Designed guanidinium-rich amphipathic oligocarbonate molecular transporters complex, deliver and release siRNA in cells

Abstract

The polyanionic nature of oligonucleotides and their enzymatic degradation present challenges for the use of siRNA in research and therapy; among the most notable of these is clinically relevant delivery into cells. To address this problem, we designed and synthesized the first members of a new class of guanidinium-rich amphipathic oligocarbonates that noncovalently complex, deliver, and release siRNA in cells, resulting in robust knockdown of target protein synthesis in vitro as determined using a dual-reporter system. The organocatalytic oligomerization used to synthesize these co-oligomers is step-economical and broadly tunable, affording an exceptionally quick strategy to explore chemical space for optimal siRNA delivery in varied applications. The speed and versatility of this approach and the biodegradability of the designed agents make this an attractive strategy for biological tool development, imaging, diagnostics, and therapeutic applications.amphipathic co-oligomers | nanoparticles | oligonucleotide delivery | biodegradable oligomers | organocatalysis R NA interference (RNAi) is an emerging technology that is revolutionizing many strategic approaches to biochemical pathway analysis, drug discovery, and therapy (1-6). As part of the RNAi pathway, small interfering RNAs (siRNAs) induce post-transcriptional, sequence-specific gene silencing utilizing endogenous intracellular machinery to selectively suppress gene expression and, thereby, reduce target protein synthesis (7). The net effect is equivalent to protein inhibition without the use of small molecule inhibitors. The specificity of RNAi also allows one to make inhibitors against previously undruggable targets. Both the ubiquity of the RNAi pathway within the body and the ease with which siRNA can be used to suppress a specific target of interest have made siRNAs a promising class of molecules for the treatment of cancer, viral infections, ocular disorders, and genetic diseases (5). In 2004, the first siRNA-based therapy entered Phase 1 clinical trials (4). Since then, several other RNAi-based therapies have reached clinical evaluation for a number of indications including cancer, viral infections, and genetic skin disorders (5,8,9). Notwithstanding this progress, formidable challenges remain for the application of RNAi technology in basic research and therapy, the most fundamental of which is delivery of siRNA across biological barriers.The siRNAs are double-stranded RNA molecules typically consisting of a 19-23 base-paired region with two 3′ overhanging nucleotides. It is polyanionic, polar, and large (ca. 13 kDa), compared to small molecule therapeutics. These physical properties suppress or prevent its unassisted passage through nonpolar membranes and, thus, its access to the intracellular RNA-induced silencing complex (RISC) components required for target protein knockdown (6). This problem is further exacerbated by siRNA's susceptibility to enzymatic degradation (i.e., RNases) (3). To address these problems, two strategies have been pursued: d...

Charge-altering releasable transporters (CARTs) for the delivery and release of mRNA in living animals

“…GPC analysis of cationic diblock 11b after exposure to pH 7.4 PBS showed a diminished molecular weight (4.3 kDa) that was nearly identical to the homooligomer 10 (4.6 kDa), suggesting, in line with the proposed mechanism, that at physiological pH the cationic portion of the CART degrades whereas the lipophilic block remains intact. Complexes formed with α-amino ester homooligomer A 13 9 induced no EGFP expression, consistent with our prior work on amphipathic oligocarbonates for which a hydrophobic domain was necessary for siRNA delivery (46). Contrasting their efficacy in delivering siRNA, complexes formed with guanidinium-functionalized oligocarbonates D 4 :G 4 12 and D 13 :G 12 13 resulted in no detectable EGFP expression.…”

“…Previous work on nucleic acid delivery has highlighted the importance of lipophilic domains on delivery vehicles to facilitate cargo binding and membrane interaction leading to cellular internalization (46)(47)(48). This requirement is readily addressed with our living OROP approach, because both lipophilic and charged blocks can be incorporated without additional synthetic steps.…”

“…Contrasting their efficacy in delivering siRNA, complexes formed with guanidinium-functionalized oligocarbonates D 4 :G 4 12 and D 13 :G 12 13 resulted in no detectable EGFP expression. Relative to the rapid self-immolative rearrangement (t 1/2 = 2 min) of CARTs, oligocarbonates 12 and 13 degrade slowly by passive hydrolysis (t 1/2 = 8-12 h) (46,49), establishing a strong correlation between transporter degradation rate and mRNA expression. Collectively, the exceptional performance of the CARTs is consistent with our initial hypothesis that endosomal escape and cytosolic mRNA release can be attributed to the rapid charge-altering transformation of cationic amines to neutral amides.…”

PEGylated and Functionalized Aliphatic Polycarbonate Polyplex Nanoparticles for Intravenous Administration of HDAC5 siRNA in Cancer Therapy

“…The guanidine function, cationic at neutral pH, is necessary for both siRNA binding and interactions with the negatively charged plasma membranes. [26][27] The morpholine function, weak base and ionizable in acidic pH, confers buffer capacity to the polymer and helps to escape from the endosome using the "proton sponge" effect. 28 In addition to these functionalized blocks, the polymer contains a hydrophobic chain of poly(trimethylene) carbonate (PTMC), bringing an amphiphilic character to the polymer, and thus allowing it to form nanoparticles in aqueous solution.…”

Non-Nucleosidic Analogues of Polyaminonucleosides and Their Influence on Thermodynamic Properties of Derived Oligonucleotides

“…Many laboratories try to improve nucleic acid delivery to cells by synthesis of cationic bioconjugates [1][2][3], bioconjugate analogues with reduced polyanionic character [4][5][6], or a great variety of polymeric transfection media [7][8][9][10][11][12]. On the other hand, there are studies to elucidate a OPEN ACCESS specific mode of small cationic molecular drugs interactions with biomolecules (e.g., nucleic acids, proteins) useful in developing their more active analogues [13].…”