In this study, we synthesized, characterized and tested a mannosylated pegylated PEI for potential delivery of siRNA. Pegylation has been previously shown to be of significant value in increasing the circulation time of nanoparticles and polyplexes (Brus et al., 2004
; Owens and Peppas, 2006
; Yamaoka et al., 1994
). Mannose has previously been demonstrated to significantly increase binding of particles to cells that express the mannose receptor (Diebold et al., 1999a
; Diebold et al., 1999b
; Diebold et al., 2002
; Hashimoto et al., 2006
; Jiang et al., 2009a
; Park et al., 2008b
; Zhou et al., 2007a
). To our knowledge, this is the first study to evaluate mannosylated pegylated PEI for siRNA delivery and it is the first study to characterize the effect of the location of the mannose ligand in mannosylated pegylated PEI constructs on knockdown efficiency.
PEG and mannose were either both directly conjugated onto the PEI backbone or mannose was conjugated to the PEI via a PEG spacer. 1
H NMR spectra’s confirmed that both constructs had PEI, PEG and mannose present and the peaks strongly corresponded to previously reported values (Handwerger and Diamond, 2007
; Sagara and Kim, 2002
). The signal for mannose at 7 ppm was weak due to the relatively low proportion of mannose in the overall construct composition. For this reason, we used the resorcinol assay to quantify the amount of mannose present in each construct.
The surface chain density of PEG is a critical factor in improving stealth shielding of nanoparticles and polyplexes. The PEI to PEG ratio of PEI-PEG was 3.704, which suggests that every 25kDa PEI chain has 3.45 chains of 2kDa PEG. PEI-PEG-mannose had a 1.23 PEI/PEG ratio indicating 10.16 PEG chains per PEI. The 0.962 PEI/PEG ratio of mannose-PEI-PEG suggests that there are 13.3 PEG chains for each PEI. PEG chains have a larger range of motion at low surface coverage, that can lead to gaps in the PEG protective layer (Storm et al., 1995
). For PEG chains to fully cover the surface of PEI/siRNA polyplexes, six short PEGs (5kDa) or one long PEG (20kDa) are needed (Brus et al., 2004
; Petersen et al., 2002
). Therefore, 3.45–13.3 chains of 2kDa PEG is expected to provide a satisfactory level of pegylation for steric stabilization. The 0.19μmol/mg of mannose in mannose-PEI-PEG and 0.12μmol/mg in PEI-PEG-mannose represent an average modification of 4.7 and 3.0 molecules of mannose per PEI, respectively. This quantity of mannose is expected to be sufficient for selective binding of mannose receptors on cells.
Polymer/siRNA polyplexes exhibited sizes in the range of 169.10nm and 357.33nm. This particle size range is suitable for efficient endocytosis by RAW264.7 cells. Larger particles outside of this range observed by SEM are likely clusters or aggregates of these smaller particles. The zeta potentials of PEI-PEG-Man/siRNA polyplexes showed positive values that were approximately 21.63mV and similar to unmodified PEI and pegylated PEI. This result suggested that PEI-PEG-Man could form stable polyplexes with siRNA. The zeta potential of Man-PEI-PEG/siRNA polyplexes was relatively low compared to PEI-PEG-Man/siRNA polyplexes. This could explain the lower cellular uptake of Man-PEI-PEG observed in our intracellular trafficking studies. The lower zeta potential could lead to weaker interactions with siRNA and the cell surface, which in turn could lead to decreased endocytosis of the polyplexes.
Branched PEI was selected as the backbone for our system because the complexation of branched PEI with siRNA has been reported to exceed that of linear PEI (Breunig et al., 2008
). As a result, in gel retardation assays, no reduction in siRNA condensation properties was found with PEGylated PEIs. Overall, complete binding of siRNA was achieved with all the various constructs at N/P ratios of 3 and higher.
We selected RAW264.7 cells for evaluation of polyplex uptake, trafficking and knockdown because the RAW264.7 cells are a murine macrophage cell line that are known to express mannose receptors and that are considered to be typically hard to transfect. Macrophages are also a potential target for our pegylated mannosylated PEI delivery system (Diebold et al., 2002
In confocal images, the cells endosomes/lysosomes were stained with Lysotracker Green™
. Co-localization of the green signals with red signals associated with the Cy-3 labelled siRNA indicated that polyplexes were being internalized by endocytosis, which is consistent with previous reports on uptake by PEI/nucleic acid polyplexes (Lecocq et al., 2000
). In addition, separate red signals seen in some cells were most likely due to siRNA that had released from the endosomal compartments. Thirty minutes after incubation, polyplexes had been internalized by cells and localized in cytoplasm. From another cellular uptake study using green fluorescent-labeled (Oregon Green 488) polymers and red fluorescent-labeled (Cy3) siRNA, we observed the two signals were separated in the cytoplasm (data not shown) 2 hours after transfection. This result indicated that siRNA complexes had been released from the lysosomal vesicles as well as from the polymers and were distributed in the cytosol. This release of siRNA is purported to be due to the proton sponge effect, which causes rupture of the endosomes because of the PEI’s strong buffering capacity (Boussif et al., 1995
). Intracellular trafficking plays an important role in the fate of siRNA polyplexes because their spatial distribution does not correspond to simple diffusion (Jen and Gewirtz, 2000
). Perinuclear localization of siRNA, as seen in , is required for successful gene silencing by interaction with RISC to induce RNAi. Interactions with RISC dictate siRNA localization even when siRNA is conjugated to cell-binding ligands such as the TAT peptide (Chiu et al., 2004
). It suggests that the mannosylated pegylated PEI polymers developed in this study successfully protected siRNA during endocytosis and lysosomal escape in order to integrate siRNA into the RISC complex for correct RNAi processing.
Mannosylation and pegylation of PEI did not reduce gene knockdown efficiency relative to unmodified PEI showing no significant difference in relative gene expression. The advantages of pegylating polyplexes and incorporating cell binding ligands for in vivo
applications have been well established (Beyerle et al., 2010
; Ogris M, 1999
; Owens and Peppas, 2006
; Sagara and Kim, 2002
; Webster et al., 2007
; Yamaoka et al., 1994
). Furthermore, not only did pegylation have no adverse effect on knockdown efficiency, but it also reduced toxicity. This reduction in toxicity was slightly offset by the mannosylation but not significantly so. In addition to observing a reduction in cellular toxicity, we anticipate that pegylation of the polyplexes will also reduce toxicity at the systemic level by reducing aggregation of the polyplexes and therefore the capillary embolism that has been associated with the use of unmodified PEI (Intra and Salem, 2008
; Ogris M, 1999
One aspect of mannosylated pegylated PEI delivery systems that has not been explored before is the importance of the location in which mannose is bound to the construct. In our studies, PEI-PEG-mannose/siRNA polyplexes resulted in higher toxicity and higher knockdown efficiency than mannose-PEI-PEG. Furthermore, qualitatively we observe that PEI-PEG-mannose/siRNA polyplexes have more stable uptake by RAW264.7 cells than mannose-PEI-PEG/siRNA polyplexes. This observation is not clearly understood but could be explained by the structural difference between two constructs. The PEI-PEG-mannose construct has mannose moieties exposed at the tip of PEG chain whereas the mannose in mannose-PEI-PEG could be hindered by the PEG chains. Thus the mannose ligand-receptor interaction could be obstructed by the shielding effect of PEG chains. The use of a PEG chain can impair cell binding by shielding not only PEI but also the ligand (Kunath et al., 2003a
). We carried out cellular uptake time-lapse studies at various time points including 0.5, 1, 2, 4, 8, and 24 hours (data not shown). Throughout the course of experiment, we observed that PEGylated PEI had delayed endocytosis relative to PEI alone. The reason that PEG-PEI without mannosylaton at an N:P ratio of 3 still has better luciferase knockdown than either the mannose-PEI-PEG or PEI-PEG-mannose constructs could be attributed to its better cytotoxicity profile or the use of an optimized degree of pegylation for the PEI-PEG construct.
The endogenous knockdown generated by our modified PEIs was significantly less effective than RNAiMax at targeted mRNA reduction. In addition, in our hands, TransIT-TKO can routinely provide 80–90% knockdown, which is significantly more effective than the modified PEIs (data not shown).
The modified PEIs were more efficient at luciferase knockdown than that of another commercial transfection reagent, siLentFect, which was used following the manufacturers guidelines. Moreover, both mannose and PEG have well established attributes for in vivo delivery such as selected cell binding, reduced systemic toxicity and enhanced circulation. In particular, mannose ligands have shown significant potential for binding antigen presenting cells (APCs) such as mouse macrophages and dendritic cells. Furthermore, PEGylated PEI/siRNA complexes have been reported to have decreased random uptake into non-specific organs including liver and spleen compared to unmodified PEI.
Finally, the determination of the optimal location of cell binding ligands in delivery construct in this study is expected to have important implications in the design of several plasmid DNA, oligonucleotide and siRNA delivery systems currently in development that utilize alternative cell binding ligands and alternative cationic backbones such as chitosan.