Nebulization of liposomal rh-Cu/Zn-SOD with a novel vibrating membrane nebulizer.

Liposomes are potential drug carriers for pulmonary drug delivery: They can be prepared from phospholipids, which are endogenous to the respiratory tract as a component of pulmonary surfactant, and at an appropriate dose liposomes do not pose a toxicological risk to this organ. Among the various categories of drug that benefit from liposomal entrapment is the anti-inflammatory enzyme superoxide dismutase, thus prolonging its biological half-life. The delivery of liposomes by nebulization is hampered by stability problems, like physical and chemical changes that may lead to chemical degradation and leakage of the encapsulated drug. Here we present data of liposomes aerosolized with a novel electronic nebulizer based on a vibrating membrane technology (PARI eFlow), which amends drawbacks like liposomes degradation and product release. The data acquisition included aerosol properties such as aerodynamic particle size, nebulization efficiency, and liposome leakage upon nebulization. In conclusion, this study shows the ability of the PARI eFlow to nebulize high amounts of liposomal recombinant human superoxide dismutase with reduced vesicle disruption tested in an enclosing experimental protocol.

Topically applied liposome encapsulated superoxide dismutase reduces postburn wound size and edema formation.

The overproduction of biochemical mediators, and activation of leukocytes and endothelial cells, generated in thermally injured tissue, gives rise to both local and distant effects. The formation of short-lived, highly reactive metabolites, such as oxygen free radicals, increases with increasing tissue ischemia, and causes further cell damage. Human recombinant Cu/Zn-superoxide dismutase (rh-Cu/Zn-SOD), an enzyme which captures these radicals, may have a beneficial effect on the postburn inflammation processes. In this study, the influence of rh-Cu/Zn-SOD application to thermally injured tissue of rabbit backskin was examined. Three different delivery strategies were compared, pure or liposomally encapsulated enzyme, or intralesionally injected rh-Cu/Zn-SOD. For control, one animal group was treated with plain gel and another group was kept untreated. At 24 h following trauma a statistically significant difference in lesion sizes between the enzyme treated and control groups was observed. After 72 h tissue swelling had diminished significantly more in the rh-Cu/Zn-SOD treated groups as compared to the control animals. The best results were achieved by spreading liposomes encapsulating the enzyme onto the wounds. Our results suggest that local treatment of burn wounds with enzymatic radical scavengers such as rh-Cu/Zn-SOD has a beneficial effect on the extent of the postburn damage.

Determination of liposome size distribution by flow cytometry.

BACKGROUND: An essential parameter that describes the quality of liposome suspensions is the mean size, respectively the size distribution. Currently several analytical methods including laser light scattering techniques (LLST) are being employed. METHODS: Here we present an alternative technique using flow cytometry (FCM) to characterize uni- and polydisperse suspensions. As model liposomes preparations containing dipalmitoylphosphatidylcholine (DPPC) were used. A constant number of particles (1,500/s) in the fluid stream and a representative number of 10,000 particles of each sample was measured. Fluorescence-labeled latex beads were measured identically, and their side scatter signals were calibrated and correlated to the results obtained with liposome vesicles. RESULTS: Evaluation of the measurement and validation of the FCM results in comparison to LLST confirm the reliability of results obtained with our method. Latex beads in the range of 100-1000 nm were used for calibration to classify liposomes. Although measurement characteristics and calculation in both methods are basically different, very good agreement of the results was achieved. CONCLUSIONS: Demonstration of stability, reproducibility, and reliability of results make the employment of this method acceptable for an adequate routine analysis technique.

Analysis of mitogenic activity of proteins after separation by gel electrophoresis.

We have used a combination of gel electrophoresis and a cell culture assay in microplates to analyse mitogenic activity in tissue extracts. The procedure is a modification of the method described by Kuo et al. The proteins were separated by native gel electrophoresis or isoelectric focusing. The gel was sliced and defined pieces were transferred into tissue culture inserts fitting in 96 well microplates, which contained the test cells. The proteins diffused from the gel slices directly into the culture supernatant and the mitogenic effects were evaluated by a colorimetric assay (MTT or phosphatase activity). Human interleukin 2 was used to demonstrate the feasibility of the method by evaluating the mitogenic effect on the cell line CTLL-2. Extracts of bovine pituitary glands were separated by native gel electrophoresis and isoelectric focusing and several protein bands could be identified which showed a distinct mitogenic effect on human endothelial cells. The method is very sensitive and allows rapid screening of protein mixtures for bioactive fractions.

Human interleukin-2 (IL-2) expressed by transfected mammalian cells.

cDNA for human interleukin-2 (IL-2) was cloned into the pRc/RSV vector for expression in animal cells. Baby hamster kidney (BHK-21) cells and Chinese hamster ovary (CHO) cells were transfected several times using calcium phosphate and electroporation methods with the construct pRc/RSV SIGIL2. Different transfection efficiencies were obtained. The biological test on CTLL-2 (mouse cytotoxic lymphocytes) showed that the kinetics of cell proliferation were different from those of rIL-2 (recombinant IL-2) expressed in bacteria and in BHK cells. When high concentrations of rIL-2 were applied, an inhibitory effect on CTLL-2 was observed when bacterial product was used, whilst rBHK interleukin caused no inhibition. Recombinant BHK IL2 induced a slower response of CTLL-2 cells at the beginning of the cultivation, however, prolonged activity was detected at the later stage of the experiment.

Flocculation: an alternative process to ion-exchange chromatography: (A scale-up study using recombinant human superoxide dismutase as model protein).

Flocculation using pellicular charged flocculants was investigated as an alternative process to conventional chromatography in purification of recombinant proteins using human recombinant superoxide dismutase expressed in E. coli as a model system. The removal of pyrogens, proteins, debris and the yield were determined. At laboratory scale, the starting conditions were optimized to yield a stable solution and the flocculation process fitted into a purification scheme. 100 L fermentation broth was the initial volume at pilot scale. The process parameters determined at the laboratory scale were tested and the results were compared to the pilot scale. The method was also compared to an ion-exchange chromatography.