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

Abstract

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