--- arxiv_id: PMC12888636 title: "Lung-targeted RNA delivery systems: strategies and therapeutic applications." authors: - Xu S - Li M - Wang T - Chen R - Zhou M - Tang Z - Liu Q - Hu L - Li Z difficulty: Advanced tags: - RNA Delivery - Lung - Nanoparticle - LNP - Therapeutics published_at: 2026 Jan 1 flecto_url: https://flecto.zer0ai.dev/papers/PMC12888636/ lang: en --- > Journal of Nanobiotechnology (2026) 24:142 **Authors**: Xu S, Li M, Wang T, Chen R, Zhou M, Tang Z, Liu Q, Hu L & Li Z ## Abstract ### Abstract Pulmonary diseases—including asthma, lung cancer, COPD, and pulmonary fibrosis—are major contributors to global morbidity and mortality, placing enormous burdens on healthcare systems worldwide. RNA-based therapeutics have emerged as promising tools for modulating disease at the molecular level, yet achieving efficient, lung-specific RNA delivery remains a critical challenge limiting clinical translation. Unlike previous reviews that primarily catalog delivery system performance metrics, this review uniquely integrates structure-function design principles with clinical translation insights. It provides mechanistic understanding of how specific physicochemical parameters govern pulmonary tropism and therapeutic efficacy. The authors systematically examine both synthetic and biologically derived carriers, with focus on lung-targeted delivery strategies including inhalation, intravenous targeting, and local pulmonary administration. They critically analyze lessons from clinical trial failures (ALN-RSV01 and MRT-5005) to identify key barriers, and discuss translational outlook including formulation stability, immunological compatibility, and scalable manufacturing. ## Introduction ### Introduction Pulmonary diseases—including COPD, asthma, idiopathic pulmonary fibrosis (IPF), and lung cancer—rank among the leading causes of global morbidity and mortality, profoundly compromising patient quality of life. These conditions arise from complex multifactorial interactions involving genetic susceptibilities, environmental pollutants, and microbial pathogens, resulting in heterogeneous disease manifestations that challenge therapeutic intervention. The COVID-19 pandemic underscored the vulnerability of the respiratory system and exposed critical deficiencies in existing treatments. Conventional therapies largely focus on symptomatic management rather than addressing disease etiology, limiting long-term efficacy. RNA-based therapeutics —including antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), circular RNA (circRNA), and messenger RNAs (mRNAs)—offer a fundamentally different approach by modulating gene expression to restore functional proteins or suppress pathogenic protein synthesis at the molecular level. ### RNA Therapeutic Modalities ### Messenger RNA (mRNA) Translated by host ribosomes to produce functional proteins. Enables protein replacement therapy , vaccination, and genome editing applications. The COVID-19 mRNA vaccines demonstrated the transformative potential of this modality. ### Small Interfering RNA (siRNA) Double-stranded RNA duplexes that load into the RNA-induced silencing complex (RISC) , guiding sequence-specific cleavage of target mRNA. Enables potent gene silencing for disease-causing proteins. ### MicroRNA (miRNA) Small non-coding RNAs that regulate gene expression post-transcriptionally. Can simultaneously modulate multiple gene targets , offering therapeutic potential for complex diseases with multifactorial pathways. ### Antisense Oligonucleotides (ASOs) Single-stranded, chemically modified oligonucleotides that hybridize to complementary RNA targets. Induce gene silencing through RNase H1-mediated degradation , steric blockade of translation, or modulation of pre-mRNA splicing. ## References ### References (selected) ## Graphical Overview ### Graphical Overview Graphical Abstract: The landscape of nanoengineered RNA delivery systems for pulmonary therapy, encompassing six major platform categories: Lipid Nanoparticles, Polymers, Peptide-Based Delivery Vectors, Viral Vectors, Exosomes, and Protein Carriers. ## Clinical Challenges ### Clinical Challenges in Pulmonary RNA Delivery ### Biological Barriers Successful RNA delivery to the respiratory tract requires overcoming multiple sequential biological barriers, each presenting distinct challenges that collectively determine therapeutic efficacy. ### Mucociliary Clearance The first line of defense: inhaled particles encounter a complex protective mucus layer enriched with mucins, antimicrobial peptides, and secretory immunoglobulins that trap and eliminate foreign materials through coordinated ciliary beating. ### Epithelial Barrier After penetrating mucus, RNA carriers must traverse the epithelial barrier, which varies from pseudostratified ciliated epithelium in conducting airways to the thin type I pneumocytes in alveoli. Tight junctions limit paracellular transport. ### Alveolar Macrophage Uptake Resident alveolar macrophages rapidly phagocytose particles reaching the alveolar space, particularly those in the 200 nm–1 µm range, representing a major clearance mechanism for inhaled nanocarriers. ### Endosomal Escape After cellular uptake via endocytosis, nanocarriers must escape the endosomal compartment before lysosomal degradation. This remains a rate-limiting step, with most carriers achieving less than 2% escape efficiency. ### RNase Degradation Ubiquitous RNases in the respiratory tract rapidly degrade unprotected RNA molecules, necessitating robust encapsulation or chemical modification strategies to preserve cargo integrity. ### Clinical Trials in Pulmonary RNA Therapeutics ### Lessons from Clinical Trial Failures ### ALN-RSV01 (Alnylam Pharmaceuticals) This inhaled siRNA targeting RSV nucleocapsid protein completed Phase II trials with excellent tolerability. However, it failed to demonstrate significant antiviral efficacy compared to placebo. Post-hoc analysis revealed that insufficient siRNA delivery to infected epithelial cells and rapid mucociliary clearance limited effective target engagement. Key lesson: Tolerability alone is insufficient—delivery efficiency to the target cell population is paramount. ### MRT-5005 (Translate Bio) This nebulized mRNA encoding functional CFTR protein for cystic fibrosis showed dose-dependent increases in ppFEV1 in Phase I/II trials. However, subsequent analysis revealed formulation instability under nebulization conditions , inconsistent CFTR expression levels across patients, and challenges with repeated dosing. Key lesson: Formulation stability under real-world delivery conditions (aerosolization stress, temperature) is critical for clinical success. ## Disease Considerations ### Disease-Specific Considerations for Pulmonary RNA Delivery Different pulmonary diseases present unique biological barriers, target cell populations, and delivery challenges. Understanding these disease-specific factors is essential for rational carrier design and therapeutic strategy selection. ## Synthetic Carriers ### Synthetic Nanocarriers for RNA Delivery ### Lipid Nanoparticles (LNPs) LNPs represent the most clinically validated platform for RNA delivery, with their success in COVID-19 mRNA vaccines establishing regulatory and manufacturing precedent. A typical LNP contains four components: an ionizable lipid (for RNA encapsulation and endosomal escape), a helper lipid (structural stability), cholesterol (membrane rigidity), and a PEG-lipid (steric stabilization and prolonged circulation). Next-generation ionizable lipids are driving a paradigm shift in organ-selective delivery. The SORT (Selective Organ Targeting) technology incorporates permanently charged lipids like DOTAP at defined molar ratios (10–50%) to achieve preferential lung accumulation after intravenous administration. This tropism shift correlates with altered protein corona composition—specifically, enrichment of vitronectin and integrin-binding proteins that redirect uptake to pulmonary endothelium. High-throughput combinatorial screening has accelerated LNP optimization. Researchers have developed barcoded LNP libraries (containing 100+ unique lipid formulations) that can be pooled and administered in a single animal, with deep sequencing of organ-specific barcode distribution revealing lung-tropic hits. The CAD (Combinatorial Aldehyde Degradable) lipid library approach synthesized 180 lipids via Schiff base reduction chemistry. For pulmonary-specific challenges, inhaled dry-powder LNP formulations offer advantages over nebulization by avoiding the shear forces that compromise LNP structural integrity. PLGA-modified LNPs have shown improved stability and sustained release properties in lung tissue. The platform nature of LNPs allows modular swapping of ionizable lipids, targeting ligands, and RNA cargo for different disease indications. Active targeting strategies include conjugation with lung-specific antibodies, peptides, or aptamers. Ligand-free approaches exploit the endogenous protein corona to achieve organ selectivity. The "PEG dilemma" remains a key challenge: PEGylation improves circulation time but can reduce cellular uptake and trigger anti-PEG immune responses upon repeated dosing. Figure: LNP engineering strategies. (A) SiLNPs with different structures demonstrating organ-specific delivery to liver, lung, and spleen. (B-D) Characterization and biodistribution of lung-targeting Si 6 -N14 LNP. (E) Combinatorial aldehyde degradable (CAD) lipid screening workflow using barcoded nanoparticles and deep sequencing. (F) Chemical synthesis of 180 CAD lipids. ### Polymer-Based Carriers Polymeric carriers for RNA delivery encompass several classes: cationic polymers (e.g., PEI, PBAE) that electrostatically complex with negatively charged RNA, biodegradable polymers (e.g., PLGA, chitosan) that offer controlled release, and dendrimers (e.g., PAMAM) with defined branched architectures for multivalent RNA binding. The mechanism of RNA complexation relies on electrostatic interactions between the positively charged polymer and negatively charged RNA backbone, forming condensed polyplexes. Intracellular delivery depends critically on endosomal escape , often achieved via the "proton sponge effect"—where buffering capacity of amine groups causes osmotic swelling and endosomal membrane rupture. Fluorine-modified PEI (F-PEI) polyplexes have demonstrated effective miRNA delivery for NSCLC therapy, functioning as a miRNA sponge to inhibit VEGF expression and suppress tumor angiogenesis. PONI-Guan/siRNA polyplexes achieve lung tropism through membrane fusion-like delivery, enabling efficient cytosolic siRNA delivery and TNF-α knockdown in LPS-challenged lungs. Key limitations of polymer carriers include cytotoxicity (especially for high-molecular-weight PEI), batch-to-batch variation, and variable transfection efficiency. Engineering solutions include fluorination, PEGylation, biodegradable linkage design, and controlled molecular weight optimization. Figure: Polymer-based RNA delivery. (A) Fluorine-modified PCC F-PEI polyplex fabrication and mechanism for NSCLC inhibition via miRNA sponge and VEGF suppression. (B) PONI-Guan/siRNA polyplexes for systemic delivery with lung tropism and TNF-α knockdown. ### Peptide-Based Delivery Vectors Peptide-based vectors leverage cell-penetrating peptides (CPPs), amphipathic helical structures, and cholesterol conjugation to achieve cellular uptake and endosomal escape. Computational approaches—including molecular dynamics simulation and machine learning—enable rational design of peptide libraries optimized for lung targeting. The DP7-C peptide (cholesterol-conjugated VQWRIRVAVIRK sequence) was identified through a two-stage screening pipeline combining computer simulation with single-cell sequencing evaluation. It demonstrated preferential lung accumulation and effective siRNA delivery in vivo, with favorable safety profiles across cytotoxicity, tissue histology, and metabolic assessments. Peptide vectors offer advantages in precision targeting and biocompatibility but face challenges including rapid enzymatic degradation, limited cargo capacity, and lower transfection efficiency compared to LNPs. Strategies such as D-amino acid substitution, cyclization, and PEGylation can improve stability and pharmacokinetics. Figure: Peptide-based delivery vector design. (A) Two-stage screening pipeline: computer simulation (Stage 1) followed by single-cell sequencing evaluation (Stage 2) with DP peptide sequences. (B–E) DP7-C biodistribution, organ accumulation, imaging, and therapeutic efficacy data. ## Biological Carriers ### Biological & Non-Synthetic Carriers ### Viral Vectors Viral vectors represent biologically evolved delivery systems that exploit natural viral mechanisms for cellular entry and gene expression. Four major types are used for pulmonary RNA delivery, each with distinct advantages and trade-offs. ### Adenoviral Vectors Pros: High transduction efficiency, large cargo capacity (~36 kb for helper-dependent), well-characterized biology. Cons: Strong immunogenicity, transient expression, pre-existing immunity in many patients. Helper-dependent adenoviruses (HDAd) reduce immunogenicity by removing all viral coding sequences. ### Lentiviral Vectors Pros: Stable genomic integration for long-term expression, ability to transduce non-dividing cells, regulated promoter systems (tetracycline-inducible). Cons: Insertional mutagenesis risk, complex manufacturing, limited lung tropism without pseudotyping. Effective for alveolar macrophage transduction. ### Adeno-Associated Virus (AAV) Vectors Pros: Low immunogenicity, long-term expression without integration, multiple serotypes with different tissue tropisms (AAV5, AAV6, AAV9 for lung). CRISPR/Cas9 delivery demonstrated in NIH SCGE project. Cons: Small cargo capacity (~4.7 kb), pre-existing neutralizing antibodies, manufacturing complexity. ### Retroviral Vectors Pros: Stable integration for permanent genetic modification, well-established technology. Cons: Only transduce dividing cells (limiting lung applications), insertional mutagenesis risk, lower efficiency than other viral platforms for pulmonary delivery. Figure: Lentiviral vector design for pulmonary gene therapy. (A) Construct architecture with NF-κB-responsive and tetracycline-inducible promoter elements. (B–D) In vivo experimental workflow demonstrating regulated gene expression in alveolar macrophages following intratracheal delivery. Figure: AAV-based somatic cell gene editing in lung tissue. (A–B) NIH SCGE delivery project using AAV5 and scAAV5 for SpCas9 delivery. (C–D) Histological analysis confirming gene editing in airway cells. (E–G) Sftpc locus targeting and oncogene modeling with AAV-KPL vectors. ### Exosome-Based Delivery Exosomes are natural cell-derived extracellular vesicles (30–150 nm) that offer inherent biocompatibility and low immunogenicity for RNA delivery. Surface engineering enables targeted delivery—for example, decoration with EGFRvIII antibody fragments redirects exosomes to EGFR-overexpressing tumors. Cargo loading methods include electroporation, sonication, and incubation-based approaches. Key advantages include natural ability to cross biological barriers, intrinsic cell-to-cell communication capacity, and potential for blood-brain barrier crossing. However, challenges remain in scalable manufacturing , batch-to-batch variability, relatively low RNA loading efficiency compared to synthetic systems, and difficulty maintaining vesicle integrity during surface modification. CRISPR/Cas9-modified exosomes have shown promise for anti-metastasis therapy. Figure: Exosome-based RNA delivery. (A) Schematic of exosome loading and surface decoration with targeting ligands. (B) In vivo biodistribution showing enhanced tumor accumulation with EGFR-targeted exosomes vs unmodified exosomes over 48 hours. ### Protein-Based Carriers Protein carriers leverage natural protein structures for RNA encapsulation and targeted delivery. Albumin-based systems exploit the most abundant serum protein (HSA) to create nanoparticles with inherent biocompatibility. CTX-HSA nanoparticles combined with TGF-β-1 siRNA LNPs demonstrate combination therapy approaches for lung cancer, achieving tumor-targeted drug delivery and gene silencing simultaneously. Ferritin nanocages offer self-assembling protein shells (~12 nm) that can encapsulate RNA cargo in their hollow interior. Their surface is amenable to genetic or chemical modification for targeting. Transferrin receptor-targeted protein-polymer hybrids combine the specificity of transferrin binding with the loading capacity of polymeric components. Emerging technologies include engineered protein capsids derived from non-viral sources, offering the advantages of viral-like cellular entry mechanisms without the immunogenicity concerns of viral vectors. These systems represent a promising convergence of biological and synthetic design principles. Figure: Albumin-based combination therapy. CTX-HSA nanoparticles conjugated with cabazitaxel combined with TGF-β-1 siRNA LNPs for lung cancer treatment, demonstrating tumor-targeted delivery through the bloodstream and intracellular gene silencing. Figure: Engineered exosomes for anti-metastasis therapy. (A) CRISPR/Cas9-modified exosomes for targeted S100A4 gene silencing. (B–E) Quantification of tumor-to-liver ratio, in vivo and ex vivo imaging, and tumor radiance showing superior performance of siRNA-L 2 over jetPEI nanoparticles. ## Comparative Analysis ### Comparative Analysis of Delivery Platforms The comparative analysis supports a context-dependent approach to delivery platform selection rather than identification of a universally optimal carrier. Each platform category has distinct strengths that align with specific clinical scenarios. ### Synthetic Carriers (LNPs & Polymers) Strengths: Scalable GMP manufacturing, tunable physicochemical properties, regulatory precedent (COVID-19 vaccines), modular platform design. Limitations: Dose-dependent toxicity, anti-PEG immunity on repeated dosing, endosomal escape efficiency <2%. Best for: Acute interventions requiring transient high-level protein expression, vaccination, established disease targets. ### Biological Carriers (Exosomes & Viral Vectors) Strengths: Natural biocompatibility, evolved cellular entry mechanisms, long-term expression (viral), barrier crossing ability (exosomes). Limitations: Manufacturing scalability, batch variability, pre-existing immunity (viral), low loading efficiency (exosomes). Best for: Genetic disorders requiring durable correction, conditions where natural targeting is advantageous. ### Peptide & Protein Systems Strengths: High targeting precision, biocompatibility, rational computational design, modular surface chemistry. Limitations: Rapid enzymatic degradation, limited cargo capacity, lower transfection efficiency, stability challenges. Best for: Precision targeting of specific lung cell populations, combination strategies with other carriers. ### Strategic Framework for Platform Selection Platform selection should be guided by the following considerations rather than a one-size-fits-all approach: Disease type and target cell population: Acute inflammatory conditions favor LNPs; genetic disorders requiring permanent correction favor viral vectors; cancer applications may benefit from exosome or protein-based targeting. RNA modality: mRNA requires cytoplasmic delivery only; siRNA needs RISC loading; gene editing cargo (CRISPR) demands nuclear access—each favoring different carrier types. Delivery route: Inhalation requires aerosolization-stable formulations (dry-powder LNPs excel); IV delivery benefits from SORT-modified LNPs; local administration opens options for direct tissue injection. Manufacturing scalability: LNPs have established GMP infrastructure; exosomes and viral vectors face significant scale-up challenges for clinical-grade production. Regulatory pathway: Platforms with existing clinical precedent (LNPs) offer faster regulatory pathways; novel carriers (engineered protein capsids) face longer approval timelines. ## Conclusions ### Conclusions and Future Perspectives The development of targeted RNA delivery systems has significantly advanced pulmonary medicine, offering new opportunities to address the molecular underpinnings of diverse lung pathologies. Considerable progress has been achieved in both synthetic and non-synthetic delivery platforms, yet several critical challenges must be addressed for clinical translation. ### Critical Challenges ### Toxicity LNPs exhibit dose-dependent cytotoxicity and inflammatory responses mediated by pattern recognition receptors. Certain polymer compositions trigger cytotoxicity depending on molecular weight and degradation products. Viral vectors with integrating capacity carry insertional mutagenesis risks. ### Immunogenicity LNP components can activate innate immune pathways. Peptide-based vectors may elicit responses to non-self sequences. Viral vectors face robust humoral and cellular immunity, with pre-existing neutralizing antibodies in substantial patient populations severely limiting repeated dosing. ### Manufacturability Exosomes and biological vesicles face significant hurdles in large-scale production, batch-to-batch variability, and heterogeneous cargo loading. Surface modification without compromising vesicle integrity further complicates clinical translation. ### Stability LNPs exhibit insufficient circulation stability in biological fluids. Peptide-based vectors undergo rapid enzymatic degradation with abbreviated circulation half-life. Polymer systems show variable performance, and protein carriers often exhibit poor stability under physiological conditions. ### Delivery Efficiency LNPs are constrained by non-specific tissue distribution. Polymer carriers exhibit variable transfection efficiency. Exosomes show relatively low RNA loading efficiency compared to synthetic systems. Protein carriers demonstrate limited endosomal escape capacity. ### Future Directions The convergence of advances in lipid chemistry, polymer science, and biological carrier engineering suggests that next-generation pulmonary RNA therapeutics will increasingly employ rationally designed hybrid platforms integrating complementary strengths of multiple delivery modalities. Cell-type specific targeting: Exploiting lung cellular heterogeneity (AT2 cells, macrophages, fibroblasts) with precision ligands for disease-appropriate cell populations. Mucosal penetration strategies: Engineering carriers with optimized size, surface chemistry, and mucoadhesive properties to overcome the mucus barrier and mucociliary clearance. Physicochemical optimization: Fine-tuning particle size (100–500 nm optimal), surface charge, and release kinetics for enhanced alveolar deposition and retention. Combinatorial delivery strategies: Hybrid approaches combining LNPs with receptor-specific ligands or exosome-derived systems with synthetic polymers for synergistic targeting. Advanced preclinical models: Organ-on-chip technology, high-throughput screening, and advanced imaging for detailed biodistribution analysis and systematic evaluation of delivery strategies. Personalized medicine: Tailoring delivery systems based on patient-specific disease characteristics, genetic profiles, and immune status for optimized therapeutic outcomes. The continued evolution of RNA delivery technologies, coupled with expanding knowledge of pulmonary disease mechanisms and patient-specific factors, will be pivotal in unlocking the full therapeutic potential of RNA-based treatments, ultimately transforming the landscape of pulmonary medicine.