Polymeric Nanomedicines for siRNA Delivery in Hepatocellular Carcinoma: Emerging Therapeutic Strategies, Combination Approaches, and Translational Perspectives

Abstract

Hepatocellular carcinoma is the sixth most common cancer worldwide and a leading cause of cancer related mortality. Risk factors include chronic hepatitis B/C, obesity, alcohol abuse, diabetes and metabolic disorders. In advanced stages, normal treatments such as surgery, transplantation and chemotherapy are not as effective as tumors develop resistance and do not target oncogenic pathways well. Small interfering RNA (siRNA) therapeutics have the potential to change treatment paradigms by silencing specific biological pathways, thus suppressing tumor progression. Polymeric nanoparticles (PNPs) such as poly(lactic-co-glycolic acid) (PLGA), polyethyleneimine (PEI) and stimuli-responsive copolymers have attracted attention as effective delivery systems for siRNA. These carriers protect siRNA from enzymatic degradation and facilitate its cellular uptake. Furthermore, the surface of the carriers can be modified with targeting ligands, eg, galactose or ASGPR antibodies, to improve tumor-specific delivery. The PNPs can be tuned to change their physical and chemical properties, are more stable and responsive to signals from the tumor microenvironment for regulated release. This means they perform better than lipophilic or metallic carriers. Hepatocyte specific absorption pathways even enhance the liver tropism. This review focuses on recent advances in siRNA based therapeutic strategies against hepatocellular carcinoma, with a special focus on PNPs mediated drug delivery systems. It describes significant oncogenic pathways like β-catenin and STAT3 and formulation techniques including nanoprecipitation and layer by layer assembly. The review also reports the increasing importance of artificial intelligence in siRNA design, formulation optimization, and personalized therapeutic methods to improve the clinical applicability of HCC treatment.

Graphical Abstract

Introduction

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and a leading contributor to cancer-related mortality worldwide, with rising incidence driven by chronic hepatitis infections, non-alcoholic fatty liver disease, and metabolic disorders. Standard therapeutic approaches such as tumor excision, liver grafting, radiofrequency-based tumor destruction, and systemic anticancer drugs often face major challenges, including diagnosis at later stages, frequent tumor relapse, and adverse systemic effects, all of which limit their effectiveness in patients with advanced disease.¹ These therapeutic shortcomings have prompted extensive investigation into targeted molecular strategies designed to precisely regulate cancer-related signaling pathways involved in the onset and advancement of HCC. Small interfering RNA (siRNA) has received significant interest among these methods due to the capacity to target genes in a sequence-selective way enabling direct and direct inhibition of abnormal oncogenes and signaling factors, which facilitate tumor growth and survival. Indicatively, concurrent silencing of multiple oncogenic pathways, including β-catenin and STAT3 has been reported to effect synergistic activation of apoptosis and repression of proliferation in liver cancer models, which illustrates the therapeutic advantage of multi-targeted gene knockdown approaches.² However, a number of biological challenges have deterred the clinical translation of siRNA therapeutics, such as rapid enzymatic degradation in circulation, ineffective cellular uptake and ineffective endosomal escape upon internalization.³ To overcome these hurdles, non-viral delivery systems have been especially recognized as multi-purpose vectors with the ability to protect siRNA from nucleases, improve pharmacokinetic stability and deliver tumor tissues.⁴ Bio-compatible and bio-degradable polymers such as polylactic -co-glycolic acid (PLGA), polyethyleneimine (PEI), chitosan and poly(β-amino esters) have been widely studied in siRNA encapsulation and delivery due to their tunable physicochemical properties, ability to form stable complexes with nucleic acids and they can be functionalised with targeting ligands.⁵ For example, PLGA-based nanoparticles have demonstrated significant potential for protecting siRNA from enzymatic degradation and offering controlled and sustained release, whereas chitosan-based carriers are recognized for efficient cellular uptake and low toxicity in liver targeting applications.⁶ Besides protecting siRNA and enhancing its cellular uptake, polymeric delivery vehicles can be selective by incorporating targeting ligands or using receptor-mediated endocytosis to become tumor targeted and reduce non-targeted effects. The nanoprecipitation and layer-by-layer assembly of fabrication techniques allow for precise control over particle size, surface properties, and loading capacity factors which have a significant impact on biodistribution, cellular uptake, and avoiding endosomal compartmental trapping.⁷ Furthermore, multifunctional polymeric nanoparticles can simultaneously transport siRNAs together with anticancer drugs or multiple gene-silencing sequences, supporting coordinated regulation of pathways like STAT3, β-catenin, VEGF, and AFP that contribute to HCC progression⁸ performance of silencing genes more, support optimization of formulations by predicting polymer siRNA interactions, and individualize dosing plans depending on tumor-specific properties.⁹ Together with more sophisticated polymeric vehicles, these computationally directed approaches are a strong move in the direction of tailored, pathway-directed therapy of HCC. Altogether, the polymer-based siRNA delivery systems offer a prospective path forward to addressing the most significant therapeutic challenges by increasing the molecular stability, improving the delivery efficiency, targeting pathways precisely, and making gene-specific therapy translationable to the clinic.¹⁰ In addition, these approaches maximize the silencing efficiency of the sequences, predict the interactions between polymers and siRNAs to help optimize the formulations, and tailor the dosages according to specific tumor characteristics. Together with advanced polymeric vectors, these approaches herald a shift towards personalized and pathway-specific therapies for HCC. Polymeric platforms for siRNA delivery provide an efficient approach to overcoming the challenges of HCC treatment by increasing siRNA stability and uptake, specific knockdown of oncogenic cascades, and furthering the clinical translation of gene therapies.¹¹ This review intends to critically discuss the recent advances in engineering polymeric nanomedicines for siRNA delivery in hepatocellular carcinoma with emphasis on their design strategies, targeting mechanisms and therapeutic potential.

Cell Signalling

The development of HCC is driven by dysregulation of critical signaling pathways such as RTK, RAS/RAF/MEK/ERK, PI3K/AKT/mTOR, Wnt/β-catenin, JAK/STAT, Hedgehog (Hh) and Hippo. Receptor tyrosine kinases (RTKs) and their downstream signaling networks such as RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways are central players in tumor progression, metastasis, and therapeutic resistance.¹² The Wnt/β-catenin pathway is involved in cell mobility, invasiveness and maintenance of stem-like tumor cells, whereas JAK/STAT signaling is involved in cancer cell growth, formation of new blood vessels and interactions with the immune system, making it a useful target for combined therapeutic approaches. Increased Hedgehog signaling has been associated with metastatic properties and decreased sensitivity to chemotherapy. The Hippo pathway regulates cell growth and apoptosis. Interference in these signaling pathways is a promising strategy for improving therapeutic outcome in hepatocellular carcinoma as shown in (Figure 1).¹³

Figure 1.

Schematic representation of polymeric nanomedicine loaded with siRNA targeting key oncogenic signaling pathways involved in HCC.

Notes: Created in BioRender. Habeeb, M. (2026) https://BioRender.com/h4q00q1.

VEGF Signaling Pathway

HCC proliferation and dissemination are angiogenesis-dependent, predominantly mediated by vascular endothelial growth factor. One form, VEGF-A, is important because it activates receptors called VEGFR1, VEGFR2 and VEGFR3 that regulate the behaviour of the cells lining the vessels. VEGFR2 is essential to stimulate the formation of neo-vessels.¹⁴ Tumors with low oxygen inside also increase VEGF levels and help the cancer to keep progressing. Researchers are trying to block this pathway with gene-silencing techniques. For example, siRNA against VEGF can reduce its expression and inhibit vascularization. Delivery carriers, such as lipid nanoparticles, protect these molecules and improve their delivery to the tumor tissue, resulting in tumor-reducing effects in experimental work.¹⁵ In addition, VEGFR2 siRNA can block proliferation and migration of vessel forming cells. Some studies show that combining these approaches with drugs such as sorafenib may be more effective than using one of the treatments alone.¹⁶

EGFR Signaling Pathway

The epidermal growth factor receptor (EGFR) is associated with HCC progression by regulating proliferation, survival and motility of liver cells. High EGFR expression is frequently associated with rapid tumor growth and worse clinical outcome. Certain activating molecules such as HB-EGF can further stimulate cancer cell multiplication, invasion into surrounding tissue, and the formation of new blood vessels. Gene silencing approaches have been explored to disrupt this signaling system. When EGFR expression is reduced using siRNA, the activity of downstream pathways such as RAS/RAF/MEK/ERK and PI3K/AKT is also diminished, which weakens signals that support tumor development shown in (Figure 1).¹⁷ Experimental work has shown that lipid-based nanoparticles carrying EGFR-targeted siRNA can suppress tumor growth. The inhibition of the EGFR can also be done by silencing the ligands, such as HB-EGF, and this results in the inhibition of angiogenesis and cell migration. Researchers are also considering using carriers such as exosomes and biodegradable micelles to deliver EGFR targeted siRNA to tumors in a more direct manner and minimize the undesirable effects on healthy tissues.¹⁸

FGF/FGFR Signaling Pathway

Fibroblast growth factor (FGF) signaling and its receptors (FGFRs) have an important role in HCC because they influence tumor growth, blood vessel formation, and resistance to treatment.¹⁹ In many cases, FGF2 and FGF19 are over-expressed, and crosstalk between FGF19 and FGFR4 has been highly associated with disease progression. This signaling system can also cross-talk with other cancer related pathways, like Wnt/β-catenin and STAT3, that can augment tumor promoting activity. Experimental gene-silencing studies suggest that blocking this pathway could inhibit tumor progression. For example, downregulating FGF19 expression can inhibit cell proliferation and angiogenesis, and lipid-based delivery of FGF-targeted siRNA has shown promising results in experimental models of liver cancer. A comparable approach to target FGFR4 can block FGF19–FGFR4-driven signaling and may be useful to overcome resistance to therapy.²⁰ In some studies, the synergistic antitumor effect of siRNA targeting FGFR with other drugs targeting associated pathways is also shown, which may be an option for the treatment of more aggressive tumors.²¹

HGF/c-Met Pathway

The HGF/c-Met signaling pathway has also been shown to facilitate tumor cell proliferation, angiogenesis and metabolic switch of HCC.²² Increased c-Met levels are usually associated with poor clinical outcome and low response to therapeutic interventions including sorafenib. Blocking this pathway is being investigated as one way to disrupt this pathway, for example with siRNA targeted against HGF or c-Met.²³ This approach can reduce c-Met, and thus the survivability and invasiveness of tumor cells. HGF can also be applied to inhibit c-Met activation, and to break off associated signaling pathways including PI3K/AKT and STAT3.²⁴ These siRNA molecules are often delivered in lipid-based carriers to improve stability and delivery. Some studies also reported that c-Met targeted siRNA combined with drugs such as defactinib can have stronger anticancer effects, indicating the possibility of overcoming resistance and improving treatment response.²⁵

IGF Signaling Pathway-

Insulin like growth factors have been shown to contribute to tumour behaviour in liver cancer by promoting tumour cell proliferation, angiogenesis and metastasis.²⁶ Abnormally high levels of IGF-1R and excessive production of IGF-2 are often found in many cases of HCC, which are associated with more aggressive disease. Researchers have been looking at ways to silence genes to reduce IGF signaling.²⁷ Downregulation of IGF-1R by siRNA has been shown to inhibit tumor growth and encapsulating siRNA into nanoparticles enhances its tumor targeting ability. Blocking IGF-2 can also upset the internal signaling that cancer cells need and slow their growth and spread.¹² IGF-targeted siRNA in combination with either chemotherapy or anti-inflammatory agents has also been shown to enhance the responsiveness to treatment most especially in the tumors that are not particularly responsive to conventional therapy.²⁸

PDGFR Signaling Pathway

Platelet derived growth factor receptors (PDGFRα and PDGFRβ) are involved in several processes related to progression of liver cancer, such as cell transformation, blood vessel formation and spread of tumour cells.²⁹ PDGF signaling activation can also increase VEGF, which promotes further vascular growth of the tumor. To counteract these effects researchers have tested siRNA aimed at reducing PDGFR activity.³⁰ Reduced PDGFRα expression has been shown to inhibit cell growth and vessel formation. This kind of siRNA has been delivered by nanoparticle carriers to reduce the level of VEGF and size of tumor in experimental models. Some data also indicate that combination of PDGFR-targeted siRNA with other anti-angiogenic drugs can improve the response to treatment.³¹

RAS/RAF/MEK/ERK Pathway

Dysregulation of components of MAPK cascade including the RAS/RAF/MEK/ERK pathway is a common event in proliferation, survival and metastasis of HCC cells. Aberrant activation of this pathway is associated with a maladaptive prognosis and therapy resistance³² siRNA of crucial elements in this signalling including RAS or MEK can sufficiently suppress oncogenic signalling siRNA against MEK reduces ERK activation and this can slow down the growth of cancer cells and bring about apoptosis.³³ Lipid-based nanoparticles protect this siRNA and improve its cellular entry. It was also shown in studies that a combination of MEK-targeted siRNA and sorafenib may lead to enhanced inhibitory effects on hepatocellular carcinoma, suggesting a possible benefit for patients who do not respond well to standard therapy, as illustrated in (Figure 1).³⁴

PI3K/AKT/mTOR Pathway

The PI3K/AKT/mTOR signaling is also significantly impaired in HCC, which leads to tumor evolution, survival, and resistance to treatments.³⁵ The PI3Ks are activated which causes the AKT to be phosphorylated leading to the activation of mTOR complexes stimulating cell growth and inhibition off apoptotic processes. This is also a pathway that facilitates lipid biosynthesis and autophagy, which are essential in tumor metabolism. In almost half of HCC cases, dysregulation, such as mTOR overexpression and abnormal PDK1 activity is detected.³⁶ siRNA to this pathway provides a highly specific approach to silence tumor-favouring signaling and silence PI3K or AKT downstream signalling impairs cell growth and induces cell death. A case in point involves pan-PI3K siRNA which downregulates all isoforms whereas certain siRNA such as p110α result in greater specificity results in downregulating of mTOR activity and inhibition of tumor growth.³⁷ Furthermore, the antitumor effect is further improved by the use of siRNA targeting mTOR and autophagy or angiogenesis inhibitors. Inhibitors with third generation that inhibit both mTORC1 and mTORC2 efficacies are promising in combination with siRNA to increase therapy resistance.³⁸

Wnt/β-Catenin Pathway

WNT/ β -catenin pathway is commonly implicated in the progression of liver cancer, as it along with its ability to promote cell survival, cell division and invasiveness in cells.³⁹ Mutations of β-catenin cause its accumulation in the nucleus which activates genes that facilitate cancer development in most tumors. The pathway is also able to interplay with other pathways such as EGFR-related pathways and tissue-degrading enzymes that in combination can facilitate the progression of disease. This has been interfered with using gene-silencing approaches. As an example, β-catenin siRNA can decrease its concentration and decrease its translocation into the nucleus, leading to reduced gene activation that helps tumor cells survive and could increase their response to chemotherapy. Carriers such as micelles or liposomes are often used to improve delivery and stability. The inhibition of Wnt signaling can also be achieved by targeting upstream elements, such as the co-receptors LRP5 or LRP6, prior to the activation of β -catenin. Suppression of this pathway has been found experimentally to decrease tumor growth, migration, and invasion (Figure 1).⁴⁰

JAK–STAT Pathway

The JAK 2–STAT signaling pathway is significant in the regulation of inflammation, immune responses, and cancer cell survival in HCC.⁴¹ Hyperactivity of this pathway, particularly involving STAT3, is frequently associated with HCC progression, metastasis and resistance to treatment. STAT3 is able to regulate genes that inhibit cell death, and thus contribute to the survival of tumor cells. Scientists have thus experimented on siRNA which can inhibit the activity of STAT3. By reducing the levels of STAT3, it is possible to reduce its activation and diminish tumor development supporting signals.⁴² This siRNA is frequently administered in lipid-based carriers to enable it to target tumor tissue and minimize undesirable effects elsewhere. The upstream inhibition of JAK2 can also be used to inhibit STAT signaling. Certain researchers indicate that a combination of STAT3-targeted siRNA and chemotherapy or immunotherapy can enhance the effect of treatment, especially in cases that are hard to treat.⁴³

Hedgehog (Hh) Signaling Pathway

The Hedgehog signaling pathway is a normal signaling pathway involved in the regulation of early development and tissue repair, but has been observed to be abnormally active in certain cases of hepatocellular carcinoma.⁴⁴ The increased activity is associated with tumor growth, angiogenesis and decreased treatment response. This pathway is mediated by binding of Hedgehog proteins to Ptch receptors, relieving the repression of Smoothened (Smo) which then can activate Gli transcription factors. When this signaling pathway is disrupted, it can promote tumor invasion, dissemination and recurrence. Some researchers have tried gene silencing strategies to mess with this pathway.⁴⁵ By reducing Smo expression with siRNA, downstream signaling can be interrupted and activation of genes that support tumor development can be lowered. Blocking Gli1 in a similar manner may block it from entering the nucleus and decrease its ability to regulate gene expression, which may inhibit cell growth and survival.⁴⁶ These siRNA molecules are often delivered more efficiently to the tumor tissue using lipid-based carriers. Other experimental models have also shown improved outcomes by combining Hedgehog-targeted siRNA with small molecule drugs such as vismodegib, including decreased metastasis and increased sensitivity to radiotherapy in liver cancer models.⁴⁷

Hippo Signaling Pathway

Hippo signaling pathway is a pathway that is involved in the regulation of cell growth, cell death, and tissue balance. Changes in this pathway have been specifically associated with the tumor development, metastasis and decreased drug resistance in chemotherapy in HCC.⁴⁸ Elevated amounts of YAP within the nucleus are commonly observed in more aggressive tumors and are associated with less favorable clinical outcomes. Due to this link, gene silencing strategies targeting YAP expression or inhibiting its downstream signaling have been investigated. When YAP levels are lowered, genes that promote tumor cell growth and survival are not activated. siRNA molecules are frequently encapsulated into nanoparticle carriers to enhance delivery. The carriers help to stabilize the siRNA and facilitate their accumulation into tumour tissue. Experimental evidence also indicates that the combination of YAP-directed siRNA and anticancer drugs, including doxorubicin, may improve the responsiveness to the treatment. Furthermore, nanoparticle formulations of verteporfin, which disrupts YAP-TEAD interactions, have demonstrated a more potent antitumor effect in combination with YAP-targeted siRNA, indicating potential utility in advanced cases.⁴⁹

Comparative Analysis of siRNA-Mediated Signaling Pathway Targeting in HCC

The therapeutic relevance of siRNA targets in HCC varies according to their position and connectivity within oncogenic signaling networks. While targeting EGFR, RAS/RAF/MEK/ERK and PI3K/AKT/mTOR primarily suppresses tumor-cell proliferation and survival, targeting VEGF/PDGFR/FGFR mainly affects angiogenesis, whereas Wnt/β-catenin, JAK/STAT3 and Hippo/YAP signaling can additionally regulate invasion, stemness, immune responses and therapeutic resistance.⁵⁰ Importantly, extensive crosstalk among these pathways may allow compensatory signaling following single-target inhibition, thereby limiting therapeutic durability (Table 1). Therefore, simultaneous targeting of complementary pathways may provide greater benefit than isolated gene silencing, particularly in heterogeneous HCC. This is also evident from studies on co-delivery of siRNA with anticancer agents like sorafenib or doxorubicin wherein coordinated pathway inhibition can improve antitumor activity.⁵¹ Overall, the therapeutic potential of siRNA should be evaluated not only for gene-silencing efficiency but also for target specificity, pathway connectivity, resistance mechanisms and delivery efficiency. Thus, the novel approach of network-directed and combination siRNA therapy, facilitated by targeted polymeric nanocarriers, could provide a more effective approach to circumvent pathway redundancy and therapeutic resistance in HCC. However, the predominantly preclinical evidence and differences in experimental models and delivery systems remain important barriers to clinical translation.⁵²

Table 1.

siRNA Targets and Rational Combination Strategies for Hepatocellular Carcinoma Therapy

siRNA Target	Major HCC Effect	Potential Advantage	Major Limitations	Rational Combination	References
VEGF, VEGFR2	Angiogenesis	Strong anti-angiogenic effect	Tumor cells can activate alternative angiogenic pathways	Antiangiogenic drugs	[50]
EGFR, HB-EGF	Proliferation, survival, migration	Suppresses upstream signaling	Bypass signaling and resistance	EGFR + MAPK/PI3K inhibition	[51]
FGF19, FGFR4	Growth, angiogenesis, resistance	Addresses FGFR4-driven HCC	Strong pathway crosstalk	Wnt/β-catenin or STAT3 targeting	[52]
RAS, MEK	Proliferation and survival	Direct inhibition of MAPK signaling	Compensatory survival signaling	Sorafenib/MAPK-directed therapy	[53]
PI3K, AKT, mTOR	Survival, metabolism, autophagy	Broad downstream control	Physiological functions and pathway redundancy	Autophagy/angiogenesis inhibitors	[54]
β-catenin, LRP5/6	Stemness, invasion, survival	Targets an important HCC driver	Crosstalk with EGFR and other pathways	STAT3/EGFR targeting	[55]
STAT3, JAK2	Survival, inflammation, immune regulation	Links tumor growth and immune signaling	Redundant cytokine signaling	Immunotherapy/chemotherapy	[56]
SMO, Gli1	Growth, invasion, metastasis	May affect aggressive phenotypes	Context-dependent pathway activation	Small-molecule Hedgehog inhibitors	[57]
YAP	Growth, survival, resistance	Targets a major transcriptional regulator	Complex upstream regulation	YAP/TEAD-directed therapy	[58]

Manufacture of siRNA Loaded Polymeric Nanomedicines for Blocking the HCC

The design of siRNA-loaded polymeric nanomedicines depends on the proper choice of polymers, siRNA loading strategies, fabrication methods and surface properties. PLGA, chitosan, poly(β-amino esters) and polyaspartamide are examples of biodegradable polymers that can protect siRNA from enzymatic degradation, enhance cellular uptake, promote endosomal escape and allow intracellular release.⁵⁹ siRNA can be loaded by physical encapsulation or electrostatic complexation with cationic or ionizable polymers. The major manufacturing approaches include but are not limited to nanoprecipitation, solvent displacement, emulsion-based approaches, controlled sonication, polymeric micelle formation and assembly of RNA nanostructures based on rolling circle transcription. Double-emulsion approaches involve successive emulsification followed by solvent removal to incorporate siRNA within a polymeric matrix, whereas amphiphilic block copolymers can self-assemble into polyion complex micelles via electrostatic interactions with siRNA. RNA nanostructures containing siRNA sequences and targeting elements can be generated by rolling circle transcription, and cyclodextrin-assisted assembly can enhance structural stability and co-delivery of anticancer drugs (Table 2). Surface functionalization with targeting ligands can improve receptor-mediated uptake and increase the selectivity for HCC. Also, stimuli-responsive polymers can lead to a controlled release of siRNA in intracellular pH, redox or enzymatic environments. Multifunctional systems can be used to deliver siRNA and anticancer drugs such as sorafenib simultaneously for synergistic therapeutic effects. Characterization includes particle size, polydispersity, zeta potential, morphology, siRNA loading/ encapsulation efficiency, release profile, siRNA integrity, cellular uptake, gene silencing and cytotoxicity. Manufacturing processes for clinical translation should be scalable, reproducible, batch-to-batch consistent, stable, sterile and have well defined critical quality attributes.⁶

Table 2.

Comparative Advantages and Limitations of Polymeric Materials Used for siRNA Delivery in Hepatocellular Carcinoma

Polymer System	Major Advantages	Limitations	Potential Application in HCC siRNA Delivery	Reference
PLGA	Biodegradable and biocompatible; protects siRNA from degradation; supports controlled and sustained release; well-established nanoparticle fabrication approaches.	Relatively limited intrinsic cellular uptake and endosomal escape; surface modification may be required for efficient targeting.	Suitable for sustained siRNA delivery and combination gene–chemotherapy.	[60]
Chitosan	Biocompatible; low toxicity; positively charged groups facilitate siRNA complexation and cellular uptake; easily functionalized with targeting ligands; supports stimuli-responsive systems.	Variable physicochemical properties; relatively weak stability under some physiological conditions; endosomal escape may require additional modifications.	Attractive for liver/HCC targeting, dual-ligand systems, and gene–drug co-delivery.	[61]
Polyethyleneimine (PEI)	Strong electrostatic interaction with siRNA; high nucleic-acid loading; efficient cellular uptake and endosomal escape through proton-sponge effects.	High molecular-weight/cationic PEI may cause cytotoxicity and nonspecific interactions; requires structural modification to improve safety.	Useful where strong siRNA condensation and intracellular delivery are required.	[62]
Poly(β-amino esters) (PAEs)	Biodegradable; tunable structure; pH-responsive behavior; facilitates intracellular siRNA release; adaptable for targeted delivery.	Formulation and polymer composition require careful optimization; long-term in vivo safety and translational evidence remain comparatively limited.	Promising for tumor-targeted and stimuli-responsive siRNA therapy.	[47]
Polyaspartamide/ PHEA-based polymers	Functionalizable backbone; galactose modification enables ASGPR-mediated targeting; relatively selective HCC delivery; reduced off-target toxicity reported.	Additional synthesis and ligand-functionalization steps may increase formulation complexity; clinical translation remains limited.	Particularly suitable for hepatocyte/HCC-selective siRNA delivery.	[63]
Polymeric micelles/polyion complex micelles (PICMs)	Small nanoscale structures; efficient nucleic-acid complexation; suitable for co-delivery of siRNA and small-molecule drugs; can incorporate targeting and stimuli-responsive functions.	Possible structural instability after dilution in biological fluids; loading capacity and premature cargo release require optimization.	Useful for multifunctional siRNA–drug co-delivery and tumor-microenvironment-responsive therapy.	[64]
PEG-containing polymers (eg, PEG–PLGA)	Improved colloidal/systemic stability; prolonged circulation; reduced nonspecific interactions; supports controlled drug/siRNA delivery.	PEG may reduce cellular uptake in some systems; additional formulation complexity; potential need for optimized PEG density.	Useful for systemic delivery and improved tumor accumulation.s	[65]
Stimuli-responsive polymers	pH-, redox-, enzyme- or GSH-responsive release; enables site-specific intracellular/tumor release; can improve therapeutic selectivity.	More complex synthesis and characterization; heterogeneous tumor microenvironment may affect responsiveness; scalability requires validation.	Promising for precision HCC therapy and overcoming drug resistance.	[66]
Polypeptide-based nanoparticles	Biodegradable and structurally tunable; can be engineered for tumor-microenvironment modulation and combination immunotherapy.	More complex synthesis and characterization; limited clinical validation compared with established polymers.	Potential for siRNA delivery combined with immune-checkpoint therapy.	[67]
Thermosensitive polymeric hydrogels	Prolonged local release; can maintain nanoparticles at the administration site; supports sustained siRNA exposure.	Mainly suited to localized or depot-type administration; formulation viscosity and release kinetics require optimization.	Potential for prolonged/sustained siRNA delivery and reduced dosing frequency.	[68]

siRNA-loaded nanoparticles are generally prepared by encapsulation of the RNA within biodegradable carriers such as PLGA or by the formation of stabilized RNA assemblies using agents such as cyclodextrin to improve durability and targeting potential.⁶⁹ Appropriate dimensions and efficient cellular internalization of the particles are achieved by employing production methods such as double emulsion fabrication, controlled sonication and rolling circle transcription. These formulations protect siRNA from early degradation and allow its release in the intracellular milieu facilitating its therapeutic application in hepatocellular carcinoma. Rolling circle transcription can produce porous RNA nanospheres with EpCAM aptamers, siRNA fragments, and probe binding regions within long RNA strands.⁷⁰ The strands can assemble into nanoscale structures with the help of amino-cyclodextrin modified DNA probes that help keep the structure intact. Cyclodextrin is also a hydrophobic host which allows the incorporation of poorly soluble drugs such as sorafenib into the cyclodextrin cavity and improving their physicochemical stability. Amino-cyclodextrin is positively charged and can therefore electrostatically interact with negatively charged RNA, thus promoting condensation into compact nanostructures. After intravenous injection, PRS@SF nanospheres can circulate in the body and preferentially accumulate in the tumor sites via aptamer-guided recognition. Such a system provides coordinated gene silencing and chemotherapeutic delivery while helping to limit off-target exposure and enhance delivery precision (Figure 2).⁵²

Figure 2.

Schematic representation of sorafenib (SF) loaded porous RNA nanospheres (PRS@SF). (a) Illustration of the cyclodextrin-assisted assembly process and the Dicer-mediated disintegration of PRS@SF. (b) Depiction of the therapeutic actions of PRS@SF, including circulation in the bloodstream following intravenous administration, cellular uptake by target cells, and subsequent release of siRNA and sorafenib for combined anticancer therapy. (Figure 2) is reproduced from the Ref.52

A well-defined ABC triblock copolymer was synthesized and systematically characterized to evaluate its suitability for siRNA-loaded polymeric micelle formation. Completion of the pSar-b-pGlu(OBn)-b-pLys ABC-triblock copolymer occurs by sequential ring-opening polymerization of NCAs, in which neopentylamine is used as the initiator (Figure 3A). The GPC analysis indicates a downward shift in the elution volumes with an addition of each block, indicating an indication of a rise in the molecular weight and the extension of the chain (Figure 3B). A constant distribution of molecular weights suggests controlled polymerization without any side reactions, whereas the occurrence of slight broadening of peaks most probably suggests secondary structures in peptide segments. DOSY NMR verifies that proton signals from all blocks belong to a single species, validating the integrity of the triblock copolymer (Figure 3C). Boc removal from the pLys segment yields free amines, which enhance siRNA complexation via electrostatic interaction after protonation (Figure 3A). NMR is used to verify complete deprotection and GPC is used to demonstrate backbone stability in such conditions. The block lengths selected prudently maximize solubility and encapsulation competence of drug and siRNA, and circumvent solubility difficulties such as drug aggregation or poor encapsulation. (Neo)pSar₁₉₀-b-pGlu(OBn)₁₂-b-pLys₈ is the optimal triblock copolymer display high complexation of siRNA at N/P 3. This can then be followed by ensuing dual centrifugation to load drugs and siRNA into polymeric micelles (PICMs) as shown by the schematic representation (Figure 3D), size and zeta potential (Figure 3E), normalized strength (Figure 3F), siRNA filling efficiency (Figure 3G), and micelle encapsulation efficiency (Figure 3H). These descriptions uphold a robust synthesis and optimal formulation of siRNA delivery through block copolymer micelles.⁵³

Figure 3.

(A) Illustrates the synthetic route of the (Neo)pSar-b-pGlu(OBn)-b-pLys triblock copolymer via sequential ring-opening polymerization (ROP) of NCAs followed by Boc deprotection to expose amine groups for siRNA complexation. (B) shows gel permeation chromatography (GPC) elugrams confirming controlled polymer chain growth during the synthesis, while (C) presents 1H DOSY NMR spectra verifying the structural integrity of the triblock copolymer. (D) depicts the formation of polyion complex micelles (PICMs) co-loaded with siRNA and desloratadine (DES), stabilized by electrostatic and hydrophobic/π-π interactions. (E–G) illustrate the dynamic light scattering (DLS) data indicating monodisperse PICMs of ~120 nm size with near-neutral zeta potential in HEPES buffer. Lastly, (H) quantifies the high drug loading efficiency (~85%) of DES in the siRNA/DES PICMs using ultra-performance liquid chromatography (UPLC). These results collectively demonstrate successful synthesis and formulation of well-characterized, stable multicompartment PICMs for efficient co-delivery of siRNA and DES to enhance endosomal escape and therapeutic efficacy in hepatocellular carcinoma treatment. FIGURE 3 is reproduced from the Ref.53

Mechanism of siRNA Loaded Polymeric Nanomedicines for Hepatocellular Carcinoma Therapy

The therapeutic mechanism of siRNA-loaded polymeric nanomedicines for HCC. Polymeric nanoparticles loading siRNA can passively target HCC lesions through the enhanced permeability and retention effect after systemic delivery through leaky tumor vessels and defective lymphatic drainage, Simultaneously, targeted therapy with surface functional groups that react to overexpressed receptors on HCC cells can be used to achieve active targeting that leads to receptor-mediated endocytosis and increased tumor selectivity.^54,55 After the cellular internalization, the polymeric carriers are used to mediate endosomal escape due to proton sponge effects, membrane destabilization, or pH-sensitive polymer behavior, which releases siRNA into the cytoplasm. The liberated siRNA is integrated into the RNA-induced silencing complex (RISC) in which it instructs sequence-specific destabilization of matching mRNA, which leads to effective gene silencing illustrated in (Figure 4). Oncogenic and pro-tumorigenic genes that promote proliferation, angiogenesis and metastasis are silenced and this results in inhibition of the growth of tumors, inhibition of metastatic invasion and inhibition of tumor angiogenesis. This figure, overall, underscores how polymeric siRNA nanomedicines combine targeting, intracellular delivery and RNA interference to effectually formulate molecular therapy of HCC.⁵⁹

Figure 4.

Various types siRNA loaded polymeric nanomedicines for treating HCC.

Notes: Created in BioRender. Habeeb, M. (2026) https://BioRender.com/g6i0j4q.

Targeted Polymeric Carriers and Ligand Modifications

Ligand functionalized surface polymeric carriers have emerged as a promising strategy to upscale selective delivery of siRNA to HCC cells, with minimal off-target toxicity. These nanocarriers are used to enhance the cellular uptake and tumor specificity by incorporating ligands such as TAT peptides, glycyrrhetinic acid, lactobionic acid and galactose to take advantage of receptor-mediated endocytosis.⁷¹ Polymeric scaffolds like chitosan, PLGA and polyaspartamide also offer structural stability, controlled release and enzyme protection of siRNA. The stimuli-responsive changes such as redox sensitive disulfide linkages can be exploited to facilitate the intracellular release of drugs for enhancing the efficacy of therapy in the tumor microenvironment. Altogether, these sophisticated targeted polymeric systems hold great promise to overcome biological barriers and improve the efficacy of gene silencing in HCC therapy. Chen et al (2024) developed a chitosan-based polymeric micelle (TAT-C-SS-P@SF@PDL1-siRNA) for co-delivery of sorafenib and PDL1-siRNA in hepatocellular carcinoma (Table 3). The system consists of TAT peptide to improve cellular uptake and disulfide bonds to promote redox-responsive drug release. Nanoparticles (~150 nm) were prepared by dialysis and then electrostatic complexation for siRNA loading. The formulation resulted in a loading efficiency of sorafenib of 34.2%. PDL1-siRNA inhibits immune escape and sorafenib inhibits angiogenesis via downregulation of VEGF. It also reduces drug resistance by decreasing the expression of P-gp and MRP1. The nanocarrier bypasses major barriers such as poor bioavailability, cellular uptake limitations and multidrug resistance. High intracellular GSH level triggers disulfide cleavage and release in tumor cells. This induces ROS generation and disrupts redox balance, promoting apoptosis. The system showed enhanced efficacy with ~73% apoptosis and ~90% tumor inhibition in vivo.⁶⁰ In another research they designed a dual-receptor-targeted chitosan nanoparticle system (GCGA) to carry siRNA against PAK1 in the hepatocellular carcinoma (HCC). This dual-ligand system, known to use glycyrrhetinic acid (GA) and lactobionic acid (LA) to a significant degree increased uptake of nanoparticles in the HCC cells relying upon the receptor-mediated endocytosis. This selective transfer of siPAK1 was able to silence the expression of PAK1 in cells which resulted in a decrease of cell proliferation, migration and invasion of cells and an increase in apoptosis. In vivo experiments showed significant tumor growth suppression in the xenograft models where the therapeutic effect was attributed to inhibition of the PAK1/ MEK/ERK signal pathway. This way of working emphasizes how dual-ligand nanoparticles can be used as a way of a precise gene therapy in HCC.⁶¹ Pho-iam and they team in 2021 developed PLGA nanoparticles that entrap α-fetoprotein (AFP) siRNA to be targeted to HCC therapy. These nanoparticles were very specific to the HCC cells, therefore, decreasing cell viability to a great extent, and resulting to apoptosis. The system used together with doxorubicin increased apoptosis two-fold compared to each treatment alone, which showed that the system had excellent synergistic therapeutic potential. The new method highlights the importance of siRNA-loaded nanoparticles in enhancing the efficacy of traditional chemotherapy without affecting biosafety.⁶² In another investigation Perrone and they colleagues fabricated galactosylated polyaspartamide nanoparticles (PDPG) to target HCC-related gene silencing with siRNAs. The system was based on galactose residues to specifically target asialoglycoprotein receptors (ASGPR) overexpressed in HCC cells (Table 3). This approach allowed the targeted delivery of siRNAs against eEF1A1, eEF1A2 and E2F1, resulting in significant downregulation of these oncogenes. The therapy decreased cell viability and tumor growth in vitro and in xenograft models and showed little off-target effects and toxicity against normal hepatocytes. In this paper we show the potential of ASGPR-targeted nanocarriers for precise and effective gene therapy of liver cancer.⁴⁷

Table 3.

Recent Advances in Polymeric Nanomedicines for siRNA Delivery and Liver Tumor Suppression

Type of polymeric Nanocarrier	Surface Modification	Target Gene	Formulation Method	Size (nm)	Biological Barriers Addressed	Tumor Microenvironment Interaction	Advanced Features	Reference
Polymeric micelle	TAT peptide, disulfide linkage	PD-L1, VEGF	Dialysis-assisted polymeric micelle self-assembly followed by electrostatic complexation	150	Cellular uptake, drug resistance	GSH-responsive release (high intracellular GSH triggers disulfide cleavage), ROS increase, redox imbalance, VEGF inhibition, immune checkpoint modulation	Drug/gene co-delivery	[60]
Graphdiyne oxide nanosheets	SP94-PEG	SLC7A11	GDYO oxidation and PEG functionalization followed by π–π stacking-mediated sorafenib loading and electrostatic siRNA adsorption	50	Poor tumor accumulation, drug resistance	Targets HCC via SP94 binding; EPR-enhanced passive targeting; disrupts mitochondrial ROS/lipid peroxidation/GSH in TME; in vivo tumor accumulation peaks at 24h (Cy5.5 imaging)	Hemo-gene synergism	[70]
Nanospheres	SP94 peptide	Survivin	Self-assembly via coprecipitation and sonication/dialysis, followed by EDC/NHS-mediated SP94 conjugation and electrostatic siRNA complexation.	178 ± 0.5	siRNA degradation, endosomal escape	SP94 binds GRP78 receptor on HCC cells; EPR effect (100–200 nm optimal); enhanced tumor accumulation (3.3x fluorescence at 24h vs non-targeted)	Dual-modal imaging	[72]
Reduction-responsive polymeric NPs	PEG, G0-C14	CFL1	Nanoprecipitation (solvent displacement) followed by ultrafiltration purification.	100	Systemic delivery, endosomal escape	Responds to high intratumoral GSH (2–10 mM) for rapid cargo release and GSH scavenging to boost ROS	Sorafenib sensitization	[66]
Chitosan-based nanocomplex	Lactobionic acid	VEGFR2	Chitosan coating with stimuli-response	165.5 ± 8.2	Targeted delivery to HCC and sustained drug release	Reduced angiogenesis; suppressed IL-6/IL-8/NF-κB	CRISPR-Cas9 therapy	[67]
Poly (β-amino esters) nanoparticles	None	circMDK	Polymer-based encapsulation	120	Enhanced siRNA delivery to tumor cells	Reduced tumor growth and metastasis	Biomarker-targeted therapy	[73]
Polyion Complex Micelles (PICMs)	PEGylation + DES incorporation	MFAP-5	Dual centrifugation	100	Endosomal escape	Reduced CAF activity; angiogenesis inhibition	Multicompartment RNAi	[53]
Carotenoid-based nanovector	β-carotene	FOXC1	Dendrimer complexation	30	Nuclear delivery	EMT suppression; metastasis reduction	Enhanced cellular uptake	[68]
Porous RNA nanospheres	Cyclodextrin	EpCAM	Rolling circle transcription	120	siRNA protection	EpCAM silencing; tumor stemness reduction	Biodegradability; synergistic therapy	[52]
Dual-ligand chitosan NPs	GA + LA ligands	PAK1	Ionic gelation	170	Cellular uptake and RNA stability	Promoted apoptosis via PAK1 silencing	Dual-receptor targeting; apoptosis	[61]
PLGA nanoparticles	None	AFP	Double emulsion solvent evaporation	150	Reduced systemic toxicity and drug resistance	Enhanced sensitivity to doxorubicin	Synergistic gene and chemo-therapy	[62]
Galactosylated polyaspartamide	Galactose	eEF1A1, eEF1A2, E2F1	Covalent attachment to PHEA backbone	200	Specific targeting to HCC cells	Downregulated key oncogenes; reduced toxicity	Selective targeting; minimal toxicity	[47]
PLGA	None	AFP	Double emulsion solvent	150	Reduced degradation, improved uptake	Enhanced caspase-mediated apoptosis	Synergistic gene/chemo-therapy	[74]
PLGA-PEG	PEG	circROBO1	Double emulsion solvent	160	Increased systemic stability	Suppressed miR-130a-5p/CCNT2 pathway	Efficient tumor targeting; non-toxic	[75]
Galactosylated Albumin	Galactose	p53-SREBP1-CFASN	Desolvation	170	Enhanced tumor targeting; pH-responsive release	Targeted lipogenesis inhibition via molecular pathways	Reduced toxicity; specific tumor release	[76]
PEG-PLGA (AIE-Mito-TPP)	Peptide coating	cGAS-STING pathway	Single emulsion	180	Immune activation	Sustained mtDNA-mediated immune stimulation	Immunotherapy enhancement; innate immunity activation	[77]
CMCS-pH-responsive NPs	CMCS	Tim-3	Single emulsification	200	Immune checkpoint barrier	Enhanced siRNA release and tumor-specific sorafenib activation	Dual delivery for immune activation and angiogenesis suppression	[69]
Hydrogel-encapsulated NPs	Cholesterol-DP7 peptide	Pin1	Electrostatic complexation	36	Sustained release, improved endosomal escape	Prolonged silencing of Pin1; liver-specific targeting	Injectable thermosensitive hydrogel for long-term effect	[78]
Polymeric NPs	PEG2000	NET-1	Thin-film hydration + extrusion	675	Enhanced uptake, ultrasound-triggered release	NET-1 suppression via sonodynamic ROS generation	Ultrasound-activated apoptosis; sonodynamic therapy integration	[79]
Hybrid cell membrane-coated polymeric micelles	HCC cell membrane + macrophage membrane	Xkr8	Electrostatic complexation	100	Prolonged circulation, tumor-specific delivery, siRNA protection and cellular uptake	Xkr8 silencing suppresses phosphatidylserine externalization and inhibits efferocytosis; sorafenib induces immunogenic cell death and enhances DAMP-mediated antitumor immunity	Sorafenib/siRNA co-delivery; hybrid membrane-mediated homologous targeting; immune microenvironment modulation; efferocytosis inhibition	[80]

Multifunctional and Stimuli Responsive Designs

Multifunctional and stimuli responsive polymeric nanocarriers have become one of the new platforms to enhance the accuracy and effectiveness of siRNA-based therapy in HCC.⁶³ Such systems incorporate various functionalities like redox, pH, and enzyme-responsive release systems to allow site specific release of payloads in the tumor microenvironment. Synergy Co-delivery of siRNA with chemotherapeutics or gene editing agents this concept increases therapeutic synergy by altering oncogenic signatures and overcoming drug resistance simultaneously.⁶⁴ PEGylation, chitosan surfaces, and amphiphilic polymer have been used to enhance systemic stability, extended circulation and delivery efficiency to the intracellular environment. Taken together, these intelligent nanoplatforms are a second-generation approach to combinatorial and microenvironment-responsive HCC therapy.⁶⁵ To overcome sorafenib resistance in hepatocellular carcinoma (HCC), Li et al (2023) developed reduction-responsive polymeric nanoparticles (MeO-PEG-S-S-PLGA + G0-C14) for the co-delivery of siCFL1 and sorafenib. These NPs include PEGylation with MeO-PEG for stealth and prolonged systemic circulation and cationic lipid-like G0-C14 for siRNA complexation and endosomal escape. Formulated by nanoprecipitation mixing siCFL1, G0-C14, sorafenib, and MeO-PEG-S-S-PLGA in DMF/DMSO, followed by dropwise addition to water and ultrafiltration—the NPs achieve ~100 nm size with sorafenib encapsulation efficiency of ~40% and siCFL1 at ~80% (Table 3). They address systemic barriers through PEG-enabled long circulation and 7x higher tumor accumulation versus naked siRNA, while G0-C14 facilitates endosomal escape. The system responds to tumor microenvironment GSH (2–10 mM), cleaving disulfide linkers for rapid cargo release and GSH scavenging to amplify sorafenib-induced ROS. This targeted CFL1 silencing disrupts serine synthesis/metabolism, enhancing sorafenib sensitivity via metabolic reprogramming. Advanced features include GSH/reduction-responsiveness and dual-drug synergy.⁶⁶ An investigation was conducted by designing a pH-controllable chitosan nanocomplex to co-deliver CRISPR/Cas9 elements against VEGFR2 and paclitaxel, which developed a synergistic gene-chemo therapeutic model of hepatocellular carcinoma. The system has interrupted angiogenesis in tumors by knocking VEGFR2 with Cas9 and increasing chemosensitivity with paclitaxel, which led to extensive tumor growth inhibition. The nanocomplex suppressed IL-6/IL-8/NF-κB pathway and minimized inflammation, as well as enhanced the biosafety, and chitosan assists in the release of the complex and target delivery. This dual approach highlights the promise of combining CRISPR-based gene editing and chemotherapy to use in precision oncology applications.⁶⁷ Schneider et al in 2024 they built multicompartment polyion complex micelles (PICMs) that deliver siRNA to microfibrillar-associated protein 5(MFAP-5) in hepatocellular carcinoma (HCC)-associated cancer-associated fibroblasts (CAFs). The MFAP-5 knockdown effect mediated by the PICMs through co-encapsulation of anti-MFAP-5 siRNA with the cationic amphiphilic drug desloratadine (DES) resulted in reduced CAF-mediated tumor angiogenesis. SiMFAP-5/DES PICMs showed strong synergy with RNAi-drug in reducing hepatic tumor burden by 80% in syngeneic HCC models and provide strong evidence that MFAP-5 is a feasible stromal target of antistromal therapy.⁵³ A carotenoid functionalized nanoparticle to deliver siRNA against FOXC1, which is a metastasis-associated transcription factor in HCC. The nanovector attained an efficient knockdown of FOXC1 in vitro of the order of 90%, higher cellular uptake, and low cytotoxicity than traditional transfection agents. This was a successful inhibitory method of HCC progression by inhibiting FOXC1-driven pathways, which provides a biosafe method to silence genes in liver cancer.⁶⁸ Song et al in 2022 developed a pH-responsive nanoparticle system (CMCS@Tim-3 siRNA and Sorafenib@mPEG5K-PAE) for co-delivery to treat hepatocellular carcinoma. The CMCS coating enhanced stability and enabled tumor-specific drug release under acidic conditions. Tim-3 siRNA promoted immune activation and sorafenib inhibited angiogenesis, resulting in synergistic therapy. The ~200 nm nanoparticles demonstrated increased cytotoxicity with decreased IC50 and significant inhibition of tumor growth. The antitumor activity of the injected CC@SR&SF@PP nanoparticles was evaluated in vivo by testing the nanoparticles on an orthotopic H22 liver tumor mouse model. Mice were injected 5 times (after 3 days) in the tail-vein with the following doses Tim-3 siRNA (2.5 mg/kg) and sorafenib (10mg/kg). Single or partial treatments were less effective in tumor suppression compared to control groups, which contained non-nanoparticle formulations of PBS and other nanoparticles (Figure 5). There was moderate inhibition of tumor growth in the case of free combination of sorafenib and siRNA. The opposite was true with the CC@SR&SF@PP nanoparticles which inhibited tumor progression to the effect of almost causing stagnant tumor growth. The better therapeutic effect of this combined nanoparticle system was further proven by Bioluminescense imaging. The end liver analysis showed the minimal tumor burden in mice accorded to CC and SF respectively with the usage of the PP nanoparticles, which indicates their high synergistic antitumor activity (Figure 5).⁶⁹

Figure 5.

(a) Real time imaging of tumors in the H22 orthotopic tumor-bearing mouse in vivo. On day 2, 5, 8, 11, and 14 (a day after drug injection), bioluminescense images were obtained by intraperitoneal injection of 150 0L of 15 mg/mL D- Luciferin potassium salt solution on day 2, 5, 8, 11 and 14 respectively. (b) Curves of tumor volume increase of mice following different treatments (n = 5). The intensity of bioluminescence on the Xenogen IVIS SPECTRUM small animal imaging system measured tumor sizes. (c) Photos of representative livers of the mice at the conclusion of day 16 following the various treatments. FIGURE 5 was reproduced from the Ref.69

In another research they synthesized a novel thermosensitive hydrogel system with DP7-C siRNA nanoparticles to deliver Pin1 as the target gene in the sustained and targeted delivery of silencing proceeding of Pin1, one of the primary regulators of hepatocellular carcinoma (HCC). The DP7 cholesterol-modified peptide was shown to stabilize siRNA and deliver it to the liver through N-acetylgalactosamine (GalNAc) targeting as well as the hydrogel of PDLLA-PEG-PDLLA supported the release of siRNA over 21 days with a high transfection volume (Table 1). This platform was successful in inhibiting tumor, and enhancing survival in an orthotopic HCC model through silencing of Pin1 expression, indicating a great therapeutic efficacy with reduced systemic toxicity. A core size of about 36 nm and a zeta potential of +37 mV was observed and the nanoparticles had an IC50 value of about −0.45 −1g/mL, which was significantly lower than the untreated controls, indicating a good silencing effect and cytotoxicity.⁷⁸

Emerging Combination Strategies

New combination approaches utilize polymeric nanomedicines to combine to silencing of oncogenic drivers, such as circMDK, survivin, and AFP, with chemotherapeutics, ferroptosis inducers, photothermal ablation radiation therapy, and immunotherapies in HCC.⁸¹ With multifunctional properties, such as PAE nanoparticles, SP94-targeted nanospheres, and GDYO nanosheets, they improve tumor targeting, drug resistance, and by reorganizing the immunosuppressive tumor microenvironment, they enhance the antitumor efficacy. Through co-delivery of siRNAs targeting the major pathways such as, PI3K/AKT/mTOR, PD-L1/NR2F6 and administration of agents such as sorafenib, DOX, or sonodynamic ROS generators) they induce tumor ablation in orthotopic, PDX, and metastatic models of HCC with a low toxicity rate. This emerging paradigm is expected to deliver radical, precision therapies by enhancing gene knock down when using multimodal interventions.⁸² Du et al, in 2022 examined the oncogenic property of circMDK in hepatocellular carcinoma (HCC) and found that it is a newly discovered type of circular RNA in the process of upregulation through m6A modifications. Mechanically, circMDK enhanced tumorigenesis by miR-346 / 874–3p -ATG16L1 axis, which stimulated PI3K / AKT / mTOR signaling to increase cell proliferation, migration, and invasion. To overcome this, poly (3-aminopropylamido acrylate) (PAEs) in nanoparticles were used to deliver circMDK siRNA to counter this with high stability and efficiency in vivo. This nanotherapy method was very effective in suppressing tumor growth and metastasis in several models of HCC such as subcutaneous, metastatic, orthotopic and patient-derived xenograft (PDX). The results identified circMDK as a biomarker and therapeutic target in HCC, which presents a good approach to treatment.⁷³ In 2022, Zhang et al, developed cationized amylose (CA)/superparamagnetic iron oxide (SPIO)/tetraphenylethene (TPE) nanospheres (CSPTPEsiRNA-SP94) for targeted survivin siRNA delivery to hepatocellular carcinoma (HCC). The SP94 peptide, specific for HCC via GRP78 receptor binding, was conjugated through EDC/NHS amidation at pH 5. Nanospheres self-assembled via SPIO coprecipitation, CA coating, TPE entrapment (sonication/dialysis), and electrostatic siRNA complexation at N/P ratio 20, yielding 178 ± 0.5 nm size (PDI 0.25) and −5.8 ± 0.6 mV zeta potential. They enabled high siRNA condensation (>NP 8) with 79.8% cumulative release at 24 h (pH 7.4), overcoming RNase degradation, renal clearance, cell membrane penetration, and endosomal barriers via cationic surfaces and endocytosis (Table 1). SP94 targeting, combined with EPR effect (optimal 100–200 nm), boosted tumor accumulation (3.3× fluorescence at 24 h vs non-targeted). Dual-modal imaging leveraged AIE-fluorescence from TPE and T2-MRI from SPIO (r₂ = 31.9 mM⁻¹s⁻¹), with superparamagnetic properties and high biocompatibility.⁷² A research in 2024, developed a SP94-targeted graphdiyne oxide (GDYO) nanosheets ultrathin 2D carbon-based lamellae (~3 nm thick per AFM)—for synergistic HCC treatment via ferroptosis induction. The multifunctional nanosheets were conjugated with SP94-PEG (EDC/NHS amide bonds) for HCC-specific targeting, and co-loaded with DOX-Fe2+ (chelation), sorafenib (π-π stacking) and siRNA SLC7A11-i (electrostatic adsorption). They have nanosheet morphology (TEM) and size <100 nm (hydrodynamic, increasing with payloads) and are prepared by GDY oxidation (HNO3/H2SO4), stepwise functionalization and drug/gene loading. The platform overcomes the nonspecific distribution, short half-life, drug resistance and poor solubility of sorafenib through SP94-mediated endocytosis, EPR accumulation and lysosomal release. In HCC tumor microenvironment, SP94 targets receptors for active targeting, while payloads interfere SLC7A11 cystine transport, depletes GSH, and increases mitochondrial ROS/lipid peroxidation, with maximum tumor accumulation at 24h (Cy5.5 imaging). siSLC7A11 inhibits GPX4, amplifying iron-dependent ferroptosis synergistically with DOX/sorafenib chemo-effects. Advanced features include biocompatibility, 5–21-day stability, minimal off-target toxicity, and multi-modal action (targeting + gene interference + chemo-ferroptosis).⁷⁰ To evaluate PAE-siRNA’s therapeutic potential in vivo HCC, they used an orthotopic hepatic tumor model mimicking the tumor microenvironment (Figure 6A). Antitumor efficacy was assessed via systemic administration (40 µg siRNA/mouse from 150 µL of 20 µM stock; (Figure 6B), with no body weight changes indicating no systemic toxicity (Figure 6C). PET/CT scans with 18F-FDG showed lower liver accumulation in PAE-siRNA-treated mice (Figure 6D and E). PAE-siRNA significantly suppressed liver tumor growth vs saline (Figure 6F). H&E staining revealed more necrotic areas in PAE-siRNA livers (Figure 6G), and ATG16L1 IF (green) was markedly reduced, confirming target knockdown.⁷³ A Cu-doped layered double hydroxide (LDH)-based nanoplatform was developed for hepatocellular carcinoma therapy by integrating photothermal therapy (PTT) with siRNA-mediated silencing of PD-L1 and NR2F6. By remodeling the tumor microenvironment (TME), the PTT successfully turned immunologically cold tumors into immunologically hot ones, whereas the dual targeting of NR2F6 and PD-L1 generated strong antitumor immunity. Not only did this multifunctional nanoplatform improve immune responses, but it was also potentially imaging and combination therapy. The paper has demonstrated the clinical benefits of combining PTT with immune checkpoint inhibition to mediate antitumor immunity as a new treatment option of HCC.⁸³ In the year 2023 a study entitled “Epigallocatechin Gallate Potentiates the Effect of AFP-siRNA Nanoparticles” investigated the application of AFP-siRNA nanoparticles encapsulated with epigallocatechin gallate (EGCG) to the treatment of hepatocellular carcinoma. Co-delivery system demonstrated high reduction in cell viability of HepG2 in comparison with monotherapy, a 2.5-fold rise in caspase-3/7 activity which demonstrates greater induction of apoptosis. His procedure demonstrated great degree of biocompatibility and stability as well as offered a prospective synergistic method to improve siRNA-based therapy of liver cancer.⁷⁴ PLGA-PEG nanoparticles were utilized to deliver circROBO1 siRNA for hepatocellular carcinoma therapy by targeting a circular RNA associated with HCC progression. Silencing of circROBO1 disrupted the miR-130a-5p/CCNT2 regulatory pathway, resulting in reduced cell proliferation, colony formation, and tumor growth in vivo. The nanoparticles have shown to be an efficient system of delivery of siRNA and this is highly stable in the circulation of the body and even has limited toxicity to the main body organs, which can be seen as an appreciable advantage in terms of treating HCC using the nanoparticles as therapeutic body agents.⁷⁵ Polymeric nanoparticles incorporating angiogenesis inhibitors were developed for the delivery of AFP-siRNA as a therapeutic strategy against alpha-fetoprotein (AFP)-associated hepatocellular carcinoma. The formulated nanoparticles efficiently encapsulated AFP-siRNA and successfully silenced AFP mRNA expression in HepG2 cells. The system was found to be more therapeutically effective when used in combination with angiogenesis inhibitors, including sorafenib and sunitinib, with which the system showed synergistic effects in reducing cell viability and AFP expression. The hydrodynamic size of the nanoparticles was approximately 242 nm (mean) with a neutral zeta potential, which facilitated cellular uptake and systemic stability. siRNA encapsulation could release siRNA longer than 14 days with therapeutic benefits, and degradation reduced. This co-delivery method was found to have low levels of toxicity in normal fibroblast cells and this demonstrates its biocompatibility and safety. The article highlights the role of polymeric nanoparticles in combination therapy to improve silencing of genes and chemotherapy to treat HCC.⁸¹ Kim et al, in 2024 constructed a platform using polypeptide nanoparticle to deliver siRNAs against TGFβ and Cox2 to overcome immune exclusion in hepatocellular carcinoma (HCC). The novel approach increased the penetration of both the CD4+ and the T-cells to the tumor core, which essentially transforms the immune exclusive cold tumors into immune inflamed hot tumors. SiRNA therapy conducted in combination with PD-L1 immune checkpoint inhibitors had a substantial beneficial effect on the therapeutic outcomes, such as tumor regression and long-term survival, in preclinical models (Table 1). Another aspect that is favorable about the platform was its favorable safety profile with less systemic toxicity but high efficacy. These results suggest the possibility of this method to redefine the tumor microenvironment and increase the efficacy of immunotherapies against more complex cases of HCC.⁸⁴ Lipid-derived NET-1 siRNA nanoparticle was proposed as a gene therapy approach for hepatocellular carcinoma by combining siRNA delivery with sonodynamic therapy (SDT) to enhance therapeutic efficacy. Nanoparticles were prepared by a thin-film hydration and extrusion procedure and their mean particle size was approximately equal to 675 nm and their zeta potential was approximately equal to −39 mV. The NET-1 siRNA effectively suppressed the NET-1 gene, which is a key element in HCC growth and invasion, and SDT magnified the therapeutic impact of initiating the production of the reactive oxygen species (ROS) when activated by ultrasound and apoptotic cancer cells were selectively induced. Such combination therapy was a potent inhibitor of tumor growth and extended survival in vivo with better efficacy than alternative treatments. IC50 of NET-1 siRNA nanoparticles using SDT was 0.28 µg/mL, which revealed an increased tumor suppressing and therapeutic effect.⁷⁹

Figure 6.

Illustrates an antitumor effect of PAE-siRNA complex in orthotopic model. (A) Scheme of the establishment of orthotopic tumor model. HepG2 cells were injected into the lower lobe of liver of Balb/c nude mice to form orthotopic tumors. (B) Schematic illustration of orthotopic model establishment and treatment. (C) Changes of mouse body weight during experimental period. Data are shown as means ± SD (n = 6). (D) Schematic cartoon of PET/ CT detection in mice. (E) Representative images of PET/CT scans from each group. Images of mice were acquired 24 h after intravenous injection of 18F-FDG. Shown from left to right are the axial, coronal, and lateral views. The white circles and red arrows indicated the regions of hepatic tumors. Standardized uptake values (SUV) were examined by 18F-FDG. (F) Representative images of the HepG2 orthotopic HCC tumors. The white dotted lines indicate tumor regions (n = 6). (G) Representative images (left) and quantification (right) of liver tumor sections stained with H&E (top) and immunofluorescence (bottom) of ATG16L1. *p < 0.05; **p < 0.01. Scale bars are 50 µm. FIGURE 6 depicted from the Ref.73

AI in siRNA Loaded Polymeric Nanomedicine for HCC Targeting

Artificial intelligence (AI) has been a strong tool for promoting siRNA-loaded polymeric nanomedicine in hepatocellular carcinoma (HCC) therapy. AI computational strategies can rationally design, optimize and predict the performance of nanoparticles for improved delivery efficiency and therapeutic precision. AI is combined with machine learning, deep learning and multi-omics analysis for targeted gene silencing and personalized treatment strategies. These advances accelerate the clinical translation of polymeric siRNA nanocarriers for effective HCC management. The schematic represents a combined artificial intelligence (AI) assisted platform of designing, optimizing, and therapeutically utilizing PNPs to deliver siRNA in the treatment of HCC. The central part of the platform is the organization of various steps of siRNA nanomedicine formation through AI algorithms, which allows an accurate targeting of delivery and maximum therapeutic action. First, polymer-siRNA affinity, compatibility, and stability of the complex are predicted by AI-based molecular interaction analyses,⁸⁵ such as Schrodinger Suite, AutoDock Vina, and machine-learning-assisted scoring functions.⁸⁶ These findings influence the sensible choice of biodegradable polymers and formulation parameters. Simultaneously, transformer neural networks, graph neural networks and support vector machines (SVMs) are used to design and optimize siRNA sequences to be more efficient in silencing genes and reduce immunogenicity and degradation.⁸⁷ AI-driven predictive modeling models, like LightGBM, Bayesian optimization, and deep neural networks, are then used to regulate particle size, surface charge, encapsulation efficiency, and release kinetics by constructing polymeric siRNA nanoparticles.⁸⁸ Computational analysis of off target effects is done through alignment tools (Bowtie/BWA) with AI filters and deep learning models such as CRISPRnet, DeepCRISPR, making sure that the modifications are high-specificity targets and less unintended gene modulation (Figure 7).^89,90 To monitor diseases and stratify treatment approaches, AI-based imaging models such as U-Net, 3D U-Net, nnU-Net, DeepLabV3+ and MONAI provide reasonable liver tumor detection and segmentation.⁹¹ Multi-omics data integration with deep learning, SPADE, and t-SNE clustering are used at the cellular level to estimate the apoptosis and measure the response at the pathway level.⁹² Moreover, mechanistic physiologically inspired pharmacokinetic (PBPK) modeling coupled with machine learning can be used in conjunction with virtual screening, dose optimization, and predicts in vivo nanoparticle biodistribution and clearance.⁸² The conventional approach for the development of siRNA-loaded polymeric nanomedicines depends on experimental screening and optimization of polymer type, polymer/siRNA ratio, particle size, surface charge, encapsulation efficiency and release characteristics. While these methods offer direct experimental evidence, they are time-consuming and resource-intensive.⁹³ Conversely, AI and machine learning approaches can evaluate multidimensional formulation and biological datasets to understand the relationships between polymer characteristics, nanoparticle properties, cellular uptake, siRNA release, and therapeutic responses. In polymer selection traditional approaches require the preparation and testing of several candidate polymers while artificial intelligence can rank promising materials based on available physicochemical and biological data. Similarly, formulation optimization is generally an iterative experimental process, but artificial intelligence can predict the effect of several formulation parameters, and recommend possibly optimal combinations. AI-assisted approaches can also help in prioritizing targeting ligands and polymer-ligand combinations for HCC-associated receptors such as ASGPR. This AI-based polymeric siRNA nanomedicine platform enables pathway-specific targeting of critical oncogenic drivers in HCC, increases treatment efficacy, increases therapeutic precision, complements computational intelligence with advanced polymeric drug delivery systems, and facilitates clinical translation.⁹⁴

Figure 7.

Represents the AI Driven polymeric siRNA nanomedicines for hepatocellular carcinoma therapy.

Notes: Created in BioRender. Habeeb, M. (2026) https://BioRender.com/g6i0j4q.

Future Challenges and Perspectives

There are still various obstacles to be overcome for clinical translation despite considerable advances in polymeric nanomedicines for siRNA delivery in hepatocellular carcinoma (HCC). Off-target gene silencing is a major concern because partial sequence complementarity can lead to unintentional modulation of other genes, and nanoparticle-mediated delivery may also increase exposure of siRNA in non-target tissues.⁹⁵ Therefore, there is a need for better siRNA sequence selection, transcriptome-wide assessment and computational prediction. Another important consideration is immunogenicity. siRNA molecules, polymers and surface modifications can induce innate immune responses, influencing therapeutic efficacy and safety.⁹⁶ A systematic evaluation of the cytokine responses, complement activation, immune-cell interactions, and effects after repeated administration is needed.⁹⁷ Likewise, long-term safety and biodegradation are not well characterized, especially in the case of nanoparticle retention or accumulation in organs. Therefore, long-term pharmacokinetic, biodistribution, degradation, histopathological and toxicity studies are needed. Another challenge is the heterogeneity of tumor targeting, because HCC tumors present different receptor expression, vascularization, genetic features, and tumor microenvironment. This variability in delivery between patients and tumor regions can therefore be attributed to targeting ligands. Multi-receptor targeting, tumor specific biomarkers, adaptive targeting and patient stratification may improve targeting precision. Differences between conventional pre-clinical models and human HCC also limit clinical validation. More clinically relevant models could improve prediction of therapeutic outcome. Moreover, scalable and reproducible manufacturing, batch to batch consistency, stability, sterility, and regulatory characterization should be defined. Recent regulatory views highlighted that the complexity of polymeric nanotherapeutics, the variability of their physicochemical properties, the limited predictability of their in vivo performance and the absence of harmonized nanospecific standards are still important obstacles for clinical translation.⁹⁸ In the future, the integration of artificial intelligence, multi-omics and pharmacokinetic modeling may support formulation optimization, biodistribution prediction and patient specific treatment. To develop polymeric siRNA nanomedicines for HCC that are safe, reproducible and clinically effective, addressing these obstacles will be essential.

Conclusion

Polymeric nanomedicines have emerged as promising platforms for siRNA delivery in hepatocellular carcinoma (HCC) by protecting siRNA from degradation, enhancing cellular uptake, promoting endosomal escape, and enabling targeted and controlled delivery. This flexibility allows the incorporation of different polymers, targeting ligands, stimuli-responsive mechanisms, and therapeutic agents, which can improve gene silencing and address the multifactorial nature of HCC. However, clinical translation remains challenging due to differences in nanoparticle size, surface properties, loading efficiency, degradation and manufacturing processes that can alter biodistribution, pharmacokinetics and therapeutic outcomes. Reproducible and scalable manufacturing, as well as rigorous quality control, is therefore required. Moreover, long-term stability, biodegradability, immunogenicity, off-target effects and possible accumulation in non-target organs need to be carefully considered. In addition, the predictive value of experimental findings may be limited by differences between preclinical models and patients, including tumor heterogeneity, tumor microenvironment, liver function, and immune status. Future work should focus on standardized assessment of pharmacokinetics, biodistribution, toxicity, efficacy and long-term safety in clinically relevant models. Machine learning and artificial intelligence approaches can also assist formulation optimization, prediction of polymer–siRNA interactions and biodistribution, and patient stratification. Success in translation will require coordinated advances in nanocarrier design, biological validation, manufacturing, regulatory science, and personalized treatment strategies.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

We declare no competing interests.