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Calcium phosphate/poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles with a diameter below 200 nm, loaded with either nucleic acids or proteins, were synthesized by a water-in-oil-in-water (W 1 /O/W 2 ) emulsion solvent evaporation technique. The particles were stabilized by polyvinyl alcohol (PVA) and had a negative charge (zeta potential À26 mV). By the addition of calcium phosphate into the inner aqueous phase of the W 1 /O/W 2 -emulsion, the encapsulation efficiency of siRNA was increased to 37%, of DNA to 52%, and of bovine serum albumin to 78%, i.e. by a factor of 3 to 10 compared to PLGA nanoparticles without calcium phosphate. Total loadings of 8 mg siRNA, 5 mg DNA and 280 mg fluorescein isothiocyanate-labelled bovine serum albumin (FITC-BSA) per mg of PLGA nanoparticles were achieved by this method. The addition of an outer layer of either chitosan or polyethyleneimine (PEI) reversed the charge of the particles (zeta potential > +30 mV) and improved the cellular uptake as well as the endosomal escape of these particles as demonstrated by confocal laser scanning microscopy. Calcium phosphate-PLGA nanoparticles loaded with DNA encoding for enhanced green fluorescent protein (eGFP-DNA) showed a good transfection efficiency for epithelial cells (HeLa). Gene silencing with HeLa cells expressing eGFP gave knockdown efficiencies of 53% for anionic nanoparticles, of 68% for chitosan-coated cationic nanoparticles, and of 89% for polyethyleneimine-coated cationic nanoparticles.

The incidence of cardiovascular and metabolic diseases has increased over the last decades and is an important cause of death worldwide. An upcoming ingredient on the nutraceutical market are anthocyanins, a flavonoid subgroup, abundant mostly in berries and fruits. Epidemiological studies have suggested an association between anthocyanin intake and improved cardiovascular risk, type 2 diabetes and myocardial infarct. Clinical studies using anthocyanins have shown a significant decrease in inflammation markers and oxidative stress, a beneficial effect on vascular function and hyperlipidemia by decreasing low-density lipoprotein and increasing high-density lipoprotein. They have also shown a potential effect on glucose homeostasis and cognitive decline. This review summarizes the effects of anthocyanins in in-vitro, animal and human studies to give an overview of their application in medical prevention or as a dietary supplement.

SummaryAs reported in the literat ure, oral endotracheal intubat ion of rats is considered to be very difficult. Specialised equipment and complicat ed techniques have been described to perform this procedure. In our experiment we adopted a simple method, which allowedÐwithout any complicated equipmentÐthe insertion of a relatively wide tube into the trachea of rats, allowing drug administrat ion.Keywords Endotrac heal intubat ion; rat; large tube; intrat racheal aerosol drug administrat ion Endot racheal intubation in small laborat ory anim als is often used for studies of pulmonary absorption and the bioavailabi lity of various macromolecules (Patton e t a l. 1994, Smith e t a l. 1994, Niven e t a l. 1995, Lizio e t a l. 2000 ). Endotrac heal intubation allows the delivery of test substances both in¯uid form (solution or suspension) as well as by aerosol (Osaier & Oberdo Èrster 1997 ). Endotracheal intubation of rats is problemati c because of the narrow oral cavity and glottis, the dif®culty in visualizing the trachea, and the mobility of the larynx even under narcosis. Several techniques have been developed for endotracheal intubation in rats but most of them require special skill, e.g. blind oral tracheal intubation (Stark e t a l. 1981 ); or elaborat e and specially designed equipm ent, e.g. a miniature laryngoscope (Proctor & Fernando 1973 ), a ®bre-optic laryngoscope (Costa e t a l. 1986 ), or an otoscope (Weksler e t a l. 1994 ). Most of these methods are associated with a high failure rate and require considerable practice to ensure successful intubati on without oropharyngeal or endotracheal damage. Moreover, for endotracheal aerosol drug delivery, large tubes are needed in order to reduce aerosol condensation or deposition on the tube's walls. T his ensures tight contact between the tube and trachea, which avoids loss of air or aerosol during application. A relatively simple intubation can be performed using a very thin cannula, as described by Yasaki and Dyck (1991) but this method is only applicable for endotracheal instillation and can produce tracheal traum a and bleeding. We have therefore developed a simple, fast and reliable method for endotracheal intubat ion in the rat that utilizes readily available normal laboratory utensils, and which allows instillation as well as delivery of aerosol for pulmonary drug studies.

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