Co-delivery of small interfering RNA and plasmid DNA using a polymeric vector incorporating endosomolytic oligomeric sulfonamide
Introduction
RNA interference (RNAi) has been recognized as a promising therapy to induce sequence-specific and post-transcriptional silencing via the cleavage of homologous mRNA [1], [2] to treat cancers, infectious diseases, and CNS diseases [1]. RNAi is most often mediated by short hairpin RNAs (shRNA) or small interfering RNAs (siRNA) [2]. The shRNA is expressed after nuclear delivery of shRNA-expressing plasmid DNA (pDNA) and the duration of shRNA expression depends on the use of viral or non-viral vectors [2], [3]. The transient expression of shRNA carried by non-viral vectors is strongly influenced by dilution of the vector following cell proliferation and by the stability of siRNA in the cytosol [2], [3]. The use of siRNA has several advantages over shRNA. siRNA delivery avoids the barrier of the nuclear membrane as it acts in the cytosol, unlike shRNA-expressing pDNA [2], [3]. More importantly, siRNA duplexes are readily available from various sources via custom or pre-designed siRNAs targeting a particular mRNA of interest.
As unaided delivery of naked siRNA is very limited due to its anionic nature and resulting low permeability to plasma membranes [2], [4], polymer-based (polyplex and polymersome) or lipid-based (lipoplex and liposome) non-viral vectors are often used to aid in siRNA delivery [2], [5], [6], [7], [8]. While lipoplexes often aggregate or are unstable over time [9], polymeric siRNA vectors are more attractive due to their chemical diversity and ease of modifications [4], [10] for cell targeting, endosomal escape [11], and cytosolic release [8], [12], [13] of siRNA.
However when cationic polymers are used, siRNA frequently results in more loosely condensed and larger complexed particles than are seen under similar complexing conditions when used for pDNA polyplexes of 100–200 nm in size. Along with size concerns for use in in vivo tests, these less stable polymeric siRNA polyplexes were prone to premature dissociation of siRNA from the counterpart polycation [14]. This is the result of the siRNA chain being short and rigid [14], unlike long and flexible pDNA. In general, the electrostatic strength between polycations and polyanions (here, nucleic acids) increases with higher molecular weights (MW) [14], [15]. These short siRNAs (usually duplexes of 21–23 nucleotides) may result in one hundredth of the electrostatic strength of pDNA (more than 2000 base pairs) in an association with a given polycation [16]. Thus, to form stable and nanoscale siRNA polyplexes, one should adopt higher molecular weight (MW) polymers or high polycation/siRNA ratios [10]. However, excess polycation induces polycation-mediated cytotoxicity [17]. Alternatively, siRNA can be elongated by concatemerization with sticky overhangs [16] or multimerization via chemical linkages [18]. To avoid off-target silencing effects of lengthy siRNAs, these multimeric siRNAs can be designed to be cleaved into monomeric siRNAs in the cytoplasm.
Instead of modifying siRNAs, some groups have incorporated polyanions as an additive [19], [20]. The Huang group added calf thymus DNA or hyaluronic acid (HA) when formulating siRNA polyplexes with protamine and then the siRNA/protamine/DNA (or HA) nanoparticles were encapsulated in liposomes [19], [20]. This strategy resulted in a particle size reduction by 10–30% and improved silencing efficiency 20–80% when compared with siRNA formulations without polyanion additives [5], [19], [20], [21].
In this study, polymer-based siRNA carriers were used to exclude the instability of liposome-based formulations. Poly (l-lysine) (PLL) was selected as a representative polycation. Oligomeric sulfonamides (OSA) with known endosomolytic activity that have been developed by our group [22] were also introduced into the PLL-based siRNA complexes. In addition, to enhance the stability of polymeric siRNA complexes as well as to examine the potential of including multiple therapeutics in a single drug carrier, a reporter pDNA was employed as a lengthy polyanion instead of calf thymus DNA or HA. This study aimed to elucidate how the addition of pDNA in PLL/siRNA-pDNA and PLL/siRNA-pDNA-OSA complexes modulates their physicochemical, biological, and therapeutic characteristics.
Section snippets
Materials and cell culture
Poly(l-lysine) hydrogen bromide (PLL∙HBr; MW 24 kDa), branched polyethyleneimine (bPEI; Mw 25 kDa, Mn 10 kDa), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dulbecco’s Modified Eagle’s Medium (DMEM), Ca2+-free and Mg2+-free Dulbecco’s phosphate buffered saline (DPBS), dimethyl sulfoxide (DMSO), 4-(2-hydroxy-ethyl)-1-piperazine (HEPES), d-glucose, sodium bicarbonate, ethidium bromide (EtBr), aphidicolin, heparin sodium salts (149 USP units/mg), 4′,6-diamidino-2-phenylindole
Physicochemical properties and transfection of PLL/siRNA complexes
Prior to the investigation of PLL/siRNA-pDNA complexes, the physicochemical and transfection properties of the PLL/siRNA complexes were studied as a reference. PLL was used to condense siRNA over a complexation range of 2 ≤ C/A (cation/anion) ≤ 20 (Fig. 1(A)). The PLL/siRNA complexes were retained well when run on an agarose gel. However, a weak EtBr intensity (approximately 10–20% of the EtBr intensity when EtBr is intercalated with all of the siRNA) from the polyplexes was still detected.
Conclusion
This study was designed to produce stable and dense nanocomplexes by complexing PLL with siRNA and pGFP at low C/A ratios. The resulting PLL/siRNA-pGFP complexes were compact and induced effective gene silencing. OSA-containing PLL/siRNA-pGFP complexes were better at inducing silencing than their OSA-free counterparts. In addition to silencing a target mRNA, the polyplexes were able to express GFP. Thus, the polyplexes investigated in this study may have the potential to silence an mRNA of
Acknowledgements
This work was partially supported by National Institutes of Health, USA (NIH GM82866).
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