Reductive nanocomplex encapsulation of cRGD-siRNA conjugates for enhanced targeting to cancer cells

Abstract

In this study, through covalent conjugation and lipid material entrapment, a combined modification strategy was established for effective delivery of small interfering RNA (siRNA). Single strands of siRNA targeting to BRAF V600E gene (siMB3) conjugated with cRGD peptide at 3′-terminus or 5′-terminus via cleavable disulfide bond was synthesized and then annealed with corresponding strands to obtain single and bis-cRGD-siRNA conjugates. A cationic lipid material (CLD) developed by our laboratory was mixed with the conjugates to generate nanocomplexes; their uniformity and electrical property were revealed by particle size and zeta potential measurement. Compared with CLD/siBraf, CLD/cRGD-siBraf achieved higher cell uptake and more excellent tumor-targeting ability, especially 21 (sense-5′/antisense-3″-cRGD-congjugate) nanocomplex. Moreover, they can regulate multiple pathways to varying degree and reduce acidification of endosome. Compared with the gene silencing of different conjugates, single or bis-cRGD-conjugated siRNA showed little differences except 22 (5/5) which cRGD was conjugated at 5′-terminus of antisense strand and sense strand. However bis-cRGD conjugate 21 nanocomplex exhibited better specific target gene silencing at multiple time points. Furthermore, the serum stabilities of the bis-cRGD conjugates were higher than those of the single-cRGD conjugates. In conclusion, all these data suggested that CLD/bis-conjugates, especially CLD/ 21 , can be an effective system for delivery of siRNA to target BRAF V600E gene for therapy of melanoma.

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

“…This result matches our previous work showing that cRGD-modified siRNA partially enhanced the α v β 3 -positive A375 cellular uptake. 24 The effects of cRGD conjugates with different linkers on cellular uptake were also compared ( Figure 3 A). The cellular uptake abilities of conjugates increased with extended linker lengths until RL2As/S.…”

“…The cyclo(Arg-Gly-Asp-D-Phe-Lys) peptide (cRGD) acts as a tumor-target group by recognizing the α v class of integrins upregulated in tumors, 22 usually covalently conjugating to siRNA, 23 and is also a bulky group that can block RNAi when conjugated at the 5′-terminus of the antisense strand. 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.…”

Current Aspects of siRNA Bioconjugate for In Vitro and In Vivo Delivery

“…They claimed that the new construct can overcome the low membrane permeability of siRNA (better endosomal escape) and showed sufficient gene silencing efficiency at final concentration of 250 nM in the absence of any transfection agent [45]. Similarly, peptide siRNA conjugates such as LHRH-siRNA or cRGD-siRNA have been complexed with cationic polymer PEI or lipid to form the targeting nanoparticle for better endosomal release and delivery [46,47].…”

Systemic Delivery of Small Interfering RNA Therapeutics: Obstacles and Advances

“…NGR guided these inactive particles to CD13+ tumor cells where near-infrared (NIR) light illumination activated them by uncaging the lysine from the photosensitive groups (Yang et al, 2015). Cyclic RGD (cRGD) has provided extensive systemic siRNA delivery both in vitro and in vivo (Khatri et al, 2014;Huang et al, 2015;He et al, 2017;Zhou et al, 2017).…”