Non-viral nucleic acid vectors are commonly generated by the complexation of negatively charged nucleic acids with cationic polymers or cationic lipids. Thus electrostatic interactions hold the polyplex together. However - especially in vivo - the polyplex is faced with polyanions, e.g. negatively charged plasma proteins like soluble glycosaminoglycans or heparan sulfates of the extracellular matrix. Interaction of polyplexes with this polyanions can lead to vector unpackaging before the nucleic acid carrier reaches the target cell [52]. To ensure extracellular stability a siRNA delivery system containing covalently attached siRNA was synthesized. As linkage a bioreversible disulfide bond was chosen. After reaching the cytosol the intracellular reductive environment could break the bond and thus enable release of the siRNA. The siRNA was linked to PLL-PEG-DMMAn-Mel, which in the previous in vitro experiments turned out to be the most promising siRNA carrier. A schematic structure is shown in Scheme 3.
Scheme 3: Schematic structure of PLL-PEG-DMMAn-Mel-siRNA
Synthesis of PLL-PEG-DMMAn-Mel-siRNA was carried out similarly to the PLL-PEG- DMMAn-Mel synthesis. In brief, thiol-modified luciferase siRNA (sense strand modified at the 5’ end) was mixed with PLL-PEG-PDP. After reaction of thiol-siRNA with PDP-linkers, DMMAn-Mel was added to the mixture. To avoid aggregation of siRNA and DMMAn-Mel with PLL-PEG-PDP during synthesis, a high salt concentration (1.5 M NaCl) was applied. As previously mentioned PEG (MW 5000) could not be separated from branched PEI (25 kDa) by SEC. Because it was questionable if unbound siRNA (MW 13400) could be separated from the conjugate by size exclusion chromagraphy, a mock synthesis without covalent linkage was carried out. Unmodified siRNA was mixed with PLL-PEG-PDP and DMMAn-Mel.
After coupling of DMMAn-Mel the mixture containing PLL-PEG-DMMAn-Mel-PDP, free siRNA, DMMAn-Mel and low molecular weight impurities was loaded onto a Superdex 75 column. Fig. 33 and 34 clearly show that separation of the mixture was possible.
Fig. 33: Chromatogram of the PLL-PEG-DMMAn-Mel and siRNA-mixture. The mixture containing the siRNA-conjugate was loaded onto a Superdex 75 HR 10/30 column. The conjugate was eluted with 1M NaCl, 20mM Hepes, pH 8.2; flow rate: 0.5 ml/min. x-axis: elution volume; y-axis: absorption (dotted line: absorption at 260 nm. Solid line: absorption at 280nm). Peak 1: product conjugate; peak 2: unbound siRNA(see Fig. 34)
As expected mixing of siRNA and PLL-PEG-DMMAn led to complex formation (Fig. 34, lane 2). However electrostatic interactions could be eliminated by heparin (Fig. 34, lane 3). Free siRNA was only present in peak 2 (Fig. 34, lanes 6 and 7) and hence could be separated from the PLL-PEG-DMMAn-Mel conjugate by SEC.
After establishing the reaction and purification protocol, synthesis was conducted analogously with the thiol-modified siRNA. First thiol-modified GL3 luc siRNA was mixed with
1 2 21 bp (siRNA) mAU 4000 3000 2000 1000 0 mAU 4000 3000 2000 1000 0
Fig. 34: Agarose gel electrophoresis of peaks 1 and 2 from Fig. 33.1: 0.5 µg siRNA; 2: PLL-PEG-DMMAn- Mel siRNA polyplex (w/w 2); 3: like 2 plus heparin; 4: Peak 1; 5: Peak 1 plus heparin; 6: peak 2; 7: peak 2 plus heparin
PLL-PEG-PDP. After 1 h released pyridine-2-thione was measured at 343 nm to determine the degree of modification with siRNA (approximately 1.5 siRNA / 1 PLL). Subsequently DMMAn-Mel was coupled to the remaining PDP-groups and the siRNA-conjugate was loaded onto a Superdex 75 size exclusion column for purification. Fig. 35 shows the
chromatogram of the SEC purification.
Fig. 35: Chromatogram of the PLL-PEG-DMMAn-Mel-siRNA purification. The mixture containing
the siRNA-conjugate was loaded onto a Superdex 75 HR 10/30 column. The conjugate was eluted with 1M NaCl, 20mM Hepes, pH 8.2; flow rate: 0.5 ml/min. x-axis: elution volume; y-axis: absorption (dotted line: absorption at 260 nm. Solid line: absorption at 280nm). Peak 1: product conjugate; peak 2: unbound siRNA
In comparison with the chromatogram obtained after synthesis with unmodified siRNA (Fig. 33) a significant smaller amount of free siRNA (peak 2) was found, which is due to conjugation of a large part of thiol-modified siRNA to PLL-PEG-PDP. The fractions containing the conjugate were pooled and snap-frozen in liquid nitrogen. The purified PLL-PEG- DMMAn-Mel-siRNA conjugate had a molar ratio of approximately 1 / 1 / 7,5 / 1,5 (PLL / PEG / DMMAn-Mel / siRNA). This batch was used for the transfection experiments. A second batch was synthesized analogously, however after SEC-purification the conjugate was frist desalted by dialysis against HBG (pH 8.2). Not before desalting, this conjugate was snap- frozen in liquid nitrogen.
Additional, a conjugate containing Dharmacon’s non-targeting control#3 siRNA was synthesized for transfection experiments. This control-conjugate was purified and handled like the first batch. Briefly, after SEC-purification it was snap-frozen in liquid nitrogen without further desalting. The composition was comparable to the GL3 luc conjugate: 1 / 1 / 6 / 1.3 (molar ratio of PLL / PEG / DMMAn-Mel / siRNA).
1 2 mAU 4000 3000 2000 1000 0 mAU 4000 3000 2000 1000 0
1 2 3 4 5 6 7 8 9 10 11 12 13 1415
Covalent attachment of siRNA or rather absence of free siRNA in the conjugate were checked by agarose gel electrophoresis (Fig. 36).
Fig. 36: Agarose gel electrophoresis of purified PLL-PEG-DMMAn-Mel-siRNA conjugate. 1: 0.25 µg siRNA; 2: 0.5 µg siRNA; 0.5 µg siRNA + heparin + TCEP; 4: free lane; 5: PLL-conjugate (2.4 µl of peak 1, Fig. 35) 6: peak 1 plus TCEP; 7: peak 1 plus heparin; 8: peak 1 plus heparin and TCEP; 9: free lane; 10: PLL/siRNA-polyplex (w/w 2); 11: PLL/siRNA-polyplex plus TCEP; 12: PLL/siRNA- polyplex plus heparin; 13: PLL/siRNA-polyplex plus heparin and TCEP; 14: free lane; 15: 20µl of peak 2, Fig. 35
From untreated conjugate (Fig. 36, lane 5) no siRNA is released during agarose gel electrophoresis. Neither TCEP nor heparin pretreatment alone could induce a release of siRNA (Fig. 36, lane 6 and 7, respectively). Only if reducing conditions and heparin treatment are combined siRNA is released from the PLL-PEG-DMMAn-Mel-siRNA conjugate (Fig. 36, lane 8). In contrast, heparin treatment alone could already unpackage a conventional electrostatic complexed PLL/siRNA polyplex (Fig, 36, lane 12).