Commercially Supplied Amine-Modified siRNAs May Require Ultrafiltration prior to Conjugation with Amine-Reactive Compounds

Conjugation of siRNA to macromolecules such as serum albumin has multiple potential benefits, including enhanced extravasation via albumin-mediated transcytosis across endothelial cells and reduced renal clearance. In attempting to conjugate siRNA to albumin, we used commercially sourced amine-modified siRNA and reacted it with the heterobifunctional linker succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) to introduce a maleimide group suitable for conjugation to the thiol group of the surface-exposed cysteine residue (Cys 34) within albumin. We found the conjugation of the SMCC-treated siRNA to bovine serum albumin (BSA) to be very inefficient and investigated the cause of the low yield of conjugate. Ultrafiltration with phosphate-buffered saline prior to activation with SMCC dramatically increased the yield of siRNA-albumin conjugate (~15-fold). Communication with the commercial supplier revealed that ammonium acetate buffer was used in a desalting step as part of the siRNA purification process prior to supply, likely resulting in ammonium counterions to the siRNA polyanion, which would interfere with conjugation by consuming the SMCC. After ultrafiltration, a greatly reduced amount of SMCC could be used to affect conjugation, without significant reduction in yield. These data indicate that amine-modified siRNA sourced commercially may require ultrafiltration or dialysis prior to use in conjugation reactions

Investigation of systemic delivery of naked siRNA and albumin as carrier in vivo

Significant efforts have been made on transforming small interfering RNA (siRNA) into genetic therapy, however, insufficient systemic delivery has hindered its applications. In the current study, it was demonstated that systemically administered naked siRNA exhibit limited uptake and silencing in organs with continuous endothelia. Therefore, the present study aimed to overcome the endothelial barrier by harnessing the transcytosis mechanism of serum protein albumin to enhance extravasation. IGF-IR was used as the exemplar gene target for evaluation of silencing in vivo and efficiency of the albumin delivery strategy. IGF-IR mRNA knockdown (84.27 ± 1.29% at 30 nM) and protein reduction (95.71 ± 4.29% at 72 hr) induced by the current sequence were considerably more potent than other IGF-IR siRNA sequences (Oussedik et al., 2010; Pavan et al., 2010). In the current set up, prolonged transfection times did not facilitate RNAi, but utilisation of dicer-substrate siRNA and non-modified sequence enhanced knockdown by >50%. The first approach to enhance extravasation involved direct conjugation of amine-modified siRNA to cysteine 34 residue within albumin via the succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) linker. Significant batch-to-batch variability in conjugation efficiency (4-20%) was recorded and was attributed to the residing ammonium ions from purification acting as counterions for the SMCC linker. Ammonium counterions were removed from amine-modified siRNA by repeated ultrafiltration with PBS and resulted in a 15-fold increase in siRNA-albumin conjugation yield. Consequently, SMCC:siRNA conjugation ratio can be lowered from 40:1 to 10:1 without alteration in yield but with cost of synthesis lowered. Intravenously administered siRNA-albumin conjugate demonstrated reduced plasma clearance and circulating half-life extended to ~75 min, comparable to siRNA-cholesterol conjugation (Soutschek et al., 2004). The enhanced “window of opportunity” may have facilitated extravasation for conjugate uptake into the myocardium which resulted in localisation and significant target IGF-IR knockdown (~40%, 1 mg/kg over 4 days) in cardiac tissues. The conjugate did not induce silencing in kidney tissues and were hypothesised the conjugate size (80 kDa) have prevented siRNA reaching the glomerular filtrate. In contrast, the unconjugated siRNA (~14 kDa) did not induce knockdown in the heart but silencing was observed in kidney attributed to glomerular filtration. The second approach involved albumin coating lipopeptide (LP3) condensed siRNA complex. An N:P (siRNA:LP3) ratio of 1:2.5 was used to construct positively charged complexes, and steric stabiliser F127 was utilised with slow mixing method to prevent complex aggregation as ζ-potential approaches neutral. Confirmed by cryo-TEM and dynamic light scattering, the final siRNA-LP3-F127-albumin complex was ~300 nm with -6.47 ± 2.12 mV surface charge. Albumin binding onto the siRNA complex was demonstrated by the increased particle size with decreased ζ-potential of the complex without albumin. Albumin bound siRNA complexes exhibited enhanced uptake into myocardium whilst reducing deposition into organs with fenestrated/sinuisoidal endothelia. However, these complexes did not result in silencing in myocardium as the conjugate had. Further development and optimisation of the complex formulation is required. The characterisation of albumin induced extravasation of the conjugate, cellular uptake, and the fate of the conjugate in cytoplasm should be examined for future research. Despite the lack of silencing from the siRNA complex, the current siRNA-albumin conjugation strategy might prove to be more suitable for siRNA therapeutics, with effective silencing in myocardium and not kidney tissues

Investigation of systemic delivery of naked siRNA and albumin as carrier in vivo

Significant efforts have been made on transforming small interfering RNA (siRNA) into genetic therapy, however, insufficient systemic delivery has hindered its applications. In the current study, it was demonstated that systemically administered naked siRNA exhibit limited uptake and silencing in organs with continuous endothelia. Therefore, the present study aimed to overcome the endothelial barrier by harnessing the transcytosis mechanism of serum protein albumin to enhance extravasation. IGF-IR was used as the exemplar gene target for evaluation of silencing in vivo and efficiency of the albumin delivery strategy. IGF-IR mRNA knockdown (84.27 ± 1.29% at 30 nM) and protein reduction (95.71 ± 4.29% at 72 hr) induced by the current sequence were considerably more potent than other IGF-IR siRNA sequences (Oussedik et al., 2010; Pavan et al., 2010). In the current set up, prolonged transfection times did not facilitate RNAi, but utilisation of dicer-substrate siRNA and non-modified sequence enhanced knockdown by >50%. The first approach to enhance extravasation involved direct conjugation of amine-modified siRNA to cysteine 34 residue within albumin via the succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) linker. Significant batch-to-batch variability in conjugation efficiency (4-20%) was recorded and was attributed to the residing ammonium ions from purification acting as counterions for the SMCC linker. Ammonium counterions were removed from amine-modified siRNA by repeated ultrafiltration with PBS and resulted in a 15-fold increase in siRNA-albumin conjugation yield. Consequently, SMCC:siRNA conjugation ratio can be lowered from 40:1 to 10:1 without alteration in yield but with cost of synthesis lowered. Intravenously administered siRNA-albumin conjugate demonstrated reduced plasma clearance and circulating half-life extended to ~75 min, comparable to siRNA-cholesterol conjugation (Soutschek et al., 2004). The enhanced “window of opportunity” may have facilitated extravasation for conjugate uptake into the myocardium which resulted in localisation and significant target IGF-IR knockdown (~40%, 1 mg/kg over 4 days) in cardiac tissues. The conjugate did not induce silencing in kidney tissues and were hypothesised the conjugate size (80 kDa) have prevented siRNA reaching the glomerular filtrate. In contrast, the unconjugated siRNA (~14 kDa) did not induce knockdown in the heart but silencing was observed in kidney attributed to glomerular filtration. The second approach involved albumin coating lipopeptide (LP3) condensed siRNA complex. An N:P (siRNA:LP3) ratio of 1:2.5 was used to construct positively charged complexes, and steric stabiliser F127 was utilised with slow mixing method to prevent complex aggregation as ζ-potential approaches neutral. Confirmed by cryo-TEM and dynamic light scattering, the final siRNA-LP3-F127-albumin complex was ~300 nm with -6.47 ± 2.12 mV surface charge. Albumin binding onto the siRNA complex was demonstrated by the increased particle size with decreased ζ-potential of the complex without albumin. Albumin bound siRNA complexes exhibited enhanced uptake into myocardium whilst reducing deposition into organs with fenestrated/sinuisoidal endothelia. However, these complexes did not result in silencing in myocardium as the conjugate had. Further development and optimisation of the complex formulation is required. The characterisation of albumin induced extravasation of the conjugate, cellular uptake, and the fate of the conjugate in cytoplasm should be examined for future research. Despite the lack of silencing from the siRNA complex, the current siRNA-albumin conjugation strategy might prove to be more suitable for siRNA therapeutics, with effective silencing in myocardium and not kidney tissues

Edaravone containing isoindoline nitroxides for the potential treatment of cardiovascular ischaemia

A novel antioxidant for the potential treatment of ischaemia was designed by incorporating an isoindoline nitroxide into the framework of the free radical scavenger edaravone. 5-(3-Methyl-pyrazol-5-ol-1-yl)-1,1,3,3-tetramethylisoindolin-2-yloxyl 7 was prepared by N-arylation of 3-methyl-5-pyrazolone with 5-iodo-1,1,3,3-tetramethylisoindoline-2-yloxyl 8 in the presence of catalytic copper(I)iodide. Evaluation of 7, its methoxyamine derivative 10 and 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (CTMIO) against edaravone 1 in ischaemic rat atrial cardiomyocytes revealed significant decreases in cell death after prolonged ischaemia for each agent; however the protective effect of the novel antioxidant 7 (showing greater than 85% reduction in cell death at 100 μM) was significantly enhanced over that of edaravone 1 alone. Furthermore, the activity for 7 was found to be equal to or greater than the potent cardioprotective agent N6-cyclopentyladenosine (CPA). The methoxyamine adduct 10 and edaravone 1 showed no difference between the extent of reduction in cell death whilst CTMIO had only a modest protective effect