Therapeutic delivery of siRNA for the management of breast cancer and triple-negative breast cancer

Abstract

Breast cancer is the leading cause of cancer-related deaths among women globally. The difficulties with anticancer medications, such as ineffective targeting, larger doses, toxicity to healthy cells and side effects, have prompted attention to alternate approaches to address these difficulties. RNA interference by small interfering RNA (siRNA) is one such tactic. When compared with chemotherapy, siRNA has several advantages, including the ability to quickly modify and suppress the expression of the target gene and display superior efficacy and safety. However, there are known challenges and hurdles that limits their clinical translation. Decomposition by endonucleases, renal clearance, hydrophilicity, negative surface charge, short half-life and off-target effects of naked siRNA are obstacles that hinder the desired biological activity of naked siRNA. Nanoparticulate systems such as polymeric, lipid, lipid-polymeric, metallic, mesoporous silica nanoparticles and several other nanocarriers were used for effective delivery of siRNA and to knock down genes involved in breast cancer and triple-negative breast cancer. The focus of this review is to provide a comprehensive picture of various strategies utilized for delivering siRNA, such as combinatorial delivery, development of modified nanoparticles, smart nanocarriers and nanocarriers that target angiogenesis, cancer stem cells and metastasis of breast cancer.

Plain Language Summary

Breast cancer is the leading cause of cancer-related deaths among women globally. The difficulties with anticancer medications, have prompted attention to alternate approaches to address these difficulties. RNA interference (RNAi) by small interfering RNA (siRNA) is one such approach, which has several advantages, including the ability to quickly modify and suppress the expression of the target gene and display superior efficacy and safety. However, there are many hurdles that hinder the desired biological activity of naked siRNA. For addressing various problems pertaining to the delivery of naked siRNA, nanoparticles are employed to carry siRNA to the target cells. We have attempted to outline a broad variety of nanocarriers carrying various siRNAs developed for the therapy of breast cancer and triple-negative breast cancer.

Plain language summary

Article highlights.

• RNA interference by small interfering RNA (siRNA) is a promising approach to treat cancer.

• siRNA can quickly modify and suppress the expression of the target gene.

• Naked siRNA is unstable hence requires novel delivery platforms.

• Site-specific delivery of siRNA is possible through smart nanocarriers.

Nanocarriers, considerations & mechanisms for the siRNA delivery

• Various polymeric, metallic, lipid-based nanoparticles were explored to deliver siRNA.

• Nanocarriers prevent degradation and enhanced therapeutic efficacy of loaded siRNA.

Delivery of siRNA through the nanoparticulate systems

• siRNA loaded nanoparticles and its effect against breast cancer are discussed.

Combinatorial siRNA delivery

• siRNA in combination with either drugs or other siRNAs demonstrated better effects.

Modified nanosystems for siRNA delivery

• Enhanced uptake and serum stability were observed with modified nanosystems.

• Nanocarriers modified using various ligands demonstrate better anti-cancer effects.

Smart nanocarriers as a platform for delivering siRNA

• Various smart nanocarriers with distinct characteristics facilitate the release of siRNA at the target site.

siRNA for targeting TNBC

• Studies demonstrating the effectiveness of siRNA in triple-negative breast cancer, an aggressive form of breast cancer were summarised in this section.

Miscellaneous strategies for siRNA delivery

• The quantum dot-lipid nanocarriers and aptamer functionalized nanocarriers with their anti-cancer effects were briefly discussed.

Targeting some key processes involved in breast cancer through siRNA

• siRNA loaded nanocarriers demonstrated anti-angiogenetic effects, controlled metastasis and targeted stem cells associated with breast cancer.

• Various in vitro and in vivo models were employed to exploit the mechanisms involved in cancer progression.

Clinical aspects & lack of clinical translation of si-RNA-based therapies

• siRNA based therapies demonstrated effectiveness in various models of breast cancer.

• Limited clinical translation could be attributed to several challenges.

• Addressing these challenges would lead to successful clinical translation.

1. Introduction

siRNA or small interfering RNA is a non-coding type of dsRNA of 21–23 nucleotide base pairs in length [1]. While miRNA and siRNA both serve the same purpose, siRNA selectively binds to a single gene target region while numerous genes can be regulated by miRNA through incorrect base pairing. Using the enzyme Dicer, siRNA is produced from short hairpin RNA and dsRNA (RNAase endonuclease) [2].

siRNA has been explored to be a successful strategy for inhibiting certain genes [3,4,5]. Employing siRNA technology for cancer treatment has been extensively researched [6,7,8,9,10,11]. Transportation of siRNA to the tumor in vivo has been demonstrated to be difficult due to siRNA's short blood half-life and inadequate cellular absorption [12]. Because of their physicochemical and pharmacokinetic features, bare siRNAs cannot be administered systemically in humans and as it is frequently cleared by the kidneys, resulting in ineffective accumulation in the targeted tissues [13]. The therapeutic efficacy of siRNA has number of difficulties, unmodified siRNA molecules cannot easily penetrate because of their size and polyanionic nature [14]. Numerous studies have pointed to the need for the creation of efficient, secure and siRNA-targeted delivery systems using both viral and non-viral vehicles [15,16,17].

Mechanism of siRNA involves cleavage and processing of long dsRNA (double-stranded RNA) into small interfering RNA (siRNA) which typically include 3′ end of each strand, there is usually a 2-nucleotide overhang, this is the initial step in the RNAi. Dicer is an endo-ribonuclease (RNase III enzyme) that is responsible for this processing. When siRNA is generated, they are compelled by RISC (RNA induced silencing complex) a multiprotein component complex. The RISC is made up of several different components. The strand with the more stable 5′-end is usually incorporated into the active RISC complex after the siRNA strands have been separated. The RISC complex is then aligned and guided on target mRNA by catalytic RISC protein and the antisense single-stranded siRNA component, an argonaute family member, breaks the mRNA (Ago2) [18]. We can overcome the problems of complexation, entrapment and conjugation by using the siRNA delivery (Figure 1) [19,20].

Figure 1.

Mechanism of RNAi through siRNA through RISC and its effect on translation and inhibition of protein formation. Adapted with permission from [20] with permission from Elsevier, copyright 2022.

Breast cancer is key causes of cancer-related mortalities among females worldwide. Cancer statistics 2023 predict approximately 300,000 breast cancer cases and 43700 deaths in united states alone [21,22,23]. Various treatment options for breast cancer include radiation therapy, surgery, endocrine therapy, adjuvant and neoadjuvant therapy [24,25,26,27]. For the targeted therapy of breast cancer, drugs belonging to various classes such as anti-HER2 inhibitors such as pertuzumab, lapatinib, tyrosine kinase inhibitors, mTOR inhibitors such as everolimus, PI3K inhibitors like Buparlisib, CDK4/6 inhibitors such as Palbociclib, Abemaciclib, Ribociclib, immune checkpoint inhibitors such as Pembrolizumab etc. were developed [28,29]. In addition to the above mentioned, the targeting of various processes involved in breast cancer such as stemness, metabolism, metastasis, could offer some benefit in the therapy of breast cancer [30,31,32]. However, there exists various limitations for the above-mentioned therapies. For example, the limitation of surgery is the recurrence of breast cancer due to the existence of some undetected remains [33] and some of the main drawbacks of chemotherapy include damage to healthy cells, non-specific distribution, anemia, fatigue etc. [24]. Even though such shortcomings are addressed through nanocarrier-based drug delivery strategies, the problem of drug resistance arises due to the strategies adopted by the cells to prevent death and resist to chemotherapy by anti-apoptotic pathway activation [34,35,36,37]. Not just this, several mechanisms attributed to the development of drug resistance in breast cancer include enhanced stemness by overexpression of AMPK which leads to doxorubicin resistance [38], stabilization of YB-1 which mediates chemoresistance [39], TGF β mediated modulation of EMT, stemness, apoptosis, 14,15-EET overexpression which leads to cisplatin resistance [40] etc. For example, in a study by Zheng et al., it was demonstrated that NLRP3 enhanced the gemcitabine resistance through the IL-1β/EMT/Wnt/β-catenin signaling [41,42]. To overcome the drug resistance, siRNA is one of the considerable strategies [43]. For example, Wu et al., co-delivered doxorubicin and P-gp siRNA through PECL3 micelles and it was demonstrated that the DOX and P-gp siRNA reversed multi-drug resistance and inhibited tumor growth [44]. Similarly in another study, inhibition of MDR1 gene expression and increased chemosensitivity to resistant cells was observed when treated with siRNA duplex [45].

Due to existence of various shortcomings in anticancer drugs such as poor targeting, higher doses, toxicity to healthy cells and side effects, has shifted focus on alternative strategies to overcome them and one such strategy is RNA interference through the siRNA [24,34,46,47]. siRNA has demonstrated effectiveness against breast cancer as demonstrated in various research studies [6,13,48,49]. siRNA targets processes of breast cancer development such as angiogenesis, metastasis and breast cancer stem cells (BCSC) and hence it could be employed as a potential treatment opinion.

Various strategies for delivering siRNA using nanocarriers developed for effective gene silencing and targeting BC and TNBC are emphasized in this review. Various strategies utilized for delivering siRNA for example, siRNA with chemotherapeutics as well as peptides, modified nanoparticles, use of smart nanocarriers and others like antibody and quantum dots-based systems have been discussed in detail. In addition, several research studies demonstrating the applicability of nanosystems developed and tested against angiogenesis, cancer stem cells and metastasis of breast cancer have also been reviewed in this manuscript.

2. Nanocarriers, considerations & mechanisms for the siRNA delivery

siRNA offers significant advantages over small molecules such as the inhibition of target gene expression [50]. However, obstacles like decomposition by endo nucleases of the plasma, renal clearance, hydrophilicity, negative surface charge, short half-life and off-target consequences of the naked siRNA hinder desired biological activity [51,52]. Delivery of the siRNA through the nanocarriers is considered one of the most encouraging approaches and cyclodextrin-based material for delivering siRNA was the first to go into human trials for melanoma therapy [53]. Nano vehicles such as polymeric [54], lipid [55] lipid-polymeric, metallic [56,57], mesoporous silica nanoparticles [58] and several other nanocarriers were applied to successfully deliver siRNA and knockdown several genes (Table 1). The incorporation of siRNA into nanocarriers offers advantages such as the shielding of drugs from the degradation of enzymes and preventing recognition by immune system. Furthermore, the nanoparticles portray greater penetration efficiency through the membranes of cells and offer sustained and controlled delivery of the siRNA. In addition to the above, the ability of surface modifications to target the biomarkers, escape from RES and co-delivery with other therapeutic moieties are other added advantages of loading the siRNA into a nanocarrier system [59].

Table 1.

Details of various nanocarriers loaded with siRNA and their overall effects.

Nanocarrier	Si RNA	Cells / animal model used	Summary	Ref.
Lipidoid nanoparticles	HoxA1 siRNA	M6 and M6C cell lines and transgenic FVB C3(1)-SV40Tag mice	Silencing of HoxA1, decreases the mammary cell proliferation, tumor incidence and estrogen and progesterone receptor expression loss was observed on nanoparticle treatment	[173]
Lipid nanoparticles	Cy3-labeled siRNA	MDA-MB-468	The developed nanoparticles depicted better uptake by the cells, down-regulation of CDK4 protein expression and ∼13.8%s G1 cell cycle arrest.	[174]
Barium salt nanoparticles	p53 siRNA, MAPK siRNA, AF 488 as a negative siRNA	MCF-7 and 4T1 cells	The barium salt nanoparticles demonstrated efficient uptake, increased transgene expression, suppression of MAPK and AKT expression and activation	[175]
Phosphatidylcholine–sodium cholate-based nanoparticles	Stealth™ RNAi Actin.	MCF7 cells	Reduced cytotoxicity and improved efficiency were observed upon treatment	[176]
Polymeric nanoparticles	Stat-3	4T1 and MDA-MB-231 cells, nude mice	Suppression of tumor growth, maximum accumulation at the tumor site was observed.	[177]
Liposomes and lipoplexes	siGAPDH (Glyceraldehyde-3-phosphate dehydrogenase)	MDA-MB-231 cells	Various formulations were tested and it was shown that anionic formulation was found to be effective for the delivering siRNA	[73]

Low toxicity, increase vascular permeability, offer stability in serum, permit immune invasion, with stand clearance from the renal system, allow entry into cells are some of the key considerations when designing an efficient nanocarrier for delivering siRNA [60,61]. Furthermore, the shape, size and surface charge of the nanocarriers have been considered. Studies suggested that the size of the carriers varies depending on type of cancer. While some studies show 10–50 nm as an ideal size, others show a size of <200 nm as the ideal size. In a similar way both charge and shape play a key role in the effective delivery of siRNA [62,63]. The route of siRNA administration depends on whether the target site is local or systemic. For example, if the target site is skin, eyes etc, the siRNA is administered locally, however when there is a need to target genes which are crucial for genetic dysfunctions, systemic delivery through oral, intravenous or intraperitoneal routes are preferred [64]. It was mentioned that infusion injection and intravenous administration are the preferred routes for cancer therapy because of advantages such as the bypassing of hepatic metabolism, the ability to enhance bioavailability, improving the half-life of siRNA etc [65]. The mechanism involved in the efficient delivery of siRNA through various nanoparticles also plays a crucial role. The mechanism involved in lipid-based systems such as liposomes and lipoplexes is a reduction in toxicity as well as an improvement in the pharmacokinetic profiles. Similarly, peptide and polymer-based nanoparticles aid in endosomal escape through the proton-sponge effect [66].

Scaling up the production of siRNA-loaded nanoparticles involves overcoming challenges in manufacturing complexity, cost, reproducibility and long-term safety. Ensuring consistent production and stringent quality control is difficult due to variations in synthesis and the high cost of raw materials and specialized equipment. Reproducibility issues arise from maintaining nanoparticle stability and minimizing biological variability in interactions with different cell types. Long-term safety concerns include assessing toxicity, bioaccumulation, immune responses and ensuring nanoparticles degrade into non-toxic components for safe clearance. Addressing these challenges through innovative approaches and rigorous testing is essential for advancing the clinical use of siRNA-loaded nanoparticles [67,68,69].

3. Delivery of siRNA through the nanoparticulate systems

Nanoparticles composed of chitosan were used as vehicles for encapsulating siRNA and targeting CXCR4 which is linked to breast cancer. These siRNA nanoparticles significantly reduced the CXCR4 expression in both protein and mRNA levels which led to enhanced sensitivity to cisplatin [70,71]. Chitosan-based nanoplexes containing multiple siRNAs (siVEGF-A, siVEGFR-1, siVEGFR-2 and siNRP-1) were injected intratumorally to breast cancer-bearing sprague-dawley rats demonstrated ∼97% decline in the tumor volume and reduced VEGF protein levels [72]. In a study by Kapoor et al., anionic liposomes (DOPG/DOPE), Ca²⁺ ions and siRNA were used to construct the lipoplexes. The lipoplexes demonstrated a 70% gene silencing effect in in vitro experiments [73]. Taking advantage of polymeric and lipid nanoparticles, the hybrid lipid-polymeric nanoparticles were formulated to load the siRNA. One example is the IGF-1R targeting siRNA delivery to the MCF7 cells through the lipid/polymeric hybrid nanoparticles in which the polymer and the lipid (PLA-PEG-PLA and cationic lipid) were combined via sonication. The prepared HNPs were ∼48 ± 2 nm in size, the zeta potential of 12 ± 4 mV. About 80% encapsulation efficiency was observed in the HNPs and showed greater biocompatibility in comparison with lipid nanoparticles alone and caused the suppression of about 70% IGF-1R gene [74].

Apatite-based synthetic carriers such as hydroxy apatite and carbonate apatite were used to deliver genes and plasmids [75]. Uddin et al., designed calcium apatite nanoparticles loaded with multiple siRNAs (TRPM7, TRPC6, SLC41A1, TRPM8, ORAI1, SLC41A2, ORAI3 and ATP2C1) for intravenous delivery. Results demonstrated a cytotoxic effect of 57.06 ± 3.72% in 4T1 cells and 59.83 ± 2.309% in MCF7 cells with the use of TRPC6, SLC41A2, MAGT1 and TRPM8 siRNAs. western blot analysis depicted the downregulation of Akt pathway [76]. Although the in vitro results are encouraging, this study hasn't included any in vivo testing. In another study, the siRNA delivered through carbonate apatite nanoparticles silenced the egfr1 and erbb2 genes and produced apoptosis and reduced the tumor burden in vivo [77].

Metal-based nanocarriers like gold nanorods were also explored for delivering therapeutic cargoes, such as siRNA for cancer targeting [74]. One such example is utilizing gold nanorods to inhibit metastasis of breast cancer via siRNA in which siRNA opposed to PAR-1 (protease-activated receptor-1) through the electrostatic interaction was conjugated to nanorods and the RT-PCR and the flow cytometric studies depicted the reduction of protein and mRNA levels of PAR-1 and reduced cancer cell metastasis [78].

4. Combinatorial siRNA delivery

Delivering siRNA alongside various drugs through the nanoparticulate carriers has depicted effectiveness in achieving better anticancer effects than either siRNA or the drug alone [79]. A system incorporating siRNA and DOX was constructed which displayed high drug loading ability and P-gp inhibition which is encoded by the MDR-1 gene in MCF-7/MDR cells to greater extent [80]. DOX along with other siRNA was delivered in combination using PLGA nanoparticles. The DOX + siRNA–NPs or the native DOX + siRNA demonstrated higher suppression of cell growth compared with DOX alone and higher cytotoxicity was observed in the DOX+ siRNA–NPs in comparison with native DOX + siRNA. Further evaluation revealed that greater apoptosis was observed in the combination treatment group than in all other groups and MMPS loss [81]. In another study, biodegradable cationic micelles with paclitaxel and VEGF siRNA simultaneously, significantly reduced the amount of VEGF [82]. Co-delivering siRNA and epigallocatechin-3-gallate (EGCG) is one such instance. The Nrf2 siRNA and EGCG demonstrated synergistic effects as evidenced by considerable activation of apoptosis and further enhancement of BAX, caspase-3,8,9 protein expression [23,83].

Multiple siRNAs could be co-delivered. For example, the multi-delivery of Se, anti-β-tubulin III and anti-P-gp siRNAs through layered nanoparticles demonstrated that P-gp and β-tubulin III expression are considerably downregulated by Se@LDH-pooled siRNAs effective silencing of genes. Furthermore, these can alter Bcl-2/Bax expression, activate MAPK/ERK, PI3K/AKT/mTOR pathways and caspase-3. This can cause cellular ROS levels to increase, disrupt cell shape and cause apoptosis in cells [84]. The synergistic effect of peptides with siRNA was reported. In a study, PR39, a porcine cathelicidin could quickly cross cell membranes and was exploited as a membrane-penetrating peptide for the delivery of siRNA for targeting STAT3 and its downstream pathways. According to the study, the combination of PR39 and STAT3 siRNA inhibits 4T1 cells' ability to invade and migrate [85,86].

5. Modified nanosystem for siRNA delivery

Several obstacles to directing therapeutic moiety to the specific cells include non-specific targeting, reduced intracellular uptake, shorter half-life in the blood, aggregation of the nanoparticles, requirement of high doses and adverse effects lead to the development of various strategies for overcoming these barriers and one such approach is the surface modification of the nanoparticles [87,88]. While some modifications are done to make nanoparticles recognize their targets, such as surface modifications of the nanoparticles with monoclonal antibodies [89], folate [90], hyaluronic acid [91], peptides [92] and more [93] others are done to improve their stability (Figure 2). For example, nanoparticle modification with polymers that are nonionic in nature aid in the stabilization of the nanoparticles. For example, positive charge functionalization led to higher uptake into the cells because of electrostatic interaction with the membrane of the cell [94], As a result, surface modification is crucial to the cells and nanoparticles interaction [95]. The section below focuses on targeting breast cancer through various modified nanoparticulate systems intended for siRNA delivery.

Figure 2.

Anti-cancer effects of the siRNA through receptor mediated drug delivery.

GGCT (γ-glutamylcyclotransferase) plays a crucial role in cell proliferation and is targeted using PEGylated hyaluronic acid-modified liposomes incorporating anti-γ- Glutamylcyclotransferase siRNA. The PEGylation with HA provides enhanced serum stability and CD44 receptor-mediated endocytosis. Cell culture and animal studies of the formulation were carried out. The formulation intended for the intravenous administration for the therapy for breast cancer that is drug-resistant revealed that significant knockdown of GGCT was achieved and depicted anti-apoptotic and anti-proliferative effects [96]. In the context of codelivery of siRNA alongside drugs, Feng et al., developed vapreotide (a somatostatin analog) modified core-shell nanoparticles for somatostatin receptor-mediated delivery of the VEGF siRNA with paclitaxel. The enhanced gene silencing activity due to the enhanced uptake in the VAP-modified groups justifies the effect of VAP modification. The VAP-modified nanoparticles (VAP-PLPC/siRNA NPs) displayed a high fluorescence signal following 6, 12 and 24 h after intravenous administration in tumors which could be due to the receptor-mediated delivery and inhibited tumor neovascularization [97].

Cationic polyethyleneimine (PEI) was used to modify silk fibroin nanoparticles for survivin siRNA and DOX delivery to provide the surface a positive charge for improved absorption by cells via electrostatic bonding. Survivin was suppressed in 4T1 cells and mice with 4T1 tumors and the dual delivery showed higher apoptosis and anti-tumor activity [98]. In another study, the PEI was modified with the lipid dioleoyl phosphatidylethanolamine (DOPE) for modulating P-gp expression through the delivery of siRNA which resulted in P-gp inhibition, greater accumulation of the DOX intracellularly and greater toxicity [99]. In addition to these alterations, folate modifications to target the overexpressed folate receptors are used for the administration of siRNA [100]. A summary of various modified nanosystem for more effective and specific delivery/codelivery of siRNA is included in Table 2 with additional details.

Table 2.

siRNA loaded modified nano formulations developed against breast cancer.

Nanocarriers	Therapeutic cargo	Modification	Purpose of modification	Process involved	Results	Ref.
Core shell nanoparticles	Anti-EGFR siRNA	T7 peptide modification	Targeting transferrin receptors	LPC/siRNA suspension and DSPE-PEG2000-T7 solution were mixed for ten min at 50°C.	• Suppression of EGFR • Blocking proliferation • Targeted accumulation in the tumor • Tumor growth inhibition	[92]
Gelatin nanoparticles	Astrocyte elevated gene-1 (AEG-1, also ca’ metadherin MTDH targeting siRNA	Surface modification with cholamine	Down regulating the AEG-1, a metastasis linked gene	Coupling the cholamine with unmodified gelatin nanoparticles	• High transfection efficiency • Promising gene supression efficiency • Silencing effect depcited on MTDH	[5]
Gold nanoparticles	Captopril and siRNA	Surface modification of the gold nanoparticles by captopril and PEI conjugate	The creation of a unique method for siRNA and drug delivery	Conjugation followed by chemical reduction with gold precursors	• Superiorangiogenesis effect anti-angiogenesis effect • Strong down-regulation of VEGF • Tumor targeting efficiency in vivo	[178]
Liposomes	Survivin siRNA	Modification of cationic liposomes with polypeptide p37	To develop a polypeptide liposome as a potential gene vector for the targeted delivery	Cationic lipid CDO14 and DSPE-PEG2000-p37 were mixed in methanol to form thin film followed by hydration and the mixing of liposomes with siRNA	• High delivery efficiencyt for survivin siRNA • Declined survivin expression • Inhibited tumor growth in xenograft model	[179]
Core-shell nanomicelles	P-gp siRNA and DOX	Folic acid modification	Delivery of the cargo through folate receptor mediated endocytosis	Synthesis of folic acid (FA)-decorated PEG-b-(PCL-g-PEI)-b-PCL triblock copolymers through click chemistry	• Prevention of aggregation, RNAse degradation and renal clearance • Reduced P-gp and IC50 values of DOX in FA functionalized groups • Inhibition of MDR tumor growth	[44]
Nano micelleplexes	Bcl-2 siRNA and DOX	Folic acid modification	Delivery of the cargo through folate receptor mediated endocytosis	Ring-opening polymerization, reversible addition-fragmentation chain transfer polymerization, PEGylation and hydrazinolysis.	• pH dependent drug release • Higher intracellular accumulation • Synergistic effect in vitro	[180]
Mesoporous silica nanoparticles	siHER2 and trastuzumab	Crosslinked polyethyleneimine–polyethyleneglycol copolymer	Delivery of siHER2 and trastuzumab	Shaking, crosslinking and conjugation	• Caused apoptotic death in vitro • Single dose of siHER2 reduced 60% of HER2 expression • Inhibited tumor growth after multiple dose administration over three weeks • Improved safety profile	[181]

6. Smart nanocarriers as a platform for delivering siRNA

The smart nanocarriers are those which aid the release of therapeutic moiety due to the characteristic pathological state at the tissue and through the stimulus given externally. These sorts of systems are widely employed to achieve intracellular delivery for delivering siRNA [101,102,103,104]. Studies demonstrating the utility of responsive nanocarriers employing nanoparticulate systems such as mesoporous silica nanoparticles, polymeric nanoparticles and micelle-plexes are summarised in this section.

To achieve the silencing of VEGF, Dalmina et al., designed a novel magnetic hybrid nanoparticle containing VEGF siRNA which in accordance with the magnetic field delivered the siRNA (100 nM) into the target tissue and a significant knockdown of VEGF (∼60%) was achieved without any potential cytotoxicity in vitro [105]. While this is a system which requires external stimulus, another system which utilizes the tumor environment for drug release such as hypoxia-responsive nanoparticles have been developed. For example, PEG-azobenzene-PEI-DOPE (PAPD) nanoparticles for P-gp siRNA and DOX dual delivery to MCF7 ADR in which azobenzene serves as a hypoxia-responsive material. The uptake was increased, a strong decrease in P-gp regulation was observed in the cells and, a 3D spheroid model with hypoxia showed the highest levels of cytotoxicity, whereas the normal conditions showed little to no impact. In summary, this system demonstrates the cooperative action of siRNA and DOX with less adverse effects via hypoxic responsive release [106]. Endogenous dual stimulus responsive (MMP2 and pH) nanocarrier was developed for inhibiting the proliferation as well as the pulmonary metastasis of breast cancer. The PEG-p-PDHA/PEIPDHA/PTX/siTwi nanoparticle-treated 4T1 tumor-bearing animal group showed a higher degree of apoptosis and a strong anti-metastatic effect with 6.4 metastatic nodules on average where the average of 105.2 metastatic nodules was observed in saline-treated group) [107]. While the above studies demonstrated the efficiency of siRNA either alone or in combination with a chemotherapeutic drug, there are studies in which two siRNAs were delivered using a single nanocarrier system (Figure 3). Two siRNAs (siSna and siTwi) and paclitaxel were delivered through a pH-sensitive PEI-PDHA/PEG-PDHA/PTX/siSna/siTwi nanoparticles. In 4T1 model in mice, the nanocarrier triggered apoptosis, downregulated snail and twist proteins expression and provided anticancer effect. The PPSTs have an additional lung anti-metastatic action which is proven by the decrease in the metastatic nodules [108]. These are some of the studies that demonstrate the significance of siRNA when delivered through a smart nanocarrier system [109,110]. Table 3 depicts various additional smart nanocarriers used for the delivery of several siRNAs along with their biological effects.

Figure 3.

The schematic representation of developing smart nanocarrier (MSNs) for the tumor microenvironment responsive release which upon endocytosis show the siRNA release and exert its activity.

Table 3.

Smart nanocarriers developed and tested in in vitro and in vivo models of breast cancer.

Type of responsive nanocarrier	Nanoparticulate system	Therapeutic moiety delivered	In vitro effects	In vivo effects	Ref.
pH and redox responsive	Mesoporous silica nanoparticles	Bcl-2 siRNA and DOX	pH and redox responsive drug release and enhanced uptake due to the folate modification, silenced the expression of Bcl-2 therefore exhibiting cytotoxic and apoptotic activities in MDA-MB-231 cells	–	[182]
Acid responsive	Polymeric nanoparticle	NgBR siRNA	Appearance of positive surface charge due to acid mediated discarding of DMMA which aided in the better siRNA uptake and supressed the NgBR expression.	The nanosystem suppressed the distant metastasis through inhibiting epithelial-mesenchymal transition and normalizing the blood vessels in 4T1 tumor bearing BALB/C nude mice	[183]
Enzyme and pH responsive	Polymeric nanoparticle	Anti-luciferase siRNA	Polymeric NPs depicted a 2.5-fold increase in the cellular internalization in the presence of MMP-7, and increase in hemolysis at pH <6.2. Enhanced luciferase knockdown R221A-Luc mammary tumor cells	–	[184]
Acid responsive	Core-shell nanoparticles	MnSOD siRNA	Greater cellular apoptosis and MnSOD expression silencing was observed in TAM-resistant MCF7-BK-TR breast cancer cells by core-shell acid responsive NPs	Reversal of TAM-resistance to apoptosis, the scrambled siRNA transplanted tumors exhibited five-times larger size than MnSOD siRNA transplanted tumors in nude mice.	[172]
pH responsive	Triple layer micelle plex	siRNA-p65 and cisplatin prodrug	150 nM concentration of siRNA-p65 about 50% of the p65 which further inhibited the MMP-9 and down regulated 70% of cyclin D1 in 4T1 cells. Further, 4.4- and 1.5-fold decline in the migration and invasion capability and inhibition of NF-κB pathway respectively was demonstrated.	The triple layer micelle plex blocked the primary tumor growth as well as the metastasis to lungs in vivo	[185]
Enzyme responsive	Cascaded targeting nanoparticles	siRNA and DOX	In MDA-MB-231 cells, elevated cytotoxicity was observed due to the knockdown of Bcl-xL and breaking the CTGF-mediated resistance to apoptosis which is induced by DOX	The nanoparticles treated animal group shown a significant suppression of Bcl-xL, cIAP1 and CTGF levels and inhibited the tumor cell division and proliferation.	[186]

7. siRNA for targeting TNBC

TNBC is one of the more aggressive kinds of breast cancer and is defined by a lack of three receptors: progesterone, estrogen and human epidermal growth factor 2 (HER2) and is characterized by very poor survival, enhanced metastasis and poor prognosis [111,112,113,114]. TNBC accounts for 15–20% of the total breast cancer cases [115]. The hormonal therapy was not effective in TNBC patients [116]. Chemotherapy is one of the methods utilized to treat TNBC. But the obstacles of conventional chemotherapeutic drugs include poor selectivity, poor bioavailability, affecting normal cells and multidrug resistance [117]. Furthermore, it is known that these drugs are effective in killing the cancer cells but have no effects on the oncogenes that are either damaged or mutated [118]. Various signaling pathways are involved in the progression, proliferation and survival of breast cancer such as PI3Kpathway, JAK-STAT pathway, TGF β pathway, NFκB pathway, PI3K/AKT/mTOR pathway, HER2 signaling pathway, Notch signaling, WNT signaling, hedgehog pathway etc. (Figure 4) [119,120,121,122]. Studies suggest the effectiveness of siRNA in targeting various oncogenes, modulating various signaling pathways and more in TNBC [123]. Even though siRNA has widely been explored in in vitro models, due to the anionic nature, tendency to increase immune responses and liability, siRNA administration in vivo is impeded. For delivering siRNA to malignant cells and getting beyond the obstacles, several nanocarriers, referred to as nano vectorization has been attempted.

Figure 4.

Schematic representation of some key pathways involved in proliferation, progression and survival of breast cancer.

To effectively distribute siRNA, to target TNBC, various types of nanocarriers were utilized [124]. qRT-PCR studies revealed the overexpression of DANCR, a long non-coding RNA (lncRNA) in TNBC cells (BT549, MDA-MB-231) [125]. Functionalized NPs were developed against DANCR and the efficiency of the formulated NPs demonstrated the efficiency of the NPs after single transfection in inhibiting the DANCR ∼80–90%. A great decline in potential of the cells to form tumor spheroids and a 30–70% decline in the cell viability was attributed to the DANCR silencing effect of the NPs. Further studies confirmed that the decline in expression of various oncotargets such as β-Catenin, surviving, ZEB1 and N-cadherin was due to the DANCR inhibiting effect of the NPs [123]. Collagen type IV alpha 2 (COL4A2) exists in the vascular basement membrane. It is expressed in TNBC and is investigated to have a crucial part in the etiology of cancer. MDA-MB-231 and MDA-MB-468 cell proliferation, migration and induction of apoptosis were effectively reduced by the administration of COL4A2 siRNA through a lentivirus vector. In addition, arrest of the cell cycle of TNBC was observed in the G2 phase [126].

Receptor-mediated delivery of siRNA has been utilized for delivering siRNA against TNBC. Because of its essential function in the pathogenesis of TNBC, targeting EGFR would be advantageous to combat TNBC. Nguyen et al. synthesized an anti-EGFR antibody which upon attachment onto the surface of SPION and PEG served as an active targeting moiety. Better internalization into cells due to the targeted receptor-assisted endocytosis and ease of endosomal scape showed an efficient siRNA delivery in TNBC cells [113]. Deng et al. developed layer-by-layer NPs in which the core was loaded with DOX) and siRNA was loaded into another layer followed by a layer of hyaluronic acid coating for tumor targeting. Due to the extended half-life, CD44 receptor-mediated active targeting and enhanced endosomal escape of these LBL NPs, these NPs depicted effective gene silencing activity against MRP1 upon intravenous administration. Histological and serum analysis have shown no sign of toxicity of the prepared NPs [127]. p53, a widely mutated gene in BC is overexpressed in TNBC. The p53 siRNA transfected Hs578T cells upon treatment with EGCG, led to an increase in apoptosis, decreased angiogenesis, cell survival decrease and a decline in autophagy [128]. A nanocomplex was constructed containing cholesterol peptide, IKBKE siRNA, cabazitaxel and modified hyaluronic acid where a synergistic activity was observed. In vivo studies on orthotopic TNBC mouse model depicted the tumor-targeted accumulation of the siRNA following intravenous administration thereby depicting a better antitumor response [129].

Smart nanocarriers have been explored for successful siRNA delivery in TNBC. Sujuan et al. designed a dual stimuli responsive microbubble formulation onto which HIF1α-siRNA was adsorbed. The formulation upon ultrasound stimulus converts into nanoparticles which upon irradiation caused the suppression of HIF1α. In the siRNA microbubble after ultrasound treatment, an 80% decrease in cell viability was observed. Cell-based and animal studies on mice demonstrated the efficiency of ultrasound and photodynamic therapy combination as a good strategy to target TNBC [130]. In another study, stimuli-responsive manganese oxide-based lanthanide nanoprobe containing S100A4 siRNA was developed onto which the peptide (RGD) was bound. The peptide can bind to overexpressed αvβ3 integrins in the tumor. The release of siRNA, whose activity is further increased by NIR irradiation and the breakdown of the core on exposure to glutathione (GSH) would be another strategy to achieve precise and targeted siRNA delivery. The concept behind this was endorsed by MDA-MB-231 bearing mice's strong cytotoxic impact and effective reduction in tumor volume [131].

Application of CPPs has been utilized in targeting TNBC [132]. Graphene oxide (GO)/polyethyleneimine (PEI)/polyethylene glycol (PEG)/cell-penetrating peptide/siRNA system was checked for its effect on cytotoxicity, apoptosis, rictor and colony formation in MDA-MB-231 cells and on tumor volume in SCID/nude mice. The GO-PEI-PEG-CPP/siRNA significantly decreased viable cell count at the IC₅₀ values of 100 and 150 μM, caused the suppression of colony formation, induction of apoptosis and inhibited the expression of rictor. Further, the tumor volume was reduced to less than 200 mm³ which was more than 600 mm³ in the control group [133]. Utilizing the concept of ultrasound-targeted microbubble destruction (UTMD), Jing et al. developed a nanobubble formulation containing EGFR-targeted siRNA (siEGFR) onto which the CPP was loaded. Inhibition of proliferation, down-regulation of the EGFR mRNA in CPP-loaded NBs and UTMD demonstrates the collaborative effect of the system [134]. Various nanoparticulate systems, combinatorial delivery, smart nanosystem as well as peptides were utilized for efficient siRNA delivery in the TNBC are summarized in Supplementary Table S1.

8. Miscellaneous strategies for siRNA delivery

Among the EGFR ligands, HB-EGF (heparin-binding epidermal growth factor-like growth factor) is highly expressed in TNBC patients. Okamoto et al. designed Anti-HB-EGF antibody-modified lipid nanoparticles that were decorated with Fab′ antibody against HB-EGF for the systemic administration of siRNA targeting polo-like kinase 1 (PLK1). Successful delivery of siRNA to targeted tumor region of mice with MDA-MB-231 carcinoma was observed following the PLK1 protein expression decline which depicts the efficiency of αHB-EGF LNP against TNBC [135]. Not only antibodies but aptamers have been used as ligands for targeting because of their high selectivity and affinity [136]. For example, the liposomes' surface was functionalized with an anti-CD44 aptamer, encapsulated with siRNA and the studies confirm luc2 inhibition to a greater extent and prolonged inhibition [137]. In another study, aptamer-QLs (anti-EGFR aptamer-lipid conjugates in Quantum Dots-lipid nanocarriers) have shown enhanced delivery and efficient silencing of genes, reduced tumor development and metastasis in combinatorial delivery of Bcl-2 and PKC-1 siRNAs through the anti-EGFR QLs [138]. Zang et al. developed Anti-EphA10 antibody-conjugated pH-sensitive liposomes which upon systemic administration bind to expressed Anti-EphA10 in breast cancer that contributes to growth and metastasis loaded with siRNA that targets the MDR1 gene. The studies on MCF-7/ADR cell xenograft nude mice conclude the effectiveness of the formulation in declining the MDR1 protein levels along with the tumor-specific delivery of siRNA [139].

9. Targeting some key processes involved in breast cancer through siRNA

Nanoparticulate systems offers numerous advantages for delivering siRNA for targeting various key processes involved in progression of breast cancer. However, very few platforms like cyclodextrin based nanoparticles were tested in clinical setting for solid tumors. Various other platform has also been tested but none of them have shown promising results and necessitates further evaluation. We summarized nanoparticulate platforms which demonstrate promise and have the potential to be evaluated in clinical phases of investigation in Supplementary Table S2.

9.1. siRNA in angiogenesis associated with breast cancer

Angiogenesis, also known as neovascularization, is an intricate process wherein new blood vessels are created from the vascular network of existing tissue. When tumors or certain stromal cells create specific endothelial cell growth factors, angiogenesis occurs (mast cells, fibroblasts, macrophages). Activated endothelial cells proliferate and migrate to generate new capillary tubes. Creation of vascular lumen completes vessel maturation as endothelial cells grow and create a new basement membrane. For vascular endothelium, VEGF is the most active, selective and potent endothelial cell growth factor [140]. siRNA has demonstrated potential against angiogenesis of breast cancer and several nano formulations have been formulated [19,141,142].

The delivery of siRNA through calcium phosphate nanoparticles loaded in polycation liposome (PLCP) can decrease angiogenesis. To assess the ability of gene silencing, various VEGF siRNA sequences were created and mediated by Lipofectamine 2000 and PLCP. MCF-7 cells were employed for testing the efficiency of PLCP-mediated siRNA delivery. Three VEGF siRNA sequences targeting VEGF mRNA were created. After 24 hs of transfection, PLCP silencing effectiveness of VEGF mRNA was substantially higher than Lipofectamine 2000. An appreciable ability of gene silencing was observed in VEGF siRNA mediated by PLCP causing notable inhibition of angiogenesis [19].

siRNA administration using chitosan as a carrier resulted in efficient reporter gene suppression. Administration of nanoplexes targeting VEGF expression caused a silencing effect and inhibited tumor development. As a result, chitosan nanoplexes containing were used a mix of siRNAs, including siNRP-1, siVEGFR 2, siVEGFR-1 and siVEGF-A [143]. Salva et al. revealed that VEGF siRNA had inhibitory effects on breast carcinoma and has anti-angiogenic potential in addition to this chitosan for targeting the siRNA. With the mismatch siRNA, a small drop in VEGF mRNA and protein can be detected. When the chitosan and siRNA were evaluated in terms of IFN-γ expression, it was discovered that neither induced an interferon-mediated immune response and anti-tumor effects compared with the implying that anti-tumors effects, tumors control group were almost all VEGF specific [72].

The μPAR/μPA system is implicated in cancer cell invasion, survival and proliferation. Simultaneously downregulation of these proteins caused by RNAi, activation of the pro-survival gene is inhibited by it. Phospho-p38 and phosphor-ERK kinases, as well as possibly inhibiting the angiogenesis factors. The μPAR/μPA system is a favorable therapeutic target for malignant research. For inhibition of these proteins, siRNA constructs were created. siRNA used a sequence driven by a CMV promoter to lower μPA expression in cell lines and its receptors, both separately and in combination, to determine the consequences [142].

Shtykalvo et al. demonstrated that endothelial cell proliferation, angiogenesis and migration can be significantly decreased by anti-VEGFR1, anti-endoglin and ani-VEGFA siRNA-induced silencing [144]. Bharathi et al. depicted mitochondrial and IL-6R/IL-6 signaling in breast cancer and offered a different strategy using a siRNA-based silencing technique to target cancer angiogenesis. By upregulating IL-siRNA, the author showed how IL-6R/IL-6 signaling inhibited angiogenesis and invasion [145]. SiH1324 may be able to prevent heparinase-related angiogenesis by adjusting the amount of bFGF released from the ECM [146].

The delivery of BV6 and STAT3 siRNA by carboxymethyl dextran trimethyl chitosan nanoparticles (CMDTMC NPs) inhibits tumor cell growth. CMD-conjugated TMC NPs were found to be able to deliver BV6 and siRNA to colorectal, melanoma and breast cancer cells. Furthermore, these cancer cell lines depicted considerable induction, suppression of proliferation, apoptosis, migration, angiogenesis and survival using anti-NIK siRNA, BV6 and anti-STAT3 siRNA loaded in carboxymethyl dextran trimethyl chitosan nanoparticles in combination with anti-NIK siRNA, BV6 and anti-STAT3 siRNA loaded in carboxymethyl dextran trimethyl chitosan [147]. Anti-RhoA and anti-RhoC siRNAs are effective inhibitors of angiogenesis against advanced breast cancer [148].

Hwang et al. demonstrated that doxorubicin and ionizing radiation treatment caused senescence in the HUVEC cell by CXCL11 regulates immune cell activation, motility and differentiation through binding to CXCR3. Treating the cells with HUVEC and CXCR3 siRNA with CXCL11 siRNA to observe that CXCR3 is involved in the effects of CXCL11 from HUVEC on migration. Levels of CXCL11 and CXCR3 were significantly reduced by CXCR3 siRNA in MDA-MB-231 cells and by administering of CXCL11 siRNA in senescent HUVEC [149].

9.2. siRNA in metastasis associated with breast cancer

Metastasis is migration and spread of malignant cells from primary tumors to other parts of the body, resulting in additional tumors. Malignant or cancerous, tumors are those that develop and spread aggressively. They can spread throughout the body and comprise the organ function if it is untreated [https://www.britannica.com/science/metastasis]. Malignant cells must experience a sequence of steps to spread from primary site to the other parts of the body. Following are the steps of metastatic cascade: 1. Developing into an invasive and migratory locally 2. Intravasating into the circulation and reaching the blood vessels. 3. Spreads through the blood flow 4. Extravasating to the distant organ and arresting 5. Getting through the initial aggressive stress 6. Co-opting the distant stroma and reinitiating outgrowth [31]. siRNA has demonstrated potential against metastasis of breast cancer and several nano formulations have been formulated. MUC1Apt-conjugated chitosan nanoparticles were created for the delivering DTX (Docetaxel) and IGF-1R siRNA to SKBR3 cells. This unique targeted co-delivery strategy increased cellular absorption of nanoparticles while significantly reducing gene expression and cell viability connected to breast cancer spread and tumor growth. In addition, Apt-conjugated nanoparticles significantly suppressed IGF-1R signal transducers and activators of VEGF and matrix metalloproteinase [136].

siRNA was utilized to disrupt the downstream signaling partners of Src, a non-receptor tyrosine kinase. MDA-MB-435S was suppressed by Src knockdown alone, as well as a combination of Stat3, cMyc, and/or Src knockdown. This inhibition causes a reduction in soft agar culture and monolayer proliferation, as well as the ability to produce the primary tumor. Furthermore, intra-tumoral injection of siRNA directly, against those signaling pathways inhibited primary tumor growth and metastasis significantly. Simultaneously suppression of Stat3, Myc and/or Src resulted in the most significant reduction of metastasis of breast cancer and primary cancer growth [150].

To defeat PTX resistance and improve breast cancer chemotherapeutic effectiveness. Chen et al. demonstrated that redox-oligopeptide liposomes can simultaneously deliver anti-survivin siRNA and PTX. Anti-survivin siRNA blocks PTX from increasing survivin expression, slowing wound healing and cell proliferation while increasing cell death. The combination of PTX, siRNA and SS-L not only slows tumor development but lowers pulmonary metastasis. As a result, combining ani-survivin siRNA with PTX to reduce metastatic breast cancer and breast cancer development could be a potent drug nanocarrier for combination tumor therapy, as could redox-sensitive oligopeptide liposomes [151].

TMC NPs (Trimethyl chitosan) successfully encapsulated HMGA-2 and DOX, suggesting that HMGA-2 and its downstream signaling molecules could be inhibited. Furthermore, inhibiting HMGA-2 mRNA gene resulted in a severe cytotoxic effect and was associated with drug sensitivity to DOX. This research depicts that DOX–siRNA–TMC NPs effective at causing tumor cell death and silencing the HMGA-2 gene [152].

HDM2-siRNA promoted genes involved in proliferation and metastasis of cancer cells. Suppression of matrix metalloproteinase expression by hdm2-siRNA may prevent cancer spread. (32) lPEI2200-SS had lower cell toxicity and good blood compatibility. Furthermore, lPE2200-SS/siRNAsur polyplexes had a strong anti-proliferative effect and impeded tumor development and metastasis [153].

9.3. siRNA in stem cells associated with breast cancer

Breast cancer stem cells (BCSCs) are tiny clusters of cells that can be found in breast (BCSC). Spheroids, also known as “mammosphere,” are formed when cells proliferate under poor adherence conditions and have a variety of stem-cell-like characteristics, such as the activation of stem-cell signaling pathways [154,155,156,157]. Pathways such as Notch, Hedgehog, Wnt and Hippo are dysregulated in BCSCs and may be involved in tumor resistance, recurrence and metastasis [158]. siRNA has demonstrated with potential against BCSCs and several nanoformulations have been formulated. Increased p53 expression was connected to HMGA1 silencing. Both E6 and HMGA1 could be used as anti-CSC targets. To direct siRNA to EpCAM overexpressing breast CSCs, they employed PEGylated EpCAM aptamers [159,160]. siRNA targeting AKT2, a key oncogene implicated in breast cancer carcinogenesis and with a specific function in CSC malignancy, was loaded into PM (Polymeric micelles). After therapy, it demonstrated a decline in cell invasion potential with strong suppression in mammosphere formation [161].

Inhibiting BCSC self-renewal, EDB-FN knockdown by siRNA has also been explored. The use of CDK4-specific siRNA and Let-7a miRNA as therapeutic agents to modify BCSCs and breast cancer cells throughout their development has been reported. HA and protamine were used to treat a Herceptin-conjugated cationic immunoliposome [162] therapeutic method in the treatment of EDB-FN (Extra domain B of fibronectin) positive BCSC-derived malignancies [163]. Sox-2 siRNA suppression reduced the expression of Sox-2, its companion genes Oct-4 and Nanog, as well as the CSC surface markers Sca-1 and AbcG2. CSC-associated gene expression and surface markers dropped after Sox-2 knockdown, as did the fraction of SP cells in the tumor cell population [164]. The Glu-NPs improved absorption of siRNA payloads into GLUT1-overexpressing MDAMB-231 cell spheroids because of the unique interaction between the Glu ligands and GLUT1. The NPs demonstrated enhanced gene silencing activity and anticancer efficacy in the MDA-MB-231 orthotopic tumor following intravenous treatment as compared with the control MeO-NPs. Therefore, Glu-NPs successfully lowered proportion of CSCs in orthotopic tumor (ALDH high cells) [165].

10. Clinical aspects & lack of clinical translation of si-RNA based therapies

It was in 2012 when the siRNA-based treatment was started and various therapeutics are in various phases of clinical trials [166]. The ligand-targeted siRNA nanoparticles based on cyclodextrin developed for the treatment of solid tumors are in clinical trials [167]. siRNA offers significant advantages over conventional therapies and several siRNA-based treatments are under clinical trials for the treatment of cancer and other diseases. Even though numerous studies summarize the effectiveness of siRNA in knockdown of various genes, in inhibition of angiogenesis and metastasis, only a very few studies are in clinical trials some of which are terminated in the context of breast cancer. Very strong optimization of the clinical trials is needed to obtain clinically acceptable results since the results obtained in the in vivo studies cannot be interpolated with the human results. Furthermore, the stability and immunogenicity aspects also hinder the clinical translation. As per the various studies, liposomes and micelles would be best options for the delivery of siRNA.

The field of siRNA-based therapies holds tremendous promise for treating various diseases, including breast cancer. However, several critical challenges have prevented many siRNA therapies from progressing beyond early-stage clinical trials and achieving widespread clinical translation. Some of the challenges with clinical translation of siRNA-based therapies include [168]: The delivery of siRNA faces numerous biological barriers, including rapid degradation by nucleases in the bloodstream and poor cellular uptake efficiency. Despite advances in nanoparticle-based delivery systems, achieving sufficient stability and efficient delivery to target tissues remains a significant hurdle. Even if siRNA nanoparticles successfully reach the target cells, they must effectively enter the cytoplasm where the RNA interference machinery operates. Ensuring this intracellular delivery without triggering unwanted immune responses or cellular toxicity is challenging. One of the primary concerns with siRNA therapy is off-target effects, where unintended genes or cellular pathways are inadvertently affected. This can lead to unpredictable biological consequences and potentially harmful side effects in patients. While siRNA sequences are designed to be highly specific to target genes, mismatches or unintended interactions with non-targeted mRNA sequences can occur. These sequence-specific effects may vary among patients and can complicate the clinical efficacy and safety profiles of siRNA therapies. siRNA molecules themselves can trigger innate immune responses through pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs). This activation can lead to inflammation, cytokine release and potentially severe immune reactions, particularly if the siRNA is recognized as foreign or if it accumulates in tissues. Prolonged exposure to siRNA could potentially induce adaptive immune responses, including the production of antibodies against the therapeutic siRNA or its delivery vehicle. This can limit the effectiveness of repeated dosing and complicate patient management in clinical settings. The safety profile of siRNA therapies must be rigorously evaluated in clinical trials, particularly concerning long-term effects and unintended biological interactions. Regulatory agencies require extensive preclinical data demonstrating safety and efficacy before approving siRNA therapies for human use. Navigating regulatory pathways for novel RNA-based therapies can be complex and time-consuming. Regulatory agencies require robust data on pharmacokinetics, pharmacodynamics and toxicology profiles, which may be challenging to obtain for siRNA molecules due to their unique biochemical properties. Developing scalable manufacturing processes for siRNA therapies and their delivery systems is crucial for commercial viability. High production costs and technical challenges associated with large-scale manufacturing can hinder the broader adoption of siRNA therapies in clinical practice. While siRNA-based therapies offer exciting potential for treating breast cancer and other diseases, significant scientific, technical and regulatory challenges have slowed their progression beyond early-stage clinical trials. Addressing these issues requires continued innovation in delivery technologies, enhanced understanding of siRNA biology and robust clinical validation of safety and efficacy. Overcoming these hurdles will be essential for realizing the full therapeutic promise of siRNA in clinical settings and improving patient outcomes [169,170,171].

11. Conclusion

For addressing various problems pertaining to the delivery of naked siRNA, nanoparticles are employed to carry siRNA to the target cells. We have attempted to outline a broad variety of nanocarriers carrying various siRNAs developed for the therapy of BC and TNBC. Also, combinatorial delivery of siRNA, several modified systems, smart nano vehicles have also been covered in this review. It was demonstrated that combinatorial delivery with other siRNAs or chemotherapeutics has shown better effect when compared with siRNA alone. Not just conventional nanocarriers but also the modification or chemical conjugation of the nanocarriers aid in achieving better results and provide receptor-mediated delivery of siRNA. Smart nanocarriers which are responsive to the local environment have been explored for site-specific siRNA delivery. The studies summarized the inhibitory effects of siRNA nanoparticles on breast cancer and TNBC via the suppression of various genes and proteins. Furthermore, a special emphasis on angiogenesis, BCSCs and metastasis is provided in this article which is crucial in development of breast cancer. The results obtained from the studies were found to be promising in preclinical models of breast cancer and TNBC.

12. Future perspective

In our opinion, this is a growing topic and the use of siRNAs still needs to be explored to a greater extent for targeting numerous genes and the knowledge from these studies can be used for translating the siRNAs into clinics in the future. Continued research into nanotechnology and biomaterials will likely lead to more sophisticated delivery systems. These may include smart nanoparticles capable of targeted delivery, triggered release mechanisms and improved biocompatibility. Integrating siRNA therapies with other treatment modalities such as chemotherapy, immunotherapy and radiotherapy holds promise. This approach can potentially overcome resistance mechanisms and improve overall treatment outcomes. Developing siRNA molecules that are less immunogenic or that trigger beneficial immune responses against cancer cells could open new therapeutic avenues. Moving from preclinical studies to robust clinical trials will be crucial. Demonstrating safety, efficacy and long-term benefits in human trials will determine the practicality and adoption of siRNA therapies in clinical settings.

Supplemental material

Supplemental data for this article can be accessed at https://doi.org/10.1080/20415990.2024.2400044

Author contributions

BM Sai: conceptualization, writing - original draft; YH Dinakar: conceptualization, writing-original draft, H Kumar: conceptualization, writing - original draft, writing - review & editing; R Jain: writing - review & editing; S Kesharwani: writing – review & editing; SS Kesharwani: writing - review & editing; SL Mudavath: writing - review & editing; A Ramakishan: writing - review & editing; V Jain: conceptualization, writing-original draft, review & editing and supervision.

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, stock ownership or options and expert testimony.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.