Publikationsserver der Universitätsbibliothek Marburg
Titel:Biodegradable amphiphilic PEG-PCL-PEI triblock copolymers designed for the self-assembly of multifunctional gene carriers
Autor:Endres, Thomas
Weitere Beteiligte: Kissel, Thomas (Prof. Dr.)
Veröffentlicht:2012
URI:https://archiv.ub.uni-marburg.de/diss/z2012/1104
DOI: https://doi.org/10.17192/z2012.1104
URN: urn:nbn:de:hebis:04-z2012-11044
DDC: Naturwissenschaften
Titel (trans.):Bioabbaubare amphiphile PEG-PCL-PEI Triblock-Copolymere zur Herstellung multifunktionaler Genvektoren
Publikationsdatum:2013-02-22
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
Quantum dot, Fluoreszenz-Resonanz-Energie-Transfer, RNS-Interferenz, siRNA-Formulierung, Micelle, Micelle, Self assembly, Gefriertrocknung, Copolymer, Quantenpunkt, Gentherapie, Lyophilisation, Cationic triblock copolymer, In vitro, In vivo

Summary:
The great promise that gene therapy holds is the opportunity of directly introducing genetic material into cells for a causal therapy of yet incurable diseases. One promising way to achieve that goal is the usage of non-viral delivery vehicles, constructed from amphiphilic block copolymers. This thesis presents the establishment of a multifunctional PEG-PCL-PEI block copolymer platform, designed for multifunctional gene delivery. Across the scientific disciplines of chemistry, chemical physics, pharmacy and biomedicine the underlying work covers all aspects of non-viral gene delivery: In a first step, block copolymers were synthesised and characterised. In a systematic approach a library of compounds with varying hydrophilic/hydrophobic ratio was established. Subsequently, polymers were assembled into gene carriers followed by a non-invasive structural characterisation. Most importantly it was noticed, that polymers hydrophilic in nature formed smaller micelle-like carriers, whereas hydrophobic polymers aggregated to larger particle-like assemblies. In that vein, carrier features such as colloidal stability and toxicity were found to depend on chemical composition. Second, the nucleic acid loading process was optimised. Herein it was the overall goal to manufacture compactly condensed carrier complexes by understanding the basic principles of the electrostatic loading procedure. It was hypothesised that a more homogeneous fusion of charges is supposed to lead to superior carrier complexes. In that line, a microfluidic mixing technique, bringing cationic polymer and nucleic acid together at a constant ratio during the entire mixing process, was found to be the most promising technique. Ultimately, gene delivery carriers with superior colloidal stability, RNA protection and transfection efficiency were manufactured and process parameters were optimised with the help of a central composite design. Third, as a prerequisite for effective in vivo usage, carriers were transferred into stable ready-to-use formulations by lyophilisation with the help of glucose as a lyoprotectant. Unloaded nano-suspensions could be restored after rehydration by addition of small amounts of glucose. Upon loading of those rehydrated carriers, no significant difference in complex size or transfection efficiency was observed as compared to freshly-prepared ones. Moreover, the stabilisation of pre-formed carrier/siRNA complexes is feasible at elevated N/P and higher glucose concentrations. Fourth, most promising carriers where tested for their in vitro and in vivo transfection efficiency. Vectors constructed from rather hydrophobic block copolymers showed superior transfection efficiency, whereas poor performance was found in case of predominantly hydrophilic ones in a good correlation in vitro and in vivo. FACS studies revealed that this might possibly be due to reduced cell uptake of carriers with thicker PEG shell preventing cell interaction. In that way a yet active vector with diminished toxicity as compared to PEI homopolymers was evaluated. Fluorescent microscopy images of murine lung tissue revealed emission predominantly in the alveolar region, rendering this carrier system as promising for local treatment of airway diseases. Finally, the feasibility of multifunctional carrier co-loading and FRET-monitored nucleic acid unpacking was approved. Double-labelled nano-carriers emitted light at the acceptor’s emission wavelength upon donor excitation, proving successful FRET-effect and hence, complex integrity. The ability of dual loading is especially useful for “theranostic” purposes or co-delivery of nucleic acids and drugs. FRET-switching functionality may be advantageous for monitoring complex stability and nucleic acid unpacking. In view of prospective experiments, to circumvent the observed charge-toxicity-relationship, carriers are supposed to be taken up by the target tissue in a selective way. This can be achieved, up to a certain degree, via targeting ligands. Rather hydrophilic carriers with thicker PEG shells, increased colloidal stability and reduced toxicity represent the ideal candidates for this modification. In the long term, required work is evident: the development of more effective non-viral vectors and conquering the yet severe toxicity effects of current delivery systems. By a deeper understanding in the mechanistic aspects of the gene delivery process plus a rational vector design we are on the right track to achieve that goal.

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