Nanocarrier-Enabled siRNA Therapy for Pulmonary Fibrosis: Pharmacological Rationale, Delivery Barriers, and Translational Opportunities

Abstract

Pulmonary fibrosis, particularly idiopathic pulmonary fibrosis (IPF), is a progressive interstitial lung disease caused by failed epithelial repair, persistent fibroblast activation, immune dysregulation, and extracellular matrix (ECM) remodeling. Nintedanib and pirfenidone slow functional decline but do not reverse established fibrosis. Small interfering RNA (siRNA) offers sequence-specific silencing of disease-driving genes, but degradation, mucus and ECM barriers, inefficient cellular uptake, and endosomal sequestration restrict its activity in fibrotic lungs. This review examines these barriers alongside the rationale for selecting siRNA targets and nanocarrier platforms. Lipid, polymeric, biomimetic, and inorganic systems are compared in terms of loading, tissue penetration, intracellular release, safety, manufacturing, and clinical feasibility. We also consider single-target and multitarget strategies, pulmonary and systemic administration, disease-model limitations, and early clinical experience. The resulting target-to-delivery framework defines the evidence required before pulmonary siRNA nanomedicines can enter informative clinical testing.

Introduction

Pulmonary fibrosis is a fatal interstitial lung disease characterized by aberrant repair, persistent fibroblast activation, and excessive extracellular matrix deposition. Idiopathic pulmonary fibrosis (IPF) is its prototypical form.^1,2 Clinically, patients present with progressive dyspnea, reduced lung compliance, and a continuous decline in pulmonary function.^3,4 Despite substantial progress in understanding genetic susceptibility, epithelial injury and repair failure, immune dysregulation, and mechanical microenvironment remodeling, treatment options for IPF remain limited.¹ The two approved drugs, nintedanib and pirfenidone, mainly slow the decline in lung function but have only limited capacity to reverse established fibrosis.^2,5 Thus, current therapy primarily delays progression rather than eliminating pre-existing fibrotic lesions.

Small interfering RNA (siRNA) offers a distinct therapeutic strategy. Unlike conventional small molecules or antibodies, siRNA acts directly on mRNA and provides high sequence specificity together with a broad range of targetable molecules.^6,7 IPF is not driven by a single pathway. Rather, it is maintained by a complex pathogenic network involving injured alveolar epithelium, profibrotic immune cells, persistently activated myofibroblasts, and a stiffened matrix. In this setting, single-target drugs often suppress only part of the process and rarely disrupt the positive feedback loops that sustain disease progression.⁸ By contrast, siRNA enables customized post-transcriptional silencing of key drivers and is therefore well suited to network-level intervention.

Clinical translation of siRNA is limited by poor in vivo stability, low membrane permeability, immunogenicity, and off-target effects.^9–11 Effective therapy therefore requires stable and selective delivery to diseased lung cells.¹² Nanocarriers can protect siRNA, alter pharmacokinetics, and improve local accumulation through control of their composition and surface properties.^13–16 Rather than cataloguing carriers or molecular targets separately, this review evaluates target selection, lesion biology, delivery barriers, platform trade-offs, and clinical development as a connected problem.

Pathogenic Circuits with Pharmacological Intervention Value in Pulmonary Fibrosis

Fibrotic lesions become self-sustaining through a linked sequence. Epithelial injury provokes abnormal repair; immune signals maintain fibroblast activation; and deposited matrix reinforces both states through mechanical feedback. The subsections separate these processes for clarity, but target selection must account for the network they form.

Injured Alveolar Epithelium as a Central Hub of Disease Initiation and Persistence

Repeated microinjury to type II alveolar epithelial cells (ATII cells) is widely regarded as a key initiating event in IPF.^17–19 Under the combined influence of genetic susceptibility and environmental stress, ATII cells undergo apoptosis, senescence, and impaired regeneration.^20,21 As a result, normal repair is replaced by persistently activated maladaptive repair. Injured epithelium also releases mediators such as TGF-β and IL-33, which drive surrounding immune and stromal cells toward a profibrotic state.²²

The epithelium is therefore not only the site of initial injury but also a signaling hub that sustains disease progression.^17,23 For siRNA-based intervention, epithelial-associated molecules are attractive targets because they may allow abnormal signal amplification to be interrupted at an early stage of disease.

Profibrotic Immune Remodeling Sustains the Lesion Microenvironment

Immune microenvironment remodeling is a major driver of persistent fibrosis. Among these changes, M2 macrophage polarization is particularly important.²⁴ Driven by signals such as IL-4 and IL-13, macrophages release profibrotic mediators including TGF-β1, CCL17, and CCL22, thereby directly promoting fibroblast activation and maintaining a Th2-skewed immune state.²⁵ In addition to M2 polarization, Th1/Th2 imbalance, dysregulation of the Treg/Th17 axis, and amplification of IL-33–ILC2 signaling also contribute to lesion remodeling.^26–30 Together, these immune circuits continuously provide chemotactic, differentiation, and matrix-promoting signals and therefore represent important entry points for siRNA-mediated cell-state reprogramming.

Matrix Stiffening and Mechanotransduction Sustain Persistent Fibroblast Activation

As disease progresses, pulmonary fibrosis evolves from maladaptive repair after injury into a state of mechanical disequilibrium. Activated myofibroblasts continuously secrete collagen, fibronectin, and elastin, which increases interstitial stiffness and distorts alveolar architecture.³¹ The stiffened matrix further enhances fibroblast activation through integrin-dependent focal adhesion kinase (FAK) signaling and nuclear translocation of YAP/TAZ, thereby creating a positive feedback loop.^32–36 Interventions targeting mechanotransduction and ECM maintenance may therefore reduce collagen production and disrupt the structural feedback that sustains disease progression.^37–40

Cellular Senescence, Defective Autophagy, and Ferroptosis as Emerging Therapeutic Nodes

The refractory nature of pulmonary fibrosis is also linked to abnormal control of cell fate. ATII cell senescence is a hallmark of IPF. In addition to proliferative arrest, senescent cells acquire a senescence-associated secretory phenotype (SASP) and continuously release mediators such as IL-6, IL-8, TGF-β, and CTGF, thereby amplifying local injury signals.⁴¹ Defective autophagy also contributes to disease remodeling. Insufficient autophagy impairs the ability of epithelial cells to clear damaged organelles and abnormal proteins and makes fibroblasts more resistant to deactivation and apoptosis.^42–44

More recently, ferroptosis has been incorporated into the pathogenic framework of pulmonary fibrosis. Ferroptosis in ATII cells can aggravate epithelial injury, whereas altered ferroptosis sensitivity in myofibroblasts may affect the clearance of diseased cells.^45,46 These emerging nodes are relevant to siRNA therapy because they lie between classical signaling pathways and terminal pathological outputs and may therefore provide more selective intervention opportunities than direct inhibition of core cytokines.

The effect of manipulating autophagy or ferroptosis depends on cell type and disease stage. Restoring epithelial homeostasis may reduce repeated injury, whereas changing stress responses in activated fibroblasts may promote their clearance. A candidate target therefore needs evidence identifying the relevant cell population and direction of modulation; the same pathway cannot be assumed to require the same intervention throughout the lung.

Key Signaling Pathways

TGF-β/Smad

After alveolar epithelial injury, activated macrophages and epithelial cells release large amounts of TGF-β1.⁴⁷ TGF-β1 binds to TβRII at the cell membrane, recruits and phosphorylates the type I receptor ALK5, and the activated receptor complex then phosphorylates Smad2 and Smad3 in the cytoplasm. Phosphorylated Smad2/3 forms a complex with Smad4, which regulates the transcription of collagen, CTGF, α-SMA, and other fibrotic genes.^48,49 This pathway links injury signals to terminal effector programs and serves as a central hub driving fibroblast activation and ECM deposition.^50,51

PI3K/Akt

Aberrant activation of PI3K/Akt promotes fibroblast proliferation and resistance to apoptosis in fibrosis and also mediates non-Smad signaling downstream of TGF-β1.^52–55 In addition, PI3K/Akt regulates autophagy through the mTOR pathway, and its overactivation is closely associated with impaired autophagy.⁵⁶ Because this pathway also influences immune-cell function, it shapes not only stromal responses but also the profibrotic microenvironment.^57–59

Stat6

Binding of IL-4 and IL-13 to their receptors activates JAK kinases, which then phosphorylate STAT6.⁶⁰ Dimerized STAT6 translocates to the nucleus and induces the expression of M2 markers and profibrotic mediators.⁶¹ In fibroblasts, STAT6 directly mediates IL-13-induced collagen synthesis.⁶² This pathway therefore links immune polarization to matrix deposition and is an important molecular basis of profibrotic immune remodeling.⁶³

Yap/Taz

In an ECM-stiffened environment, dephosphorylated YAP/TAZ translocates to the nucleus and cooperates with TEAD transcription factors to activate pro-proliferative, anti-apoptotic, and profibrotic genes.^35,64,65 YAP/TAZ can also form nuclear complexes with Smad proteins and thereby amplify fibrotic gene transcription.⁶⁶ This pathway illustrates that matrix stiffening is not only a consequence of disease but also an active driver of disease progression^67–69 [Figure 1].

Figure 1.

Fibrotic network remodeling and the tipping point in pulmonary fibrosis Repeated epithelial injury initiates abnormal repair and recruits profibrotic immune responses. Activated fibroblasts deposit ECM, and the stiffened matrix reinforces fibroblast persistence through mechanotransduction. Senescence, defective autophagy, and ferroptosis modify this network and create additional intervention points. The arrows indicate reinforcing biological relationships rather than a fixed temporal sequence.

Abbreviations: ATII, alveolar type II epithelial cell; TGF-β, transforming growth factor-β; SMAD, mothers against decapentaplegic/small; IL, interleukin; CCL, C-C motif chemokine ligand; ILC2, type 2 innate lymphoid cells; Th17, T helper 17; Treg, regulatory T cells; ECM, extracellular matrix; FAK, focal adhesion kinase; YAP, yes-associated protein; TAZ, transcriptional co-activator with PDZ-binding motif; TEAD, transcriptional enhanced associate domain; CTGF, connective tissue growth factor; SASP, senescence-associated secretory phenotype; PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; mTOR, mechanistic target of rapamycin; JAK, janus kinase; STAT, signal transducer and activator of transcription; Created with BioGDP.com.⁷⁰.

Rationale and Intrinsic Limitations of siRNA Therapy in Pulmonary Fibrosis

Distinct Advantages of siRNA Over Conventional Anti-Fibrotic Drugs

siRNA is a double-stranded RNA molecule of approximately 20–25 base pairs. After entering cells, it is loaded into the RNA-induced silencing complex (RISC), where it recognizes complementary target mRNA and promotes its cleavage, thereby achieving post-transcriptional gene silencing.⁷¹ Unlike small molecules that depend on protein binding sites, siRNA acts on mRNA and can therefore target a much broader range of molecules, including some that are traditionally considered difficult to drug.^72,73 This feature is particularly relevant to pulmonary fibrosis. Current antifibrotic drugs mainly act through relatively broad mechanisms and are clinically limited to slowing progression rather than reversing disease.⁷⁴ By contrast, siRNA can be designed against disease-driving genes and offers a more selective way to suppress aberrantly amplified pathological programs.

Pulmonary Fibrosis Is Well Suited to Network-Level Gene Silencing

Pulmonary fibrosis is sustained by multiple signaling pathways, multiple cell types, and several feedback loops.⁷⁵ Pathways such as TGF-β/Smad, PI3K/Akt, STAT6, and YAP/TAZ act in an interconnected manner across fibroblast activation, macrophage polarization, ECM production, and maladaptive repair.^76,77 In such a highly coupled system, single-target inhibition is particularly vulnerable to pathway compensation and patient heterogeneity.⁷⁸ siRNA is advantageous in this context because it can silence central pathway nodes, feedback amplifiers, regulators of collagen maturation, or cell-fate switches.⁷⁹ In addition, nanocarrier-based co-delivery allows multiple siRNAs to be integrated into a single platform, enabling simultaneous intervention across different cellular and pathological layers.^80,81 For a heterogeneous disease such as IPF, this combinatorial and stratified therapeutic logic is especially attractive.⁸²

Delivery Remains the Major Bottleneck for Clinical Translation

Delivery, rather than target plausibility, remains the main limitation of siRNA. Naked siRNA is readily degraded by nucleases in body fluids and tissues, while its negatively charged backbone and hydrophilic nature restrict membrane transport.^83,84 Material taken up by endocytosis may remain trapped in endosomes or lysosomes instead of reaching the cytosol.^83,85 siRNA can also activate innate immunity, silence unintended transcripts, or distribute to nontarget tissues.^86,87 These failures occur in sequence. Better lung deposition does not ensure cellular uptake or endosomal escape, and efficient uptake does not prevent sequence-dependent off-target effects. Carrier screening should measure deposition, cell-specific internalization, cytosolic release, and gene silencing separately.

Nanomaterials for siRNA Delivery

Biological Barriers in Fibrotic Lungs

Mucus Barrier

The mucus layer covering the airway surface consists of a highly cross-linked mucin network with pore radii typically in the range of 100–200 nm.⁸⁸ In fibrotic lungs, mucin secretion is upregulated and the physicochemical properties of mucus are altered, leading to marked increases in viscosity and elasticity.^89,90 This barrier can rapidly trap and clear conventional drug carriers, impede penetration into diseased tissue, and thus become a major obstacle to pulmonary delivery.⁹¹

Dense and Stiffened Extracellular Matrix

Collagen, fibronectin, and elastin are abnormally cross-linked and deposited within the alveolar interstitium, forming a dense and stiffened ECM.⁹² This remodeled matrix acts not only as a physical barrier that restricts nanoparticle diffusion into deeper interstitial regions but also as a signaling substrate that promotes fibroblast activation and proliferation, thereby sustaining the fibrotic state.⁹³

Enhanced Immune Clearance

In pulmonary fibrosis, large numbers of activated M2 macrophages accumulate in the alveolar and interstitial compartments, together with increased infiltration of neutrophils and dendritic cells. These immune cells express high levels of scavenger receptors and Fc receptors and display strong nonspecific phagocytic activity toward nanoparticles entering the lung.^94,95

Lysosomal Sequestration

Lysosomal sequestration refers to the subcellular barrier process whereby weakly basic lipophilic drugs or carriers that have been internalized into the acidic lysosomal lumen become protonated and are therefore retained in membrane-impermeable charged forms.⁹⁶ In pulmonary fibrosis, profibrotic signaling enhances lysosome biogenesis, luminal acidification, and lysosomal enlargement, thereby intensifying this sequestration effect.^97,98 As a result, drug carriers are confined within lysosomal compartments and subjected to enzymatic degradation, which severely limits intracellular effective drug exposure.

Design Principles for Nanocarriers

Small Size for Matrix Penetration

The highly cross-linked collagen network in the fibrotic lung interstitium is a major barrier to nanoparticle diffusion.^99,100 When particle size exceeds the average pore size of the ECM, particles are easily trapped. Designing smaller siRNA nanocomplexes is therefore a basic requirement for penetrating stiffened ECM. However, particles that are too small may undergo rapid renal clearance or display increased nonspecific distribution.^101,102 An optimal size window is therefore required.

PEGylation for Mucus Penetration

The dense mucin network in airway mucus strongly adheres to nanoparticles with hydrophobic or positively charged surfaces, causing rapid immobilization and subsequent mucociliary clearance.^103,104 Surface conjugation with polyethylene glycol (PEG) is a classic strategy to reduce interactions with mucins.¹⁰⁵ PEG forms a hydrated shell that shields surface charge and hydrophobic domains, allowing particles to diffuse through mucus in a manner closer to ideal Brownian motion.^106,107 However, although PEGylation improves mucus penetration, it may also reduce subsequent cellular uptake and endosomal escape.¹⁰⁸

Targeting Ligands to Enhance Uptake

After traversing mucus and ECM, nanoparticles must be efficiently internalized by target cells within the lesion.¹⁰⁰ Because simple PEGylation or charge-neutral surfaces often reduce cell-binding capacity, surface modification with targeting ligands has become an important strategy to improve uptake.¹⁰⁹ Covalent conjugation of ligands to the nanoparticle surface can mediate receptor-dependent endocytosis.¹¹⁰ This active targeting strategy increases siRNA accumulation at diseased sites while reducing nonspecific distribution in other cell populations.¹¹¹

pH Responsiveness for Endosomal Escape

Even after successful entry into target cells, the fate of nanoparticles depends on whether they can escape from endosomal and lysosomal compartments. During endosomal maturation, pH decreases from approximately 6.5 to 4.5, which provides a useful trigger for carrier design.¹¹² At neutral pH, carrier amines remain deprotonated, which favors siRNA binding and stability. After entry into early endosomes, protonation triggers electrostatic repulsion and counterion influx, leading to increased osmotic pressure and membrane rupture through a proton-sponge effect.^113,114 Ionizable lipid nanoparticles can also promote electrostatic fusion with anionic endosomal membranes by precise control of lipid pKa.^115,116 This strategy has already proven effective in liver delivery, and its design logic is now being adapted for pulmonary fibrosis^117,118 [Figure 2].

Figure 2.

Biological barriers and nanomaterial design principles for siRNA delivery in fibrotic lungs Extracellular barriers include mucus retention, dense ECM, and phagocytic clearance. After cellular uptake, endosomal and lysosomal sequestration can prevent cytosolic delivery. The design features shown in the figure address different steps and should not be interpreted as interchangeable solutions.

Functional Nanoplatforms

Lipid Nanoplatforms

Lipid nanoplatforms are among the most mature and clinically advanced systems for siRNA delivery. Early cationic liposomes such as DOTAP and DC-6-14 efficiently complexed siRNA through electrostatic interactions but were often associated with high immunogenicity and cytotoxicity.^119–121 Ionizable lipids have substantially improved this limitation. These materials remain close to neutral under physiological conditions, thereby reducing nonspecific interactions while still enabling efficient release.^122,123 Cheng Meiqi et al designed a cyclic lipid nanocarrier through topological engineering, and its conical structure markedly enhanced membrane-disruptive activity and improved endosomal escape.¹²⁴ The main strengths of lipid platforms include high loading efficiency, mature manufacturing processes, and relatively strong endosomal escape capacity. Their limitation is that the successful experience of LNPs in the liver cannot be directly transferred to the lung. The surface environment, matrix architecture, and cellular composition of fibrotic lungs differ substantially from those of the liver, and additional optimization is therefore required for pulmonary retention, lesion penetration, and cell selectivity.

Polymer Nanoplatforms

Polymer nanoplatforms are highly valuable for siRNA delivery in pulmonary fibrosis because of their structural tunability. For cationic polymers such as PEI, systematic modifications have been developed to reduce toxicity and introduce targeting functions. For example, the introduction of cycloalkyl derivatives can generate carriers that combine siRNA delivery with CXCR4 antagonism, whereas coupling PDGFRα or Sca-1 antibody fragments can improve delivery selectivity for specific cell populations.^125,126 Dendrimers are also important representatives. Although PAMAM G4NH2 complexes siRNA efficiently, its gene-silencing performance remains limited by insufficient intracellular release, indicating that polymer platforms must still balance complex stability against effective release.¹²⁷

Multifunctional hybrid carriers represent the frontier of the polymer field. PAMD@PFOB core–shell nanoemulsions, which use perfluorooctyl bromide as the core and PAMD as the shell, combine CXCR4 antagonism with mucus penetration.¹²⁸ Micelle-embedded polyplexes formed from hydrophilic spermine and hydrophobic oleylamine generate multilayer structures that release siRNA through both hydrophobic disruption and ionic competition, thereby improving complex stability.¹²⁹ Overall, polymer platforms allow modular design tailored to pulmonary fibrosis, but their structural complexity poses challenges for manufacturing scale-up, batch consistency, and quality control.

Biomimetic and Naturally Derived Platforms

Biologically derived carriers generally provide good biocompatibility, low immunogenicity, and intrinsic targeting potential. Albumin nanoparticles offer a stable scaffold with multiple sites for chemical modification.¹³⁰ Peptide nanoparticles, because of their low toxicity and high delivery efficiency, can support coordinated regulation across multiple cell types.⁸² Exosomes represent a particularly active frontier among naturally derived carriers. Milk-derived exosomes have attracted attention because of their abundant source and high biocompatibility. Qiu Chong’s group highlighted their lysosomal escape and collagen-penetrating properties, which make them attractive vehicles for siRNA delivery.¹³¹ Exosomes derived from human embryonic kidney cells also display low immunogenicity and good biocompatibility, and ultrasound-assisted microfluidic methods have been developed to improve siRNA loading.¹³² Engineered HEK293F exosomes expressing the targeting peptide RDYH58 on their surface can actively recognize myofibroblasts.¹³³ Despite these advantages, low loading capacity, difficulties in large-scale production and purification, and substantial heterogeneity remain major barriers to clinical translation.

Inorganic and Hybrid Platforms

Inorganic platforms remain exploratory in pulmonary nucleic acid delivery, but they offer advantages in structural stability, imaging integration, and surface modification, making them particularly useful for mechanistic studies and theranostics. Metal–polyphenol networks (MPNs) self-assemble through one-step coordination between metal ions and natural polyphenols, forming stable structures that can load multiple functional molecules. For example, an MPN constructed from zirconium ions and luteolin can simultaneously deliver siRNA, exert anti-inflammatory effects, and incorporate the radionuclide 89Zr for noninvasive PET imaging, thereby allowing real-time monitoring of biodistribution.¹³⁴

However, lung tissue is sensitive to long-term retention of foreign materials. The biodegradability and clearance route of inorganic carriers are therefore critical determinants of their translational potential. Accordingly, these systems are better suited to theranostic applications, image-guided administration, and mechanistic validation. For clinical translation, platforms should preferably be built from clearable or degradable material systems.

There is no uniformly superior platform. Lipid systems offer high loading and established manufacturing experience but often need additional lung and cell targeting. Polymers allow control of charge, release, and ligand display, although structural complexity can increase toxicity and batch variability. Biomimetic carriers are often well tolerated and may provide intrinsic targeting, but loading, purification, and product heterogeneity remain difficult to control. Inorganic systems support multifunctional designs and imaging, yet persistence and clearance limit repeated pulmonary dosing. Selection must reflect the intended route, target cell, dosing schedule, and manufacturing burden.

siRNA can be loaded by electrostatic complexation, encapsulation during particle formation, or post-production loading into biological vesicles. Loading efficiency alone is an incomplete comparator because a tightly bound cargo may withstand storage yet release poorly in the cytosol. Studies should report loading and encapsulation efficiency, leakage, nuclease protection, release under relevant intracellular conditions, and retained silencing after nebulization or storage. Although it used stavudine rather than siRNA, a recent niosome study found that surfactant choice (Span-60 versus Tween-80) altered entrapment and 12-hour release. This formulation-level evidence illustrates why carrier composition and release conditions should be reported explicitly.¹³⁵

Therapeutic Strategies Targeting the Fibrotic Network Using siRNA Nanoplatforms

Targets should be judged by four criteria: causal relevance to fibrotic progression, evidence in disease-relevant cells and models, feasibility of selective delivery, and an acceptable safety margin. TGF-β1 has strong mechanistic support but broad physiological functions. HSP47 acts closer to collagen production and has reached clinical testing, whereas IL-11 is a newer fibroblast-centered target supported mainly by preclinical evidence. Target priority should therefore reflect both the evidence base and the delivery context, rather than pathway prominence alone [Table 1].

Table 1.

Representative Nanocarrier-Enabled siRNA Strategies for Pulmonary Fibrosis. Studies Were Selected to Show Diversity in Therapeutic Targets, Carrier Classes, Administration Routes, Target Cells, and Reported Limitations. Their Order Does Not Indicate Comparative Efficacy or Clinical Priority

Target	Delivery Platform	Administration Route	Primary Target Cell(s)	Mechanistic Rationale	Key Limitations	Ref.
TGF-β1, Astaxanthin	Ionizable lipid nanoparticles	Intravenous	Alveolar epithelial type II cells, (activated) fibroblasts/myofibroblasts	Inhibit the activation of myofibroblasts and the deposition of ECM, Alleviate oxidative stress and mitochondrial dysfunction	No active targeting ligands have been introduced, and the targeting ability can be further optimized; The issue of endosome escape has not been fully resolved	[136]
TGF-β1	Ionizable lipid nanoparticles	Inhalation	(activated) fibroblasts/myofibroblasts	Inhibiting TGF-β/Smad2/3-driven epithelial-mesenchymal transition, activation of fibroblasts, expression of CTGF, and production of ECM	Lack of negative control group	[137]
TGF-β1	Peptide nanoparticles (MNM)	Intratracheal administration	Fibroblasts, M2 macrophages	Significantly reduce the expression of TGF-β1 and decrease the phosphorylation of Smad2/3, reduce collagen deposition and M2 macrophage infiltration	Lack of cell-specific targeting function;Compared to healthy lungs, the delivery efficiency decreases in fibrotic lungs	[138]
MECP2	Cationic liposomes	Intratracheal administration	M2 macrophages	Inhibit the expression of M2 macrophage markers (CD206, Arg1, Ym1) to reduce collagen deposition and fibrosis	The primary molecular mechanism (Mecp2 regulating Irf4 expression) has not been deeply studied	[139]
GDF15	Cationic liposomes	Intravenous	Aging alveolar epithelial type II cells	Inhibiting TGF-β receptor-associated epithelial-mesenchymal transition (EMT) and fibroblast activation	It has not been ruled out whether other cell types also secrete the target factor GDF15;The direct impact of GDF15 on the cellular aging process has not been thoroughly investigated	[140]
ZNF416	Cationic liposomes	Intravenous	Fibroblasts	Significantly reduce the nuclear accumulation of p-Smad2/3 to regulate fibroblast activation	No active targeting modification has been made	[141]
RUNX1	Cationic liposomes	Intravenous	Lung epithelial cells, macrophages	Inhibiting RUNX1-mediated fibroblast activation and EMT while combining anti-inflammatory treatment with dexamethasone	The translational reproducibility of selective targeting of human lung organs needs further verification	[16]
CypD, MCU	3D-ionizable lipid nanoparticles	Inhalation	Alveolar epithelial type II cells,(activated) fibroblasts/myofibroblasts	Dual inhibition reduces the release of DAMPs (such as ROS and NO) to inhibit the NF-κB/NLRP3 pathway, thereby reducing EMT to alleviate fibrosis	The optimization of dosing frequency and dosage has not been explored;The clinical translational pathway is still unclear	[142]
GTSE1	Ionizable lipid nanoparticles	Intratracheal administration	Macrophages with high expression of CD206	Inhibit the expression of EMT-related markers such as FN1 and CDH2, and reduce collagen deposition	Limitations on drug administration route: Intratracheal injection is not feasible clinically, necessitating the development of more viable inhalation formulations;The specific signaling pathway of GTSE1 in pulmonary fibrosis has not been fully elucidated	[143]
IL-11	PEI-GBZA	Inhalation	(activated) fibroblasts/myofibroblastsM2 macrophages	Disrupting fibroblast activation and downstream pro-fibrotic signaling, reducing ECM deposition and persistent matrix remodeling	The clinical translation pathway has not been explored	[144]
CTGF	PDMAEMA-b-PMAPEG	Intratracheal administration	Fibroblasts	Inhibit the activation of fibroblasts, collagen deposition, and the production of inflammatory cytokines, and reduce the progressive accumulation of ECM in fibrotic lung tissue	The clinical translation pathway has not been explored	[145]
LGALS3	Cationic liposomes	Intravenous	Aging endothelial cells,fibroblasts, macrophages	Inhibit the binding of Gal3 to TGFBR1 on fibroblasts to reduce FMT;Inhibit the activation of the NLRP3 inflammasome and suppress the expression of the upstream transcriptional regulatory factor CEBPB	The research is primarily based on HUVECs, which differ from human alveolar capillary endothelial cells;The synergistic effect of combining Gal3 inhibitors with anti-aging drugs has not been explored	[146]
Acp5	Ionizable lipid nanoparticles	Intratracheal administration	(activated) fibroblasts/myofibroblasts	Inhibiting ACP5 promotes the degradation of β-catenin and attenuates the transition of fibroblasts into myofibroblasts	The cohort of IPF patients is relatively small, with limited sample size;Due to the lack of commercial antibodies targeting key targets, mechanistic studies rely on indirect experiments;The role of ACP5 in other cells has not been fully elucidated	[147]
Cxcl10, Mmp14	Ionizable lipid nanoparticles	Intravenous	Cxcl10+/Mmp14+ monocytesCcl3+ neutrophils	Silence the expression of Cxcl10 and Mmp14, block the interaction between HBEGF–CD44 and CSF1–CSF1R ligand-receptor pairs, and reduce the release of fibrogenic factors	The role of other monocyte subpopulations in fibrosis cannot be completely excluded	[148]

Targeting Core Profibrotic Signaling Hubs

The onset and progression of pulmonary fibrosis depend on aberrant activation of multiple intracellular pathways, among which TGF-β/Smad is central. Direct targeting of TGF-β1 by siRNA can effectively inhibit fibrosis, but TGF-β1 also plays important roles in immune homeostasis, and long-term systemic suppression may therefore increase the risk of autoimmune toxicity.^{136–138,149–151} For this reason, downstream signaling nodes are generally regarded as safer alternatives. Research has gradually shifted toward more refined downstream targets such as Smad3, Smad4, and several feedback amplifiers.¹⁵² For example, targeting SRPX2, a key profibrotic factor, does not simply block the main pathway but interrupts an auto-amplification loop, thereby selectively weakening the aberrant homeostasis of the fibrotic network while preserving baseline physiological signaling as much as possible.¹⁵³

Intervention can also extend beyond TGF-β. The PI3K/Akt pathway is closely linked to macrophage polarization through MBD2 and Plekhf1.^154,155 Meanwhile, the STAT6/PPAR-γ pathway forms another independent axis of M2 polarization control through Sart1.¹⁵⁶ At the fibroblast level, the YAP/TEAD pathway participates in fibrogenesis by upregulating UHRF1 and regulating autophagy.¹⁵⁷

Overall, targeting core profibrotic signaling hubs highlights one of the key advantages of siRNA therapy: the ability to intervene precisely at critical nodes within a pathological network. These strategies can improve target selectivity, but they are also constrained by extensive pathway crosstalk and by the dual physiological and pathological roles of many of these pathways. Future strategies with stronger translational potential will likely combine pathological stratification with optimized delivery in order to achieve selective intervention at key signaling nodes.

Cell-Specific Targeting Strategies

Targeting Myofibroblasts

Myofibroblasts are the direct executors of excessive ECM deposition and the most characteristic effector cells in pulmonary fibrosis. Consequently, therapeutic strategies centered on this population have long remained a major focus. Nanocarriers now allow siRNA to accumulate more selectively in activated myofibroblasts by means of ligands such as vitamin A derivatives and FAP-targeting peptides, thereby silencing genes involved in survival, differentiation, and secretory programs.^119,121,158

If sufficiently high intracellular exposure can be achieved at the lesion site, siRNA therapy directed against myofibroblasts may produce more durable effects than simple suppression of inflammation. The major challenge is that myofibroblasts reside deep within the stiffened interstitium, making access particularly difficult. This approach therefore places especially high demands on tissue penetration and local retention.

Targeting Macrophages

Macrophages, especially profibrotic M2 macrophages, are important regulators of the lesion microenvironment. Through the release of multiple mediators, they promote fibroblast activation, collagen deposition, and amplification of aberrant repair signals. Thus, macrophage-targeted siRNA therapy is not simply anti-inflammatory; rather, it aims to reprogram local immune circuits and weaken the cell–cell crosstalk that sustains fibrosis. Nanocarriers have enabled this strategy. For example, mannose-modified albumin nanoparticles have been used to deliver siRNA to macrophages via CD206-mediated endocytosis.¹³⁰ T-cell apoptotic body membrane-coated lipid nanoparticles have also been developed to enhance macrophage uptake and induce hepatocyte growth factor secretion, thereby combining gene silencing with epithelial repair.¹⁴⁹

Compared with direct inhibition of myofibroblasts, macrophage targeting focuses more on the immune-support system of the fibrotic lesion and thus has stronger significance at the network level. The main challenge is that macrophages are highly heterogeneous and plastic, and nonselective inhibition may impair host defense and tissue clearance. Future strategies with greater translational promise should therefore emphasize pathological reprogramming rather than simple macrophage depletion.

Targeting Extracellular Matrix Remodeling

Once a stable ECM network has formed, pulmonary fibrosis acquires a stronger capacity for self-maintenance. Directly targeting molecules involved in collagen synthesis, maturation, and cross-linking therefore has special value. HSP47 is an essential chaperone for collagen folding and secretion and is one of the most representative ECM-related targets. siRNA against HSP47 not only reduces collagen output but may also weaken myofibroblast survival by inducing endoplasmic reticulum stress.^119,132,158

LOXL2 and FKBP10 participate in collagen cross-linking at a critical stage of ECM maturation. Targeting these molecules reduces insoluble collagen formation and the degree of matrix irreversibility, thereby alleviating tissue stiffness and mechanical positive feedback.^120,133 Such strategies may be particularly suitable for patients at later disease stages or for those in whom matrix stiffening is a dominant feature.

Some ECM-related molecules function beyond matrix metabolism itself and participate more broadly in the aberrant remodeling network. Matrix metalloproteinase 7, matrix-binding proteins, and nuclear stress molecules can affect epithelial–mesenchymal crosstalk, cell migration, inflammatory recruitment, and tissue architecture remodeling.^129,159 In these settings, siRNA therapy is intended to modulate abnormal remodeling programs rather than simply promote or inhibit a single matrix metabolic reaction.

Taken together, these observations suggest that pulmonary fibrosis therapy is shifting from conventional inhibition of collagen toward broader remodeling of the disease network. Nanocarriers make it possible to implement more complex intervention strategies, such as simultaneous suppression of profibrotic signaling and reduction of structural stabilization within lesions.

Multi-Target Synergistic Therapy

Within the same cell type or pathway, simultaneous silencing of PTPN13 and NOX4 in myofibroblasts can synergistically promote apoptosis and inhibit activation. Combined targeting of the transcription factors RUNX1 and Gli1 also blocks mesenchymal stem-cell differentiation into myofibroblasts more effectively. Using SAMiRNA to co-deliver siRNAs targeting AR and CTGF, Yoon et al achieved markedly stronger antifibrotic effects than single-target approaches.^81,126,160

Across different cell types or parallel pathways, Olga B. Garbuzenko’s group designed nanocarriers co-delivering three siRNAs targeting MMP3, CCL12, and HIF1A, thereby simultaneously intervening in ECM degradation, fibroblast recruitment, and hypoxic responses. Ding Weifeng et al co-silenced SPARC, CCR2, and SMAD3 to regulate fibroblasts, monocytes/macrophages, and epithelial cells in a coordinated manner, achieving more comprehensive antifibrotic effects.^80,82

siRNA can also be combined with approved antifibrotic drugs. Co-delivery of siIL-11 and nintedanib to myofibroblasts enhances suppression of fibrotic signaling pathways.¹²¹ Likewise, combining siTGF-β1 or siHSP47 with pirfenidone significantly strengthens inhibition of fibroblast differentiation and migration.^124,151 These approaches aim to preserve the activity of existing drugs while compensating for their target limitations through siRNA.

Multitarget therapy is justified when the selected genes control nonredundant parts of one pathogenic circuit or act in different cell populations that sustain each other. Such combinations may reduce compensatory signaling, but added complexity does not establish synergy. Each formulation should be compared with every single-siRNA component, with the intracellular dose ratio defined in advance. The experiments must also show that additional silencing improves efficacy without increasing off-target or immune effects [Figure 3].

Figure 3.

siRNA-based therapeutic strategies targeting the fibrotic network The diagram groups siRNA strategies by intervention level. Core signaling targets reduce profibrotic activation, cell-directed systems concentrate silencing in myofibroblasts or macrophages, and ECM-related targets alter matrix production or maturation. Multitarget combinations connect these levels but require evidence of benefit over each single component.

Abbreviations: TGF-β, transforming growth factor-β; SMAD, mothers against decapentaplegic/small; PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; STAT6, signal transducer and activator of transcription 6; PPAR-γ, peroxisome proliferator-activated receptor gamma; YAP, yes-associated protein; TEAD, transcriptional enhanced associate domain; FAP, fibroblast activation protein; LNPs, lipid nanoparticles; HSP47, heat shock protein 47; LOXL2, lysyl oxidase like protein 2; FKBP10, FK506-binding protein 10; PTPN13, protein tyrosine phosphatase, non-receptor type 13; NOX4, NADPH oxidase 4; RUNX1, runt-related transcription factor 1; Gli1, glioma-associated oncogene homolog 1; AR, amphiregulin; CTGF, connective tissue growth factor; MMP3, matrix metalloproteinase-3; CCL12, C-C motif chemokine ligand 12; HIF1A, hypoxia inducible factor-1α; SPARC, secreted protein, acidic and rich in cysteine; CCR2, CC chemokine receptor 2; Created with BioGDP.com.⁷⁰.

Translational Challenges and Clinical Prospects

Route of Administration

Advantages and Challenges of Inhalation

Inhalation increases local lung exposure, reduces systemic distribution, and avoids first-pass uptake by the liver and spleen.^161,162 It may therefore lower systemic toxicity while improving delivery to pulmonary lesions.¹⁶³ Inhaled products must nevertheless preserve siRNA integrity and particle structure during nebulization or dry-powder processing. Aerosol characterization should report mass median aerodynamic diameter (MMAD), fine-particle fraction (FPF), emitted dose, and deposition across proximal and distal lung regions. Device shear, air-liquid interfaces, excipients, and reconstitution can alter particle size, leakage, and silencing activity.^164,165 In pulmonary fibrosis, deposition within diseased regions and penetration into deep lesions remain the main challenges.¹⁶⁶

Advantages and Challenges of Intravenous Administration

Intravenous administration offers relatively mature protocols, more controllable dosing, and easier integration with existing systemic treatment paradigms.^167,168 For RNA formulations that cannot be delivered stably by inhalation, the intravenous route remains practically feasible. However, its limitations are also clear. Nanocarriers are readily trapped by the mononuclear phagocyte system in the liver and spleen, resulting in insufficient pulmonary specificity.¹⁶⁹ In addition, efficient passage across the vascular barrier into the fibrotic interstitium often requires extra organ-targeting or cell-targeting design.¹⁷⁰ Intravenous administration is therefore more suitable for platforms that already possess strong lung-targeting properties or active targeting modules.¹⁷¹

Translational Strategies

Administration route is part of delivery-system design. Inhaled formulations require aerosol stability, appropriate pulmonary deposition, and local tolerability. Intravenous formulations instead require blood stability, lung enrichment, and limited hepatic and splenic uptake.^172–174 Nebulization is clinically practical but can destabilize nanocomplexes. Dry-powder inhalers may simplify storage and transport but require control of aerodynamic properties and redispersion. Intratracheal instillation is useful for preclinical proof of concept, not routine clinical delivery. Systemic administration permits controlled dosing but needs direct evidence of lung targeting.

Safety, Immunogenicity, and Long-Term Tolerability

Core Challenges

Safety remains a major barrier to the clinical translation of pulmonary RNA nanomedicines. Repeated administration may trigger innate immune activation, inflammatory cytokine release, complement-related reactions, or local cytotoxicity.¹⁷⁵ Some cationic materials and membrane-active components may also directly damage the alveolar epithelial barrier.¹⁷⁶ These issues are particularly important in IPF, which typically requires long-term treatment rather than short-term dosing.¹⁷⁷

Lung-Specific Risks

Lung tissue is sensitive to long-term retention of foreign materials. If carriers are nondegradable, slow to clear, or persistently retained in the alveolar space, they may provoke chronic inflammation, sustained activation of phagocytes, and further impairment of alveolar structure and gas exchange.^178–182 In IPF patients, who already have diffusion impairment and structural destruction, this risk warrants special attention. Material selection should therefore prioritize degradability, the safety of degradation products, and tissue tolerability after repeated administration.¹⁸³

Safety Assessment

Safety assessment should extend beyond short-term measures such as body weight, serum biochemistry, and histological inflammation scores.¹⁸⁴ Pulmonary RNA nanomedicines also require evaluation of repeated-dose immunogenicity, complement activation, cytokine responses, lung function, and local tissue accumulation. Risk reduction measures include sequence screening, appropriate chemical modification of siRNA, control of carrier charge and degradability, and dosing schedules that maintain silencing without sustained inflammation.¹⁸⁵

Limitations of Current Animal Models

The bleomycin-induced model remains the most commonly used preclinical system for RNA therapeutics in pulmonary fibrosis.¹⁸⁶ This model is relatively mature, reproducible, and convenient for target validation and efficacy screening, and it therefore serves as the basic platform for most nano-siRNA studies.¹⁸⁷ However, its limitations are clear. Compared with human IPF, the bleomycin model usually has a faster onset, a stronger early inflammatory component, and a degree of spontaneous resolution, making it unable to fully reproduce the persistent and progressive nature of human disease.¹⁸⁸ Consequently, candidate drugs that perform well in this model may not retain the same efficacy under conditions that are more clinically relevant.¹⁸⁹

Future approaches may include repeated-injury or more chronic models to increase disease persistence, alternative models such as silica exposure to cover different pathological dimensions, and the use of human precision-cut lung slices (PCLS) for target and delivery validation.^190,191 Because PCLS preserves lung architecture and cell–cell interactions, it offers unique value for assessing local delivery, tissue penetration, and pharmacodynamic biomarkers.¹⁹²

Clinical Translation Progress and Pipeline

RNA nanomedicines that have truly entered clinical evaluation for pulmonary fibrosis remain rare, and the field is still in an early exploratory phase. A representative example is ND-L02-s0201, an siRNA lipid nanoparticle targeting HSP47, which advanced into a Phase II clinical study in IPF. This provided proof that antifibrotic siRNA nanomedicines can move from preclinical work into patient studies. However, subsequent results showed that, although safety and tolerability were supported, the expected efficacy in IPF was not achieved. Meanwhile, BMS-986263, which also targets HSP47, has shown overall tolerability and some histological improvement signals in clinical studies related to liver fibrosis.¹⁹³ These findings suggest that antifibrotic siRNA nanoplatforms do have clinical development potential, but substantial progress is still needed before efficacy in pulmonary fibrosis can be realized.

Manufacturing, Stability, and Regulatory Readiness

Clinical development requires scalable production and predefined controls for particle size, siRNA identity and integrity, encapsulation, potency, sterility, impurities, and batch reproducibility. Stability studies should distinguish chemical degradation of siRNA from physical changes in the carrier and should use the final container and delivery device. Regulatory development also requires analytical methods that connect these quality attributes with biological activity, together with a credible route to GMP production and cost-controlled manufacture.

Conclusions and Future Perspectives

Near-term work should match a biologically justified target with a carrier capable of reaching the relevant cell population at a clinically workable dose. Ligand-targeted particles and engineered exosomes may improve selectivity. Machine-learning models may assist formulation ranking, but only when trained on standardized datasets and tested prospectively.

IPF lesions differ between patients and across regions of the same lung. This heterogeneity complicates uniform target selection and helps explain why effects in simplified models may not translate to patients. Biomarker-guided stratification, human lung models, and repeated-dose studies need to be incorporated early in development.

Nanocarrier-enabled siRNA provides a way to intervene in epithelial injury, immune remodeling, fibroblast persistence, or ECM stabilization. Evidence remains predominantly preclinical, and clinical benefit in IPF has not been established. Progress depends on reproducible lung delivery and cytosolic release, acceptable repeated-dose safety, and models that better predict human response.

Immediate priorities are to test biologically justified target combinations against their single components, compare carriers using common performance measures, establish scalable and stable formulations, and link pharmacodynamic biomarkers to FUNpatient selection. These studies would distinguish candidates ready for clinical testing from systems that remain experimental.

Funding Statement

This work was supported by the Medical and Health Talent Special Project of Jilin Province (Grant No. 2024WSZX-D06 to Xin Di), and Jilin Provincial Natural Science Foundation Provincial-Local-Enterprise Joint Fund (Group B) (YDZJ202501ZYTS317).

Disclosure

Dr Zhao-Qi Lv reports Support for the manuscript from Jilin Provincial Natural Science Foundation Provincial-Local-Enterprise Joint Fund (Group B) (YDZJ202501ZYTS317), Medical and Health Talent Special Project of Jilin Province, during the conduct of the study. Dr Xin Di reports Support for the manuscript from Medical and Health Talent Special Project of Jilin Province, during the conduct of the study. The authors report no conflicts of interest in this work.