Physicochemical and biological characterization of polyethylenimine-graft-poly(ethylene glycol) block copolymers as a delivery system for oligonucleotides and ribozymes.

Two different series of polyethylenimine (PEI) block copolymers grafted with linear poly(ethylene glycol) (PEG) were investigated as delivery systems for oligodeoxynucleotides (ODN) and ribozymes. The resulting interpolyelectrolyte complexes were characterized with respect to their physicochemical properties, protection efficiency against enzymatic degradation, complement activation, and biological activity under in vitro conditions. The effect of PEG molecular weight and the graft density of PEG blocks on complex characteristics was studied with two different series of block copolymers. The resulting ODN complexes were characterized by photon correlation spectroscopy (PCS) and laser Doppler anemometry (LDA) to determine complex size and zeta potential. Electrophoresis was performed to study the protective effects of the different block copolymers against enzymatic degradation of ODN. Intact ODN was quantified via densitometric analysis. Ribozymes, a particularly unstable type of oligonucleotides, were used to examine the influence of block copolymer structure on biological activity. The stabilization of ribozymes was also characterized in a cell culture model. Within the first series of block copolymers, the grafted PEG chains (5 kDa) had marginal influence on the complex size. Two grafted PEG chains were sufficient to achieve a neutral zeta potential. Within the second series, size and zeta potential increased with an increasing number of PEG chains. A high number of short PEG chains resulted in a decrease in complex size to values comparable to that of the homopolymer PEI 25 kDa and a neutral zeta potential, indicating a complete shielding of the charges. Complement activation decreased with an increasing number of short PEG 550 Da chains. Ribozyme complexes with PEG-PEI block copolymers achieved a 50% down-regulation of the target mRNA. This effect demonstrated an efficient stabilization and biological activity of the ribozyme, which was comparable to that of PEI 25 kDa. PEGylated PEI block copolymers represent a promising new class of drug delivery systems for ODN and ribozymes with increased biocompatibility and physical stability.

Efficiency of polyethylenimines and polyethylenimine-graft-poly (ethylene glycol) block copolymers to protect oligonucleotides against enzymatic degradation.

Enzymatic instability of oligonucleotides (ON) is one of the major drawbacks of this new class of therapeutic agents. The development of safe, efficient delivery systems capable of stabilizing and protecting these molecules within the formulation, as well as during application, is a challenge in modern gene therapy. In the present study, polyethylenimine (PEI) of different molecular weights and PEGylated PEI block copolymers (PEI-g-PEG) were investigated with regard to their protective properties when complexes with chemically unmodified DNA (d-ON) and RNA (r-ON) oligonucleotides. PEI/ON complexes were incubated with different amounts of serum or nucleases. The influence of pH on the stability was studied and the integrity of the ON was determined by gel electrophoresis. The amount of stable ON within the gels was quantified via densitometric analysis. PEI homopolymers ranging from 800 to 2 kDa protected both types of ON very efficiently, whereas PEI 0.8 kDa demonstrated a slight decrease in protection. The PEGylated PEI derivatives generally protected ON as efficiently as the PEI homopolymers. In particular, the PEI-g-PEG derivative containing 100 PEG chains of 550 Da yielded the highest protection efficiency for both d-ON and r-ON. In general, the highest protection could be achieved at pH 6.7. The ratio of polymer and ON (N/P ratio) also had a great impact on ON stability with higher N/P ratios achieving a better protection. In conclusion, PEIs showed advantageous protective properties for ON. The results of this study offer indications for a rational design of PEI derivatives for the protection and the delivery of ON.

Stabilization of oligonucleotide-polyethylenimine complexes by freeze-drying: physicochemical and biological characterization.

In the present study the lyophilization of oligodeoxynucleotide-polyethylenimine (ODN-PEI) complexes was investigated regarding the maintenance of physicochemical properties and influence on biological activity. To achieve this, we used PEI of different molecular weights, in the range of 800-0.8 kDa, as complexing agents for unmodified ODN and ribozymes. The hydrodynamic diameter was measured by photon correlation spectroscopy (PCS) and the zeta potential was determined using laser Doppler anemometry (LDA) of ODN complexes with PEI derivatives of different molecular weights both before and after lyophilization. Atomic force microscopy (AFM) was used to visualize freshly prepared, stored and lyophilized complexes in solution. The biological activity of the ODN, as well as of plasmid DNA, in lyophilized PEI complexes was examined and compared to freshly prepared complexes using standard transfection assays. All PEI derivatives formed very small complexes with ODN displaying hydrodynamic diameters ranging from 15 to 30 nm. Marginal changes in size after lyophilization were observed for ODN-PEI complexes. In contrast, plasmid complexed with PEI was found to aggregate. In either cases minimal or no influence of the added amount of lyoprotectant was observed. The shape of the very small and highly condensed ODN complexes was not altered by lyophilization as seen in the AFM images. The transfection efficiency of lyophilized ribozyme-PEI complexes relative to freshly prepared complexes was approximately 100%, whereas a decrease was seen for lyophilized plasmid-PEI complexes. An additive of the lyoprotectants trehalose, mannitol or sucrose preserved biological activity. This study demonstrates the particular suitability of ODN-PEI complexes to be formulated as lyophilized systems with no loss in physical stability or biological activity.

Distribution and quantification of polyethylenimine oligodeoxynucleotide complexes in human skin after iontophoretic delivery using confocal scanning laser microscopy.

Iontophoresis may be a potentially useful technique for the delivery of oligonucleotides into the skin. To enhance intracellular uptake during iontophoresis, we investigated the dermal delivery of oligodeoxynucleotides (ODN) as a polyelectrolyte complex with polyethylenimine (PEI). Perpendicular cross-sectioning was performed to visualize and quantify the penetration properties of double labeled PEI/ODN complexes across full thickness human skin. Due to the net positive charge of the complexes, anodal iontophoresis was expected to enhance skin delivery by electrorepulsion compared to passive diffusion. Confocal laser scanning microscopy demonstrated that non-complexed ODN could penetrate the skin after 1 h of cathodal iontophoresis but not by passive diffusion or anodal iontophoresis. However, extensive degradation occurred as documented by a dramatic decrease of fluorescence intensity within viable skin tissue after 10 h. Anodal iontophoresis of the complexes led to a deep penetration of both the TAMRA-labeled ODN and the Oregon Green-labeled PEI. A constant increase in fluorescence indicated a protective effect of the polymer against nuclease degradation. Co-localization of red and green fluorescence was noted within numerous nuclei of epidermal keratinocytes. In contrast, passive diffusion of the complexes did not lead to successful uptake into keratinocytes and was limited to the stratum corneum. Complexation of ODN by PEI, therefore, seems to be a promising method to enhance both the transport of charged complexes into the skin and to facilitate intracellular uptake, which may potentially be useful for the local treatment of skin diseases using ODN.