Transfection of 3′,3′′-bis-peptide-siRNA conjugate by cationic lipoplexes mixed with a neutral cytosin-1-yl-lipid

siRNA Packaged with Neutral Cytidinyl/Cationic/PEG Lipids for Enhanced Antitumor Efficiency and Safety In Vitro and In Vivo

“…These optimized formulations induced robust firefly gene silencing when the siRNA concentration was only 25 nM and could also inhibit about 80% endogenous gene expression (BRAF V600E mRNA in A375 cells), by encapsulating siMB3, at a siRNA concentration of 5 nM (Figure 2b), while in our previous reports, that of the bis-peptide-siMB3 was 100 nM. 29 Furthermore, formulations with less CLD were evaluated (formulations s1−s8, Table S2, Supporting Information). Results showed that siRNA delivered by DNCA alone could permeate into cells, although the uptake ability was weaker than the other groups.…”

“…The mixture of lipid formulation with a DNCA/CLD mol ratio of 8:3 (formulation 2) has been reported to successfully deliver bispeptide-siMB3. 29,30 Thus, formulation 2, a single dose of CLD (formulation 8), 28 and commercial liposome lipofectamine 2000 were used as positive controls.…”

“…27,28 Later, with the aid of DNCA, the mixed siRNA conjugate delivery system (CLD/DNCA, molar ratio 3:8, N/P 3/1, base ratio 2/1) exhibits relatively better gene activity at the cellular level with a lower N/P ratio (3:1) and lower toxicity at 100 nM. 29,30 In this case, siRNA transfection by DNCA only (base ratio 2/1) has been achieved, silencing 50% target mRNA at 25 nM. Further, the optimal formulation of DNCA/CLD with PEG-DSPE (0.7%) (molar ratio 3:2, N/P ∼3/1, base ratio ∼0.5/ 1) 31,32 gave compact and noncationic nanoparticles by multiple kinds of molecular interactions (Figure 1b), which permeated into the cell through the nonlysosome cellular pathway with robust gene silencing with a siRNA targeting to BRAF V600E at 10 nM in vitro, showing a superior antitumor effect, longer circulation time, and much lower toxicity in vivo by IV administration.…”

“…Continuous optimization by mixing DNCA with dioleoyl-3,3′-disulfanediylbis-[2-(2,6-diaminohexanamido)]­propanoate (CLD) [molar ratio 28:3, N/P 1.5/1, the ratio of DNCA to the single nucleotide­(base ratio) 3.5/1] gives the ideal gene silencing activity of the antisense oligonucleotide G3139 analog at ∼100 nM . Meanwhile, CLD (a cationic lipid with cysteine as the skeleton structure) has been applied (Figure a) to encapsulate the 3′,3″-bis-peptide-siRNA conjugate (N/P 5/1). , Later, with the aid of DNCA, the mixed siRNA conjugate delivery system (CLD/DNCA, molar ratio 3:8, N/P 3/1, base ratio 2/1) exhibits relatively better gene activity at the cellular level with a lower N/P ratio (3:1) and lower toxicity at 100 nM. , …”

Feasibility of cRGD conjugation at 5′-antisense strand of siRNA by phosphodiester linkage extension

“… 24 In this study, cRGD-siRNA conjugates at the 5′-terminus antisense strand via phosphodiester linkage have been reported, which were encapsulated with neutral cytidinyl lipid DNCA and the gemini-like cationic lipid CLD, both developed by our group ( Figure S1 ). 25 , 26 , 27 , 28 , 29 , 30 DNCA could provide hydrogen bond and π-π stacking with siRNA. 31 Overall, with a relative longer flexible linker, the siRNA conjugates effectively silenced the target mRNA in vitro and inhibited tumor proliferation in vivo .…”