Proliposome Powders for the Generation of Liposomes: the Influence of Carbohydrate Carrier and Separation Conditions on Crystallinity and Entrapment of a Model Antiasthma Steroid

Formulation effects on the entrapment of beclometasone dipropionate (BDP) in liposomes generated by hydration of proliposomes were studied, using the high-density dispersion medium deuterium oxide in comparison to deionized water (DW). Proliposomes incorporating BDP (2 mol% of the lipid phase consisting of soya phosphatidylcholine (SPC) and cholesterol; 1:1) were manufactured, using lactose monohydrate (LMH), sorbitol or D-mannitol as carbohydrate carriers (1:5 w/w lipid to carrier). Following hydration of proliposomes, separation of BDP-entrapped liposomes from the unentrapped (free) BDP at an optimized centrifugation duration of 90 min and a centrifugation force of 15,500g were identified. The dispersion medium was found to have a major influence on separation of BDP-entrapped liposomes from the unentrapped drug. Entrapment efficiency values were higher than 95% as estimated when DW was used. By contrast, the entrapment efficiency was 19.69 ± 5.88, 28.78 ± 4.69 and 34.84 ± 3.62% upon using D2O as a dispersion medium (for LMH-, sorbitol- and D-mannitol-based proliposomes, respectively). The similarity in size of liposomes and BDP crystals was found to be responsible for co-sedimentation of liposomes and free BDP crystals upon centrifugation in DW, giving rise to the falsely high entrapment values estimated. This was remedied by the use of D2O as confirmed by light microscopy, nuclear magnetic resonance ((1)HNMR), X-ray diffraction (XRD) and entrapment studies. This study showed that carrier type has a significant influence on the entrapment of BDP in liposomes generated from proliposomes, and using D2O is essential for accurate determination of steroid entrapment in the vesicles

Buccal micronucleus cytome assay

The Buccal Micronucleus Cytome (BMCyt) assay is a minimally invasive method for studying DNA damage, chromosomal instability, cell death and the regenerative potential of human buccal mucosal tissue. This method is increasingly used in molecular epidemiological studies for investigating the impact of nutrition, lifestyle factors, genotoxin exposure and genotype on DNA damage, chromosome malsegregation and cell death. The biomarkers measured in this assay have been associated with increased risk of accelerated ageing, cancer and neurodegenerative diseases. This protocol describes one of the current established methods for buccal cell collection using a small-headed toothbrush, the generation of a single-cell suspension, slide preparation using cytocentrifugation, fixation and staining using Feulgen and Light Green for both bright field and fluorescence microscopic analysis. The scoring criteria for micronuclei and other nuclear anomalies are also described in detail. The protocol in its current form takes approximately 4 h to complete from the time of buccal cell collection to the generation of stained slides for microscopic analysis.Philip Thomas, Nina Holland, Claudia Bolognesi, Micheline Kirsch-Volders, Stefano Bonassi, Errol Zeiger, Siegfried Knasmueller & Michael Fenec