Publikationsserver der Universitätsbibliothek Marburg

Titel:Biodegradable multifunctional nanocarriers for pDNA and siRNA delivery
Autor:Zheng, Mengyao
Weitere Beteiligte: Kissel, Thomas (Prof. Dr.)
Veröffentlicht:2012
URI:https://archiv.ub.uni-marburg.de/diss/z2012/0769
DOI: https://doi.org/10.17192/z2012.0769
URN: urn:nbn:de:hebis:04-z2012-07697
DDC:610 Medizin
Titel (trans.):Bioabbaubare multifunktionelle Nanotransporter für pDNA- und siRNA-Delivery
Publikationsdatum:2012-10-05
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
siRNA, DNA, Gene delivery, DNA, Gen Transfektion, Gene transfection, siRNA

Summary:
In this thesis, biodegradable non-viral polymeric nucleic acids delivery vectors were characterized concerning biophysicochemical parameters. In the first part of this research, to answer the questions: why the principle of DNA transfection cannot be directly applied for siRNA transfection, we investigated the complexation and aggregation mechanism of nucleic acids/polycations on the atomic and molecular scale. The MD and ITC data showed us the different nature and the different hierarchical mechanism related polycation-siRNA and polycation-pDNA complexes. All our results emphasized one point: lower N/P-ratios are especially effective for polycationic nanocarrier-based siRNA delivery, because siRNA aggregation results in a more uniform and stable complex formation at low N/P ratios already, which lead to increased siRNA delivery efficiency. This could have broad implications for the delivery of siRNA as less toxic and yet efficient delivery systems have been the bottle-neck for the translation of this promising approach into the clinical arena. In chapter 3, novel biodegradable amphiphilic copolymers hy-PEI-g-PCL-b-PEG were prepared by grafting PCL-b-PEG chains onto hyper-branched poly(ethylene imine) as non-viral gene delivery vectors. With the question: how can the graft densities of PCL-b-PEG chains influence the in vitro DNA delivery efficiency, our study began with the characterization of physico-chemical properties and expected that with the introducing of the grafted PCL-b-PEG chains, the in vitro DNA delivery efficiency with the grafted PCL-b-PEG chains could be improved. Of all the experimental results, buffer-capacity has almost exactly the same tendency as transfection efficiency. We assume that in all processes of DNA transfection, the endosomal escape has a really important and rate-limiting role. This opens new perspectives to advance the rational design of new gene delivery systems. The further investigation of these biodegradable grafted amphiphilic copolymers hy-PEI-g-(PCL-b-PEG)n as potential siRNA delivery vectors was showed in chapter 4. The purpose in this section was to enhance the in vivo blood circulation time and siRNA delivery efficiency of biodegradable copolymers polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol) (hyPEI-g-PCL-b-PEG) by introducing high graft densities of PCL-PEG chains. Our study indicated that the effect of PEG on prolonged circulating depends not only on its content in a copolymer (length or percentage), but also on the structure or the shape of the amphiphilic copolymer. We demonstrated that polymeric micelles, which are formed with amphiphilic block polymers have advantages especially for in vivo siRNA delivery, and that the graft density of the amphiphilic chains can enhance the blood circulation, which is a key parameter to promote the development of safe and efficient non-viral polymeric siRNA delivery in vivo. Although the copolymers hy-PEI-g-(PCL-b-PEG)n showed positive results as pDNA and siRNA delivery vectors in chapter 3 and 4, the delivery of gene materials with these non-targeted copolymers is achieved mainly passively by the passive targeting. Therefore, to optimize these polymeric gene delivery vectors with targeting function, in chapter 5, folate conjugated PEI-g-PCL-b-PEG was examined for targeted gene delivery. Lower cytotoxicity was observed for PEI-g-PCL-b-PEG-Fol than PEI-g-PCL-b-PEG and the cellular uptake of polyplexes was enhanced by PEI-g-PCL-b-PEG-Fol in FR over-expressing KB cells compared with those by PEI-g-PCL-b-PEG. Importantly, this enhancement was inhibited by free folic acid, while did not appear in FR-negative A549 cells. All these suggested the specific cell uptake of PEI-g-PCL-b-PEG-Fol/pDNA polyplexes via folate receptor-mediated endocytosis. Consequently, PEI-g-PCL-b-PEG-Fol/pDNA polyplexes revealed higher transfection than PEI-g-PCL-b-PEG/pDNA. Additional studies on gene transfection in vivo and utilizing these described folate-conjugated copolymers for targeted siRNA delivery are in proceeding. In Chapter 6, the novel siRNA delivery systems based on hyperflexible generation 2-4 triazine dendrimers was identified by correlating physico-chemical and biological in vitro and in vivo properties of the complexes with their thermodynamic interaction features simulated by molecular modeling and the influence of dendrimer flexibility has systematically been investigated and discussed. In this study, molecular modeling helped to understand experimental parameters based on the dendrimers’ structural properties and molecular imaging non-invasively predicted the in vivo fate of the complexes, both techniques can efficiently support the rapid development of safe and efficient siRNA formulations that are stable in vivo.

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