A Comprehensive Review of Small Interfering RNAs (siRNAs): Mechanism, Therapeutic Targets, and Delivery Strategies for Cancer Therapy

Abstract

Small interfering RNA (siRNA) delivery by nanocarriers has been identified as a promising strategy in the study and treatment of cancer. Short nucleotide sequences are synthesized exogenously to create siRNA, which triggers RNA interference (RNAi) in cells and silences target gene expression in a sequence-specific way. As a nucleic acid-based medicine that has gained popularity recently, siRNA exhibits novel potential for the treatment of cancer. However, there are still many obstacles to overcome before clinical siRNA delivery devices can be developed. In this review, we discuss prospective targets for siRNA drug design, explain siRNA drug properties and benefits, and give an overview of the current clinical siRNA therapeutics for the treatment of cancer. Additionally, we introduce the siRNA chemical modifications and delivery systems that are clinically sophisticated and classify bioresponsive materials for siRNA release in a methodical manner. This review will serve as a reference for researchers in developing more precise and efficient targeted delivery systems, promoting ongoing advances in clinical applications.

Keywords: bioresponsive materials; cancer; chemical modifications; delivery systems; small interfering RNA.

Graphical abstract

Figure 1

Mechanism of microRNA (miRNA) and small interfering RNA (siRNA) action. miRNA: The miRNA gene is transcribed into pri-mRNA in the cell nucleus and then processed by Drosha and DGCR8 to form pre-miRNA. The pre-miRNA is transported to the cytoplasm by Exportin 5, where it undergoes further processing by Dicer to remove the stem-loop structure, forming mature miRNA. The miRNA is then loaded into the RISC that comprises Argonaute 1–4 (Ago1-Ago4), and the passenger strand is discarded. The miRNA-RISC pairs with the target mRNA in an imperfectly complementary manner, leading to mRNA degradation or translational repression. siRNA: The precursor of siRNA, such as double-stranded RNA (dsRNA), is first recognized and processed by Dicer. Dicer cleaves the precursor molecules into small siRNA fragments, typically 21–23 nucleotides in length. These siRNA fragments are then incorporated into the RNA-induced silencing complex (RISC). Once formed, the siRNA-RISC complex binds to a specific target mRNA molecule through base pairing between the siRNA and the mRNA sequence. The siRNA-RISC complex, specifically the Argonaute-2 endonuclease (Ago2) within RISC, then induces cleavage of the target mRNA at a specific site. This cleavage prevents the target mRNA from being translated into protein, ultimately leading to a reduction in target protein expression. The icons (cell, mitochondria, endoplasmic reticulum, miRNA gene, genomic DNA, and mRNA) were Created with BioRender.com.

Figure 2

The targeting pathways of siRNA in cancer treatment. The inner circle illustrates the diverse pathways involved in cancer treatment, encompassing: cell cycle regulation, proliferation, cancer metastasis, angiogenesis, anti-apoptotic processes, DNA damage repair, cancer immune escape, cancer metabolism, oncogene, and cancer tyrosine kinase. The outer circle represents the specific siRNA interference targets corresponding to these pathways, which have been explored in preclinical research. The tumor cells in the center were Created with BioRender.com.

Figure 3

Barriers to the delivery of siRNA in vivo. After administration, siRNAs can encounter several obstacles. Sequestration and excretion: siRNAs may be sequestered or excreted by organs like the liver and kidneys, limiting their distribution and reducing their effectiveness in reaching target tissues. Extracellular barriers: nucleases present in the bloodstream can degrade siRNA molecules, rendering them ineffective before they can reach their intended targets; macrophages are capable of recognizing and engulfing foreign particles, including siRNAs, hamper their delivery to target cells and reduce their availability; siRNA is a negatively charged molecule, and it faces significant challenges in crossing biological membranes. Intracellular barriers: once internalized by target cells, siRNAs may encounter challenges in escaping the endosomal-lysosomal pathway, potentially hindering their ability to reach the desired intracellular target mRNA and diminishing their efficacy; off-target effect led to unwanted toxicities. Created with BioRender.com.

Figure 4

Chemical modifications commonly used in siRNA therapeutics. According to the structure of the RNA nucleotide, it can be classified into phosphate (pink), ribose (purple), and base modifications (yellow). Besides, Ligands (green, such as GalNAc, antibodies, and peptides) enable specific cell types delivery.

Figure 5

Delivery systems for delivering siRNAs to target tissues and cells can be classified into (A) viral and (B–E) non-viral vectors. Structure of the triantennary GalNAc used in several drug candidates from Alnylam Pharmaceuticals. The icons were Created with BioRender.com.

Figure 6

Bioresponsive materials. (A) Schematic diagram of siRNA delivery systems with dual tumor-targeting and pH-responsive capabilities, which can break through biological barriers and penetrate deep into tumors to achieve better tumor therapeutic effects. International Journal of Nanomedicine 2022;17:953–967’ used with permission from Zhang XY, Qin B, Wang M et al. Dual pH-responsive and tumor-targeted nanoparticle-mediated anti-angiogenesis siRNA delivery for tumor treatment. Int J Nanomedicine. 2022;17:953–967. Copyright 2022 Dove Medical Press Ltd. (B) Schematic diagram of reduction-sensitive PEG-SS-PEI-PE and GSH-mediated PEG de-shielding, and proposed mechanisms for intracellular stimulus-sensitive siRNA delivery. Reproduced with permission from Mutlu Agardan NB, Sarisozen C, Torchilin VP. Redox-triggered intracellular siRNA delivery. Chem Commun (Camb). 2018;54(49):6368–6371. Copyright 2018, The Royal Society of Chemistry. (C) Schematic diagram of triazolium amphiphiles capable of binding siRNA and releasing the nucleic acid payload via enzyme-responsive. Reproduced with permission from Hollstein S, Ali LMA, Coste M et al. A triazolium-anchored self-immolative linker enables self-assembly-driven siRNA binding and esterase-induced release. Chemistry. 2023;29(8):e202203311. Creative Commons CC BY license. Copyright 2022, The Authors, published by Wiley-VCH GmbH. (D) Schematic diagram of hypoxia-induced siRNA uptake and silencing using a nanocarrier consisting of PEG2000, azobenzene, PEI (1.8 kDa), and DOPE units (named PAPD). Reproduced with permission from Perche F, Biswas S, Wang T, Zhu L, Torchilin VP. Hypoxia-targeted siRNA delivery. Angew Chem Int Ed Engl. 2014;53(13):3362–3366. Copyright 2014, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. (E) Schematic diagram of self-assembly of PEI1.8k-PBA/PEI1.8k-α-CD supramolecular polymer and release of siRNA through multimer disassembly under ATP stimulation. Reproduced with permission from Jiang C, Qi Z, Jia H et al. ATP-responsive low-molecular-weight polyethylenimine-based supramolecular assembly via host-guest interaction for gene delivery. Biomacromolecules. 2019;20(1):478–489. Copyright 2019, American Chemical Society.