The landscape of nanoparticle-based siRNA delivery and therapeutic development

Abstract

Five small interfering RNA (siRNA)-based therapeutics have been approved by the Food and Drug Administration (FDA), namely patisiran, givosiran, lumasiran, inclisiran, and vutrisiran. Besides, siRNA delivery to the target site without toxicity is a big challenge for researchers, and naked-siRNA delivery possesses several challenges, including membrane impermeability, enzymatic degradation, mononuclear phagocyte system (MPS) entrapment, fast renal excretion, endosomal escape, and off-target effects. The siRNA therapeutics can silence any disease-specific gene, but their intracellular and extracellular barriers limit their clinical applications. For this purpose, several modifications have been employed to siRNA for better transfection efficiency. Still, there is a quest for better delivery systems for siRNA delivery to the target site. In recent years, nanoparticles have shown promising results in siRNA delivery with minimum toxicity and off-target effects. Patisiran is a lipid nanoparticle (LNP)-based siRNA formulation for treating hereditary transthyretin-mediated amyloidosis that ultimately warrants the use of nanoparticles from different classes, especially lipid-based nanoparticles. These nanoparticles may belong to different categories, including lipid-based, polymer-based, and inorganic nanoparticles. This review briefly discusses the lipid, polymer, and inorganic nanoparticles and their sub-types for siRNA delivery. Finally, several clinical trials related to siRNA therapeutics are addressed, followed by the future prospects and conclusions.

Graphical abstract

Huang and colleagues summarize the unique properties of various nanocarriers, discuss current challenges, and highlight their prospects in siRNA delivery. They also review various clinical studies as well as preclinical studies of siRNA therapeutics, which provides a comprehensive view and multidisciplinary insights regarding nanocarriers for effective siRNA delivery.

Introduction

RNA interference (RNAi) is a unique mechanism of gene silencing within the cells, and, in this way, the responsible gene for the disease can be targeted and silenced. At first, Andrew Fire and Craig Mello published a seminal paper regarding the mechanism of post-transcriptional gene silencing (PTGS) in Caenorhabditis elegans and named it RNAi in 1998.¹ In 2006, Craig Mello and Andrew Fire were awarded a Nobel Prize for discovering the RNAi mechanism. Afterward, two groups of researchers stated that 21–22 nucleotide-based dsRNA known as small interfering RNA (siRNA) could efficiently silence the gene in mammalian cells.^2,3 They also observed that double-stranded RNA (dsRNA) is more effective in gene silencing than single-stranded RNA (ssRNA). On the other hand, double-stranded siRNA targeted the mRNA with the help of the enzyme Dicer, which is involved in the RNAi pathway. Then, the antisense RNA that is complementary to the targeted mRNA headed to the RNA-induced silencing complex (RISC). Afterward, the RISC loading complex (RLC) targets the specific mRNA, followed by gene silencing to inhibit the translation process.⁴ The genes that are mainly involved in the disease can be targeted and silenced efficiently. In the last decade, several clinical trials have been conducted and achieved some major breakthroughs in the field of RNAi. Several US Food and Drug Administration (FDA)-approved siRNA therapeutics have led researchers to work on siRNA to develop more therapeutics to treat various ailments in the future.⁵ Patisiran (Onpattro) was the first siRNA-based therapeutic approved by the FDA in 2018 for the treatment of polyneuropathy in patients with hereditary transthyretin (TTR)-mediated amyloidosis.⁶ In 2019, another siRNA-based drug, givosiran (Givlaari), was also approved by the FDA for treating acute hepatic porphyria.⁷ The third siRNA-based drug, lumasiran, was also approved by the FDA for the treatment of primary hyperoxaluria.⁸ Alongside this, several siRNA-based therapeutics are in clinical trials. So far, five siRNA-based therapeutics have been approved by the FDA. Thereby, siRNA therapeutics are the most important breakthrough for developing reliable therapeutics for the treatment of any disease.

These siRNA therapeutics faced several extracellular and intracellular barriers; hence, they need modification to their chemical structure or delivery systems, or both, to get better transfection efficiency.^9,10,11,12 The chemical modifications for overcoming the siRNA delivery barriers are briefly discussed in this review. Figure 1 represents the structures of five FDA-approved siRNA therapeutics. The first approved siRNA drug, patisiran (Onpattro), is a lipid-based nanoparticle formulation of siRNA, and the other four therapeutics (givosiran, lumasiran, inclisiran, and vutrisiran) are designed with the ligand N-acetylgalactosamine (GalNAc). In addition, the combination of 2′-O-methyl (2′-OMe) and 2′-deoxy-2′-fluoro (2′-F) were used for patisiran; phosphorothioate (PS), 2′-OMe, and 2′-F were employed for inclisiran. Hence, for better siRNA delivery, non-viral nanocarriers are gaining more attention from researchers, including nanoparticles, receptor-targeted nanocomplex (RTN), and liposomes.¹⁷ These nanoparticles, especially lipid-based nanoparticles, polymer-based nanoparticles,¹⁸ poly (lactic-co-glycolic acid) (PLGA) nanoparticles, and inorganic nanocarriers, have been explored for the delivery of siRNA to the target site.^18,19,20,21 These nanoparticle-based siRNA therapeutics showed better in vivo stability, target specificity, and internalization of the siRNA to the cytosol with less cellular toxicity and minimum immune response.²²

Figure 1.

Several types of nanoparticles belong to lipid, polymer, and inorganic classes

FDA-approved siRNA therapeutic structures are presented.

(A) Structure of patisiran. Reproduced and adapted from Torre et al. under the Creative Commons Attribution (CC BY) license.¹³ (B) Structure of givosiran. Reproduced and adapted from Torre et al. under the Creative Commons Attribution (CC BY) license.¹⁴ (C) Structure of lumasiran. Reproduced and adapted from Torre et al. under the Creative Commons Attribution (CC BY) license.¹⁵ (D) Structure of inclisiran. Reproduced and adapted from Torre et al. under the Creative Commons Attribution (CC BY) license. (E) Structure of vutrisiran.¹⁶

Several studies have been published separately in the past decade regarding nanoparticle-based siRNA therapeutics, discussing the individual types of nanoparticles in different papers.^23,24,25 In this review, we highlighted all the nanoparticle-based siRNA formulations with a promising activity that were investigated by several clinical trials. The characteristics, compositions, advantages, and biomedical applications of several nanoparticles-based siRNA are discussed in detail. First, we highlight the comparison of several types of RNAi therapeutics and then briefly address the mechanism of action of siRNA in detail. Afterward, we discussed the delivery barriers siRNA faced and possible solutions to overcome these barriers. Finally, the nanoparticle-based siRNA delivery strategy is addressed briefly, followed by the clinical trials and future prospects.

Comparison of siRNA with other RNA therapeutics

Several RNA therapeutics have been used for various ailments, including siRNA, microRNA (miRNA), short hairpin RNA (shRNA), ribozyme, antisense oligonucleotides (ASOs), and mRNA. Among these therapeutics, siRNA, miRNA, and shRNA act by following the RNAi mechanism to break down the targeted mRNA. miRNA is short, 19–23 base pairs of RNA oligonucleotides, and works by establishing the miRNA-induced silencing complex. These also present naturally in the body and have a particular impact on several diseases. Some miRNAs are biomarkers for specific ailments, and some are tumor suppressors, such as miR-155 and miR-17-92. Besides, miRNA-based therapeutics can target several diseases by degrading the targeted mRNA, hence inhibiting the translation process responsible for the disease progression.

On the other hand, shRNA is an artificial RNA molecule with a tight hairpin turn. Depending on the promoter, it enters the nucleus and is transcribed by polymerase II or polymerase III. Then, it is transported into the cytoplasm and loaded into the RISC for RNAi activity. These three siRNA, miRNA, and shRNA can target genes, but their delivery barriers restrict their use in the clinical pipeline. However, several advancements/modifications have been employed in order to overcome these barriers. Several modifications for better siRNA activity and delivery are discussed in extracellular barriers section. Readers are encouraged to read the appropriate reference for shRNA and miRNA modification, as their explanation is beyond the scope of this review.^26,27,28

Other RNA therapeutics include mRNA therapeutics and ASOs, and ribozymes are also crucial in treating clinical diseases.^29,30,31 ASOs are single-stranded nucleic acid polymers that target the specific mRNA to silence a gene.³² One drug, named exondys 51 (Eteplirsen), was FDA approved as an ASO-based therapeutic on 19 September 2016 and used in patients with a confirmed mutation of the Duchenne muscular dystrophy (DMD) gene, amenable to axon 51 skipping.³³ The drug targeted dystrophin pre-mRNA and facilitated exon 51 skipping.³⁴ The FDA approval for ASO-based medicines has garnered hope for developing new therapeutics in this area.³³ All other ASO-based approved drugs are discussed in Table 1. In addition, the RNA vaccines are also a vital strategy to combat disease, as in the case of the current viral pandemic, COVID-19.⁴⁴ Several RNA-based vaccines are in clinical trials, and two got approval by FDA authorization BNT162 (BioNTech/Pfizer) and mRNA-1273 (Moderna) for COVID-19. Janssen COVID-19 vaccine has also received FDA emergency authorization against COVID-19.⁴⁵ Several vaccines are in phase 3 clinical trials, and a number of others are in phase 1/2 clinical trials for the treatment of COVID-19. The delivery of mRNA vaccines can be facilitated by carriers such as lipid nanoparticles (LNPs), polymers, and peptides.^46,47 siRNA-LNP formulation of Onpattro (patisiran) has a 3-year shelf life at 2°C–8°C.⁴⁸ However, these mRNA vaccines need to be supplied in freezing or ultra-freezing conditions due to the instability of mRNA rather than LNP instability.⁴⁸ The comparison is briefly addressed in Table 2.

Table 1.

ASO-based approved therapeutics

ASO-based therapeutic	Disease	Target	Functional mechanism	Clinical status	Company
Fomivirsen35,36	CMV retinitis	CMV IE-2 mRNA	downregulates IE2	initially approved in 1998 but Novartis stopped marketing in 2002 in Europe and 2006 in US	Ionis Pharmaceuticals, Novartis
Inotersen37	hereditary transthyretin-mediated amyloidosis	mutant and wild-type transthyretin mRNA	downregulates transthyretin mRNA	approved in 2018	Ionis Pharmaceuticals
Eteplirsen33	DMD	exon 51 on DMD pre-mRNA	splicing modulation	approved in 2016	Sarepta Therapeutics
Nusinersen38	SMA	exon 7 on survival motor neuron-2 pre-mRNA	splicing modulation	approved in 2016	Ionis Pharmaceuticals, Biogen
Mipomersen39	HFH	Apo-B-100 mRNA	downregulates Apo-B	approved in 2018	Ionis Pharmaceuticals, Kastle Therapeutics, Genzyme
Golodirsen40	DMD	induce exon 53 skipping	splicing modulation	approved in 2020	Sarepta Therapeutics
Milasen41	Batten’s disease	CLN7	splicing modulation	approved in 2018	Boston’s Children Hospital
Casimersen42	DMD	exon 45 of DMD	splicing modulation	approved in 2021	Sarepta Therapeutics
Viltolarsen43	DMD	exon 53 of DMD	splicing modulation	approved in 2020	Sarepta Therapeutics

CMV, cytomegalovirus; DMD, Duchenne muscular dystrophy; SMA, spinal muscular atrophy; HFH, homozygous familial hypercholesterolemia; Apo-B-100: apolipoprotein-B-100.

Table 2.

Comparison of different RNA-based therapeutics

RNAi therapeutics	Composition	Basic mechanism of action	Advantages	Disadvantages/barriers
siRNA49	double-stranded RNA, short length, the duplex of 20–24 nucleotide base pairs	it acts within the cytoplasm by establishing the RLC. That eventually targets the mRNA and blocks the protein synthesis followed by silencing of the gene	high transfection efficiency with suitable vectors including liposomes or polymers it can transfect into several types of cells of different organs cellular uptake by passive diffusion most barriers can be bypassed by modification of the siRNA and delivery vectors	intravascular degradation renal clearance activation of the immune system protein binding with siRNA MPS entrapment membrane impermeability endosomal escape off-target effects
shRNA26	artificial RNA molecule with a tight hairpin turns	at first, shRNA entered into the nucleus, transcribed by polymerase II or polymerase III, which depends on the promoter, then is transported into the cytoplasm and loaded into the RISC for RNAi activity	higher transduction efficacy long-term effect it can be pooled inducible expression	toxic effects produced by viral vectors technologically challenging off-target effects
miRNA50	short (19–23 base pairs) RNA oligonucleotides	first, miRNA is loaded into the argonaute enzyme and destabilizes the mRNA	target a wider range of mRNA as extensive base pairing is not required naturally occurring miRNA can be detected as potential biomarkers one miRNA can target several genes	off-target effects nuclease degradation
ASOs51	short single-stranded synthetic RNA (or DNA) 12–30 nucleotides long	the mechanisms of action of ASOs are very diverse. They can target the AUG start site of the open reading frame followed by blocking the association of ribosomal subunits. They can also bind with the sequences present on exons or introns and lead to pre-mRNA splicing	highly specific highly efficient in knocking down the genes longer responses	off-target effects. degradation by nucleases
Ribozyme52	an RNA tertiary structure that can act as a protein enzyme to catalyze the biochemical reaction	ribozymes suppress gene expression by targeting mRNA in cis or trans. This happens by acid-base catalysis or depends on the type of ribozyme. This immediately suppresses the protein synthesis site-specific cleavage of RNA	binds more precisely than other therapeutics cleaves mRNA in a specific manner	poor stability
mRNA53	mRNA is a single-stranded RNA molecule that is complementary to the DNA for a specific gene	mRNA is internalized into the cytoplasm by cell-specific mechanism and in vitro transcription mRNA is translated by protein synthesis machinery	improved efficiency replacement therapy (supply therapeutic proteins) vaccination	can cause adverse effects; most common are fatigue, muscle aches, and headaches immunogenicity, stability
RNA aptamers54	short single-stranded oligonucleotides	RNA aptamers bind to the target in a shape-fitting manner through three-dimensional interaction. They can inhibit various targets, including proteins, nucleotides, peptides, small molecules, antibiotics, and cells	target specificity superior affinity for the target easily modified and smaller size higher reproducibility	RNA aptamers are not exactly complementary to their target, so they can interact with other biomolecules prone to quick degradation in the systemic circulation

Mechanism of action of siRNA

RNAi is a unique mechanism in which siRNA silences the specific gene responsible for the disease. That is why the siRNA works by following the RNAi pathway. At first, the dsRNA paired completely, ranging from 15 base pairs (bp) to 30 bp. A length of less than 15 bp cannot be part of the RNAi process, and, on the other hand, a length greater than 30 bp can cause severe complications, such as toxicity and protein kinase R (PKR)-induced impediments.⁵⁵ Those siRNAs longer than 21 bp can easily interact with the Dicer RNAi enzymes. Besides, siRNA shorter than 21 bp can interact with the Tar RNA binding protein (TRBP) and be set up to build the RNA-induced silencing RLC. Before the maturation of RISC, the RLC selects the guide strand among the sense and antisense strands. The guide strand is 100% complementary against the targeted mRNA. After loading the antisense (guide) strand, the Dicer and TRBP dissociate from the RISC.⁵⁶ In addition, the Ago2 is an enzyme that cleaves the mRNA and inhibits the translational activity of the mRNA. The gene expression regulates the disease, and, by degrading the specific mRNA involved in the protein translation of the particular disease, the gene expression can be downregulated.⁵⁷ On the other hand, miRNAs are not fully complementary to their target and hence can silence several targets simultaneously. Figure S1 represents the mechanism of action of siRNA and miRNA.

Delivery barriers

Extracellular barriers

Extracellular barriers to delivering siRNA therapeutics include intravascular degradation (by plasma nucleases and lysosomal nucleases), renal clearance, plasma protein binding, mononuclear phagocyte system (MPS) entrapment, and membrane impermeability.¹¹

After transfecting the siRNA into the systemic circulation, the first barrier is intravascular degradation, in which the plasma nucleases degrade the naked or unmodified siRNA and reduce the bioavailability. These A-type nucleases may present in intracellular or extracellular space. The siRNA possesses a small molecular weight of 13 kDa and a small size of 7 nm in length, making it susceptible to fast renal clearance.⁹

Protein binding is also one of the critical extracellular barriers in delivering siRNA therapeutics. The carriers for siRNA usually have a positive charge, and proteins possess a negative charge. In this way, it can easily be opsonized by blood complement proteins or cause unspecific responses by interacting with proteins. Opsonization happens within the systemic circulation, and their completion time is from seconds to days. After opsonization, the opsonized complex may enhance the MPS entrapment and cause severe inflammation.⁵⁸ This mechanism ultimately reduces the bioavailability of the siRNA.

Furthermore, MPS entrapment is one of the most critical barriers, and MPS-rich organs include the liver, spleen, and bone marrow. Nanoparticle carriers greater than 100 nm can easily be trapped by MPS and degraded by monocytes and macrophages. Several siRNA and carrier modifications can bypass these barriers.⁵⁹ Coating of the nanoparticle with hydrophilic molecules such as polyethylene glycol (PEG) can also play an important role in the ability of the siRNA delivery carrier to evade the immune system and associated phagocytes.¹⁰

Another extracellular barrier is membrane impermeability for siRNA therapeutics. Naked or unmodified siRNAs are exposed to a complex environment filled with omnipresent nucleases and immune cells, leading to swift degradation and elimination.⁶⁰ For efficient delivery and therapeutic activity, siRNA should be delivered with suitable carriers. Chemical modifications, such as PS, 2′-O-methyl (2′-OMe), glycol nucleic acid (GNA), and 5′-(E)-vinylphosphonate [5′-(E)-VP]) and 2′-deoxy-2′-fluoro (2′-F), also enhanced the activity and stability of siRNA (Figure 2).⁶¹ The strategy to bypass the extracellular barriers is briefly discussed in Table 3.

Figure 2.

Structural illustration of chemical modifications for siRNA and ASO therapeutics

According to the modification site, these can be classified into three categories: phosphonate modification, ribose modification, and base modification, which is indicated in red, purple, and blue. R = H or OH. (S)-cEt-BNA, (S)-constrained ethyl bicyclic nucleic acid; PMO, phosphorodiamidate morpholino oligomer. Modified from Hu et al.²⁴⁹

Table 3.

Modifications of siRNA itself or with carriers to improve the delivery against extracellular barriers

Barrier	Modifications	Potential outcomes	Reference
Intravascular degradation and renal clearance	Modification of siRNA itself: •internal uridine to 2,4-difluorotoluylribonucleoside substitution. •2′-deoxy-2′-fluoro-β-D-arabino, 2′-O-MOE modification, 2′-O-Me modification •phosphorothioate modifications Modification with carriers: •cholesterol-siRNA •PEG-siRNA •cationic comb-type copolymers	•nucleases resistance •increased half-life in the systemic circulation •increased half-life of up to 90 min and resist renal clearance •reduced the renal clearance by 50% •enhanced the siRNA stability and resistance toward serum nucleases	Xia et al.275 Dowler et al.276 Amarzguioui et al.277 Ambardekar et al.278 Iversen et al.279 Sato et al.280
Innate immune system activation	modification of siRNA itself: •avoiding uridine and guanosine •replacing the 2-hydroxyl uridines with 2-fluoro, 2-O-methyl, or 2-deoxy uridines	•mimic the immune response in sequence-specific manner •reduced immune system activation	Varley et al.281 Robbins et al.282
Protein binding	modification with carriers: •surface modification; coating with hydrophilic positively charged polymers such as PEG •coating with PLGA nanoparticles	•reduced opsonization •reduced protein adsorption by 75%	Zhou et al.20 Zhou et al.20
MPS entrapment	modification with carriers: •coating with polyethylene oxide and poloxamers	•increased half-life and remained in blood circulation	Du et al.283
Membrane impermeability	modification with carriers: •coating with polymers or lipid carriers •coating with positively charged nanoparticles, polyamidoamine dendrimers •Attached immunoglobulins, aptamers	•hide the siRNA net negative charge and helps to cross the membrane •interaction of positively charged carrier and negatively charged membrane causes internalization of siRNA •interact with specific cell receptors and causes internalization	Mainini and Eccles284 Patil et al.285 Zhou and Rossi, Zhao et al.286,287

Another critical barrier is delivering the siRNA into the target site within the body. There is an enhanced permeation and retention effect (EPR) attributed to the leaky vasculature in tumors, which is used for passive tumor targeting of siRNA. In addition, modification with targeting ligands on nanoparticles, such as antibodies, peptides, and aptamers, has been reported to promote site-specific accumulation of nanoparticles containing siRNA. Givosiran , an FDA-approved siRNA, is conjugated with GalNAc. This molecule targets the a sialoglycoprotein receptor (ASGPR), enhancing the accumulation of siRNA in liver. In this way, the specific organs and tissues can be targeted by surface ligand modifications.⁷ In addition, several types of tumors can be targeted by cyclic arginine-glycine-aspartic acid (RGD), and it binds to the avβ3 and avβ5 integrins, which are overexpressed in angiogenic tumor cells.⁶² The antibody (F105)-siRNA complex targeted HIV cells and gave better results.⁶³

Intracellular barriers

After internalization of the siRNA through endosomes, the major problem is endosome entrapment. The endosomes can be disrupted before the release of the siRNA into the cytoplasm by using the endosomolytic agents, including polymers, small molecules, and peptides. The proton sponge effect can also be employed, in which the chloride influx into the endosomes leads to endosomal membrane disruption and releases endosomal contents. For this purpose, polyethylenimine (PEI) is used with siRNA to produce the proton sponge effect. However, cytotoxic effects have been reported with PEI that limit this molecule’s use.⁶⁴ Benjaminsen et al. measured the lysosomal pH as a function of PEI content and found that PEI does not affect the lysosomal pH.⁶⁵ Chen et al. found that the membrane destabilization after internalization of the polymer-siRNA complex is attributed to the cleavage chains of PEI.⁶⁶ Another mechanism of protonation can also be employed in which amino acids, especially lysine and arginine , get protonated in low pH and cause membrane disruption. Poly-L-arginine (PLA) can also help in disrupting the endosomal membrane by the proton sponge effect for efficient siRNA release into the cytoplasm.⁶⁷ Zhang et al. designed an amphiphilic peptide 2KH7-TAT containing seven arginines that promoted proton sponge effect extensively and help endosome escape of the drug.⁶⁸ Another molecule, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), can be used as a fusogenic lipid carrier for siRNA delivery and to induce endosomal escape.⁶⁹

Moreover, siRNA also triggers the innate immune system and the production of cytokines within the body. The immune response can be a Toll-like receptor (TLR) - dependent or - independent pathway. In the TLR-dependent pathway, three receptors have been reported, namely TLR3, TLR7, and TLR8.⁷⁰ Among these, TLR3 acts in a sequence-independent manner and the other two act in a sequence-dependent manner against siRNA response. TLR3 receptors are present on the human endothelial cells of the aorta, lung, and umbilical vein. In a sequence-dependent manner, TLR7/8 recognized the uridine and guanosine with UG or 50′-UGU-30′ nucleotides. On the other hand, plasmacytoid and monocytes express TLR7 receptors.⁹ In the TLR-dependent pathway, different cytoplasmic RNA sensors include melanoma differentiation-associated gene 5 (MDA-5), PKR, and retinoic acid-inducible gene 1 (RIG-1, also known as DDX58). RIG-1 binds to the RNA and mediates the production of interferons.⁹

Designed nanoparticles for siRNA transfection

As discussed earlier, siRNA delivery possesses several intracellular and extracellular barriers, so it is necessary to develop delivery vehicles that both facilitate accumulation in the target tissue and protect siRNA from degradation in systemic delivery. The best strategy to deliver siRNA is by using a nanoparticle system whose size is less than 100 nm. These nanoparticles help to reduce the immunogenicity, prolonged duration in systemic circulation to reach their site of action, and the route of administration. These nanoparticle carriers can deliver the synthetic siRNA to the target site more efficiently.^{71 ,72}

Moreover, these nanoparticles also possess some drawbacks, such as unnecessary and insufficient accumulation to the target site, toxicity, and stability issues, especially in liposomes with specific lipid structures. However, these limitations can be diminished by selecting specific lipids and polymers with more stability and fewer toxicity features.⁷¹ The text briefly discusses all the pros and cons of each delivery system.Figure 3 represents several nanocarriers for effective siRNA delivery.

Figure 3.

Several nanocarriers for the delivery of siRNA

Lipid-based nanocarrier delivery system

Liposomes

Lipid-based nanoparticle delivery carriers such as liposomes have gained the utmost importance in delivering DNA, ASOs, and siRNA.^73,74 Patisiran, the first FDA-approved siRNA-based drug, is delivered by lipid-based carriers.⁷⁵ Liposomes are structurally spherical vesicles containing an aqueous core adjunct with a bilayer of phospholipids. Liposomes are biologically compatible as well as biodegradable. Liposomes possess an amphipathic nature, so they are carriers of choice for several hydrophobic and hydrophilic therapeutics.⁷⁶ Liposomes are usually fused with the biological membranes, and the encapsulated siRNA is released in the cytoplasm by endocytosis. Cationic liposomes encapsulate the siRNA by interacting with their negative charge and forming the lipoplex complex. Besides, their positive charge can cause cytotoxicity and interactions with serum proteins having a negative charge on their surface. To overcome this barrier, a clinical trial was performed by formulating the neutral lipids with siRNA, as 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), to target EphA2 and got better results with reduced toxicity.⁷⁶ Recently, research was conducted to check siRNA delivery by viscous core liposomes. They concluded that the positively charged viscous core liposomes encapsulated siRNA internalized into the cells and targeted the green fluorescent protein (GFP) expression without causing toxicity.⁷⁷ Huang et al. discovered an innovative siRNA delivery carrier to treat hepatitis B. In this experiment, they prepared novel ionizable lipidoid nanoparticle (RBP131), and siRNA was chosen by following the Investigational New Drug (IND) application requirements to target the hepatitis B virus. RBP131 was used to encapsulate the siRNA and termed RB-HBV008. After intravenous injection, the siRNA was transfected into hepatocytes with an median effective dose of 0.05 mg/kg. The viral RNAs and antigens (HBsAg and HBeAg) along with viral DNA were suppressed in a dose- as well as time-dependent manner after transfecting the formulation into transgenic and transient mouse models. Moreover, the toxicity evaluation suggested safety outcomes 10 times the therapeutic window. Thus this liposomal formulation provides an effective nucleic acid delivery for the treatment of hepatitis B.⁷⁸ Yang et al. proposed an excellent delivery strategy for mRNA delivery without any toxicity. In this experiment, the mRNAs encoding luciferase and erythropoietin (EPO) were prepared by in vitro transcription and later formulated with ionizable LNP (iLNP). This iLNP was based on iBL0713 lipid for in vivo and in vitro expression of desired proteins using codon-optimized mRNAs. Finally, the formulation was prepared and named mRNA-iLNP171. The maximum expression of luciferase and EPO was noted 6 h post administration in both hepatocellular carcinoma cells and hepatocytes. Thus, the experiment provides the basis for the development of nucleic acid therapeutics.⁷⁹

Specific modifications can combat the barriers posed by liposomes to reduce the cellular toxicity and enhanced stability. These can be done by adding fusogenic lipids, including DOPE. In other cases, the cationic lipids with ester bonds can be used to reduce the toxic effects and increase the chances of endosomal escape.⁸⁰ For targeting nanoparticle delivery, P-selectin can be attached covalently to the liposomes, and PEGylation can stabilize these liposomes by 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)-2000] (DSPE-PEG2000). This also induces the siRNA concentration within the systemic circulation to contact their target site and efficiently produces an effect.⁸¹ PEG coating can decrease the opsonization process along with unspecific binding.⁹ Theoretically, it is considered that cationic liposomes have better stability and efficiency than neutral liposomes. However, another study demonstrated that the neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine-based liposomes in tumors of murine models were safe with 10- to 30-fold more effectiveness as compared to naked/unmodified siRNA and cationic liposomes.⁸² Another study developed a liposome-polycation-DNA (LPD) nanoparticle and anionic LPD to deliver siRNA to multi drug resistance (MDR) tumors. Injection of siRNA with a dose of 1.2 mg/kg with doxorubicin showed efficient inhibition of tumor growth.⁸³ Figure S2 represents the liposome-mediated siRNA delivery.

Solid LNPs

Solid LNPs (SLNs) are composed of non-toxic lipids, highly biocompatible with a size range of 50–1,000 nm.⁸⁴ These have been investigated in delivering several therapeutics and cosmetics.^85,86 SLN is composed of a lipid core surrounded by a lipid membrane, as shown in Figure S3.⁸⁷ Several methods can develop the SLNs, including sonication, solvent evaporation, spray drying, supercritical fluid extraction of emulsions, ultra-sonication, and hot or cold homogenization.^88,89 Hydrophobic therapeutics can be incorporated with the core of SLN and give a sustained-release effect. SLNs can be used to deliver siRNA by adding cationic lipids to develop the electrostatic interaction, and these deliveries can be beneficial for targeting cancer and liver diseases.^90,91

Furthermore, the hydrophobic ion-pairing (HIP) approach can be used to incorporate an siRNA in the core of SLN.⁹² This method involves the formation of an ion complex between siRNA and cationic lipids. Then, this complex is encapsulated into the core of SLN; the example includes siRNA-1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) incorporated in the triolein core.⁹³ This method successfully showed a sustained-release effect of siRNA in the mice over 10 days.⁹² In addition, another experiment was performed using betamethasone that was incorporated with siRNA into the core using SLNs and reduced the induction of inflammatory cytokines within the blood of mice.⁹⁴ The sustained-release effect can be controlled by fluctuating the ratio of core lipids to surface lipids.⁹⁵ SLNs are more biocompatible as compared to polymers due to the avoidance of unnecessary organic solvents. In addition, using physiological lipids to prepare SLN makes it more stable and avoids the risk of systemic toxicity. Apart from their advantages, there are some cons, including unexpected crystallization of drugs and drug expulsion, unpredictable particle growth, and gelation tendency.⁹⁶

Nanostructured lipid carriers

Nanostructured lipid carriers (NLCs) are the modified version of SLNs in which mainly the lipid phase may comprise solid or liquid forms at ambient temperature.⁸⁸ NLCs have an advantage over SLNs because of their greater loading capacity and stability but with bio-toxicity relevance, as shown in Figure S4. NLCs have several advantages, including better physical stability, controlled particle size, and better entrapment of hydrophilic and lipophilic drugs.⁹⁷ There are also some barriers possessed by NLCs, including cytotoxic effects related to the matrix, their concentration, and the irritative action of some surfactants.⁹⁷ Several studies have shown that the NLCs have the ability to deliver nucleic acid therapeutics within the body. In one study, researchers prepared a multifunctional NLCs system to deliver siRNA against doxorubicin or paclitaxel cellular resistance to treat lung cancer. They concluded that the NLC-mediated siRNA delivery enhanced the antineoplastic activity of the drugs.⁹⁸ Another study was conducted in which they developed multifunctional NLCs to co-deliver tacrolimus and siRNA to treat psoriasis. They noted the synergistic effect of siRNA and tacrolimus reduces the tumor necrosis factor (TNF)-α cytokine expression by 7-fold. In this way, NLCs play an essential role in delivering the TNF-α-siRNA to treat psoriasis.⁹⁹ NLCs can also act as sustained-release drug delivery systems by doing some modifications. For example, the degradation time of NLCs can be manipulated, leading to the sustained release of siRNA for up to 9 days.¹⁰⁰

LNPs

The basic principle of LNP formation is self-assembly, an integral property of smart nanosystems. Several lipids of different chemical nature have been explored for siRNA delivery due to their reduced toxic effects and biodegradable properties. Two chemical modifications may be employed to lipids to enhance the transfection efficiency of siRNA; the first includes multiply unsaturated alkyl chains promoting significant destabilization of intracellular membrane bilayer.¹⁰¹ Second, protonation of the ionizable dimethylaminopropyl group only during liposome maturation improves LNP initial fusion with the membrane.¹⁰² Varun et al. explored the potential of PEG density on the LNP surface, and they observed that increased density of PEG could potentially reduce the immune-stimulatory effect of LNP. It can also minimize hemolytic activity, resulting in the formation of a steric barrier. This study suggested a better modification strategy for LNP to enhance the transfection efficiency with safety profiles.¹⁰³ Huang et al. prepared ionizable lipid-like nanoparticles to treat hyperlipidemia. They designed four panels of lipid formulations, namely A1-B3-7, A1-D1-5, A2-C1-8, and A3-C1-8/D1-7, but A1-B3-7 was stable at 40°C. The optimized formulation, ionizable lipid-assisted nucleic acid delivery (iLAND), showed a dose- and time-dependent gene-silencing pattern with a median effective dose of 0.18 mg/kg. In addition, the serum cholesterol and triglyceride were reduced by targeting the apolipoprotein C3 (APOC3) and angiopoietin-like 3 by transfecting the siRNAs into high-fat-diet-fed mice and human APOC3 transgenic mice. This experiment showed excellent results with better safety profiles by using siRNA-iLAND that was prepared with thermostable ionizable lipid A1-D1-5, resulting in hyperlipidemia therapy and prevention of metabolic diseases.¹⁰⁴ In another experiment, Huang et al. reported that ionizable LNP iLP181 encapsulated psgPLK1, the best-performing plasmid expressing Cas9, and singe-guided RNA (sgRNA) targeting Polo-like kinase 1 (PLK1). The newly designed formulation iLP181-psgPLK1 showed neutral zeta potential at pH 7.4 and effectively triggered editing of the PLK1 gene with more than 30% efficiency in HepG2-Luc cells. It was revealed that this nanoformulation can be accumulated at the tumor site for more than 5 days. It showed better efficacy in reducing the tumor load as compared to other nucleic acid drugs such as siRNA. Hence, it was proved that lipid nanocarrier can be confidently used to deliver the CRISPR-Cas system but also constitutes a potential cancer treatment regimen based on DNA editing of oncogenes.¹⁰⁵

Several scientists have explored LNPs for their ability to deliver siRNA therapeutics efficiently and safely to the target site.^106,107 Tomohiro et al. developed LNPs conjugated with cell-penetrating peptides (CPPs) and examined the transfection and safety profile. They observed that CPP-modified LNPs were protected against serum nucleases and efficiently internalized into B16F10mrine melanoma cells. The internalization process was also examined without CPP, resulting in poor internalization into these cells. This modified CPP-LNP-siRNA was internalized into B16F10 cells overexpressed with luciferase and HT1080 human fibrosarcoma cells expressing GFP. This complex efficiently internalized into these cells and reduced the expression to a certain level. This study suggested that CPP-modified LNPs can be the potential carrier for siRNA delivery into the cytoplasm, resulting in efficient gene silencing.¹⁰⁸ Huang et al. designed an imaging-guided nanocarrier for the targeted delivery of siRNA into the cells. They fabricated multifunctional iLNP for siRNA delivery along with an MRI contrast agent. The iLNP consists of DSPC, cholesterol, PEGylated lipid, contrast agent DTPA-BSA (Gd), and ionizable lipid named iBL0104. In addition, the tumor-targeting cyclic peptide termed GARP with sequence (c(GRGDSPKC)) was also decorated with iLNP-siRNA for targeted therapeutic agent delivery. GARP/iLNP-siRNA rapidly and effectively escaped from endosome and lysosome after internalization. GARP-siPLK1 showed better tumor inhibition than those without GARP. The research reveals the importance of iLNP and the tumor-targeting cyclic peptide for the targeted delivery of siRNA.¹⁰⁹ Figure S5 represents this whole mechanism of the ionizable LNP-based siRNA delivery to target tumors.

A recent study developed LNP-based siRNA and delivered it into mice to target severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Surprisingly, it successfully suppressed the virus progression in the lungs and can be an adjunct therapy with current vaccines.¹¹⁰

Micelles

Polymeric micelles, self-assembling nano-constructs of amphiphilic copolymers with a core-shell structure, have been used as versatile carriers for delivery of drugs as well as nucleic acids.¹¹¹ Polymeric micelles of siRNA developed to date are categorized into two categories depending on their structure: (1) micelles formed through direct conjugation of PEG via degradable or nondegradable linkages to siRNA and further condensation of PEG-siRNA with an siRNA condensing agent (e.g., polycations) to micellar structure; (2) polymeric micelles formed by complexation of an amphiphilic block copolymer containing a polycation (and/or lipid) segment with siRNA followed by micellization of block copolymer/siRNA complex.¹¹¹

PEGylation is the common strategy to improve the blood circulation and the pharmacokinetic properties of a nanocarrier to successfully passive target solid tumors by EPR effect.¹¹² PEG, as a steric barrier, can effectively prevent rapid opsonization of micelles by the MPS, prolongs its circulation time, which give micelles more opportunities to accumulate slowly in the tumor through the leaky vasculature.^111,113,114

Micelles can also be used in active targeting delivery via incorporation of targeting ligands into the micelle shell, which provides control of biodistribution and site-specific cellular uptake of micelles.¹¹² The commonly used targeting ligands include lactose (to bind asialoglycoprotein receptors in hepatocytes), cyclic RGD peptide (to bind αvβ3 integrin receptors expressed in several cells, including cancerous), transferrin, and hyaluronic acid.¹¹² Micelles have been used to deliver siRNA and are delineated in Table 4.

Table 4.

Polymeric micelles for siRNA delivery

Micelle composition	siRNA dose	Target gene/protein	Potential outcomes
PEG-SS-siRNA/PEI115	100 nM	VEGF	these micelles encapsulated siRNA successfully transfected into the prostate carcinoma cells (PC-3) and silenced the VEGF gene expression up to 96.5%
Lactose-PEG-siRNA/PLL116	100 nM	luciferase	this micelle-based siRNA was delivered into the hepatoma cells, and effective up to 100 times gene-silencing activity was observed
6PEG-siRNA-Hph1/KALA117	75 pmol	GFP	this siRNA combination with micelles is effective in delivery and protected against enzymatic degradation. It inhibited the GFP gene expression in MDA-MB-435 cells
LHRH-PEG-SS-siRNA/PEI118	50 nM	VEGF	these micelles showed increased cellular uptake compared to those without LHRH and caused effective VEGF gene silencing
PEG-SS-siRNA/PEI119	100 nM	VEGF	these nanocarriers delivered by intratumoral route and silenced the VEGF expression without any inflammatory response in vivo
PDDT-Ms/siPLK1120	50 nM	PLK1	PDDT-Ms/siPLK1 efficiently targeted PLK1 gene expression in HepG2-xenograft highly malignant patient-derived xenograft models by promoting the release of siRNA into the cytosol. Figure S6illustrates this therapeutic strategy

Exosomes

Exosomes are small membrane vesicles (30–120 nm) of endocytic origin released into the extracellular environment by various cell types, which play a vital role in intercellular communication and carry a net negative surface charge under physiological conditions.^121,122,123 Compared to conventional delivery systems such as LNPs, exosomes have the following advantages: (1) more stable in body fluid than LNPs, which can be easily removed by MPS; (2) possessing relatively low cytotoxicity and immunogenicity due to their endogenous source and high biocompatibility; (3) providing better drug protection than LNPs, in which drug appeared outside and are easier to degrade; (4) delivering both hydrophobic and hydrophilic molecules; (5) showing effective homing ability to tumor sites; (6) crossing the blood-brain barrier and reaching the brain tissue owing to their small size and other characteristics.¹²³

Moreover, the exosomes can be used as a targeted therapy of siRNA. For this reason, specific ligands can be introduced onto the surface of exosomes to select the target site. Alvarez-Erviti et al. developed dendritic cells producing exosomes expressing rabies viral glycoprotein (RVG) peptide to target the neuronal cells.¹²⁴ Several exosome studies revealed that artificial exosomes or their mimetics can be developed to deliver siRNA. Researchers developed exosome membrane-coated nanoparticles and conjugated siRNA siS100A4, collectively called SA/siS100A4@exosome, for targeted delivery to lung pre-metastatic niche. The in vivo data revealed that the affinity of SA/siS100A4@exosome was higher compared to the SA/siS100A4@liposome and inhibited the growth of metastatic breast cancer.¹²⁵

Polymer-based nanocarriers

Lipid-polymer hybrid nanoparticles

These lipid-polymer hybrid nanoparticles (LPHNPs) were developed to overcome several barriers that independent lipid or polymer nanoparticles possess for siRNA delivery. These LPHNPs have shown better encapsulation efficiency, well-defined release kinetics, stability in serum, and targeting capacity with the attached ligand. Several ligands can be attached, such as transferrin, RGD, folate, and an antibody that depends on the target site.¹²⁶ Furthermore, LPHNPs are gaining recognition by combining the advantages of lipids and polymers, such as targeted drug deliveries, cancer gene therapy, diagnostic imaging methods, and vaccine development.¹²⁷ In one experiment, these LPHNPs were used to deliver siRNA-REV1, and siRNA-REV3L with cisplatin to target tumors. siRNA transfected efficiently and suppressed both genes to reduce the tumor mass. In addition, these nanoparticles showed a synergistic effect on gene inhibition in human lymph node carcinoma of the prostate xenograft mouse model.¹²⁸ Tao and coworkers developed LPHNPs and siRNA that target lesional macrophages as a potential treatment for atherosclerosis. The formulation targeted the Ca²⁺/calmodulin-dependent protein kinase γ (CaMKIIγ) for the treatment of advanced atherosclerosis and showed excellent knockdown of CaMKIIγ in atherosclerotic mice models; hence, it was proved to be effective in the treatment of atherosclerosis.¹²⁹

Dendrimer

Dendrimers have recently been investigated in delivering siRNA therapeutics to the target site safely and efficiently. Dendrimers are well-defined globular structures of multi-branched polymers that are characterized by a central core, branches of repeating units, and an outer layer of multivalent functional groups.¹³⁰ These functional groups can electrostatically interact with siRNA, whereas the hydrophobic inner cavities can encapsulate uncharged, non-polar molecules through a number of interactions.¹³⁰ There are different generations of dendrimers, each of which has a crucial role in delivering siRNA. Third-generation dendrimer poly(amidoamine) (PAMAM) has been used for siRNA delivery. The dendrimer and siRNA interaction or adaptation depends on the dendrimer’s generation and pH for the back-folding. Fourth-generation dendrimer (G4) shows better adaptation with siRNA, and G6 acts as a rigid sphere with consistent loss in the binding affinity. Besides, G5 demonstrated hybrid behavior with both rigid and flexible aspects, but the properties depend solely on the pH.¹³¹ Amine-terminated PAMAM dendrimers have several advantages in siRNA delivery. Because of surface amine groups, these are protonated and can bind to the siRNA. PAMAM dendrimers are non-immunogenic and safe, thus overcoming the barriers posed by non-viral vectors.^132,133 At the same time, several challenges are also associated with these carriers. High-generation dendrimers possess high transfection efficiency and cytotoxicity.

On the other hand, low-generation dendrimers have low transfection efficiency and cytotoxicity. That is why there is an urgent need to break up this correlation to reduce these side effects in delivering siRNA.^134,135 Another barrier is the endosomal escape, and, for this reason, siRNA cannot be helpful in silencing gene expression.¹³⁵ Last, another big challenge is the release of siRNA from the dendrimer-siRNA complex after cellular uptake.¹³⁶ Cell receptor-targeted delivery of siRNA with PAMAM has been investigated in which they synthesized a conjugate of PAMAM and paclitaxel. The complexation was formed by the interaction of cationic charges of a dendrimer with siRNA to minimize the systemic degradation and avoid the side effects proposed by chemotherapy. In addition, they can easily be internalized by cancerous cells and their modified form with neutral surface showed low cytotoxicity.¹³¹

Moreover, dendrimers possess a synthetic analog of luteinizing hormone-releasing hormone (LHRH) that acts as an anticancer-targeted moiety. The experiment revealed that the targeted form of dendrimer-siRNA could silence the BCL2 gene, but the untargeted could not silence.¹³⁷ It has been suggested that the silencing activity of the complex not only depends on modifying the chemical structure of the dendrimer but using the medium with low ionic strength to form a stable small dendrimer-siRNA complex to achieve better silencing activity, as shown in the case of siRNA-G7 but not with lower-generation dendrimers.¹³⁸ Dendrimer-conjugated magnetofluorescent nanoworms (dendriworms) were synthesized as a source to deliver siRNA in vivo. This combination induced the endosomal escape efficiently to target protein degradation after in vivo delivery. Dendriworms can easily be internalized into the cells and cause endosomal escape that is not seen in the case of nanoworms or dendrimers alone. Dendriworms were able to reduce the epidermal growth factor receptor (EGFR) expression by 70%–80% in human glioblastoma cells, which is far better than the cationic lipids. It is well tolerated in the mouse brain after 7 days of loading doses and in the EGFR-mediated mouse model.¹³⁹ Another study employed several strategies to minimize the barriers in order to deliver siRNA into the body. This study suggested that fluorinated dendrimers showed enhanced siRNA loading capacity with high electronegativity. Phosphate dendrimers showed a hydrophobic backbone with a hydrophilic surface that eventually enhanced the cell permeability to target tissues.¹⁴⁰ Lipid-based dendrimers have been investigated thoroughly and gave better results regarding their efficient siRNA delivery and cell permeability. In addition, specific tissues can be targeted by coupling the dendrimers with RGD, specific antibodies, thereby avoiding unnecessary off-target effects.¹⁴¹ These suggested that the surface-engineered dendrimers can be the potential source to deliver siRNA efficiently and avoid any barrier in delivering siRNA to the target site.¹⁴⁰ In another critical study, researchers reported two peptide dendrimers for efficient siRNA transfection named DMH13 and DMH18. Protonation of the dendrimer is also the most critical factor in developing siRNA-dendrimer conjugation to escape the endosome through the proton sponge effect. These peptide dendrimers showed better efficacy and cell permeability, followed by an endosomal escape to silence the specific gene.¹⁴²

PEI and PLL-modified nanoparticles

PEI and poly(l-lysine) (PLL) were extensively used in conjunction with siRNA to deliver to the target site in the body, which are both cationic polymers. These were the earlier candidates for the delivery of siRNA, and their conjugates were also developed to achieve better siRNA delivery. PLL-siRNA can pass through the biological membrane, but their endosomal escape property is very poor.¹⁴³ PEI nanoparticle showed high transfection efficiency and better endosomal escape inside the cytosol, but it showed cytotoxicity in vitro.^82,144,145 Zhang et al. designed a reactive oxygen species (ROS)-activatable polyplex consisting of PEGylated polymer, photosensitizer Ce6, ROS cleavable linker, and anti-RRM2-siRNA named PPTC-siRNA. Upon irradiation of near-infrared light, the ROS degraded the ROS-sensitive linker, resulting in destabilization of the cell membrane followed by endosomal escape of siRNA. PPTC-siRNA efficiently inhibited hepatocellular carcinoma growth by repressing the RRM2 expression and promoting apoptosis. This study successfully delineated the importance of this polyplex in delivering siRNA into the cytosol with better safety profiles.¹⁴⁶

PEI can be chemically modified with stearic acid (StA) before conjugation with siRNA to enhance the silencing activity. The PEI-siStA complex showed promising results after reducing the expression of STAT3 by 40% and the expression of vascular endothelial growth factor (VEGF) and tumor volume by 38% in a mouse model.¹⁴⁷ However, the cytotoxicity associated with PEI limits their use in siRNA delivery. Anderson et al. revealed that, after the injection of PEI/DNA in a mouse model, the muscle cells were damaged around the injection area, which led to spot calcification.¹⁴⁸ Several researchers suggested that the cytotoxicity attributed to PEI may be due to their chemical structure, ionic strength, zeta potential, and molecular weight.¹⁴⁹

Moreover, the toxic effects of PEI can be reduced by chemical modifications. The degradable PEI was synthesized with acid labile imine linkers by reacting low-molecular-weight PEI with glutaraldehyde. The resulting PEI can efficiently degrade into low-molecular-weight PEI, having reduced toxic effects with higher transfection efficiency.¹⁵⁰ Lian et al. recently explored fluorinated PEI for reduced cytotoxicity, better transfection efficiency, and biodistribution to organs. Fluorinated PEI showed reduced cytotoxicity in MDA-MD-231 cells. In addition, they observed that fluorinated PEI was accumulated in the liver and non-fluorinated PEI in the lungs. These suggested that fluorination may be the better way to reduce the toxic effects of cationic polymer to induce transfection activity efficiently.¹⁵¹ In addition, introducing PEG into PEI can efficiently alleviate the cytotoxicity and improve the transfection efficiency of PEI.¹⁵² Thus, PEGylated PEI can reduce the cytotoxicity posed by PEI but simultaneously show reduced transfection efficiency. Wen et al. enhanced the transfection efficiency of PEGylated PEI by synthesizing a tri-block copolymer, PEG-g-PEI-g-poly(dimethylaminoethyl L-glutamine) (PEG-g-PEI-g-PDMAEG). PEG-g-PEI-g-PDMAEG showed better transfection efficiency, 2-fold more than PEI 25k/DNA formulations in vitro, suggesting that a tri-block copolymer can be the potential carrier for gene delivery with higher transfection efficiency and lower cytotoxicity.¹⁵³

Chitosan-based nanoparticles

Chitosan is in the class of polysaccharides and is used to deliver nucleic acid therapeutics, which are far better biopolymers than other existing ones because of their flexible nature and effective gene delivery. Its flexibility is the structural heterogeneity and several functional groups, including hydroxyl, carboxyl, and amine, making it a good pocket for adding therapeutic moieties to enhance their stability and reduce delivery barriers. The presence of primary amino groups (pKa ≈ 6.5) makes chitosan a polycation, which promotes association and polyplex formation with nucleic acids.¹⁵⁴ Chitosan is biodegradable, biocompatible, and showed lower cytotoxicity inside the body.¹⁵⁵ Katas et al. experimented with evaluating the efficacy and pharmacokinetic-pharmacodynamics profile of chitosan-based nanoparticles in delivering siRNA in vitro. They prepared chitosan nanoparticles using ionic cross-linking (simple complexation) and ionic gelation by using sodium tripolyphosphate (TPP). They conducted in vitro experiments using CHO K1 and HEK293 cells and demonstrated that the preparation method significantly affects the gene-silencing activity of siRNA. The investigation revealed that chitosan-TPP-entrapped siRNA possessed better gene-silencing activity than the simple chitosan-siRNA complex, possibly due to higher binding efficiency and loading capability.¹⁵⁶

Moreover, it is reported that the complex efficiency can be up to 83%–94% by electrostatic interaction between siRNA and chitosan. This phenomenon depends on the degree of deacetylation.^157,158 Their complex has been used extensively to target cancer. Another study demonstrated that the 69.6% FHL2 gene expression was reduced by transfecting the chitosan nanoparticle-based FHL2 siRNA therapeutic close to the lipofectamine-mediated efficiency of 68.8%. It also inhibits the growth and cell proliferation of human colorectal cancer LoVo cells.¹⁵⁹ It is suggested that 20-nm chitosan nanoparticles can be used to deliver siRNA, and complex can be formed by the ion gelation method. The complex was internalized efficiently into the neuronal Neuro2a cells. Chitosan nanoparticle-mediated siRNA delivery was successful into the neuronal tissues in vivo.¹⁶⁰

In an experiment, scientists prepared a complex of unmodified siRNA with chitosan nanoparticles with core-shell poly(alkyl cyanoacrylate) to deliver by intravenous route. This was delivered to reduce the oncogene related to papillary thyroid carcinoma, and the antisense strand of siRNA with chitosan significantly reduced the tumor growth after intravenous injection.¹⁶¹ In addition, the targeted delivery of siRNA can be achieved by using chitosan with Arg-Gly-Asp (RGD) peptide. This can selectively deliver the siRNA to the animal models with ovarian cancer after intratumoral delivery. Multiple tumor growth-inducing genes such as POSTN, FAK, and PLXDC1 can be targeted by siRNA-chitosan labeled with RGD.¹⁵² In another study, the PLXDC1 was targeted by siRNA with chitosan-RGD and delivered into the integrin-positive tumor in A2780-tumor-possessing mice.¹⁶²

Furthermore, chitosan coupled with TAT-peptide tagged to polyethylene glycol to deliver siRNA to the neuronal 2a cells to target the SCA1 overexpression in vitro. After 48 h of transfection, the significant suppression of SCA1 was observed, proving the efficiency of chitosan-based nanoparticles for siRNA delivery.¹⁶³ Furthermore, researchers developed modified collagen-chitosan nanoparticles to deliver the siRNA to the fibrotic liver cells in vitro and accumulated selectively to the target site. This study suggested that chitosan nanoparticles with specific ligands can be used to deliver siRNA can be used as a targeted therapy.¹⁶⁴

Dextran and cyclodextrin nanoparticles

These are biocompatible, and their lipid-modified form is usually used against multi-drug resistance MDR1 siRNA delivery. An experiment was conducted using MDR osteosarcoma cell lines and treated with MDR1 siRNA nanoparticles. In addition, the combination therapy of MDR1 siRNA-loaded nanoparticles with enhanced concentration of doxorubicin was analyzed. It is suggested that the MDR1 siRNA-loaded dextran nanoparticles progressively suppressed P-gp expression in a drug-resistant osteosarcoma model. This method can reverse the drug resistance mechanism by inducing drug accumulation in MDR cell lines.¹⁶⁵ In another experiment, targeted siRNA was developed, named CALAA-01, and used in phase 1 human clinical trials. This agent contains cyclodextrin polymer, PEG steric stabilizing agent, and also attached a human ligand transferrin for targeted therapy that is overexpressed in cancer cells. These loaded nanoparticles were administered by the intravenous (i.v.) route, and, after treatment, there was reduced protein and mRNA expression suppressed by the RNAi mechanism. This method favors the use of dextran or cyclodextrin nanoparticles for targeted siRNA delivery.¹⁶⁶

Moreover, dextran hydroxymethyl methacrylate (dex-HEMA) nanogels have also been used in siRNA delivery. In an experiment, researchers modified it by the PEGylation process because of the less blood circulation time shown by the drug. After PEGylation of these dextran nanogels bearing siRNA showed better uptake by HuH-7 human hepatoma cells and A431 human epithelial carcinoma cells. Furthermore, these modified nanoparticles also suppressed the EGFP expression in HuH-7_EGFP without toxicity.¹⁶⁷ Spermine-modified acetylated-dextran (Spermine-Ac-DEX) nanoparticles were developed as acid-sensitive, biocompatible delivery systems for siRNA that suppressed the gene with minimal toxicity.¹⁶⁸ Dextran-based nanoparticles were developed for targeting the myeloid cells possessed by the liver, and their surface amines were modified with the PEGylation method. These nanoparticles were transfected into the myeloid liver cells and successfully knocked down the gene expression with minimal toxicity.¹⁶⁹

Inulin-based nanoparticles

Inulin (INU) is a natural polysaccharide under the class of fructan-type and has been investigated to deliver siRNA. It is biocompatible with hydrophilic character and can be modified chemically because of its reactive functional groups, especially the hydroxyl group. INU possesses a very flexible structure, and that is why it is a more competent candidate for siRNA delivery. The different step-based approach has been investigated to get INU polycation by adding several elements such as spermine, ethylenediamine, and diethylenetriamine.^170,171,172 The first generation of INU polycation-based nanoparticles were used to deliver siRNA. INU-diethylenetriamine polyplex was found to be more effective when taken by cells in acidic endosomal pathways, including clathrin-mediated endocytosis. By this mechanism, the siRNA activity was also higher with this internalization pathway as compared to the non-acidic endosomal pathway in non-tumoral 16HBE cells.¹⁷³

Moreover, a second-generation INU-based system was developed to reduce barriers including stability, retention of siRNA, siRNA loading capacity, and endosomal escape. The INU-DETA was coupled with imidazole, which increased the chances of endosomal escape and retention of the siRNA by hydrogen bonds.¹⁷² In addition, a third-generation inulin-based system was established. In this system, the first element was introduced as epidermal growth factor (EGF), termed INU-IMI-DETA-EGF copolymer, and the second element was added as PEG, termed INU-IMI-DETA-PEG. Both were mixed together to get EP-ICONs. It is suggested that the EGF significantly enhanced the siRNA coating efficiency and is dose dependent.¹⁵⁴

INU-based polycation was also studied for its ability to coat superparamagnetic iron oxide nanoparticles (SPIONs). INU-EDA was synthesized using ethylenediamine to obtain INU-EDA encapsulated SPIONs (IC-SPIONs) and get the complexation of IC-SPIONs with siRNA.¹⁷⁴ In a recent study, two copolymers of INU-IMI-DETA were prepared by chemically functionalizing with EGF and PEG. Their complex with siRNA was also designed and checked their activity. MCF-7 cells internalized the complexes with siRNA on the EGF factor and accumulated to the target site. It is demonstrated that it can also be beneficial for inducing the anticancer activity of other therapeutics, especially doxorubicin.¹⁷⁵

PLGA nanoparticles

PLGA is a biocompatible, biodegradable, FDA-approved polymer. PLGA nanoparticles have been investigated and used to deliver siRNA and plasmid DNA (pDNA) inside the body to treat various ailments. GFP-siRNA was encapsulated into the PLGA nanoparticles to be delivered into the 293T cells that showed improved stability compared to the other delivery carriers, which can be the potential carrier for siRNA therapeutics.¹⁷⁶ Another method was used for the encapsulation of siRNA into the PLGA nanoparticle core named the double-emulsion evaporation method. In this way, the encapsulation efficiency is increased by 57% by modifying the water-oil interface, PLGA concentration, and sonication time for the first-time emulsification. The major problem with using PLGA nanoparticles is endosomal escape. Due to this, the siRNAs cannot be released on time to show their activity. Due to this reason, PLGA nanoparticles have to be modified chemically to induce endosomal escape.¹⁷⁷

PLGA nanoparticles have been investigated to deliver siRNA, especially to treat cancer. PLGA nanoparticles have also been used to combat the problem of drug resistance in cancer therapy. In this regard, the PLGA nanoparticles used to deliver paclitaxel along with the P-gp-targeted siRNA reduce the paclitaxel resistance in cancer treatment. For targeting purposes, the siRNA was functionalized with biotin. Besides, higher cytotoxicity was noted when PLGA nanoparticles delivered P-gp-siRNA in conjunction with the paclitaxel as compared to the paclitaxel alone in vitro. On the other hand, in vivo studies revealed that increased inhibition of drug-resistant tumor growth was observed when treated with biotin-functionalized nanoparticles siRNA with paclitaxel. Even the dose of paclitaxel was ineffective when given alone without using gene silencing.¹⁷⁸ In an experiment, PLGA nanoparticles were loaded with pDrive-sh AnxA2 plasmid DNA and used as a sustained-release delivery of siRNA. After that, successful downregulation of AnxA2 expression was observed. These nanoparticles were delivered to xenograft prostate cancer nude mice, and successful tumor growth inhibition was noted.¹⁷⁹ A recent study demonstrated the efficacy of PLGA nanoparticles encapsulated the CX3XR1-siRNA in targeting neuropathic pain in spinal nerve ligation (SNL)-induced rats. After successful delivery, it reduced the neuropathic pain in SNL induced rats by downregulating the pro-inflammatory cytokines and microglial activity.¹⁸⁰

Moreover, PLGA nanoparticles are also used to deliver siRNA locally, giving better results. Inhalation therapy has been investigated, and these nanoparticles can be prepared by spray drying method. PLGA nanoparticles possess a negative charge and, for modification toward a positive charge, the PEI can be added. PEI modifies the PLGA surface and makes it suitable to bind with siRNA for efficient delivery.^181,182 In addition, the PLGA nanoparticles can also be modified by using chitosan, which ultimately helps in getting a positive charge. This combination helps to encapsulate the siRNA, providing better loading capacity, higher efficiency, and advanced delivery. Chitosan-PLGA nanoparticles can also resist the serum nucleases, hence protecting the siRNA.¹⁸³

Inorganic nanocarrier delivery systems

Gold nanoparticles

Gold nanoparticles (AuNPs) have been the most commonly studied nanoparticles for siRNA delivery in the last two decades. AuNPs have specific physical and chemical properties that make them good for gene therapy. Their core is stable and biocompatible, and, in this way, these are used in siRNA delivery. In addition, their surface modifications can also be done to control drug release, which is why their excessive use in siRNA delivery. The siRNA can be conjugated with AuNPs in two ways: covalent and without covalent chemistry.¹⁸⁴

Several conjugates have been used to deliver siRNA, including cell-penetrating ligands such as lipids, peptides, or other small-molecular ligands. However, these conjugates have limitations, including serum instability, premature release of siRNA, and reduced circulation time affecting gene-silencing activity. On the other hand, these barriers can be overcome by nanoparticle-based siRNA delivery. The direct method of conjugation of siRNA to AuNPs is a thiol-gold covalent bond attaching to the nanoparticle’s surface. The first conjugation of siRNA with AuNPs was studied in 2006, and, in that experiment, they developed 15-nm AuNPs with thiol-PEG5000-PAMA7500 polymer and thiol-siRNA conjugate. These AuNPs showed 65% gene-silencing efficacy in human hepatoma HuH-7 cells.

Furthermore, more modifications have shown better gene-silencing activity, such as coating of CPPs to AuNPs/siRNA.¹⁸⁵ Mirkin et al. synthesized polyvalent nucleic acid AuNPs (pNA-AuNPs) by functionalizing AuNPs covalently attached with thiol-modified oligonucleotides and applying them to siRNA-based gene silencing.¹⁸⁶ Furthermore, additional experiments demonstrated that pNA-AuNPs efficiently escaped endosomes.¹⁸⁷ They further experimented and revealed with the help of cyanine, 5-labeled pNA-AuNPs accumulated in endosomes after 1 h, and these can be seen in all over the cytoplasm after 4 h¹⁸⁸

Moreover, several experiments have been performed by using AuNPs to deliver siRNA therapeutics to treat various ailments. Reich et al. developed AuNPs/siRNA and then coated the surface with CPPs, resulting in efficient release from the endosome at the target site. This conjugate is internalized into human embryonic stem cells (hESCs) and efficiently knocks down Oct4 mRNA.¹⁸⁹ In addition, Anderson et al. developed siRNA/AuNPs conjugates and then coated them with polymer poly (β-amino ester) to enhance the gene-silencing activity of siRNA. This conjugate was able to silence the luciferase expression by 90% in HeLa cells and proved the efficiency.¹⁹⁰ Song et al. synthesized PEI-capped AuNPs/siRNA for better cellular uptake and transfection efficacy. They concluded that these NPs efficiently internalized into the cells and targeted GFP expression MDA-MB-435s cells. It also targeted PLK1 gene expression and induced cell apoptosis without cellular toxicity. This experiment showed better cell internalizing efficacy by using PEI-capped AuNPs/siRNA.¹⁹¹ Kim et al. synthesized dendronized AuNPs as G0, G1, and G2-AuNPs conjugated siRNA to target β-galactosidase (β-gal). G2-AuNPs-siRNA knocked down β-gal by 50% with reduced toxicity.¹⁹² Rahme et al. prepared AuNPs capped with PEI and conjugated with targeted ligand folic acid to target folate receptors in prostate cancer. They developed a complex AuNPs-PEI-FA-siRNA and internalized it into LNCaP cells over-expressing prostate-specific membrane antigen (PSMA). This complex efficiently targeted the folate receptors and knocked down the gene expression in prostate cancer cell lines. It has shown promising efficacy in treating prostate cancer.¹⁹³ Kong et al. prepared cationic lipid AuNPs for delivery of siRNA and lipid content compose of 3β-[N-(N′,N′-dimethylaminoethane)-carba-moyl]-cholesterol (DC-Chol, 54% w/w), L-α-dioleoyl phos-phatidylethanolamine (DOPE, 27% w/w), and cholesterol (19% w/w). They conjugated siRNA with L-AuNPs complex and transfected into MDA-MB-435 and A549 cells to target GFP gene expression. The L-AuNPs/siGFP significantly knocked down GFP gene expression and reduced the expression to 47.7% in MDA-MB-435 cells. Furthermore, they also transfected L-AuNPs conjugated with siVEGF into PC-3 cancer cells and suppressed the VEGG gene expression. These results suggested lipid AuNPs can be the potential carrier for siRNA delivery to target gene over-expressions without causing cellular toxicity.¹⁹⁴ Guo et al. and his team developed two complexes of AuNPs with targeted ligands transferrin (TF) and folic acid (FA). Two different polymers were used, PEI and PEG. Both complexes as AuNPs-PEI-FA and AuNPs-PEG-TF were conjugated with siRNA. The complex with TF was transfected into PC-3 prostate cancer cells with over-expressing TF receptors, and the other with FA was transfected into LNCaP prostate cancer cells with over-expressing PSMA. These targeted complexes efficiently reduced the gene expression in both cancer cell lines.¹⁹⁵ Shaabani et al. developed chitosan-coated AuNPs layer by layer to protect siRNA. This complex LBL-CS-AuNPs-siRNA was transfected into H1299-eGFP lung epithelial cells and it protected the siRNA against enzymatic degradation and serum instability and induced endosomal escape to facilitate the siRNA release to the target site. Finally, these nanoparticles reduced the gene expression efficiently and showed better stability and efficacy.¹⁹⁶ These all experiments have shown the potential of AuNPs to protect and deliver the siRNA in an efficient manner without any significant cytotoxicity.

Magnetic nanoparticles

Magnetic nanoparticles (MNPs) have played an essential role in delivering siRNA inside the body to the target site. SPIONs emerged as drug delivery systems due to their specific properties. Their larger surface area enables the conjugation with siRNA and other targeting ligands for targeted therapy. Moore et al. reported an innovative synthesis of the dual-purpose probe for delivering siRNA to the target site, especially tumors. This probe is based on SPIONs conjugated with Cy5.5 dye and myristoylated polyarginine peptide for translocation through the membrane. These nanoparticles can be combined with siRNA for targeted gene therapy.¹⁹⁷ In addition, MNPs-mediated siRNA delivery can be tracked by using MRI. Cheon et al. conjugated the siGFP on MNPs, then coated with serum albumin, followed by the addition of RGD peptide to target α_vβ₃ integrin, which is overexpressed in metastatic cancer cells. These nanoparticles efficiently target the GFP expression in MDA-MB-435 cells.¹⁹⁸

Moreover, surface modifications of MNPs can also be done by using lipids or cationic polymers that facilitate better siRNA encapsulation. Daxiang et al. modified MNPs by conjugating amphiphol polymer and protamine peptide that converted into cell-penetrating MNPs (CPMNPs). These prepared CPMNPs were conjugated with eGFP-siRNA and delivered into the cells. Daxiang et al. concluded that CPMNPs efficiently internalized into the cells and silenced the eGFP expression better than lipofectamine and PEI, even at a lower siRNA concentration of 5 nm.¹⁹⁹ Zhang et al. modified iron oxide NPs with PEI and conjugated with siRNA to target B cell lymphoma-2 BCL2 and baculoviral IAP repeat-containing 5 (BIRC5) into Ca9-22 oral cancer cells. These PEI-modified NPs efficiently reduced the BCL2 and BIRC5 overexpression in Ca9-22 oral cancer cells.²⁰⁰ Huang et al. modified MNPs by conjugating with different generations of PAMAM and antisense survivin oligodeoxynucleotide (asODN). This asODN dendrimer-MNP is efficiently internalized into MCF-7 and MDA-MB-435 cells within 15 min and knocks down the gene expression to a greater extent.²⁰¹ All these experiments suggested that MNPs can be the potential carriers for siRNA delivery and help to internalize into the cell and knock down the gene expression to a greater extent.

Mesoporous silica nanoparticles

Mesoporous silica nanoparticles (MSNPs) have been extensively used in delivering several types of molecules, including genes, small molecules, and proteins. MSNPs possess a large surface area that makes them eligible for better encapsulation of siRNA through the covalent or non-covalent bond. Nel et al. modified MSNPs with PEI and encapsulated GFP-siRNA to target GFP overexpression in HEPA-1 liver tumor cells. These PEI-MSNPs-siGFP were delivered into HEPA-1 liver tumor cells and efficiently reduced GFP expression without significant toxicity.²⁰² Gu et al. modified MSNPs by encapsulating siRNA into pores of mesoporous silica and surface coated with PEI and fusogenic peptide to facilitate endosomal escape. These nanoparticles were delivered into tumor-bearing mice to target VEGF to evaluate this modification. They efficiently reduced the expression of VEGF and reduced tumor growth.²⁰³ Gray et al. designed 47-nm MSNPs conjugated with PEI and polyethyleneglycol copolymer to accommodate siRNA against HER2 oncogene. They transfected this construct into trastuzumab-resistant HCC1954 xenograft. The construct efficiently internalized and knocked down the gene expression by 60% and inhibited tumor growth.²⁰⁴ Elahian et al. developed MSNPs construct by modifying these nanoparticles with ammonia (NH₂-MSNPs) and loading them with MDR1-siRNA. The remaining negative surface was covered with chitosan coating for siRNA protection against multiple barriers during drug delivery. Targeting moieties, including TAT and folate, were also conjugated with chitosan. This construct was delivered into EPG85.257-RDB and HeLa-RDB lines and significantly knocked down MDR1 expression in both cell lines. This experiment suggested that this construct can be the potential carrier for targeted gene therapy.²⁰⁵

Calcium phosphate nanoparticles

Calcium phosphate is inorganic and found in bones and teeth. Due to its biocompatibility and stability, it is extensively used in drug delivery systems. Researchers have also used calcium phosphate nanoparticles (CaPNPs) for siRNA delivery. However, there are limitations to using these nanoparticles for siRNA delivery because of poor endosomal escape under physiological conditions.²⁰⁶ Giger et al. modified CaPNPs by coating with PEG and stated that these particles can be internalized into cells more efficiently, as presented in Figure S7.²⁰⁷ Denkbas et al. prepared calcium phosphate nanoparticles and conjugated them with arginine amino acids to enhance siRNA loading capacity. They stated that, by this conjugation, these CaPNPs could efficiently deliver siRNA to treat cancer without cytotoxicity.²⁰⁸

Liang et al. designed CaPNPs by conjugating with polycation liposome, termed and polycation liposome encapsulated CaPNPs, for siRNA delivery to suppress VEGF expression in MCF-7 cells. They transfected this construct into MCF-7 cells, and 60%–80% gene inhibition was observed. This suggested that this construct can be the potential carrier to deliver siRNA to the target site and further knock down the gene expression. In addition, by using this construct, tumor growth and angiogenesis inhibition were also observed in MCF-7 xenograft mice.²⁰⁹ Recently, Bakan et al. developed three different types of CaPNPs conjugated with arginine to deliver two specific siRNAs to silence survivin and cyclin B1. It can be seen in Figure S8. This complex as CaP-Arg-siRNA activity was observed by transfecting into A549 non-small-cell lung cancer cells. They found that significant gene knockdown by Cap-Arg-mediated siRNA delivery and remarkable tumor growth reduction and induced apoptosis were also observed in this experiment.²¹⁰

These CaPNPs have been used in several experiments by different scientists for delivering siRNA to the target site. All these experiments suggested that these nanoparticles can be better carriers for siRNA delivery, and other barriers can be overcome by modification of these nanoparticles by conjugating with specific conjugating agents such as arginine, as discussed earlier. Therefore, these CaPNPs appeared to be the most promising siRNA delivery carriers inside the body.

Carbon nanotubes

Carbon nanotubes (CNTs) are cylindrical molecules composed of carbon atoms organized in thin graphite sheets of condensed benzene rings rolled up into a hollow cylinder. CNTs are potential nanocarriers to deliver nucleic acid therapeutics because of their increased systemic circulation time and stability in the biological fluid. They can easily cross the biological membranes into the cytoplasm without inducing cell death.²¹¹ According to their diameter, length, and presence of walls, CNTs are classified into four categories: single-walled carbon nanotubes (SWCNTs), double-walled carbon nanotubes, triple-walled carbon nanotubes, and multi-walled carbon nanotubes (MWCNTs). Most importantly, the outer membrane of the CNTs is usually tailored to enhance the unique carrier properties.^212,213 In addition, functionalized SWCNTs are preferably used as a potential carrier to target cancer. CNTs functionalized with PEI are used to deliver siRNA to the target site. Mohammadi et al. investigated the role of SWCNTs functionalized with RNA aptamer and siRNA against breast cancer. Epithelial cell adhesion molecule (EpCAM) is a biomarker for solid tumors and is overexpressed in cancer. They prepared the complex consisting of SWCNTs-aptamer-siRNA and transfected it into MCF-7 cell line with positive EpCAM. The prepared complex induced more than 20% apoptosis in MCF-7 cell line, which shows the gene-silencing potential of the complex.²¹⁴ Recently, Wen et al. investigated the role of MWCNTs in effective siRNA delivery. They used MWCNTs to deliver sorafenib and EGFR-siRNA to treat liver cancer. After transfection, the complex MWCNTs-Sor-siRNA effectively induced cell apoptosis in HepG2 cells. The complex showed better antitumor activity both in vitro and in vivo.²¹⁵

Conjugate-siRNA delivery system

As discussed above, siRNA possesses several intra- and extracellular delivery barriers, so chemical modification or conjugating the siRNA with a suitable carrier is essential for efficient delivery to the target site. These conjugate complexes include lipid-siRNA conjugate, peptide-siRNA conjugate, and antibody-siRNA conjugate.²¹⁶

In lipid-siRNA conjugation, the cholesterol-siRNA conjugation has been investigated thoroughly for efficient delivery to the target site. Studies revealed that the cholesterol-siRNA conjugate showed higher RNAi activity than naked siRNA. Several researchers have investigated this conjugation to target several diseases. Craig et al. characterized simple cholesterol-siRNA conjugate to target tumor necrosis factor (TNF) mRNA in liver macrophages for the treatment of non-alcoholic fatty liver disease (NAFLD). They observed the overall reduction of almost all hallmarks of NAFLD, such as steatosis, inflammation, and fibrosis in the murine NAFLD model.²¹⁷ Besides, more clinical data and the inclusion of in vivo tests are recommended for better and specific results for further development of the efficient delivery system.

Moreover, the antibody-siRNA conjugate has also been investigated in several experiments.²¹⁸ Wang et al. conducted an experiment in which they developed a conjugate composed of anti-programmed death-ligand 1 antibody (αPD-L1) with siRNA against PD-L1 mRNA through a photocleavable linker. They transfected the conjugate in HCT116 cells and mice bearing subcutaneous HCT116 tumors. After internalization, siPD-L1 is released into the cytosol and degrades PD-L1 mRNA, boosting immune cell activity. In addition, it also causes effective cancer suppression both in vitro and in vivo.²¹⁹ Tao et al. developed peptide-based siRNA micelle-plexes (P^A7R@siPD-L1) for normalizing vascular-immune crosstalk to establish a positive feedback loop in potentiating antitumor immunotherapy. The main purpose of this study is to turn cold tumors into hot ones to generate systematic immune responses. This developed conjugate can eliminate solid tumors and trigger immunogenic cell death. This nanomaterial conjugate provides the basis for further investigation of the conjugate drug delivery systems.²²⁰ In addition, linear peptides and cyclic peptides have also been investigated thoroughly by researchers. These have several advantages, including biocompatibility, biodegradability, high flexibility, and bio-functional diversity.²²¹ CPPs and cyclic cell-penetrating peptides (CCPPs) have been used to deliver siRNA. CPPs are generally composed of 6–30 amino acid residues. Langel and coworkers reported that stearylated transportan (TP)-10 (stearyl-TP10; TP-10 is a shorter version of transportan) efficiently delivered a splice-correcting phosphorothioate 2′-O-methyl RNA (2′-OMe ON) into cells. Besides, more in vitro and in vivo experiments are needed to evaluate the efficiency and stability profiles.^222,223

FDA-approved siRNA therapeutics and updated clinical trials

Several siRNA-based therapeutics are currently in clinical and preclinical trials toward clinical development. Five siRNA-based therapeutics are already approved by the FDA, namely patisiran, lumasiran, givosiran, inclisiran, and vutrisiran, to treat TTR-mediated amyloidosis, primary hyperoxularia, acute hepatic porphyria, hypercholesterolemia, and amyloidosis, respectively. These siRNA therapeutics in clinical trials are briefly discussed in Table 5.

Table 5.

Clinical trials for siRNA-based therapeutics

Drug name	Delivery system	Modification	Route of administration	Disease	Target	Clinical status/year	Company	NCT number
Patisiran6,224	LNP	2′-OMe, 2′-F	i.v.	TTR-mediated amyloidosis	TTR	approved/2018	Alnylam Pharmaceuticals	NCT03862807 NCT03997383
Givosiran7,225	GalNAc	PS, 2′-OMe, 2′-F	s.c.	acute hepatic porphyria	ALAS1	approved/2019	Alnylam Pharmaceuticals	NCT02452372 NCT02949830
Inclisiran226,227	GalNAc	PS, 2′-OMe, 2′-F	s.c.	hypercholesterolemia	PCSK9	approved/2021	Alynylam Pharmaceuticals	NCT03814187 NCT03851705
Lumasiran8,228	GalNAc	PS, 2′-OMe, 2′-F	s.c.	primary hyperoxaluria type 1	HAO1	approved/2020	Alnylam Pharmaceuticals	NCT03905694 NCT03681184
Vutrisiran229	GalNAc	PS, 2′-OMe, 2′-F	s.c.	amyloidosis	TTR	approved/2022	Alnylam Pharmaceuticals	NCT04153149
Fitusiran230,231	GalNAc	PS, 2′-OMe, 2′-F	s.c.	hemophilia A and B	thrombin (coagulation factor IIa)	phase 3	Sanofi	NCT03549871 NCT03974113
Nedosiran (DCR-PHXC)232,233	GalNAc	undisclosed	s.c.	primary hyperoxularia	LDHA	phase 3	Novo Nordisk	NCT04042402
Teprasiran (QPI-1002)234	NA	2′-OMe	s.c.	delayed graft function	p53	phase 3	Quark Pharmaceuticals	NCT02610296 NCT03510897 NCT00802347 NCT02610283
QPI-1007235	NA	2′-OMe	intraocular/subretinal/subconjunctival	nonarteritic anterior ischemic optic neuropathy	caspase-2	phase 3	Quark Pharmaceuticals	NCT01965106 NCT01064505
Tivanisiran236	NA	undisclosed	topical	dry eye disease ocular pain	TRPV1	phase 3	Sylentis	NCT02455999 CT01776658 NCT01438281 NCT03108664
Sepofarsen (QR-110)237	NA	undisclosed	intravitreal	congenital blindness	CEP290 RNA	phase 2/3	ProQR Therapeutics	NCT04855045
Cemdisiran238	GalNAc	PS, 2′-OMe, 2′-F	s.c.	complement-mediated diseases	C5	phase 2	Regeneron Pharmaceuticals	NCT03999840
BMT10158	cp-asiRNA	undisclosed	intradermal	hypertrophic scar	CTGF	phase 2	Hugel	NCT04012099 NCT03133130
PF-655 (PF-04523655)239	NA	2′-OMe	intravitreal	choroidal neovascularization, diabetic retinopathy, diabetic macular edema	RTP801	phase 2	Quark Pharmaceuticals	NCT01445899
RXI-109240	NA	undisclosed	intravitreal	hypertrophic scar	CTGF	phase 2	RXi Pharmaceuticals	NCT02599064 NCT01780077 NCT02030275 NCT02079168
siG12D-LODER241,242,243	polymeric matrix	undisclosed	intratumoral	pancreatic ductal adenocarcinoma, pancreatic cancer	KRAS G12D	phase 2	Silenseed	NCT01188785 NCT02956317
Bamosiran (SYL040012)244	NA	undisclosed	eye drops	ocular hypertension, glaucoma	ADRB2	phase 2	Sylentis	NCT01227291 NCT00990743 NCT02250612
Cobomarsen245	LNA	undisclosed	s.c., i.v., intratumoral	blood cancers (cutaneous T cell lymphoma, adult T cell lymphoma/leukemia, diffuse large B cell lymphoma, chronic lymphocytic leukemia, mycosis fungoides)	microRNA-155	phase 2	miRagen Therapeutics	NCT03713320 NCT03837457 NCT02580552
MRG-201246	NA	2′-OMe, 2′-F mismatch, PS, Chol (microRNA-29b mimic)	intradermal	pathologic fibrosis (cutaneous fibrosis, idiopathic pulmonary fibrosis, keloid, etc.)	CTGF	phase 2	miRagen Therapeutics	NCT03601052 NCT02603224
ALN-HBV02247	GalNAc	PS, 2′-OMe, 2′-F	s.c.	hepatitis B	HBV gene	phase 1/2	Alnylam Pharmaceuticals	NCT03672188
ALN-AAT02248	GalNAc	PS, 2′-OMe, 2′-F	s.c.	alpha-1 liver disease	AAT	phase 1/2	Alnylam Pharmaceuticals	NCT03767829
ARO-AAT249	GalNAc	PS, 2′-OMe, 2′-F, inverted base	s.c.	alpha-1 antitrypsin deficiency	AAT	phase 2/3	Arrowhead Pharmaceuticals	NCT03362242 NCT03945292
JNJ-3989250	GalNAc	PS, 2′-OMe, 2′-F, inverted base	s.c.	hepatitis B	HBV gene	phase 1/2	Arrowhead partnered with Janssen	NCT03365947
Olpasiran251	GalNAc	undisclosed	s.c.	cardiovascular disease	Lp(a)	phase 2	Arrowhead Pharmaceuticals partnered with Amgen	NCT03626662
HSP47252	LNP, vitamin A	undisclosed	i.v.	idiopathic pulmonary fibrosis	HSP47	phase 2	Bristol-Myers Squibb	NCT03241264 NCT02227459 NCT01858935 NCT03538301
AB729231	GalNAc	undisclosed	s.c.	hepatitis B	HBV gene	phase 2	Arbutus Biopharma Corporation	NCT04980482
ARO-APOC3253	GalNAc	PS, 2′-OMe, 2′-F, inverted base	s.c.	hypertriglyceridemia, familial chylomicronemia	ApoC3	phase 1	Arrowhead Pharmaceuticals	NCT03783377
ALNAGT254	GalNAc	PS, 2′-OMe, 2′-F, GNA	s.c.	hypertension	AGT	phase 1	Alnylam Pharmaceuticals	NCT03934307
ARO-ANG3255	GalNAc	PS, 2′-OMe, 2′-F	s.c.	hypertriglyceridemia	ANGPTL3	phase 1	Arrowhead Pharmaceuticals	NCT03747224
DCR-HBVS256	GalNAc	undisclosed	s.c.	hepatitis B	HBV gene	phase 1	Dicerna Pharmaceuticals	NCT03772249
SXL01257	NA	undisclosed	s.c.	advanced cancers	AR	phase 1	Institut Claudius Regaud	NCT02866916
siRNA-EphA2-DOPC258	Liposome	undisclosed	i.v.	advanced cancers	EphA2	phase 1	M.D. Anderson Cancer Center	NCT01591356
TD101259	NA	undisclosed	intralesional injection	pachyonychia congenital	keratin 6A N171K mutan	phase 1	Pachyonychia Congenita Project	NCT00716014
SLN124260	GalNAc	undisclosed	s.c.	non-transfusion-dependent thalassemia, low-risk myelodysplastic syndrome	TMPRSS6	phase 1	Silence Therapeutics	NCT04176653 NCT04718844
APN401257	ex vivo siRNA electroporated PBMCs	undisclosed	i.v.	solid tumors	Cbl-b/DC	phase 1	Wake Forest University Health Sciences	NCT02166255 NCT03087591
STP705 (cotsiranib)261	NA	undisclosed	intralesional injection	hepatocellular cancer	COX2/PTGS2 TGF-β and superfamily	phase 1	Sirnaomics	NCT04676633 NCT04844983
ARO-ENaC262	TRiM (EpL-siRNA conjugate)	PS, 2′-OMe, 2′-F, iB	inhalation	cystic fibrosis	αENaC	phase 1	Arrowhead Pharmaceuticals	NCT04375514
ARO-MUC5AC263	NA	undisclosed	inhalation	chronic obstructive pulmonary disease	mucin 5AC (MUC5AC)	preclinical	Arrowhead Pharmaceuticals	NCT05292950
ALG125755264	GalNAc	undisclosed	s.c.	hepatitis B	hepatitis B surface antigen (HBsAg)	preclinical	Aligos Therapeutics	NA
ALN-F12265	GalNAc	undisclosed	s.c.	hereditary angioedema	coagulation Factor XII	preclinical	Alnylam Pharmaceuticals	NA
ALN-APP	NA	PS, 2′-OMe, 2′-F	intrathecal	cerebral amyloid angiopathy	APP	preclinical	Alnylam Pharmaceuticals	NA
ALN-KHK	NA	undisclosed	NA	diabetes mellitus, type II	ketohexokinase	preclinical	Alnylam Pharmaceuticals	NA
ALN-HTT	NA	undisclosed	intracerebral/cerebroventricular	Huntington’s disease	huntingtin	preclinical	Alnylam Pharmaceuticals	NA
ALN-PNP	NA	undisclosed	NA	non-alcoholic steatohepatitis	PNPLA3	preclinical	Alnylam Pharmaceuticals	NA
AOC 1044	NA	undisclosed	NA	DMD	dystrophin gene	preclinical	Avidity Biosciences	NA
OLX-301	cp-asiRNA	undisclosed	NA	age-related macular degeneration/retinal fibrosis	CTGF	preclinical	OliX Pharmaceuticals, Thea	NA
si-PT-LODER	polymeric matrix (LODER polymer)	undisclosed	intratumoral	prostate cancer	HSP90	preclinical	Silenseed	NA
ARO-DUX4	NA	undisclosed	i.v.	muscular dystrophy	DUX4	preclinical	Arrowhead Pharmaceuticals	NA
DCR-AATsc	GalNAc	undisclosed	NA	antitrypsin deficiency, liver disease	AAT	preclinical	Dicerna Pharmaceuticals	NA
BA-434	NA	undisclosed	NA	spinal cord injury	PTEN	preclinical	BioAxone BioSciences	NA
BB-301	NA	undisclosed	intramuscular	muscular dystrophy	PABPN1	preclinical	Benitec Biopharma	NA
ARO-AMG1	TRiM	PS, 2′-OMe, 2′-F, iB	NA	undisclosed	undisclosed	preclinical	Arrowhead Pharmaceuticals	NA
SYL116011	naked siRNA	undisclosed	NA	allergic conjunctivitis (ophthalmology)	Orai1	preclinical	Sylentis	NA
Coronavirus Vaccine (Sirnaomics)	NA	undisclosed		COVID-19 prevention	SARS-CoV-2	preclinical	Sirnaomics	NA
DUET-02	NA	undisclosed	NA	prostate cancer	STAT3 transcription factor/TLR9	preclinical	Scopus BioPharma	NA
GalXC RNAi Program (Dicerna/Alexion)	NA	undisclosed	s.c.	autoimmune disorders	complement pathway	preclinical	AstraZeneca	NA
GalXC-Plus RNAi Program	NA	undisclosed	s.c.	autoimmune disorders	complement pathway	preclinical	Novo Nordisk	NA

2′-OMe, 2′-methoxy group substitution; 2′-F, 2′-fluoro substitution; TTR, transthyretin; GalNAc, N-acetyl-D-galactosamine; PS, phosphorothioate linkage; ALAS1, delta-aminolevulinate synthase 1; PCSK9, proprotein convertase subtilisin/kexin type 9; HAO1, hydroxyacid oxidase 1; LDHA, lactate dehydrogenase A; LNP, lipid nanoparticle; TRPV1, transient receptor potential cation channel subfamily V member 1; C5, complement component 5; CTGF, connective tissue growth factor; RTP801, (Ddit4) DNA-damage-inducible transcript 4; cp-asiRNA, asymmetric siRNA; KRAS, Kirsten rat sarcoma viral oncogene homolog; ADRB2, adrenoceptor beta 2; LNA, locked nucleic acid; HBV, hepatitis B virus; AAT, alpha-1 antitrypsin; Lp(a), lipoprotein (a); HSP, heat shock protein; ApoC3, apolipoprotein C3; COX2, cyclooxygenase 2; PTGS2, prostaglandin-endoperoxide synthase 2; TGF-β, transforming growth factor β; TLR9, Toll-like receptor 9; PNPLA3, patatin-like phospholipase domain-containing protein 3; CEP290, centrosomal protein of 290 kDa; KHK, ketohexokinase; NA, not available.

These siRNA-based therapeutics have shown promising results in different diseases and can target the disease at a genetic level. The disease can be treated by degrading the particular mRNA, thus inhibiting the translation process. Besides, other RNA therapeutics, including miRNA and ASOs, are also gaining prominence in clinical research. However, for the past few years, much research has been done to develop siRNA therapeutics and have shown better results. In addition, the manufacturing cost of siRNA therapeutics is less than others, especially monoclonal antibodies.²⁶⁶ Furthermore, these siRNA therapeutics offer convenient dosing and are self-administrable as subcutaneous and topical products.²³⁴

Challenges and future prospects

In the future, RNA-based therapeutics, especially siRNA, holds the potential for future therapeutic strategies and can treat any disease by silencing specific genes related to disease progression. These siRNA therapeutics provide specific gene selectivity, which is very difficult with other current therapeutic strategies. Due to these RNA-based therapeutics, the inaccessible targets can now be targeted selectively.²⁶⁷

There are several challenges in delivering siRNA, such as membrane impermeability, because of the negative charge of siRNA and of membranes. In addition, endosomal escape and off-target effects are significant barriers to siRNA delivery. Another major barrier is the displacement of siRNA from the RISC and disruption of the RNAi process.²⁶⁸ As discussed earlier, these hurdles can be tackled by modifying siRNA chemically or developing feasible delivery systems.

To date, GalNAc conjugation enables efficient hepatic delivery of siRNA. Next, the barriers of extrahepatic delivery need to be overcome urgently. Central nervous system (CNS) and eye-targeted delivery platforms have been established, and relevant therapeutics are undergoing clinical investigation. However, it is difficult to identify another extrahepatic delivery platform similar to the GalNAc-ASGPR pair.

Several delivery systems have been developed for effective extrahepatic delivery of siRNA and to avoid the RNases after systemic administration. Moreover, additional modification of nanoparticle surface with small molecules and peptides, such as FA, cyclic-RGD, and CPPs, targeting receptors overexpressed specifically in tumor cells can enhance the tumor accumulation and effectively inhibit the tumor progression. Most importantly, lipids and polymer-based approaches are the most effective delivery systems for siRNA targeting specific sites. These can deliver siRNA effectively and without any toxicity. Lipid-based carriers possessed effective delivery with protection against nucleases and targeted therapy by conjugating targeting ligands. These can reduce the off-target side effects and thus give better outcomes to treat the disease.²⁶⁹ Several nanoparticle-based siRNA therapeutics are in preclinical and clinical trials.^270,271 Besides, several difficulties are posed by nanoparticle-based systems, including large particle size by conjugating with different ligands and carriers such as PEG that can affect the overall delivery efficacy. That is why better design of these nanoparticles is required for effective siRNA delivery. Finally, the safety and efficacy profile of several nanoparticle-based siRNA therapeutics has been evaluated byseveral in vitro and in vivo studies.^{24,215,272,273,274} However, future studies and research must require actual in vivo efficacy and safety evaluation to reduce cytotoxicity and immune activation. It is also necessary to develop effective nanoparticle-based siRNA therapeutics, which should be biodegradable, stable, biocompatible, and safe.

Conclusions

RNA therapeutics, especially siRNA, can silence any gene for a specific disease by following the RNAi mechanism in the process. These siRNA-based therapeutics were discovered almost two decades ago and possessed several limitations. Several research studies have been done to address these challenges and garnered hope for using siRNA therapeutics to target any disease by silencing the specific gene. To counter these challenges, siRNA modifications and other delivery strategies were implicated for effective siRNA delivery. For this purpose, nanoparticle-based siRNA delivery systems are designed to minimize delivery barriers and off-target effects. These include lipid carriers, polymer and inorganic nanoparticles, and better siRNA delivery without toxicity. The first FDA-approved siRNA, patisiran, was delivered using LNPs, which garnered research more focused on the nanoparticle-based approach for siRNA delivery.

Cationic liposomes are extensively used to deliver siRNA and mask the poly-anionic surface of siRNA to facilitate membrane permeability. In addition, several modifications and carriers are conjugated with siRNA to induce endosomal escape inside the cytosol. Several studies revealed that PEGylation of nanoparticles helps to hide from macrophages and enhances the systemic circulation time. Besides the implications of several modifications and nanocarriers to siRNA, the final bioavailability of siRNA is still very low for the destruction of targeted mRNA to reduce gene expression. Inorganic nanoparticles have also played an important role in efficiently delivering siRNA to the target site, but more in vivo studies are required to evaluate their efficacy with safety profiles. All these studies suggested that there is still significant scope to develop a nanoparticle-based system to target mRNA effectively, enhance endosomal escape, and reduce off-target effects by conjugating with targeting ligands.