared to current cationic lipids.

Well-defined nanoparticles formed by hydrophobic assembly of a short and polydisperse random terpolymer, amphipol A8-35.

Amphipols are short amphilic polymers designed for applications in membrane biochemistry and biophysics and used, in particular, to stabilize membrane proteins in aqueous solutions. Amphipol A8-35 was obtained by modification of a short-chain parent polymer (poly(acrylic acid); PAA) with octyl- and isopropylamine, to yield an amphiphilic product with an average molar mass of 9-10 kg x mol(-1) (sodium salt form) and a polydispersity index of 2.0 to 3.1, depending on the source of PAA. The behavior of A8-35 in aqueous buffers was studied by size exclusion chromatography, static and dynamic light scattering, equilibrium and sedimentation velocity analytical ultracentrifugation, and small angle neutron scattering. Despite the variable length of the chains and the random distribution of hydrophobic groups along them, A8-35 self-organizes into well-defined assemblies. The data are best compatible with most of the polymer forming compact assemblies (particles) with a molar mass of approximately 40 kg x mol(-1), a radius of gyration of approximately 2.4 nm, and a Stokes radius of approximately 3.15 nm. Each particle contains, on average, four A8-35 macromolecules and 75-80 octyl chains. Neutron scattering reveals a sharp interface between the particles and water. A minor (approximately 0.1%) mass fraction of the material forms much larger aggregates, whose proportion may increase under certain conditions of preparation or handling, such as low pH. They can be removed by gel filtration.

Nucleic acid complexing glycosyl nucleoside-based amphiphile.

A neutral amphiphile derived from uridine featuring two oleyl chains and one glucose for DNA binding was prepared using a convenient four-step synthetic route. The nucleic acid binding capabilities of this amphiphile were investigated by UV-vis, quasi-elastic light scattering (QELS), transmission electronic microscopy (TEM), gel electrophoresis, 31P NMR, IR, and circular dichroism (CD). Amphiphile-nucleic acid complex formation is a consequence of the amphiphilic character of the molecule, phosphate-sugar, and nucleobase-nucleobase interactions. This work presents for the first time a glyco-nucleo-amphiphile capable of binding efficiently the nucleic acid double helix structure.

Charge-reversal amphiphiles for gene delivery.

Delivering a missing gene or a functional substitute of a defective gene has the potential to revolutionize current medical care. Of the two gene delivery approaches, viral and synthetic vectors, synthetic cationic vectors possess several practical advantages but suffer from poor transfection efficiency. A new approach to gene delivery using charge-reversal amphiphiles is described. This synthetic vector transforms from a cationic to an anionic amphiphile intracellularly. This amphiphile performs two roles: first, it binds and then releases DNA, and second, as an anionic multicharged amphiphile, it destabilizes lipid bilayers. A charge-reversal amphiphile was synthesized and fully characterized, including the supramolecular complex it forms with DNA. Enhanced gene transfection was observed using these vectors compared to current cationic amphiphiles.

O cuidado para familiares de clientes em período pré-operatório de cirurgia cardíaca: implicações para a enfermagem hospitalar / The care addressed to the clients' familiars in pre-surgery period of cardiac surgery: implications for the hospital nursing

O estudo tem como objeto a interação entre os familiares de clientes em pré-operatório de cirurgia cardíaca e os integrantes da equipe de enfermagem. Os objetivos são caracterizar as necessidades de cuidado de enfermagem no âmbito hospitalar dos familiares de clientes em pré-operatório de cirurgia cardíaca; analisar as implicações dessas necessidades para a determinação de especificidades do cuidado de enfermagem no âmbito hospitalar para esses familiares. Trata-se de pesquisa de natureza qualitativa, tipo exploratório, com abordagem da etnometodologia, envolvendo os conceitos de indexicalidade, reflexividade, descritibilidade, noção de membro ligado a contextualização e as práticas de ação social. Os instrumentos foram a observação participante e entrevista livre sendo aplicados formulários para caracterização dos familiares e clientes e questionários para caracterização dos integrantes da equipe de enfermagem.