Conjugation of Doxorubicin to siRNA Through Disulfide-based Self-immolative Linkers

Abstract

Co-delivery systems of siRNA and chemotherapeutic drugs have been developed as an attractive strategy to optimize the efficacy of chemotherapy towards cancer cells with multidrug resistance. In these typical systems, siRNAs are usually associated to drugs within a carrier but without covalent interactions with the risk of a premature release and degradation of the drugs inside the cells. To address this issue, we propose a covalent approach to co-deliver a siRNA-drug conjugate with a redox-responsive self-immolative linker prone to intracellular glutathione-mediated disulfide cleavage. Herein, we report the use of two disulfide bonds connected by a pentane spacer or a p-xylene spacer as self-immolative linker between the primary amine of the anticancer drug doxorubicin (Dox) and the 2′-position of one or two ribonucleotides in RNA. Five Dox-RNA conjugates were successfully synthesized using two successive thiol-disulfide exchange reactions. The Dox-RNA conjugates were annealed with their complementary strands and the duplexes were shown to form an A-helix sufficiently stable under physiological conditions. The enzymatic stability of Dox-siRNAs in human serum was enhanced compared to the unmodified siRNA, especially when two Dox are attached to siRNA. The release of native Dox and RNA from the bioconjugate was demonstrated under reducing conditions suggesting efficient linker disintegration. These results demonstrate the feasibility of making siRNA-drug conjugates via disulfide-based self-immolative linkers for potential therapeutic applications.

In-Vitro Application of Magnetic Hybrid Niosomes: Targeted siRNA-Delivery for Enhanced Breast Cancer Therapy

“…Considerable attention has been directed to regulate genes responsible for cancer pathology [ 15 , 16 , 17 ]. For instance, using small interfering RNA (siRNA) as an RNA interference (RNAi) mechanism enables both a mono- and combinational therapy with other therapeutics by silencing specific genes and inhibiting the expression of the respective protein [ 18 , 19 , 20 , 21 ]. To date, many studies have been conducted using synthetic siRNA to inhibit genes of interest by the degradation of their messenger RNA transcript in the cell cytoplasm, thereby inhibiting the expression of a specific protein [ 22 , 23 , 24 ].…”

Evolution of complexity in non-viral oligonucleotide delivery systems: from gymnotic delivery through bioconjugates to biomimetic nanoparticles

“…After FR-mediated endocytosis, nigericin dissociated from the linker and acted as an ionophore to transport potassium across the membrane causing endosomal swelling and improved miRNA release to the cytosol[ 48 ]. Exploiting disulphide-based conjugation chemistry also enabled effective codelivery of doxorubicin (Dox) and siRNA to cancer cells[ 50 ]. The direct conjugation solves a problem of premature dissociation of Dox and unintended toxicity that was observed in non-covalent formulations of Dox and siRNA.…”

Disulfide Bridging Strategies in Viral and Nonviral Platforms for Nucleic Acid Delivery

“…In addition, the effect of cholesterol conjugation was tested in a study that compared gene silencing and immunogenic effects of TAT (48–60)-, penetratin, and cholesterol-conjugated siRNA acting on p38 MAP kinase . Similar to the co-delivery strategy, hydrophobic drug doxorubicin was conjugated to the sense strand of an siRNA via a self-immolative linker containing a disulfide bond for targeting the EWS/Fli1 protein that is relevant to Ewing’s sarcoma . Although hydrophobization with lipids or small molecules had served as a typical procedure for enhancing cellular penetration, this could lead to the formation of nanoassemblies due to the amphiphilicity of the oligonucleotide conjugates in aqueous media, leading to reduced efficacy and voiding the original purpose.…”

“…22 Similar to the co-delivery strategy, hydrophobic drug doxorubicin was conjugated to the sense strand of an siRNA via a self-immolative linker containing a disulfide bond for targeting the EWS/Fli1 protein that is relevant to Ewing's sarcoma. 23 Although hydrophobization with lipids or small molecules had served as a typical procedure for enhancing cellular penetration, this could lead to the formation of nanoassemblies due to the amphiphilicity of the oligonucleotide conjugates in aqueous media, leading to reduced efficacy and voiding the original purpose. Inspired by the recent progress in disulfide-based systems showing interactions with cell surface thiols for direct cellular internalization, 24,25 Antisense DNA and siRNA molecules were tagged with multiple disulfide moieties at the chain end via phosphoramidite chemistry (Figure 2a).…”