Small interfering RNA (siRNA)–based therapeutics represent one of the most mature clinical applications of RNA interference (RNAi), a conserved cellular mechanism for sequence-specific gene silencing. Since the original demonstration in the late 1990s that double-stranded RNA could selectively suppress gene expression, siRNA has evolved from a molecular biology tool into a clinically validated drug modality. The defining feature of siRNA therapeutics is their ability to silence disease-causing genes at the mRNA level with high specificity, offering a fundamentally different intervention strategy from small molecules or monoclonal antibodies.
The convergence of RNA biology, chemical modification, and nanotechnology has been essential in translating siRNA into viable medicines, particularly in the form of nano-siRNA drugs that address the intrinsic instability and delivery barriers associated with naked RNA. Examples targeting colorectal cancer (CRC) combining siRNA with nanotechnology through the use of nanoparticles are noted (Aghamiri et al., 2019).
Mechanistically, siRNA consists of a short double-stranded RNA, typically 21–23 nucleotides in length, with two-nucleotide 3′ overhangs. Once introduced into the cytoplasm, the siRNA duplex is incorporated into the RNA-induced silencing complex (RISC). Within RISC, the passenger strand is removed and degraded, while the guide strand remains bound to Argonaute proteins. The guide strand directs RISC to complementary sequences in target messenger RNA, leading to site-specific cleavage and subsequent degradation of the mRNA. This process effectively prevents translation of the encoded protein. From a therapeutic standpoint, the catalytic nature of RISC-mediated cleavage allows a single siRNA molecule to silence multiple mRNA transcripts, contributing to potent and durable effects even at relatively low intracellular concentrations.
Despite the conceptual elegance of RNAi, early attempts to use siRNA as a drug encountered formidable obstacles. Unmodified siRNA is highly susceptible to degradation by serum nucleases, exhibits poor pharmacokinetic properties, and is unable to efficiently cross cellular membranes due to its size, charge, and hydrophilicity. Moreover, systemic administration of naked siRNA can provoke innate immune responses through activation of pattern recognition receptors such as Toll-like receptors. These challenges necessitated the development of sophisticated delivery systems and chemical modifications to transform siRNA into a practical therapeutic agent. Nanotechnology emerged as a central enabling discipline, providing platforms capable of protecting siRNA, controlling its biodistribution, and facilitating its cellular uptake and intracellular release.
Chemical modification of siRNA is a foundational element of nano-siRNA drug design. Modifications are typically introduced to the ribose sugar, the nucleobase, or the phosphodiester backbone. Common examples include 2′-O-methyl and 2′-fluoro substitutions on the ribose, phosphorothioate linkages in the backbone, and selective base modifications to reduce immunostimulation. These alterations enhance resistance to nucleases, improve binding affinity to the target mRNA, and reduce off-target effects without compromising RNAi activity. Importantly, chemical modification alone is usually insufficient for efficient in vivo delivery, but it synergizes with nanoparticle-based carriers to achieve clinically meaningful performance.
Nano-siRNA drugs rely on nanoscale delivery systems, generally ranging from 50 to 200 nanometers in diameter, to encapsulate or complex siRNA molecules. Among these systems, lipid nanoparticles (LNPs) have emerged as the most clinically successful. LNPs typically consist of four components: an ionizable lipid, a helper phospholipid, cholesterol, and a polyethylene glycol (PEG)–lipid conjugate. The ionizable lipid is the functional core, designed to be neutrally charged at physiological pH to minimize toxicity and nonspecific interactions, but positively charged in acidic environments such as endosomes. This pH-responsive behavior enables strong complexation with negatively charged siRNA during formulation and promotes endosomal escape after cellular uptake. The helper lipid and cholesterol stabilize the nanoparticle structure and facilitate membrane fusion, while the PEG-lipid provides steric stabilization and prolongs circulation time.
Upon systemic administration, nano-siRNA drugs formulated as LNPs exhibit a characteristic biodistribution profile, with preferential accumulation in the liver. This is largely due to interactions between the nanoparticle surface and apolipoprotein E, which mediates uptake by hepatocytes via low-density lipoprotein receptors. While this hepatic tropism initially limited the scope of siRNA therapeutics, it proved advantageous for treating liver-associated diseases, including metabolic disorders, viral infections, and genetic conditions. Indeed, the first FDA-approved siRNA drug, patisiran, exploits an LNP formulation to deliver siRNA to hepatocytes for the treatment of hereditary transthyretin-mediated amyloidosis. The clinical success of patisiran validated the nano-siRNA paradigm and demonstrated that RNAi could be harnessed safely and effectively in humans.
Beyond LNPs, a diverse array of nanocarriers has been explored for siRNA delivery. Polymeric nanoparticles, often based on cationic or ionizable polymers such as polyethylenimine, poly(lactic-co-glycolic acid), or dendrimers, can electrostatically complex siRNA and protect it from degradation. These systems offer tunable physicochemical properties and the potential for controlled release, but their clinical translation has been hindered by concerns over toxicity, batch-to-batch variability, and scalability. Inorganic nanoparticles, including gold nanoparticles and mesoporous silica nanoparticles, have also been investigated as siRNA carriers due to their structural precision and multifunctionality. However, issues related to long-term biocompatibility and clearance remain significant barriers.
A critical aspect of nano-siRNA drug function is cellular uptake and intracellular trafficking. Most nanoparticles enter cells via endocytosis, becoming sequestered within endosomes. Successful gene silencing requires escape from the endosomal compartment into the cytoplasm before lysosomal degradation occurs. Endosomal escape is widely recognized as a rate-limiting step in siRNA delivery. Nano-siRNA systems address this challenge through multiple mechanisms, including pH-responsive lipid ionization, membrane-disruptive peptides, and the so-called proton sponge effect associated with certain polymers. Optimization of endosomal escape remains an active area of research, as incremental improvements can yield substantial gains in therapeutic potency.
Targeting beyond the liver represents a major frontier in nano-siRNA therapeutics. Strategies to achieve tissue-specific delivery include surface functionalization of nanoparticles with ligands such as antibodies, peptides, or small molecules that bind to receptors expressed on target cells. An alternative and highly successful approach is the conjugation of siRNA to N-acetylgalactosamine (GalNAc), which enables receptor-mediated uptake by hepatocytes without the need for a nanoparticle carrier. While GalNAc–siRNA conjugates are not nanoparticles in the strict sense, they underscore the principle that rational delivery design is central to siRNA drug development. For extrahepatic targets such as tumors, the central nervous system, or the lungs, nanoparticle-based delivery remains indispensable, and ongoing research is focused on overcoming biological barriers such as the tumor microenvironment and the blood–brain barrier.
Clinically, nano-siRNA drugs offer several advantages over traditional therapeutics. Their mechanism of action allows direct modulation of gene expression, enabling access to targets previously considered “undruggable,” such as transcription factors or scaffold proteins. The specificity of Watson–Crick base pairing reduces the likelihood of unintended interactions, although careful sequence design and screening are required to minimize off-target silencing. Additionally, the modular nature of siRNA allows relatively rapid development of new drugs by altering the nucleotide sequence while retaining the same delivery platform, a feature that has implications for personalized medicine and rapid response to emerging diseases.
Nevertheless, nano-siRNA therapeutics also present unique challenges. Immune activation, particularly through innate immune pathways, remains a concern, although advances in chemical modification and formulation have substantially mitigated this risk. Manufacturing complexity and cost are nontrivial, as production requires precise control over RNA synthesis, nanoparticle assembly, and quality attributes such as size, encapsulation efficiency, and stability. Regulatory evaluation of nano-siRNA drugs is correspondingly complex, requiring integrated assessment of both the active pharmaceutical ingredient and the delivery system.
In summary, siRNA-based therapeutics represent a paradigm shift in drug development, enabled by a deep understanding of RNA biology and the application of nanotechnology to overcome fundamental delivery barriers. Nano-siRNA drugs, particularly those based on lipid nanoparticles, have transitioned from experimental constructs to clinically approved medicines, establishing RNAi as a viable and versatile therapeutic modality. Continued advances in delivery science, targeting strategies, and manufacturing are expected to expand the range of diseases amenable to siRNA intervention. As these technologies mature, nano-siRNA therapeutics are likely to play an increasingly prominent role in precision medicine, offering highly specific, gene-based treatments for conditions that remain inadequately addressed by conventional pharmacology.
References
Aghamiri, S., Jafarpour, A., Malekshahi, Z. V., Mahmoudi Gomari, M., & Negahdari, B. (2019). Targeting siRNA in colorectal cancer therapy: Nanotechnology comes into view. Journal of Cellular Physiology, 234(9), pp. 14818-14827 (Article)
Leave a Reply