Recent Advances in siRNA Delivery System and Drugs

Wenjie Huang

and Yichen Wu

Medicinal Chemistry, China Pharmaceutical University, Nanjing, China

Kunshan KangChiao School, Kunshan, China

Keywords: siRNA Delivery, RNA Interference (RNAi), Nanoparticles.

Abstract: siRNA drugs are an emerging class of nucleic acid drugs that can treat many diseases at the genetic level,

especially for rare diseases. siRNA has the characteristics of poor lipid solubility, high immunogenicity, poor

stability, and difficulty in in vivo delivery, which makes the development of related drugs full of challenges.

However, the continuous innovation of molecular modification methods and increasingly sophisticated

delivery strategies have brought hope to the development of siRNA drugs. This article reviews the mechanism

of action, modification, delivery technology, and research and development history of siRNA drugs, aiming

to provide feasible research and development strategies for potential siRNA drugs and continuously expand

the clinical application of siRNA drugs.

1 INTRODUCTION

siRNA drugs have opened up a new avenue for

innovative gene-based therapies. Since the approval

of the world's first siRNA drug, Patisiran, in 2018,

siRNA-based therapeutics have demonstrated

immense potential. siRNA drugs have opened up new

avenues for innovative gene therapy. Since the

approval of the world's first siRNA drug, Patisiran, in

2018, siRNA-based therapies have shown great

potential. Small nucleic acid drugs represented by

siRNA drugs have set off the third wave of drug

research and development after small molecule drugs

and antibody drugs. After siRNA drugs are made into

drugs, they have many advantages such as long-term

effectiveness and precise targeting, which makes

them have huge market potential. With the rich R&D

experience accumulated over the years and the

excellent sales results facing the market, it is proved

that siRNA drugs have withstood the test in all

aspects and have become a successful example of

new drug research and development.

1.1 Background of siRNA

In 1998, Fire et al. discovered in their study on

Caenorhabditis elegans that long double-stranded

RNA (dsRNA) could silence the expression of

specific genes. They termed this phenomenon RNA

interference (RNAi), opening the door to potent and

specific gene silencing through dsRNA (Fire et

al.,1998). siRNA was first identified by David

Baulcombe’s team in plants as part of the post-

transcriptional gene silencing (PTGS) mechanism

(Hamilton and Baulcombe,1999). Building upon

these studies, Thomas Tuschl et al. successfully

induced RNAi in mammalian cells using synthetic

small interfering RNAs (siRNAs) in 2001, without

triggering an immune response typically associated

with long dsRNA (Tuschl,2001). These

groundbreaking discoveries paved the way for the

application of siRNA in both research and therapeutic

fields.

1.2 Introduction to RNAi

RNAi refers to the process of introducing exogenous

or endogenous double stranded RNA (dsRNA) into

cells and processing it through Dicer, a member of the

RNase III-like enzyme family, to form 21~23

nucleotide (nt) short dsRNAs (Agrawal et al.,2003).

These small dsRNAs are called small interfering

RNAs (siRNAs). They form RNA-induced silencing

complex (RISC) under the action of Argonaute (Ago)

protein. At the same time, dsRNA is unzipped into

two single strands: Guide Strand and Passenger

Strand. Guide Strand exists in RISC, while Passenger

Strand is degraded. Subsequently, under the action of

the cleavage enzyme argonaute-2, RISC recognizes

and cuts the target sequence in mRNA through the

principle of base complementary pairing, promoting

the cleavage or degradation of target mRNA, thereby

Huang, W. and Wu, Y.

Recent Advances in siRNA Delivery System and Drugs.

DOI: 10.5220/0014465100004933

Paper published under CC license (CC BY-NC-ND 4.0)

In Proceedings of the 1st International Conference on Biomedical Engineering and Food Science (BEFS 2025), pages 223-228

ISBN: 978-989-758-789-4

223

interrupting the translation process of mRNA and

downregulating the translation level of specific

proteins (Elbashir et al.,2001). This discovery is an

important milestone in the field of gene therapy. It

provides feasible solutions for drug development in

multiple fields.

1.3 Advantages of siRNA

SiRNA, as a new type of gene therapy, has its unique

advantages. First, siRNA is specific. Since the

positive chain only binds to the mRNA that is

completely complementary to it, siRNA can highly

specifically identify any target gene in the human

body, with low off-target efficiency and high

silencing efficiency. Second, siRNA drugs are safe.

The gene silencing effect produced is transient and

will not be integrated into the genome, thus avoiding

the risk of host gene mutation. In addition, siRNA

drugs are long-lasting. Small interfering RNA

(siRNA) drugs have significantly improved stability

and resistance to nuclease degradation due to their

chemical modification, thereby extending the

biological half-life and achieving long-term

treatment with dosing every few months or even half

a year (Huang et al.,2025). For instance, Inclisiran

can successfully lower levels of low-density

lipoprotein cholesterol (LDL-C) by targeting PCSK9.

It only needs to be administered twice a year, which

significantly improves patient compliance. siRNA

drugs have shown the advantages of long-lasting

efficacy and low frequency of use in the treatment of

chronic diseases, providing important support for

precision medicine (Zhang and Li,2022).

2 SIRNA DELIVERY

Small interfering RNA (siRNA) has shown huge

potential in gene therapy and precision medicine

because it can specifically silence the expression of

target genes. However, siRNA itself has problems

such as easy degradation, low cellular uptake

efficiency and difficulty in crossing biological

barriers, which makes its clinical transformation face

challenges. In order to overcome the above problems,

researchers have developed corresponding vectors to

protect siRNA and deliver it to target cells.

2.1 Viral Vectors

Viral vectors achieve targeted delivery of siRNA by

simulating the natural infection mechanism of viruses,

mainly including adenovirus, lentivirus and adeno-

associated virus (AAV). Its core advantages are high

transfection efficiency and genetic stability, but

potential immunogenicity, insertion mutation risk and

production cost limit its clinical application

(Kaczmarek et al.,2017).

In recent years, AAV has become a research

hotspot due to its low pathogenicity and persistent

expression characteristics. By engineering the AAV

capsid protein (such as targeted receptor

modification), its tissue specificity can be

significantly improved and its immunogenicity can

be reduced (Srivastavaet al.,2021). However, AAV

still faces problems such as limited drug loading

capacity.

2.2 Lipid Carriers

Lipid nanoparticles (LNPs) are currently the only

FDA-approved siRNA delivery system (such as

Patisiran). They form a core-shell structure through

the self-assembly of cationic lipids and siRNA, which

protects nucleic acids while promoting endosomal

escape. The success of LNPs stems from four major

characteristics: (1) modular design facilitates

functional modification; (2) pH-sensitive lipid-

mediated endosomal escape; (3) PEGylated surface

prolongs circulation half-life; and (4) standardized

process that can be industrialized (Kulkarni et

al.,2019).

Although LNPs have been clinically applied, their

targeting specificity and long-term toxicity still need

to be optimized. The latest progress includes: (1) the

development of tissue-specific ligands (such as

GalNAc targeting hepatocytes); (2) the design of

ionizable lipids to reduce cytotoxicity; and (3) the

construction of stimulus-responsive carriers (such as

tumor microenvironment-triggered release). It is

worth noting that the nonspecific binding of cationic

lipids to serum proteins remains a major challenge for

systemic drug delivery, which has prompted

researchers to develop novel components such as

zwitterionic lipids (Hou et al.,2021).

2.3 Polymer Carriers

Polymer carriers achieve siRNA loading through

electrostatic adsorption or covalent coupling, and

their performance depends on molecular weight,

branching degree and surface charge. Common

systems include: Polyethyleneimine (PEI): High

cationic density gives it strong nucleic acid binding

ability, but high molecular weight PEI (>25 kDa)

produces cytotoxicity due to the proton sponge effect.

Solutions include: (1) coupling low molecular weight

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PEI with hydrophobic groups; (2) constructing a

degradable cross-linked structure (Wang et al.,2010).

PLGA: FDA-approved biodegradable material

that encapsulates siRNA through the double emulsion

method. Although it has excellent biocompatibility,

the negatively charged surface leads to low cellular

uptake rate, which needs to be improved by cationic

polymer (such as chitosan) coating or targeting ligand

(such as folic acid) modification (Danhier et al.,2012).

Emerging strategies focus on the development of

smart responsive polymers, for example:

temperature-sensitive Pluronic copolymers can

enhance tumor penetration, while reduction-sensitive

disulfide bond polymers can achieve intracellular

specific release.

2.4 Inorganic Non-Metallic Carriers

Inorganic non-metallic materials used for siRNA

drug delivery include carbon nanomaterials, calcium

phosphate, silica, graphene, etc. WEI Bo and his

research team used four techniques including cell

transfection, fluorescence imaging, MTT method and

hemolysis experiment to confirm that silica can

effectively deliver siRNA to primary cells of

mammals, showing excellent delivery ability, and no

obvious cytotoxicity and physiological hemolysis

were found. This provides an innovative delivery tool

for subsequent drug research (Wei et al.,2025).

2.5 Metal Nanocarriers

Metal nanocarriers achieve efficient siRNA delivery

through controllable surface modification, of which

two types of systems are the most representative:

Gold nanoparticles (AuNPs). The surface of AuNPs

can be thiolated to load dense siRNA chains (about

30-60 per 10 nm particle) to form a spherical nucleic

acid (SNA) structure. This structure is efficiently

endocytosed through the scavenger receptor SR-A1

and directly penetrates the cell membrane, avoiding

the endosomal escape barrier of traditional carriers

(Giljohann et al.,2010). However, the particle size

must be controlled at 20-50 nm to balance tumor

targeting (EPR effect) and in vivo retention time. Iron

oxide nanoparticles (IONPs). IONPs can achieve

deep tissue targeting under the guidance of an

external magnetic field. For example, the

Fe3O4@SiO2 core-shell structure can increase the

drug loading to 200μg siRNA/mg particle, and realize

tumor microenvironment-triggered release through

pH-responsive materials.

3 MODIFICATION OF SIRNA

Unmodified small nucleic acids are easily degraded

when used directly in vivo, with poor effects and easy

off-target effects. Modification of siRNA can

improve its resistance to enzymatic degradation,

maintain sequence stability and prolong half-life,

while increasing the lipid solubility of the drug and

reducing immunogenicity. Modification has become

one of the key directions of siRNA drug research and

development.

3.1 Chemical Modification

The basic skeleton of siRNA is composed of

phosphate, ribose and base. By chemically modifying

each part, its stability, nuclease resistance and gene

silencing efficiency can be increased,

pharmacokinetics and pharmacodynamics can be

improved, the interaction between nucleic acid and

protein can be strengthened, off-target effects can be

reduced, and the efficiency of small nucleic acid

drugs can be improved. The chemical modification of

siRNA mainly includes phosphate skeleton

modification, ribose modification and base

modification.

3.1.1 Phosphate Group Modification

Nuclease hydrolyzes nucleic acid by acting on the

phosphodiester bond on the nucleotide phosphate

skeleton. The center of nuclease attack is the

phosphorus atom in the phosphodiester bond.

Therefore, whether nucleic acid is resistant to

enzymatic hydrolysis depends on the change of

phosphorus atom structure. The most widely used

method is thio modification (phosphorothioate, PS).

The sulfur atom replaces a non-bridging oxygen atom

of the phosphate group to form a thiophosphate bond.

This method can make siRNA resistant to nucleases,

prolong the drug's action period in the body, increase

the specific recognition of small nucleic acids and

target cells, promote their aggregation in the cell

nucleus, and improve the drug's efficacy.

3.1.2 Ribose Modification

Nuclease acts on the 2' hydroxyl group in ribose to

achieve cleavage. Therefore, modification of the 2'

hydroxyl group can protect nucleic acids from attack

by nucleases. The current methods for ribose

modification applied to siRNA include locked nucleic

acid (LNA) and methoxy modification. LNA uses

aminomethylene bridges, thiomethylene bridges or

Recent Advances in siRNA Delivery System and Drugs

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oxymethylene bridges to connect the 2'-O and 4'

positions on the ribose into a ring, thereby forming a

restricted nucleic acid conformation. Compared with

unmodified siRNA, the use of LNA-siRNA to treat

SARS is more effective, and some LNA-siRNA drugs

have entered the clinical stage (Elmen et al.,2005).

Methoxy modification is the most commonly used

nucleic acid modification technology. The 2'-OH

group of the nucleotide is changed to 2'-OCH3. This

method can significantly enhance the affinity of

siRNA for the target gene and improve its ability to

resist nucleases (Li et al.,2014), while having little

effect on the activity of siRNA. The first siRNA drug

approved by the FDA, patisiran, significantly

improves its stability and therapeutic effect by

modifying the nucleosides on the sense and antisense

strands from 2'-OH to 2'-OCH3.

3.1.3 Base Modification

The pharmacological mechanism of small nucleic

acid drugs is based on the precise recognition of

specific hydrogen bonds between them and the target

sequence. In the molecular structure optimization

strategy, base modification technology effectively

regulates drug activity and metabolic characteristics

by reconstructing the intermolecular forces between

nucleotide bases. From the analysis of chemical

modification types, base modification of small

molecule non-coding nucleic acids mainly involves

structural substitution or introduction of functional

groups, among which the structural modification of

NTP has become an important technical path to

expand molecular functions and optimize chemical

selectivity. When selecting spatial modification sites,

the C5 position of the pyrimidine ring and the C2

position of the purine ring are commonly used targets

due to their good chemical modification properties.

Experimental results show that the thermodynamic

stability of RNA molecules is significantly improved

by adding modified nucleotides such as pseudouracil,

2-thiouracil, N6-methyladenosine (m6A) and 5-

methylcytosine, and the activation of TLR-mediated

innate immune response is also effectively inhibited.

It is worth noting that m6A modification has been

proven to have dual regulatory functions: it can

promote the maturation and processing of pri-miRNA

and enhance the epigenetic regulation of X

chromosome inactivation specific transcript (XIST).

In terms of clinical application optimization,

nucleotide site-directed modification shows unique

advantages. Zhang's team's research confirmed that

the introduction of 5-nitroindole modified

nucleotides at the 15th site of the siRNA sense strand

can reduce the off-target effect mediated by the sense

strand by 82% while completely retaining the

targeting activity of the antisense strand

(Zhang,2012). More noteworthy is the new cytosine

analog 6'-phenylpyrrolidine (PhpC), which not only

maintains the ability to accurately pair with natural

bases, but also has high thermal stability (Tm value

increased by 4.2℃) and strong fluorescence

properties (quantum yield of 0.68). The gene

silencing efficiency of the siRNA complex

constructed based on PhpC is comparable to that of

the prototype molecule (IC50=12nM), and its

endogenous fluorescent labeling function provides an

innovative research method for real-time monitoring

of drug intracellular transport and distribution.

4 SIRNA DRUGS: A

CHALLENGING RESEARCH

AND DEVELOPMENT PATH

Since the RNAi phenomenon won the Nobel Prize in

Physiology or Medicine in 2006, people have begun

to pay attention to the potential of siRNA drugs in the

field of precision treatment of diseases, and major

pharmaceutical companies have begun to develop

siRNA drugs. However, due to the difficulty of

siRNA drug development and the lack of delivery

technology, the results of the initial research and

development were not ideal. From 2008 to 2012, two

siRNA drugs for the treatment of blinding choroidal

neovascularization were interrupted in Phase III

studies due to immunotoxicity and unclear efficacy.

Ultimately, the first generation of small interfering

RNA drugs were terminated due to safety concerns

and lack of efficacy (Setten et al.,2019). With the

improvement of siRNA chemical modification

technology and the continuous innovation of siRNA

delivery strategies, from 2013 to 2014, a number of

chemically modified siRNA drugs combined with

targeted delivery systems entered clinical research

one after another, among which the most

representative is the N-acetylgalactosamine (GalNAc)

delivery system. GalNAc-modified siRNA drugs can

specifically bind to the asialoglycoprotein receptor

(ASGPR) highly expressed on the surface of

hepatocytes, deliver siRNA to hepatocytes and exert

pharmacological activity. However, in 2016, the

development of small interfering RNA drugs based

on early chemical modification and delivery

technology failed again. Revusiran is the first siRNA

drug combined with a GalNAc delivery system and

administered subcutaneously. It is designed to treat

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hereditary thyroxine-mediated amyloidosis

polyneuropathy (hATTR). However, in the Phase II

study, the drug failed to achieve the clinical preset

outcome and the study was forced to be terminated

early (Maraganore et al.,2022).

Fortunately, in 2018, the first siRNA drug

Patisiran was approved by the FDA for marketing,

marking its first successful application in siRNA drug

development. Patisiran is used to treat hATTR. It

contains 19 base pairs and uses lipid nanoparticles

(LNP) as a delivery system. By modifying the 2'-OH

of the nucleosides on the forward and reverse chains

to 2'-OCH3, the stability and therapeutic effect are

significantly improved. hATTR is caused by a gene

mutation that causes impaired function of

transthyretin (TTR) in the body. TTR protein is

mainly produced in the liver and is a carrier of

vitamin A. When TTR mutates, abnormal amyloid

TTR (ATTR) accumulates in the human body,

causing damage to human organs and tissues (such as

peripheral nerves and heart), and causing difficult-to-

treat peripheral sensory neuropathy, autonomic

neuropathy or cardiomyopathy. Patisiran can

specifically bind to the conserved gene sequence of

TTR mRNA, so it can specifically silence the

expression of TTR and inhibit the production of TTR

protein, thereby reducing the deposition of amyloid

protein in peripheral nerves and avoiding organ and

tissue damage. So far, the efficiency of siRNA drug

development has advanced by leaps and bounds. Six

siRNA medications have received FDA approval for

commercialization as of 2023.

Another well-known siRNA drug is Inclisiran. It

is used as an adjunct to diet to treat adult patients with

heterozygous familial hypercholesterolemia (HeFH)

or clinical atherosclerotic cardiovascular disease

(ASCVD). Cardiovascular disease (CVD) has always

been the leading cause of death worldwide, and

ASCVD, including hypercholesterolemia, is the main

CVD. Furthermore, dyslipidemia, which is defined

by increased LDL-C or total cholesterol (TC), is a

significant risk factor for ASCVD and is one of the

major causes of ASCVD. Therefore, controlling

LDL-C is crucial for patients with heart disease and

non-heart disease. Inclisiran is a GalNAc-coupled

siRNA drug (GalNac-siRNA) that targets liver

distribution and can directly act on the mRNA

encoding PCSK9 protein. It uses RNA interference

mechanism to induce PCSK9 mRNA degradation,

increase the expression and circulation of LDL-R on

the surface of hepatocytes, and then increase the

uptake of LDL-C and reduce the level of LDL-C in

the circulation. The medication has long-term

persistence, requires just two injections per year, and

is effective for six months following a single

administration.

Nedosiran, the most recent siRNA medication,

received FDA approval in 2023. It was created to treat

primary hyperoxaluria by Dicerna Pharmaceuticals, a

Novo Nordisk affiliate. The rare autosomal recessive

hereditary condition known as primary hyperoxaluria

(PH) is typified by the liver's overproduction of

oxalate. The kidneys ordinarily eliminate oxalate, but

when it builds up too much, it forms crystals of

insoluble calcium oxalate. Nephrocalcinosis and

kidney stones are caused by this buildup, which can

progress to chronic kidney disease. Nedosiran is a

synthetic double-stranded siRNA that is attached to a

N-acetyl-D-galactosamine (GalNAc) amino sugar

residue. Hepatocytes preferentially absorb it through

the succinate glycoprotein receptor (ASGPR)

following subcutaneous injection. It is loaded into the

RNA-induced silencing complex (RISC) inside the

cell, where it breaks down LDHA mRNA by RNA

interference, lowering the synthesis of LDH and

preventing the buildup of oxalate.

5 CONCLUSION

As siRNA research continues to advance, siRNA

medicine development has advanced significantly

and has been crucial in the prevention and

management of a number of illnesses. Since 2018, the

FDA has authorized and marketed six siRNA

medications. At present, many siRNA drugs in

different clinical stages provide new treatment

options for genetic diseases, blood diseases, eye

diseases and tumors. Continuous innovations in

chemical modification and delivery technologies

have given siRNA drugs broad prospects in clinical

applications and are expected to make important

contributions to human health.

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