Delivery of mobile dental services to dependent elderly people: results from a pilot study in rural Switzerland.

The objective of this pilot study was to provide data on the oral health status and oral health behaviour of care-dependent elderly people in the canton of Uri. The study aimed at assessing whether age and duration of living in a nursing home is associated with dental attendance, whether there is a relationship between age and the DMF-T index, and how high the risk was for participants to develop oral health problems. The study offered mobile dental services to people residing in nursing homes (NHG) and to community dwellers (CDG). Data was collected on demographics, medical history, presence of pain, ability to eat and oral health behaviour by means of a questionnaire. A clinical examination was performed and data on the oral status was collected. Statistical methods were: median tests, linear regressions and descriptive statistics. Uptake of the programme was low. 56 participants were examined. 47 participants (24 females, 23 males, average age 86.3 ± 7.36 years) resided in nursing home, nine participants (seven females, two males, average age 76 ± 12.8 years) were community dwellers. No significant differences were found between frequency of dental attendance and age (p = 0.35) or duration of stay in a nursing home (p = 0.55). The number of decayed teeth (p=0.005), missing teeth (p=0.01), and the DMF-T index (average in NHG = 26.5, CDG = 20.2, p < 0.001) increased, the number of filled teeth (p=0.02) decreased as age increased. Upon calculation of the 'Teamwerk-index', which takes into account oral health behaviour, dental caries, periodontal status, the majority of participants had a medium risk of developing oral health problems. Our pilot study in rural Switzerland provides first results of the oral health status in dependent elderly people. As people age, oral health deteriorates. More attention should be given in order to achieve better oral hygiene maintenance and more regular dental visits, to ensure a better oral health status in dependent elderly people.

The fusogenic lipid phosphatidic acid promotes the biogenesis of mitochondrial outer membrane protein Ugo1.

Import and assembly of mitochondrial proteins depend on a complex interplay of proteinaceous translocation machineries. The role of lipids in this process has been studied only marginally and so far no direct role for a specific lipid in mitochondrial protein biogenesis has been shown. Here we analyzed a potential role of phosphatidic acid (PA) in biogenesis of mitochondrial proteins in Saccharomyces cerevisiae. In vivo remodeling of the mitochondrial lipid composition by lithocholic acid treatment or by ablation of the lipid transport protein Ups1, both leading to an increase of mitochondrial PA levels, specifically stimulated the biogenesis of the outer membrane protein Ugo1, a component of the mitochondrial fusion machinery. We reconstituted the import and assembly pathway of Ugo1 in protein-free liposomes, mimicking the outer membrane phospholipid composition, and found a direct dependency of Ugo1 biogenesis on PA. Thus, PA represents the first lipid that is directly involved in the biogenesis pathway of a mitochondrial membrane protein.

Strong, low-density nanocomposites by chemical vapor deposition and polymerization of cyanoacrylates on aminated silica aerogels.

Strong polymer-silica aerogel composites were prepared by chemical vapor deposition of cyanoacrylate monomers onto amine-modified aerogels. Amine-modified silica aerogels were prepared by copolymerizing small amounts of (aminopropyl)triethoxysilane with tetraethoxysilane. After silation of the aminated gels with hexamethyldisilazane, they were dried as aerogels using supercritical carbon dioxide processing. The resulting aerogels had only the amine groups as initiators for the cyanoacrylate polymerizations, resulting in cyanoacrylate macromolecules that were higher in molecular weight than those observed with unmodified silica and that were covalently attached to the silica surface. Starting with aminated silica aerogels that were 0.075 g/cm(3) density, composite aerogels were made with densities up to 0.220 g/cm(3) and up to 31 times stronger (flexural strength) than the precursor aerogel and about 2.3 times stronger than an unmodified silica aerogel of the same density.

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.

Synthesis and application of integrin targeting lipopeptides in targeted gene delivery.

One of the main problems facing gene therapy is the ability to target the delivery of DNA to specific cells of choice. Recently, we developed a synthetic nonviral vector platform system known as LMD (liposome:mu:DNA) that was designed for further modular upgrading with tool-kits of chemical components. First-generation LMD systems were prepared from DC-Chol/DOPE cationic liposomes (DC-Chol=3beta-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol, DOPE=dioleoyl-L-alpha-phosphatidylethanolamine), mu peptide from the adenovirus core and plasmid DNA (pDNA). Here we report attempts to realise peptide-targeted gene delivery that build upon the LMD platform. Our strategy was to prepare novel lipopeptides with a lipid moiety designed to insert into the outer lipid bilayer of LMD particles whilst simultaneously presenting a peptide moiety for cell-surface receptor binding. One main functional peptide sequence was selected (PLAEIDGIELA; tenascin peptide sequence) known to target alpha(9)beta(1)-integrin proteins predominant on upper-airway epithelial cells. This sequence was investigated along with a corresponding control sequence. The syntheses of two classes (A and B) of lipopeptides are reported; the syntheses of class A lipopeptides requires a modification of Mitsunobu chemistry that could be of general utility to facilitate Mitsunobu reactions in other diverse systems. "Targeted" LMD and LD transfections with class A or B lipopeptides exhibit nonspecific peptide enhancements (up to one order of magnitude) over nonlipopeptide control transfections but few specific effects. Specific targeting effects can be seen if the overall LMD or LD particle cationic charge is lowered, but nonspecific effects are never eliminated. Whilst promising, these data now highlight the need for in vivo data and even a new modular, aqueous chemistry for the controlled adaptation of LMD particles in buffer in order for successful peptide-targeted, synthetic, nonviral gene delivery to be realised.

Lipidic carriers of siRNA: differences in the formulation, cellular uptake, and delivery with plasmid DNA.

RNA interference (RNAi) has become a popular tool for downregulating specific gene expression in many species, including mammalian cells [Novina, C. D., and Sharp, P. A. (2004) The RNAi revolution, Nature 430, 161-164]. Synthetic double-stranded RNA sequences (siRNA) of 21-23 nucleotides have been shown in particular to have the potential to silence specifically gene function in cultured mammalian cells. As a result, there has been a significant surge of interest in the application of siRNA in functional genomics programs as a means of deciphering specific gene function. However, for siRNA functional genomics studies to be valuable and effective, specific silencing of any given target gene is essential, devoid of nonspecific knockdown and toxic side effects. For this reason, we became interested in investigating cationic liposome/lipid-mediated siRNA delivery (siFection) as a meaningful and potentially potent way to facilitate effective functional genomics studies. Accordingly, a number of cationic liposome/lipid-based systems were selected, and their formulation with siRNA was studied, with particular emphasis on formulation parameters most beneficial for siRNA use in functional genomics studies. Cationic liposome/lipid-based systems were selected from a number of commercially available products, including lipofectAMINE2000 and a range of CDAN/DOPE systems formulated from different molar ratios of the cationic cholesterol-based polyamine lipid N(1)-cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine (CDAN) and the neutral helper lipid dioleoyl-L-alpha-phosphatidylethanolamine (DOPE). Parameters that were been investigated included the lipid:nucleic acid ratio of mixing, the extent of cationic liposome/lipid-nucleic acid complex (lipoplex) formation plus medium used, the lipoplex particle size, the mode of delivery, and dose-response effects. Results suggest that concentrations during siRNA lipoplex (LsiR) formation are crucial for maximum knockdown, but the efficacy of gene silencing is not influenced by the size of LsiR particles. Most significantly, results show that most commercially available cationic liposome/lipid-based systems investigated here mediate a significant nonspecific downregulation of the total cellular protein content at optimal doses for maximal specific gene silencing and knockdown. Furthermore, one pivotal aspect of using siRNA for functional genomics studies is the need for at least minimal cellular toxicity. Results demonstrate that CDAN and DOPE with and without siRNA confer low toxicity to mammalian cells, whereas lipofectAMINE2000 is clearly toxic both as a reagent and after formulation into LsiR particles. Interestingly, LsiR particles formulated from CDAN and DOPE (45:55, m/m; siFECTamine) seem to exhibit a slower cellular uptake than LsiR particles formulated from lipofectAMINE2000. Intracellularly, LsiR particles formulated from CDAN and DOPE systems also appear to behave differently, amassing in distinct but diffuse small nonlysosomal compartments for at least 5 h after siFection. By contrast, LsiR particles formulated from lipofectAMINE2000 accumulate in fewer larger intracellular vesicles.

Thermodynamic aspects and biological profile of CDAN/DOPE and DC-Chol/DOPE lipoplexes.

The DNA complexation and condensation properties of two established cationic liposome formulations, CDAN/DOPE (50:50, m/m; Trojene) and DC-Chol/DOPE (60:40, m/m), were investigated by using a combination of isothermal titration calorimetry (ITC), circular dichroism (CD), photon correlation spectroscopy (PCS), and turbidity assays. Plasmid DNA (7528 bp) was titrated with extruded liposomes (90 +/- 15 nm) and a thermodynamic profile established. ITC data revealed that the two liposome formulations differ substantially in their DNA complexation characteristics. Equilibrium dissociation constants for CDAN/DOPE (K(d) = 19 +/- 3 microM) and DC-Chol/DOPE liposomes (K(d) = 2 +/- 0.5 microM) were obtained by fitting the experimental data in a one-site binding model. Both CDAN/DOPE and DC-Chol/DOPE binding events take place with a negative binding enthalpy (DeltaH degrees = -0.5 and -1.7 kcal/mol, respectively) and increasing system entropy (TDeltaS = 6 +/- 0.3 and 6.2 +/- 0.3 kcal/mol, respectively). Interestingly, CDAN/DOPE liposomes undergo substantial rehydration and protonation prior to complexation with pDNA, which is observed as two discrete exothermic signals during titration. No such biphasic effects are seen with respect to the binding between DC-Chol/DOPE and pDNA that appears to be otherwise instantaneous with no rehydration effects. The rehydration and protonation characteristics of CDAN/DOPE liposomes in comparison with those of DC-Chol/DOPE cationic liposomes are confirmed by ITC; CDAN/DOPE liposomes have strongly exothermic dilution characteristics and DC-Chol/DOPE liposomes only mildly endothermic characteristics. Furthermore, analysis of cationic liposome-pDNA binding by CD spectroscopy reveals that CDAN/DOPE-pDNA lipoplexes are more structurally fluid than DC-Chol/DOPE-pDNA lipoplexes. CDAN/DOPE liposomes induced considerable fluctuation in the DNA structure for at least 60 min, whereas liposomes obtained from DC-Chol/DOPE lack the same effect on the DNA structure. Turbidity studies show that DC-Chol/DOPE lipoplexes exhibit greater resistance to serum than CDAN/DOPE lipoplexes, which showed substantial precipitation after incubation for 100 min with serum. Transfection studies on HeLa and Panc-1 cells reveal that CDAN/DOPE lipoplexes are superior in efficacy to DC-Chol/DOPE lipoplexes. CDAN/DOPE liposomes tend to transfect best in normal growth medium (including 10% serum and antibiotics), whereas DC-Chol/DOPE lipoplexes transfect best under serum free transfection conditions.

Synthesis and formulation of neoglycolipids for the functionalization of liposomes and lipoplexes.

Novel carbohydrate-based agents for the stabilization of ternary liposome:mu:DNA (LMD) nonviral vector systems are described. LMD vector systems comprise plasmid DNA (pDNA; D,7.5 kb) expressing a reporter gene (in this instance beta-galactosidase expressing gene) that is precondensed with the adenoviral core peptide mu (mu, M; MRRAHHRRRRASHRRMRGG) and then further packaged by means of DC-Chol:DOPE (3:2; m/m) cationic liposomes. Final optimized lipid:mu:pDNA ratio is typically 12:0.6:1 (w/w/w). We report the synthesis of a series of nine neoglycolipids prepared by coupling completely unprotected sugar monomers or oligomers (mannose, glucose, galactose, glucuronic acid, maltose, lactose, maltotriose, maltotetraose, and maltoheptaose) through their reducing-residue termini to an aminoxy-functionalized cholesterol-based lipid. Characterization of these novel neoglycolipids by (1)H NMR reveals that the coupling reaction has a major configurational preference for the beta-anomer. Unusually, even mannose coupling results in a neoglycolipid product with a predominantly beta-anomeric conformation (>85%). Formulation of neoglycolipids into LMD vector systems by incubation of LMD particles with neoglycolipid micelles results in the formation of a range of potential stabilized-LMD (sLMD) vector systems. Those potential sLMD systems prepared with longer chain neoglycolipids are found to have enhanced stabilities, with respect to aggregation in high ionic strength buffers, and enhanced transfection efficacies in comparison to the transfection properties of the naked first generation LMD vector system (i.e., gene delivery and expression). By contrast, when LMD vector systems are incubated with poly(ethylene glycol) DSPE-PEG micelles, resulting PEG-LMD vector systems are very stable with respect to colloidal instablility and aggregation in high ionic strength buffers and in serum, but are completely refractory to transfection. These data suggest that oligosaccharides could represent an alternative to PEG as a stealth polymer able to stabilize synthetic nonviral vector systems in some fluids but without impairing transfection efficiency. Furthermore, sLMD systems prepared with longer chain neoglycolipids appear to have sufficient useful characteristics to form the basis of viable second-generation LMD vector systems after further development.

Nuclear localisation sequence templated nonviral gene delivery vectors: investigation of intracellular trafficking events of LMD and LD vector systems.

The impact of a peptide that contains a nuclear localisation sequence (NLS) on intracellular DNA trafficking was studied. We used the adenoviral core peptide mu and an SV40 NLS peptide to condense plasmid DNA (pDNA) prior to formulation with 3beta-[N-(N', N'-dimethylaminoethane)carbamoyl]cholesterol/dioleoyl-L-alpha-phosphatidyl ethanolamine (DC-Chol/DOPE) liposomes to give LMD and LND vectors, respectively. Fluorescent-labelled lipid and peptides plus dye-labelled pDNA components were used to investigate gene delivery in dividing and S-phase growth-arrested cells. Confocal microscopic analyses reveal little difference in intracellular trafficking events. Strikingly, mu peptide associates with nuclei and nucleoli of cells within less than 15 mins incubation of LMD with cells, which suggests that mu peptide has an NLS function. These NLS properties were confirmed by cloning of a mu-beta-galactosidase fusion protein that localises in the nuclei of cells after cytosolic translation. In dividing cells both LMD and LND deliver pDNA(Cy3) to nuclei within 30-45 min incubation with cells. By contrast, pDNA is detected only in the cytoplasm in growth-arrested cells over the period of time investigated, and not in the nuclei. LD systems prepared from DC-Chol/DOPE cationic liposomes and pDNA(Cy3) behave similarly to LMD systems, which suggests that mu peptide is unable to influence trafficking events in this current LMD formulation, in spite of its strong NLS capacity. We further describe the effect of polyethyleneglycol (PEG) on cellular uptake. "Stealth" systems obtained by post-coating LMD particles with fluorescent-labelled PEG molecules (0.5, 5 and 10 mol % fluorescein-PEG(5000)-N-hydroxysuccinimide) were prepared and shown to be internalised rapidly (mins) by cells, without detectable transgene expression. This result indicates that PEG blocks intracellular trafficking of pDNA.