Title: Docetaxel-liposomes prepared by the dual centrifugation method: effect of propylene glycol and lipid charge on liposomal characteristics

Ann Mari Holsater

University of Tromso, Norway


Dr. Ann Mari Holsæter was borne in Trondheim, Norway, 30th of January 1974. She works as Associate Professor at The University of Tromsø The Arctic University of Norway, Norway, in the Drug Transport and Delivery Research Group at the Department of Pharmacy. She is a pharmacist by background and gained her PhD within pharmaceutical technology in 2004. Her research focus today is on advanced drug delivery systems and drug targeting, and liposomes, nanofibers and hydrogels as drug delivery systems in particularly. Her publication portfolio includes per Nov 2018, 53 items, including 18 original per-review journal research articles, one review article and one book chapter.  


Docetaxel (Doc) is a highly lipophilic anticancer drug. The current Doc treatment gives several side effects that might be prevented when Doc is incorporated in liposomes. The first aim of this study was to adapt the Dual Centrifugation (DC) method as a preparation technique for Doc-liposomes, and secondly, to investigate how propylene glycol (PG) and liposomal charge affect the vesicle characteristics, namely Doc-entrapment, liposomal size, polydispersity index (PI) and zeta potential (ZP).
           First, the DC-settings were standardized with regards to time (30 min), speed (3500 rpm) and batch size (0.5 g vesicular phospholipid gel (VPG) containing 40 % phospholipids (PL)). Doc was added in a 1:10 w/w ratio to the PL content, and homogenization aid (1.4 mm zirconium beads) in a 1:1 w/w ratio to the VPG content. The DC-machine applied was a SpeedMixer™ (DAC 150.1 FVZ-K, Synergy Devices Limited, UK). After DC-homogenization, the VPGs were diluted into liposomal dispersions adding distilled water to a total volume of 2 mL. The Doc-entrapment was determined by HPLC after removing free drug crystals by centrifugation. The liposomal size, PI and ZP were determined using the Malvern Zetasizer, Nanoseries ZS. PG decreased the vesicle size when included in neutral Soy phosphatidylcholine (SPC)-liposomes; the SPC-PG-liposomes with and without Doc had a size of 97.9 ± 2.0 and 107.1 ± 6.1 nm, respectively. Moreover, inclusion of PG assured a higher Doc-entrapment as compared to the non-PG containing SPC-liposomes; 86.2 ± 8.3 % relative to 41.5 ± 3.7 %, respectively. However, PG was not beneficial in liposomes incorporating charged lipids into the membranes. When positively charged DOTAP replaced 20 % (w/w) of the SPC, the liposomal size increased to 518.0 ± 34.6 nm in the presence of PG, whereas the Doc-DOTAP-liposomes without PG had a size of 170.1 ± 0.5 nm. The Doc-entrapment was also reduced in the presence of PG both for positively charged DOTAP and negatively charged DMPG SPC-liposomes, resulting in only 40-50 % Doc-entrapment. Doc-liposomes with 20 % DOTAP and no PG was found to be the best formulation (97.3 ± 5.1 % Doc entrapment). Considering inclusion of DMPG in liposomes, all SPC:DMPG ratios gave acceptable liposomal sizes (< 200 nm). Although the 20 % DMPG formulation without PG showed an increased drug entrapment (88.0 ± 1.1 %) upon production, the Doc-entrapment was at the same level as for the neutral liposomes contain only SPC after 4 weeks of storage in the fridge.
           In conclusion, DC-homogenization proved to be a suitable and reproducible method for preparing Doc-liposomes. The effect of PG on both liposomal size and Doc-entrapment varied with the charge of lipids. The most promising formulations containing either 10 % or 20 % DOTAP and no PG, and achieved a Doc-entrapment of approximately 100 % and suitable liposomal size (< 200 nm).
Audience take away:
• The applied DC-method – dual centrifugation, is a novel and efficient small-scale preparation method with several advantages, especially in early formulation screening and when working with toxic compounds, as it does not require any sample transfer and all preparation steps take place in a closed container.
• Docetaxel is a highly lipophilic drug that has some challenging features regarding a stable entrapment within the liposomal bilayers.
• The observed positive effect of the positively charged lipid 1,2-Dioleoyl-3-trimethyl-ammoniumpropane chloride (DOTAP) with regards to docetaxel entrapment is interesting considering a clinical formulation, whereas the drastic change in liposomal size upon addition of propylene glycol is worth a closer investigation.