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Date
2025-10-21
Authors
Publisher
Philipps-Universität Marburg
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Abstract
The aim of this work was to develop and characterize different non-viral transfection systems based on positively charged polymers such as bPEI, IPEI, PEI F25 and pDMAEMA. The focus of the investigations was to uncover and demonstrate a connection between the composition of the gene vehicles, their physicochemical properties and undesirable side effects. Of particular interest was to clarify the influence of the lipid shell in the formation of lipopolyplexes from polyplexes on their physical properties such as zeta potential, particle size or shape, but also the change in toxicity and transfection efficiency.
Through physicochemical characterization (PCS, zeta potential) and imaging techniques (AFM) we were able to demonstrate the successful complex formation and integrity of the polyplexes and lipopolyplexes produced. The positively charged polymers bPEI, IPEI, PEI F25 and pDMAEMA were complexed with the luciferase expressing plasmid pCMV-luc and the polyplexes were formed. When the complexes are formed, the N/P ratio is varied and the influence on the physical parameters is monitored. At low N/P ratios, the negative charge of the plasmid predominates in all polyplexes. It is often very difficult to measure the particle size or to visualize the resulting polyplexes using AFM technology. There is a broad particle size distribution, the formation of homogeneous polyplexes is not complete, and a phase-separated distribution of polymer and plasmid can often be observed. With N/P ratios greater than 10, the polyplexes stabilize and their tendency to aggregate decreases (increased PDI). The polymers have different optimal compositions with regard to the N/P ratio in order to have the smallest possible particle size with a moderate zeta potential. Optimally, with different N/P ratios, particle sizes of 200 nm and smaller can be produced with all polyplexes. Coating the polyplexes with lipids and forming the lipopolyplexes leads more quickly (at lower N/P ratios) to stable and spherically shaped complexes. The lipid layer shields the charge and prevents aggregation. With comparable N/P ratios and the polymers used, the lipopolyplexes are always slightly larger than the corresponding polyplexes with a lower net charge. The stability is also greater.
The question of the influence of the composition (N/P ratio, lipid coating) on toxicity is quite complex and difficult to answer. Depending on the application site, different criteria for toxicity can be defined or this is important for the application. In the present study, toxicological studies were carried out on the transfection systems using the MTT and LDH assay on SKOV-3 cells, the investigation of intrinsic coagulation using the determination of the activated partial prothrombin time (aPTT) and the determination of hemolysis. All positively charged polymers show a fundamental influence on body functions that trigger more or less toxic events. The general result of this work shows that a lipid coating on polyplexes has a positive effect on their toxic properties. The lipid shell has a shielding effect, which is particularly evident in the aPTT time and hemolysis. Positive charges have a negative effect on the parameters and lipids can compensate for this through their biocompatible properties. Lipopolyplexes always showed improved toxicity in the present studies. The properties of the IPEI, PEI F25 and bPEI polymers were largely comparable both as polyplexes and as lipopolyplexes. The latent toxicity of pDMAEMA could not always be adequately compensated. All polymers showed an N/P- ratio-dependent behavior for all toxicity parameters.
Finally, the transfection efficiency of pCMV-luc in SKOV-3 cells was tested with selected formulations. Here too, an increased efficiency of the lipopolyplexes compared to the corresponding polyplexes was observed. This can be seen as a result of the reduced toxicity, but also the improved biocompatibility.
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Metadata
Contributors
Dates
Created: 2025Issued: 2025-10-21Updated: 2025-10-21
Faculty
Medizin
Language
ger
Data types
DoctoralThesis
Keywords
Gentherapie
DFG-subjects
GenvektorenGentransfektionssysteme, nichtviral
DDC-Numbers
610
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Schlote, Patrick: Toxikologische Aspekte moderner nichtviraler Gen-Transfektionssysteme. : Philipps-Universität Marburg 2025-10-21. DOI: https://doi.org/10.17192/z2025.0453.
License
This item has been published with the following license: In Copyright