Production of liposomes by centrifugation of water in oil emulsions

Prof. Dr. H. Nirschl1), Karlsruhe Institute for Technology (KIT)/Germany

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Prof. Dr. Hermann NIrschlProcess Machines, Institute of Mechanical Process Engineering and Mechanics, Karlsruhe Institute of Technology, Karlsruhe/Germany

People involved

Kirsten Ullmann (PhD fellow sponsored by the PRC) – KIT, Germany (

Dr. Gero Leneweit – Carl Gustav Carus-Institute, Germany (


Liposomes are promising carriers for active pharmaceutical ingredients (API) and can be used for multiple purpose in drug targeting, reductions of adverse effects, and diagnostics. The production of liposomes with high encapsulation efficiency for high molecular weight API, which are sensitive towards heat and aggressive conditions of pH, organic solvents or surfactants, are an unmet need of increasing interest. Novel processes using centrifugation of water in oil (w/o) emulsions were proposed, based on the use of phospholipids as emulsifiers, avoiding the use of aggressive treatments and allowing asymmetric membrane functionalities. However, severe limitations regarding emulsion stability and phase transfer of liquid droplets do not allow an industrial application so far. To enable industrial liposome production for a broad class of hydrophilic API, innovative processes are to be developed for the engineering and the stability of phospholipid w/o emulsions.1)2)3)4)5)6)

We propose to explore the feasibility of this innovative liposome production process, starting from the interfacial and molecular behavior of phospholipids, reaching to w/o emulsion stability and lipid gels, all in perspective to progressively develop a successful phase transfer and promote liposome production for pharmaceutical applications. The interfacial and molecular behavior of phospholipids of different head groups and fatty acids will be studied by tensiometry. w/o emulsion stability and the formation or avoidance of lipid gels will be tested by an analytical centrifuge, nuclear magnetic resonance (NMR) and photon correlation spectroscopy (PCS). The key elements of the proposed study cover fields which were hardly explored for phospholipids so far but bear a strong potential for large-scale application in pharmaceutical engineering.

Benefit for the community

The following benefits are expected for the scientific community of phospholipid research, industrial users and for society in the field of health care:

  • Protocols to produce nanometer-sized w/o emulsions with pharmaceutical excipients including tested short and long-term stability, e. growth rates. The screening of phospholipid headgroups and fatty acids and experiences about natural phospholipid blends will enable industrial users to select phospholipids according to their physicochemical characteristics and the requirements of the application.
  • Exploring the phase transfer process of w/o emulsions in a centrifugal system with in-situ monitoring will elucidate ongoing research activities of many research groups for new production processes of liposomes. High encapsulation efficiency is a key requirement for the advancement of liposomal drug vehicles and their expanded application in the pharmaceutical industry and will also trigger new research projects.
  • The potential to design asymmetric lipid bilayers makes the process attractive and versatile for other scientific and technical applications on smaller or larger scales.
  • Increasing knowledge about phospholipid/solvent interactions and their effect on w/o emulsion stability and gel formation will support the design of new products involving phospholipids.


The project started with the investigations of tensiometric behavior of different phospholipids as interfacial phenomena have a large influence on the final phase transfer for liposome production. The oil phase was carefully selected to avoid the formation of a gel phase at the interface as well as to meet the requirements for a pharmaceutical applicable substance. Fluorocarbons have recently been in the focus of researchers because of their inert characteristics which makes them good candidates for pharmaceuticals. By changing the hydrophobic phase from an organic oil (squalene) to a fluorocarbon, interfacial behavior had to be investigated as there are not many data on interfacial tensions between a fluorocarbon and a phospholipid suspension. With the fluorocarbon new challenges appeared – optical methods such as profile analysis tensiometry were unsuitable because of similar refractive index of both phases. Hence, during the first year the suitability of the Du Noüy ring was studied and the results compared to a second method, the spinning drop tensiometry. We were able to show that the Du Noüy ring measures interfacial tensions with equal precision as the spinning drop and presented tensiometric data between a fluorocarbon and phospholipid suspensions consisting of phospholipid with different chain lengths. Shorter chain lengths reduce the interfacial tensions further. We additionally investigated the influence of temperature on the interfacial behavior, as temperature may also have an influence on the transfer of droplets. Results reveal that above the respective transition temperature of the phospholipid interfacial tensions of different phodpholipids become equal.

During year two and three, droplet size and stabilization of w/o nano-emulsions prepared with different phospholipids (head groups, chain length, natural blends) were investigated. With the fluorocarbon as the hydrophobic phase, phospholipids were dispersed in the aqueous phase. Nano-emulsions were stable for several weeks with an average droplet size of 200 nm. Higher concentrations of phospholipids lead to smaller droplets, head groups have, except for DPPS, no significant influence on the droplet size. Furthermore, influencing effects such as gas bubbles in the hydrophobic phase were examined.

With the change of the hydrophobic phase to the fluorocarbon, a breakthrough could be achieved. In addition to minor changes of the transfer process, liposomes could be produced. We further characterized these liposomes using dynamic light scattering (DLS) and small-angle x-ray scattering (SAXS) for size determination. First experiments regarding the encapsulation efficiency were carried out and show that high encapsulation of up to 86 % is possible with the proposed centrifugation method.

To summarize, the process of liposome production was successfully established by systematically analyzing interfacial behavior between the chosen hydrophobic and hydrophilic phases, using the gained information about the area per molecule to produce nano-emulsion with the needed amount of phospholipids for stabilization. From characterization of nano-emulsions, the transfer of droplets led to the production of liposomes. A model substance was used for encapsulation and revealed an encapsulation efficiency of up to 86 %.

Visit the supervisors lab


Hildebrandt E, Sommerling JH, Guthausen G, Zick K, Stürmer J, Nirschl H, Leneweit G, 2016
Phospholipid adsorption at oil in water versus water in oil interfaces. Implications for interfacial densities and bulk solubilities
Colloid Surf. A 505, 56–63
Hildebrandt E, Dessy A, Sommerling JH, Guthausen G, Nirschl H, Leneweit G, 2016
Interactions between Phospholipids and Organic Phases – Insights in Lipoproteins and Nanoemulsions
Langmuir 32, 5821–5829
Sommerling JH, Uhlenbruck N, Leneweit G, Nirschl H, 2017
Transfer of colloidal particles between two non-miscible liquid phases
Colloid Surf. A 535, 257–264
Sommerling JH, de Matos MBC, Hildebrandt E, Dessy A, Kok RJ, Nirschl H, Leneweit G, 2018
Instability Mechanisms of Water-in-Oil Nanoemulsions with Phospholipids: Temporal and Morphological Structures
Langmuir 34, 572–584
Hildebrandt E, Nirschl H, Kok RJ, Leneweit G, 2018
Adsorption of phospholipids at oil/water interfaces during emulsification is controlled by stress relaxation and diffusion
Soft Matter 14, 3730–3737
de Matos MBC, Miranda BS, Rizky Nuari Y, Storm G, Leneweit G, Schiffelers RM, Kok RJ, 2019
Liposomes with asymmetric bilayers produced from inverse emulsions for nucleic acid delivery
J. Drug Target. 27, 681-689
Publications derived from the project:
Ullmann K, Poggemann L, Nirschl H, Leneweit G, 2020
Adsorption Process for Phospholipids of Different Chain Lengths at a Fluorocarbon/Water Interface studied by Du Noüy Ring and Spinning Drop
Colloid and Polymer Science 298, 407-417
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