Technology

Our established platform technology for biotechnologically relevant lipid-nanoparticles enables the fabrication of liposomes and lipid-nanoparticles for various approaches. Biochemical and biophysical methods for the particles and quantitation of lipids and entrapped molecules are provided as shown in figure 1.

Figure 1: Cross Flow Injection Technique and liposome characteristics

  • Figure 2

By this technique, almost all variants of liposomes and lipid nanoparticles can be produced.

In figure 2 classification of liposomes and the surface modification strategies applied in each category. (A) Conventional liposomes simply contain: neutral, anionic, and cationic phospholipids; (B) stealth liposomes are PEGylated contain a polyethylene glycol (PEG) layer; (C) multifunctional liposomes have modified surfaces in addition to carrying imaging agent for diagnostic purposes (diagnosis and treatment functions); and, (D) targeted liposomes have modified surfaces through the attachment of targeting ligands (antibody, protein, peptide, small molecule, carbohydrate) [Trang Le et.al; DOI: 10.3390/ijms20194706]

Scalable technologies are still quite rare. In particular, for large and sensitive molecules, such as proteins and DNA/RNA. Within an industrial collaboration with Polymun Scientific, Klosterneuburg, Austria, we developed a promising, meanwhile patented, technique. The so-called Cross Flow Injection Technique (CFIT) is convenient to overcome known limitations. Relevant achievements are shown in table 1.

 

Achievements

Technique

Scalability with adjustable and controllable process parameters

Proven batch consistency

Applicable for all ethanol soluble membrane forming compounds Excellent encapsulation yield

Insignificant product damage

Application

Encapsulation / Integration of almost all molecules

Particles

Uni-lamellar vesicles with excellent stability properties

Homogeneous size distribution 


Table 1: Characteristics of the Cross-Flow Injection Technique 

For very small batches and screening studies, classical film hydration is well established as shown in figure 3.

Figure 3: Depiction of the Film Hydration steps

Projects

In recent years, our group has dealt with liposomes with a wide variety of tasks, as shown in table 2. Encapsulation of various recombinantly produced proteins with different properties, but also hydrophilic and hydrophobic peptides and marker substances were entrapped into different vesicles. Lipid composition was individually designed to optimize the encapsulation yield and /or for different distinct purposes.

Project

Objectives

Achievements

Encapsulation of rh-superoxide dismutase (rh-SOD) in various formulations (Fig.: 4)

Drug delivery system Clinical grade material Method optimization Constituent batch quality

New technique

Stable liposome format

Fully characterized batches

Membrane interaction studies of rh-erythropoietin (rh-Epo) with liposomes consisting of different compounds

Identification of membrane interacted protein domain Liposome transformation Lipid trigger

Identification of der membrane affecting protein domain (Fig.: 5)

Characterization of the membrane deforming parameters (Fig.: 6)

Table 2: Project overview 

Figure 4: rh-SOD

Figure 5: Amino-acid sequence of rh- Epo. Amino acids marked in dark grey correspond to the membraneaffecting domain (MAD-E) identified by mass spectroscopy. Trypsin cleavage sites are indicated by parentheses.

Figure 6: Transmission electron micrographs of (A) ULVs incubated above the transition temperature (Tm) without rh- Epo and (B) disc-like micelles in liposomal suspension appeared after the addition of rh-Epo to uni-lamellar EPG-vesicles (EPGULVs) and incubation above Tm. The black arrows mark disc-like micelles of about 30 nm in diameter.

 

HIV-vaccine for the intra-structural help vaccination approach (Fig.: 7)

 

PhD program of BioToP

Liposomes with entrapped model peptides

Decoration with the intact gp 140 HIV spike trimer

Process development characterization

Advanced, improved peptide encapsulation

Functional attachment of gp 140  HIV spike trimer

Extensive characterization

Successful cell assays

Figure 7: HIV vaccination approach strategy

 

Exploration of the membrane organization effect on weak acid transport proteins (Fig.:8)

 

PhD program of BioToP

Development of an optimized platform

Development of suitable artificial lipid vesicles

Transport measurements  of unknown membrane transporters

 

Innovation in membrane protein analytics

 

Figure 8. (A): artificial liposomes with controlled membrane composition are produced. They can be filled with substrates for the transport protein or detection systems, such as a pH indicator, fluorescent probes etc. Some lipids are biotinylated allowing the binding of the liposomes to magnetic beads. (B): the transport protein is in-vitro translated and directly incorporated into the liposomes. This avoids all issues related to denaturation and renaturation of these proteins. (C): the magnetic beads allow an easy buffer exchange, allowing to change the environment from the translation mix to the buffer condition in which the transport phenomenon shall be analyzed. (D): the liposomes can finally be isolated and the contents and supernatants individually analyzed.

Charaterization

For the different formulations a repertoire of different methods is established. In this regard biophysical, chromatographic and sensor-based techniques are available. Most of them are easily adjustable for new tasks.

Technique Application
Flow Cytometry Size and size distribution
Dynamic light scattering Size and size distribution, zeta potential
Differential scanning calorimetry Phase transition temperature profiling
Electron microscopy Visualization of vesicle population
Chromatography (HPLC equipped
with different detectors)		 Lipid identification, quantitation and degradation
Protein encapsulation
Biolayer Interferometry Protein-Lipid-Membrane Interaction