Dual centrifugation (DC) is a new and versatile technique for the preparation of liposomes by in-vial homogenization of lipid-water mixtures. Size, size distribution, and entrapping efficiencies are strongly dependent on the lipid concentration during DC-homogenization. In this study, we investigated the detailed structure of DC-made liposomes. To do so, an assay to determine the ratio of inner to total membrane surfaces of liposomes (inaccessible surface) was developed based on either time-resolved or steady-state fluorescence spectroscopy. In addition, cryogenic electron microscopy (cryo-EM) was used to confirm the lamellarity results and learn more about liposome morphology. One striking result leads to the possibility of producing a novel type of liposome—small multilamellar vesicles (SMVs) with low PDI, sizes of the order of 100 nm, and almost completely filled with bilayers. A second particularly important finding is that VPGs can be prepared to contain open bilayer structures that will close spontaneously when, after storage, more aqueous phase is added and liposomes are formed. Through this process, a drug can effectively be entrapped immediately before application. In addition, dual centrifugation at lower lipid concentrations is found to produce predominantly unilamellar vesicles.

Dual centrifugation (DC) is an innovative in-vial homogenization and in-vial nanomilling technique that has been in use for the preparation of liposomes for more than one decade. Since then, DC has continuously been developed for preparing various liposomes and other lipid nanoparticles including emulsions and solid lipid nanoparticles (SLNs) as well as polymersomes and nanocrystals. Improvements in equipment technology have been achieved over the past decade, so that DC is now on its way to becoming the quasi-standard for the simple, fast, and aseptic production of lipid nanoparticles and nanocrystals in small and medium batch sizes, including the possibility of simple and fast formulation screening or bedside preparations of therapeutic nanoparticles. More than 68 publications in which DC was used to produce nanoparticles have appeared since then, justifying an initial review of the use of DC for pharmaceutical nanotechnology.

Dual centrifugation (DC) is a novel in-vial homogenization technique for the preparation of liposomes in small batch sizes under gentle and sterile conditions which allows encapsulation efficiencies (EE) for water soluble compounds of >50%. Since liposome size, size distribution (PDI), and EE depend on the lipid concentration used in the DC process, a screening method to find optimal lipid concentrations for a defined lipid composition was developed. Four lipid mixtures consisting of cholesterol, hydrogenated or non-hydrogenated egg PC, and/or PEG-DSPE were screened and suitable concentration ranges could be identified for optimal DC homogenization. In addition to the very fast and parallel liposome preparation of up to 40 samples, the screening process was further accelerated by the finding that DC generates homogeneously mixed liposomes from a macroscopic lipid mixture without the need to initially prepare a molecularly mixed lipid film from an organic solution of all components. This much simpler procedure even works for cholesterol containing lipid blends, which could be explained by a nano-milling of the cholesterol crystals during DC homogenization. Furthermore, EE determination was performed by time-resolved fluorescence measurements of calcein-loaded liposomes without removing the non-entrapped calcein. The new strategy allows the rapid characterization of a certain lipid composition for the preparation of liposomes within a working day.

Thermosensitive liposomes (TSLs) have great potential for the selective delivery of cytostatic drugs to the tumor site with greatly reduced side effects. Here we report the discovery and characterization of new thermosensitive small multilamellar lipid nanoparticles (tSMLPs) with unusually high temperature selectivity. Furthermore, the temperature-dependent release of the fluorescent marker calcein from tSMLPs is enhanced by human serum albumin. tSMLPs can easily be prepared through dual centrifugation (DC) at very high lipid concentrations using dipalmitoyl and distearoyl phosphatidylcholine (DPPC, DSPC) and the phospholipid dipalmitoyl-sn-glycero-phosphatidyldiglycerol (DPPG₂). The new particles have a hydrodynamic diameter of about 175 nm and a narrow size distribution (PDI 0.02). tSMLPs consist of multiple lipid membranes, which become increasingly closer packed towards the particle center, and have no visible aqueous core. The particles are highly stable due to strong hydrogen bond-based membrane interactions mediated by DPPG₂. tSMLPs can be used as carriers for water-soluble drugs (EE 25 %) entrapped within the interlamellar spaces. Based on biophysical (DSC, DLS and ITC) and morphological (cryo-EM) studies, a hypothesis is presented to explain the structural basis underlying the high temperature selectivity, as well as the unusual morphology of the new thermosensitive lipid nanoparticles.