Modified Poly(ethylene imines) for plasmid delivery: Physico-chemical and in vitro/in vivo investigations

This thesis describes the development of poly(ethylene imine) (PEI) conjugates as vector systems for plasmid delivery. Conjugates were synthesized and characterized regarding their suitability as non-viral vector systems for in vivo administration. Chapter 1 gives a detailed overview of the curren...

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Bibliographic Details
Main Author: Neu, Michael
Contributors: Kissel, Thomas (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:English
Published: Philipps-Universität Marburg 2006
Pharmazeutische Technologie und Biopharmazie
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Summary:This thesis describes the development of poly(ethylene imine) (PEI) conjugates as vector systems for plasmid delivery. Conjugates were synthesized and characterized regarding their suitability as non-viral vector systems for in vivo administration. Chapter 1 gives a detailed overview of the current status of polycationic gene delivery systems based upon PEI and PEI derivatives. Basic knowledge about PEI based vectors is imparted and the range of PEI modifications currently under investigation is described in depth. In Chapter 2, a novel gene delivery vector for lung administration is investigated. The conjugate is based upon a protein transduction domain, derived from the HIV TAT peptide, coupled to branched PEI via a PEG linker. The HIV TAT transduction domain is supposed to provide direct crossing over biological membranes with high translocation ability and, therefore, was hypothesized to also enhance cell uptake of plasmid DNA in the lungs. The novel conjugate was able to form very small and stable particles with plasmid DNA, which is favorable for airway administration. A ~600% improved gene expression in the mouse lung was observed for TAT-PEG-PEI polyplexes in comparison to unmodified PEI. Furthermore, only minor effects upon lung function were observed, with no additional inflammation compared to pDNA instillation alone. A particular advantage of this carrier is its ability to transport DNA safely into the different cell types of the lung. This new carrier fulfills most of the key requirements for lung administration, namely being non-toxic and highly efficient in transfecting the epithelial cells of the conducting and respiratory airways. These results highlight that the mechanistic investigation of PEI-coupled protein transduction domains is promising in the development of stable vectors for lung administration. In Chapter 3, stabilized polyplexes of HMW (high molecular weight) and LMW (low molecular weight) PEI were developed and investigated with regard to the molecular weight of the polymers and the formation procedure. It was theorized that crosslinking the primary amines of PEI would lead to enhanced polyplex stability suitable for intravenous administration. The polymers were crosslinked using a homobifunctional linker with intrinsic redox sensitive degradation properties. Two strategies to form the polyplexes were compared. Only crosslinking after polyplex formation was able to enhance resistance against polyanion exchange and high ionic strength. These polyplexes also displayed significant reduced interactions with major blood components like albumin and erythrocytes. These results highlight the influence of the polymer molecular weight and the formulation strategy for the formation of stable vectors. In Chapter 4, the bioreversibly surface crosslinked HMW PEI polyplexes were investigated in more detail. We postulated that the intracellular redox conditions, mainly determined by the glutathione status, would influence the release properties of the DNA from the polyplex and thereby also the transfection efficiency. Indeed, the biodegradable disulfide bonds which were introduced showed a strong susceptibility to reducing conditions. Pharmacokinetic profiles of PEI/plasmid polyplexes in mice after intravenous administration showed higher blood levels for crosslinked polyplexes, indicating successful stabilization. Unwanted lung transfection was significantly reduced, while liver transfection remained at higher levels. These studies suggest that crosslinked polyplexes are more stable in circulation and retain their transfection efficiency after intravenous administration, but careful adjustment of the stabilization degree is required. In Chapter 5, the concept of surface stabilization was combined with the shielding concept using Poly(ethylene glycol) (PEG). It was hypothesized that the charge and steric shielding effect of PEG-PEI copolymers in combination with surface crosslinking would alter the stability of the polyplexes and their pharmacokinetic behavior under in vivo conditions. Cell culture experiments revealed high transfection efficiency of copolymer polyplexes, up to 5-fold higher than PEI for the copolymers built with 30 kDa PEG. Intravenous injection into mice revealed higher blood concentration of plasmid complexed with PEI-PEG(30k) with 1 PEG chain, indicating successful polyplex shielding. These polyplexes were further stabilized by surface crosslinking using DSP. Indeed, blood levels of plasmid could be further elevated up to 125% higher as with PEI directly after injection and persisted at higher values up to 60 min (+40%). These results highlight that a combined strategy to build stable vectors for intravenous administration is possible and promising for systemic administration.
DOI:https://doi.org/10.17192/z2007.0089