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Fundamental Research

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Lyophilization of Doxorubicin (DXR)-loaded Liposomes

At this Department, the lyophilization of liposomes that encapsulate cytostatic drugs was examined as part of the PhD thesis by Dr. Katharina Böhm. Freeze-drying is generally done as a three-step procedure: First, the liposomes are frozen under the imperative condition that the final temperature of the freezing process must be lower than the eutectic temperature. In the subsequent primary drying process, the water present in the sample sublimates under vacuum and is then removedvia condensation on a cold trap. During the secondary drying process, while keeping the vacuum the temperature is increased in order to obtain a residual moisture of <1%.
The advantages of freeze-drying for liposomal preparations are their long shelf life as well as the option to quickly apply such lyophilized preparations. In addition - and different from spray-drying - the liposomes are not exposed to excessive heat, so that this method can also be applied to heat-sensitive compounds.
By applying different lyo- and cryoprotectants, we were able to optimize the stability of liposomes loaded with cytostatic drugs in the course of lyophilization.

Vesicular Phospholipid Gels (VPGs)

High entrapment efficiencies for active agents as well as a long liposomal shelf-life can be achieved by high-pressure homogenization of highly concentrated lipid dispersions that initially present themselves in the form of gels. These vesicular phospholipid gels (VPGs) can either be implanted or be used after dilution as liposomal dispersions. At this Department, Professor Dr. Martin Brandl developed such systems in the course of his qualification as a professor. They were then developed further in collaboration with the Department of Tumor Biology, Freiburg, Germany (Professors Dr. Ulrich Massing and Dr. Clemens Unger) and the Freiburg-based company Oncotest GmbH (Professor Dr. Heinz-Herbert Fiebig). Here, the efficacy of different cytostatic drugs could be optimized by their lipsomal encapsulation (PhD theses by Dr. Regina Moog, Dr. Frank Güthlein and Dr. Nicole Kaiser).

 

Kaiser N, Kimpfler A, Massing U, Burger AM, Fiebig HH, Brandl M, Schubert R: 5-Fluororuracil in vesicular phospholipid gels for anticancer treatment: entrapment and release properties, Int. J. Pharm. 256, 123-131, 2003. [Epub]

Moog R, Burger AM, Brandl M, Schüler J, Schubert R, Unger C, Fiebig HH, Massing U: Change in pharmacokinetic and pharmacodynamik behavior of gemcitabine in human tumor xenografts upon entrapment into vesicular phospholipid gels, Cancer Chemoth. Pharm. 49, 356-366, 2002. [Epub]

Bender J, Michaelis W, Schubert R: Morphological and thermal properties of vesicular phospholipid gels studied by DSC, rheometry and electron microscopy, J. Therm. Anal. Calorim. 68, 603-612, 2002.
[Epub]

 

Liposomes for Topical Applications

Contrary to earlier opinions, conventional liposomes do not penetrate into the skin. However, they can help ensure a better penetration of different drugs into the skin or to enable them to even pass through the skin to reach the systemic level. However, the mechanism(s) underlying these improvements has / have not yet been elucidated. We therefore closely examined the different lipid layers of the upper epidermal horny layer - the Stratum corneum (SC) for their structures: Isolated SC lipids form exceptional types of liposomes that could be characterized precisely by means of cryo-electron microscopy (PhD thesis by Dr. Margaret Fröhlich). In addition, the binding behavior of active agents to SC-lipid liposomes was measured so as to be able to utilize this simple system for estimating the penetration properties within the skin; when appropriately mounting such SC-lipid liposomes onto support membranes, we obtain a realistic model for assessing the penetration of drugs into the skin.
Another PhD thesis dealt with phospholipid gels for optimizing drug delivery into the skin (Dr. Bernd Ibscher, along with ratiopharm GmbH, Ulm, Germany). Yet another PhD thesis compared the efficacy of liposomal preparations with other systems such as microparticles, nanoemulsions and conventional creams (Dr. Matthias Renz, together with Schering AG, Berlin, Germany).

 

Schmidtgen M, Drechsler M, Lasch J, Schubert R: Energy-filtered cryo-transmission electron microscopy of liposomes prepared from human stratum corneum lipid, J. Microscopy 191, 177-186, 1998. [Epub]

Lasch J, Zellmer S, Pfeil W, Schubert R: Interaction of liposomes with the human skin lipid barrier: hSCLLs as model systems - DSC of intact Stratum corneum and in Situ CLSM of human skin, J. Liposome Res. 5, 99-108, 1995. [Epub]

Lasch J, Schmidt U, Sternberg B, Schubert R: Human Stratum corneum lipid-based liposomes (hSCLLs), J. Liposome Res. 4, 93-106, 1994. [Epub]

Schubert R, Joos M, Deicher M, Magerle R, Lasch J: Destabilization of egg lecithin liposomes on the skin after topical application measured by perturbed γ-γ-angular correlation spectroscopy (PAC) with 111In, Biochim. Biophys. Acta - Biomembranes 1150, 162-164, 1993. [Epub]

 

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Liposomes for Targeting Mitochondria

Mitochondria are not only important for synthesizing ATP but, among other functions, are also closely involved in the process of apoptosis. They may thus play important roles in various disorders such as Alzheimer's and Parkinson's disease that still suffer from the lack of efficacious treatments.
In her PhD thesis, Dr. Ying Zhou thus developed fusogenic liposomes that, in in-vitro experiments with isolated mitochondria, proved to be suitable drug carriers for mitochondrial targeting, which may allow for a more specific and targeted delivery of drugs into these organelles.

Polyplexes as Gene Transfer Systems

In the group of Professor Dr. Regine Süss, Dr. Stefanie Häfele investigated in her PhD thesis polyplexes as to their suitability as non-viral systems for gene delivery. For this purpose, various monodisperse polyamidoamines that had been synthesized by Dr. Laura Hartmann at the MPI in Potsdam-Golm, Germany, were aggregated with DNA. By fluorescence microscopy, their uptake was then studied on primary cells by employing markers and inhibitors of endocytosis. 

pH-sensitive Liposomes and their Endosomal Release

A special type of liposomes - pH-sensitive liposomes - is stable at neutral pH and is destabilized at slightly acidic pH (<5.5). These liposomes then tend to merge with other membranes where upon releasing their contents. The cellular uptake of liposomes occurs via endocytosis. In this process, portions of the cellular plasma membrane are pinched off as intracellular endosomes, so that the entrapped liposomes are delivered into the cell. As the pH drops within the endosome, the pH-sensitive liposomes fuse with the endosomal membrane and release their contents into the cytosol. This concept of pH-sensitive liposomes has been explored by the groups of Professors Dr. Rolf Schubert and Dr. Regine Süss. This project involved Sabine Barnert, Dr. Thomas Steenpass, Dr. Andreas Fritze, Dr. Ulrich Huth, Dr. Louma Kalie and Dr. Anmar Marouf as well as Dr. ZeljkaPavelic of the University of Zagreb, Croatia, who has been a postdoctoral fellow at our Department in 2002

 

Vanic' Z., Barnert S, Süss R, Schubert R: Fusogenic activity of PEGylated pH-sensitive liposomes, J. Liposome Res. 22(2), 148-157, 2012. [Epub]

 

 

Polymersomes

Polymersomes are membrane vesicles similar to liposomes, which can likewise be employed as drug carriers and model membranes. Moreover, polymersomes also feature a membrane consisting of a molecular double layer, with one of the layers being hydrophilic while the other layer is lipophilic. These amphiphilic molecules consist of polymers. Using different polymers, the thickness of the membrane bilayer can be modified. It is assumed that this feature may enable a better control of the release of encapsulated agents in vivo than is achievable with liposomal membranes. Moreover, the hydrophilic part of the molecules consists of polyethylene glycol (PEG), which prevents the accumulation of serum proteins after the systemic injection of polymersomes. As a result, their clearance by the phagocytes of the immune system (e.g., macrophages) - that most prominently settle in organs such as liver, spleen and lungs - occurs at much slower rates.

Consequently, polymersomes are expected to circulate in the blood for longer periods of time, which should enable their improved direction to the target tissue(s) of choice. Therefore, the group of Professor Dr. Rolf Schubert developed various methods for manufacturing polymersomes, characterized their vesicular structures by cyro-electron microscopy, and investigated their suitability as drug carriers (PhD thesis by Dr. Anja Rank). The group of Professor Dr. Regine Süss employed various polymersomes used for the purpose of gene transfer (PhD thesis by Dr. Stefanie Häfele).

Funding
This project was funded by the Volkswagen Foundation, Germany, and was carried out in close collaboration with three other groups: Professor Dr. Stephan Förster of the Institute for Physical Chemistry, University of Hamburg, Germany, was in charge of the overall project termed "Block-Copolymer Vesicles with Controlled Uptake and Release Functions for Drugs and Genes" (Volkswagen Foundation). The other participants were the groups of Professor Dr. Dr. h.c. Markus Antonietti, Max Planck Institute of Colloids and Interfaces, Potsdam-Golm, Germany, and Professor Dr. Christian Mayer, Institute for Chemistry, University of Duisburg-Essen, Germany.

 

Rank A, Hauschild S, Förster S, Schubert R: Preparation of monodisperse block copolymer vesicles via a thermotropic cylinder-vesicle transition, Langmuir 25, 1337-1344, 2009. [Epub]

Leson A, Hauschild S, Rank A, Neub A, Schubert R, Förster S, Mayer C: Molecular exchange through membranes of poly(2-vinylpyridine-block-ethylene oxide) vesicles, Small 3, 1074-1083, 2007. [Epub]

Borchert U, Lipprandt U, Bilang M, Kimpfler A, Rank A, Peschka-Süss R, Schubert R, Lindner P, Förster S: pH-induced release from P2VP-PEO block copolymer vesicles, Langmuir 22, 5843-5847, 2006. [Epub]

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