POSICIONAMIENTO ANTE LA EUTANASIA Y EL SUICIDIO MÉDICAMENTE ASISTIDO.

Preparation and characterisation of elastin

Insoluble elastin was isolated from equine ligamentum nuchae and solubilised by oxalic acid hydrolysis (17, 18). The solubilised elastin preparation was analysed by gel electropho­ resis (SDS-PAGE, 4% gel, silver staining), and gel filtration chromatography [Sephacryl S-500 column material and the reference samples ferritin (0.88 MDa), dextran blue (2 MDa), and Helix pomatia haemocyanin (9 MDa)]. The average molecular mass of solubilised elastin was about 1,100 kDa. 2D gel electrophoresis showed that the solubilised elastin had an isoelec­ tric point of around 6. Amino acid composition of insoluble elastin and solubilised elastin was similar for all amino acid residues, including desmosine and isodesmosine (both 0.5 ± 0.0 per 1000 amino acid residues) (19).

Preparation of capsules

The standard procedure for the preparation of capsules was to drop 20 Ml of a 2.0% (w/v) elastin in 0.25 M acetic acid solution in liquid nitrogen, incubate the frozen droplets at -10 to -20°C for about 3 h, and then lyophilise the sample in a Zirbus lyophiliser (Bad Grund, Ger­ many) using the program plotted in Fig. 1a in the main text. Larger volumes (e.g. 1 ml) can be applied as well. Vapour crosslinking of elastin capsules was performed in vapour of 25% glutaraldehyde: 38% formaldehyde 1:1 for 24 h, optionally followed by wet crosslinking in 0.5% glutaraldehyde in phosphate buffer pH 7.4 for 2 h. Concentrations for type I collagen (Symatese Biomatériaux, Chaponost, France) and bovine serum albumin (Sigma, St. Louis, MO, USA) were 0.25% (w/v) and for heparin (Sigma) was 1.0% (w/v).

Incorporation of fluorescent probes into elastin capsules

Fluorescent probes were incorporated in the capsule wall by the addition of 50 Mg probe per ml 2.0% (w/v) elastin solution prior to freezing and lyophilisation. Fluorescent probes in­ cluded Alexa Fluor594-labeled goat anti-mouse antibodies (150,000 Da) and Alexa Fluor488 or 594-labeled dextrans (10,000 Da). Capsule shells were crosslinked for 24 h by vapour crosslinking. Incorporation of fluorescent probes in the capsule lumen was performed by a 96 h incubation of vapour-crosslinked capsules in a solution of 50 Mg probe in either 1 ml MilliQ water (for dextrans) or 1 ml ethanol (for DiOC18; see below), followed by wet crosslinking, and three washings with MilliQ water or 100% ethanol to remove non-included probe. Probes included Alexa Fluor488 or 594-labeled dextran (10,000 Da) and 3,3'- dioctadecyloxacarbocyanine perchlorate DiOC18 (882 Da), all from Molecular Probes Europe (Leiden, The Netherlands).

Incorporation of enzymes into elastin capsules

Peroxidase-labeled IgG (Sigma) was incorporated in the capsule wall by the addition of 100 Mg enzyme per ml 2.0% (w/v) elastin solution prior to freezing and lyophilisation. Capsule shells were crosslinked for 24 h by vapour crosslinking. Alkaline phosphatase­ labeled IgG (Dako, Glostrup, Denmark) was incorporated in the capsule lumen (100 Mg enzyme per ml 50 mM Tris-HCl for 72 h at 4°C) followed by a 2 h wet crosslinking and washed three times with Tris-HCl. Enzyme activities were studied after reaction with Fast Red TR Salt for alkaline phosphatase followed by three washing steps with Tris-HCl, and a reaction with 3,3-diaminobenzidine tetrahydrochloride (DAB) for peroxidase followed by three washings with Tris-HCl (20).

Degradation studies using elastase

Enzymatic degradation of capsules containing fluorescent probes (Alexa Fluor488-labeled dextran in the capsule wall and Alexa Fluor594-labeled dextran in the capsule lumen) was assessed after incubation with elastase (Sigma) in 100 mM Tris-HCl pH 8.0 and analysed with time resolved confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Specimens were treated with 0.4 U/ml elastase and analysed up to 30 min at 22°C.

Microscopical techniques

Scanning electron microscopy (SEM): Lyophilised samples were sputtered with gold and studied with a JEOL JSM-6310 SEM apparatus with an accelerating voltage of 15 kV. Wet samples first were critical point dried using liquid CO2. Transmission electron microscopy (TEM): Vapour crosslinked samples (stabilised samples) were post-fixed with 0.5% glutaraldehyde in 10 mM PB (pH 7.4) for at least 24 h followed by 1% (w/v) osmium tetroxide in 0.1 M PB for 1 h. After a rinsing period of 3 h with 0.1 M phosphate buffer, sam­ ples were de-hydrated in an ascending series of ethanol in water solutions, embedded in ep­ oxy resin (Epon 812). Sections (60 nm) were picked up on formvar-coated grids, post stained with lead citrate and uranyl acetate, and examined in a JEOL 1010 electron microscope. Fluorescence-activated cell sorting (FACS): The average size of the stabilised capsules was analysed by flow cytometry (Epics Elite flow cytometer, Coulter, Luton, UK). Capsules were gated according to their forward and side scattering patterns. Fractions were collected on poly-D-lysine coated coverslips and left to attach for 60 min. Coverslips were critical point dried, sputtered with gold, and studied by SEM. Fluorescence microscopy: Elastin preparations (2.0% (w/v) in 0.25 M acetic acid) were placed on a glass slide and studied be­ fore and after freezing in liquid nitrogen, using a fluorescence microscope with an I3 filter (Leica, DM 6000 B, Leica Microsystems, Wetzler, Germany). Freeze microscopy: A droplet of 2.0% (w/v) elastin in 0.25 M acetic acid was put on a glass slide, frozen in liquid nitrogen and analysed in a -70°C precooled custom-built freeze microscope with temperature registration (RIVM National Institute for Public Health and the Environment, Bilthoven, The Netherlands). The temperature of the frozen sample was increased until a temperature of -18°C was reached. Confocal microscopy: Elastin capsules with incorporated fluorescent probes were deposited on poly-D-lysine coated coverslips. Confocal images were made at 488 nm and 594 nm with a BioRad MRC1024 confocal laser scanning microscope, equipped with an ar­ gon/krypton laser, using a 60x 1.4 NA oil objective and LaserSharp2000 acquisition software. Light microscopy: Capsules with incorporated enzymes were placed on a glass slide, stained as described above and studied using a Leica DM 6000 B microscope.