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Inhaled non-viral delivery systems for RNA therapeutics
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Cheng Huanga, b, Hongjian Lic, Xing Duanb, Peidong Zhangb, Shaolong Qia, d, Jianshi Dud, *, Xiangrong Songb, *, Aiping Tongb, *, Guocan Yua, c, *
Acta Pharmaceutica Sinica B | 2025, 15(5) : 2402 - 2430
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Acta Pharmaceutica Sinica B | 2025, 15(5): 2402-2430
• REVIEW •
Inhaled non-viral delivery systems for RNA therapeutics
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Cheng Huanga, b, Hongjian Lic, Xing Duanb, Peidong Zhangb, Shaolong Qia, d, Jianshi Dud, *, Xiangrong Songb, *, Aiping Tongb, *, Guocan Yua, c, *
Affiliations
  • aKey Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Department of Chemistry, Tsinghua University, Beijing 100084, China
  • bState Key Laboratory of Biotherapy and Cancer Center, Research Unit of Gene and Immunotherapy, Chinese Academy of Medical Sciences, Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China
  • cInstitute for Immunology, School of Basic Medical Sciences, Tsinghua University, Beijing 100084, China
  • dVascular Surgery Center, the Third Hospital of Jilin University, Changchun 130031, China
About Author:

E-mail addresses: (Jianshi Du),

(Xiangrong Song),

(Aiping Tong),

(Guocan Yu).

†

These authors made equal contributions to this work.

Author contributions

Cheng Huang: Writing – original draft, Conceptualization. Hongjian Li: Writing – original draft. Xing Duan: Writing – original draft. Peidong Zhang: Writing – original draft. Shaolong Qi: Writing – original draft. Jianshi Du: Writing – review & editing. Xiangrong Song: Writing – review & editing. Aiping Tong: Writing – review & editing. Guocan Yu: Writing – review & editing, Supervision, Funding acquisition, Conceptualization.

doi: 10.1016/j.apsb.2025.03.033
Outline
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RNA-based gene therapy has been widely used for various diseases, and extensive studies have proved that suitable delivery routes greatly help the development of RNA therapeutics. Identifying a safe and effective delivery system is key to realizing RNA therapeutics’ clinical translation. Inhalation is a non-invasive pulmonary delivery modality that can enhance the retention of therapeutic agents in the lungs with negligible toxicity, thereby improving patient compliance. Inhaled RNA therapeutics are increasingly becoming an area of focus for researchers; however, only several clinical trials have explored inhaled delivery of RNA for pulmonary diseases. This review presents an overview of recent advances in inhaled delivery systems for RNA therapeutics, including viral and nonviral systems, highlighting state of the art regarding inhalation in the messenger RNA (mRNA) field. We also summarize the applications of mRNA inhalants in infectious and other lung diseases. Simultaneously, the research progresses on small interfering RNAs (siRNAs), antisense oligonucleotides (ASOs), and different types of RNA are also discussed to provide new strategies for developing RNA inhalation therapy. Finally, we clarify the challenges inhaled RNA-based therapeutics face before their widespread adoption and provide insights to help advance this exciting field to the bedside.

Inhalation therapy  /  mRNA  /  Non-viral vectors  /  Lipid nanoparticle  /  Pulmonary diseases
Cheng Huang, Hongjian Li, Xing Duan, Peidong Zhang, Shaolong Qi, Jianshi Du, Xiangrong Song, Aiping Tong, Guocan Yu. Inhaled non-viral delivery systems for RNA therapeutics[J]. Acta Pharmaceutica Sinica B, 2025 , 15 (5) : 2402 -2430 . DOI: 10.1016/j.apsb.2025.03.033
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1.　Introduction
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The lung is an essential organ for respiration and gas exchange, and it is susceptible to various diseases that affect the respiratory tract, such as cystic fibrosis, asthma, and SARS-CoV-2 infection1,2. Among novel therapies that are currently under development for these deadly diseases, one promising strategy is the use of RNA-based therapies3. Combined with the current progress in inhalation research, the term ‘RNA therapeutics’ includes mRNAs, ASOs, siRNAs, and microRNAs (miRNAs). Naked RNAs injected systemically are often susceptible to degradation by serum nucleases and are rapidly cleared. As a result, viral and non-viral vectors have been applied to facilitate a more efficient delivery of RNA to cells. Viral vectors take advantage of evolutionary adaptation to encapsulate RNA and release their contents after cell binding and membrane fusion4. As an alternative, non-viral vectors mainly rely on natural or synthetic materials to complex or encapsulate RNA5. Although the efficiency of nonviral carriers still falls behind viral systems, their physical and chemical properties and surface properties can all be modified to enable more precise cell-specific targeting to reduce off-target toxicity.
The choice of administration route is crucial for developing targeted RNA therapeutics based on the anatomy and function of the lungs. Inhalation is a noninvasive pulmonary delivery route for biopharmaceuticals that offers advantages over the parenteral route, such as easy self-administration, local delivery, rapid onset of action, dose reduction, and minimized side effects6. Inhaled drugs can reach the entire bronchiolar and alveolar epithelium through the large surface area of the alveoli7,8. The advantage of inhalation, compared to intravenous or intramuscular administration routes, lies in its ability to deliver therapeutics directly to the respiratory tract, which is the targeted site of action, while requiring lower doses9. For example, ALN-RSV01, the first siRNA therapy targeting the RSV nucleocapsid protein, was tested clinically by pulmonary delivery using inhalation in 200810. Several clinical trials on inhaled RNA therapy have been initiated. However, no inhaled mRNA therapeutics have been approved for clinical use yet due to some challenges associated with the complex pulmonary environment and the efficiency of RNA delivery. This review summarizes preclinical advances in innovative materials and delivery strategies for inhaled RNA therapeutics for treating pulmonary and other diseases. This review focuses on inhaled mRNA therapy, and the research progress for other types of RNA, such as siRNA, ASO, and miRNA, are also discussed. This review will focus on non-viral platforms used in pulmonary RNA delivery, including synthetic and biological polymers, polycomplex, liposomes, exosomes, lipid nanoparticles, and polymer-based nanoparticles, and discuss their progress and challenges. Lastly, this review presented the applications of inhaled RNAs in treating diseases, such as infectious diseases, pulmonary diseases, and lung cancer. Moreover, we point out the key issues and challenges faced by the current inhaled delivery systems, offering novel insight into the design and development of inhaled RNA therapeutics11,12. The clinical trials on inhaled RNA therapy are summarized in Table 113—33. Selected recent studies with different RNA and non-viral delivery vectors that have demonstrated in vivo and ex vivo transfection in animal models are summarized in Table 234—62.
2.　Inhalation devices and barriers
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2.1　 Compared different types of inhalers
As a noninvasive drug delivery route, inhalation relies on a well-designed inhaler. These devices can be broadly classified into pressurized metered dose (pMDI)63,64, breath-actuated metered-dose (BA-MDI)65, dry powder (DPI)66,67, soft mist inhalers (SMI)68 and nebulizers69. The main idea of all the devices is to obtain an aerosol of either liquid (e.g., inhalers) or solid-state particles (e.g., DPIs) and deliver high drug deposition in the infected area. Consistent and accurate dosing significantly impacts the effectiveness and safety of treatment. An ideal inhalation device should offer consistent performance across various usage scenarios and safeguard the medication from environmental factors like temperature and humidity. User-friendly design is crucial for ensuring proper device utilization and enhancing treatment outcomes. Furthermore, desirable attributes encompass affordability and environmentally sustainable features.
In exploratory research, nebulizers are perhaps the most accessible delivery device as they turn a liquid medicine into a fine mist that can be inhaled through the mouth or nose. There are three main types of nebulizers: jet nebulizers70, ultrasonic nebulizers71, and mesh nebulizers72. Mesh nebulizers utilize ultrasonic vibration film to create small and uniform droplets from liquid, forming aerosols that can be deposited effectively in the lower respiratory tract for therapeutic purposes. They are ideal for administering nanosuspensions and unstable drugs, offering an advantage over ultrasonic nebulizers by minimizing concerns related to heat-induced drug degradation. Meanwhile, Galindo-Filho et al.72,73 reported that mesh nebulizers delivered more radio aerosol to the lungs of COPD patients than jet nebulizers74 during non-invasive ventilation. From the recently published studies, mesh nebulizers are widely used in some preclinical studies of inhaled RNA delivery. Blanchard48, Rotolo47, Kim38 and Bai75 et al. used a similar principle to the mesh nebulizer to design the nebulizer for RNA delivery. It greatly improves the efficiency of RNA delivery to the lung. For inhaled drug delivery, at present, the updated review concludes that there is no significant difference in the efficacy of therapy between inhalers and nebulizers. However, it should be noted that efficiently targeted delivery vectors, formulation components, and suitable nebulizers are also important for inhaled RNA delivery. The three types of nebulizers have different working principles, performances, and applicability. Correctly choosing an appropriate nebulizer according to the patients' conditions and the drugs’ properties is crucial for treating diseases. Table 3 compares all types of inhalers76—81. The characteristics and scope of application of the three nebulizers are summarized in Table 482—84.
2.2　 Barriers to inhalation delivery
Despite the high clinical applicability of inhalation for localized RNA administration, several anatomical and physiological hurdles need to be overcome. The respiratory system possesses various defense mechanisms, including anatomical, physical, immune, and metabolic barriers85—87, to safeguard the gas exchange process and prevent the intrusion of external agents. Researchers are investigating strategies to enhance the stability of drug-loaded nanoparticles during inhalation, considering factors such as particle size, density, and morphology that influence lung deposition88. The optimal aerosol particle size for pulmonary-targeted drug release depends on the delivery system and the desired site of action89,90. Therefore, by tuning the size of the particles from sub-100 nm to micron-scale, one can control the location of particle deposition for pulmonary drug deliveryvia inhalation (Fig. 1A and B)91,92. Besides these, challenges in effective inhalation therapy include the mucus barrier93, diverse lung cell types hindering precise targeting6,94, potential components inactivation95, and respiratory physiology such as the air flow and the mucociliary clearance (Fig. 1C and D)96,97. Developing nano-formulations that efficiently penetrate the mucus layer and reach target lung cells is crucial for maximizing the efficacy and safety of inhaled therapies for lung diseases. For example, the desired therapeutic efficacy can only be achieved if target cells in the lungs take up the inhaled nanomedicine. Besides the cellular uptake, successful endosomal escape and release of siRNA or mRNA into the cytoplasm are known to be critical prerequisites that a nanocarrier must fulfill for effective siRNA delivery and the consequent knockdown or expression of specific proteins (Fig. 1E and F)98—100. Therefore, it is important to develop and optimize the nano-formulations that can efficiently penetrate the mucus layer and be absorbed by target cells in the lungs to maximize the efficacy and safety of inhaled therapy for treating lung disease. Based on understanding lung structure and barriers, inhaled medication conditions need to meet some requirements. Firstly, the aerodynamic diameter and the drug-loaded particles must meet the ideal size. Secondly, the administration of inhaled medication has to break through the mucus barrier. Thirdly, drugs like mucosa cilia and macrophages need to escape the clearance mechanism101,102. Targeted delivery of drugs by inhalation therapy to targeted sites could be realized though their passive targeting characteristics, active targeting characteristics, and endocytosis103,104. Furthermore, factors such as the patient's age, gender, the severity of illness, or the use of different types of inhalers and nebulizers are highly influential in respiratory rhythm, inspiratory flow, the volume of inspiration, and breathing break at the end of inspiration and hand-blown coordination50. This may be the main reason for the difficulty in inhaled RNA therapeutics in current clinical practices. Notably, with the development of mRNA modification and delivery technology, its application prospect in respiratory and pulmonary diseases is becoming increasingly widespread.
3.　Therapeutic RNA payloads
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The diverse roles of RNA in the body have led to the emergence of different approaches to harnessing RNA for therapeutic use. RNA therapeutics can be broadly divided into three functional classes: (i) inhibition of gene expression (e.g., siRNA, miRNA, and ASO); (ii) protein-encoding (e.g., mRNA); and (iii) protein targeting (e.g., RNA aptamers)94,106. These RNA therapies target RNA or proteins, encode missing or defective proteins, or mediate RNA editing. Based on the diagram of the inhaled RNA therapeutics classes framework (Fig. 2A), Table 5 summarizes the characteristics of different RNA molecules for inhaled delivery and the corresponding delivery systems used for these RNAs. Oligonucleotide drugs, such as siRNAs and ASOs that utilize enzymes endogenous to eukaryotic cells, such as RNase H1 or the RNA-induced silencing complex (RISC), respectively, facilitate delivery by not requiring the delivery of large enzymes (Fig. 2B and C). One biochemical mechanism of action safely used in humans is siRNA-mediated gene silencing. These double-stranded RNAs with a molecular weight of approximately 13 kDa suppress protein translation by recruiting RISC to mRNA via Watson–Crick base pairing. In multiple pre-clinical studies, Bai et al.75 have focused on inhaled siRNA nanoparticles to improve tumor-targeting treatment of lung cancer.ASOs are a second class of RNA therapeutics oligonucleotides with a molecular weight of 6–9 kDa. ASOs have the same manufacturing advantages as siRNA and have been approved by the FDA to treat familial hypercholesterolemia105. In a recent study, Friedman et al.18 focused on SPL84, an inhaled ASO-based drug developed for treating cystic fibrosis (CF). miRNA, a small noncoding RNA, exerts post-transcriptional gene regulation activity by targeting mRNAs. Zhang and coworkers56 investigated the efficacy of inhaled let-7b miRNA treatment in lung cancer prevention and found that let-7b given via inhalation exhibited striking tumor inhibition in both the benzo[a]pyrene (B[a]P)-induced and a syngraft model of lung cancer without causing detectable side effects (Fig. 2D) mRNA therapy is a type of RNA therapeutic with several advantages over other nucleic acid therapies. It is highly efficient as it utilizes the body's natural machinery to produce proteins, resulting in higher transcription and translation rates. mRNA therapy has a favorable safety profile with short-lived molecules and limited impact on non-target tissues. It is highly adaptable and customizable for different diseases and treatment goals.
Additionally, mRNA therapy can activate the immune system, making it beneficial for cancer immunotherapy. The applications of mRNA therapy can be broadly categorized into prophylactic vaccines against infectious diseases, therapeutic vaccines targeting cancers, and protein-replacement therapeutics. Clinical and preclinical research has focused on using mRNA for inhaled RNA delivery (Fig. 2E and F). In terms of factors affecting inhalation delivery, apart from knowing the difference in RNA molecular weight, most RNAs are loaded via electrostatic binding; however, there is not currently a large number of potentially relevant studies discussing the impact of different RNA types on the efficacy of inhalation delivery. The current focus lies in the exploration of non-viral carriers. Most research discussed augmenting inhaled delivery efficiency through carrier screening and formulation optimization. Therefore, the significance of these RNA differences in the engineering of the delivery vectors and inhalation of these formulations should be further investigated.
4.　Inhaled RNA delivery systems
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Pulmonary drug delivery systems necessitate tailored design approaches that account for the nuances of administration pathways and therapeutic agents. Many specialized vectors have been engineered for the pulmonary delivery of RNA molecules, designed to enhance cellular uptake of the RNA and shield it from degradation during delivery. This section provides an exhaustive overview of the contemporary landscape of inhalation delivery vectors, encompassing exosomes, polymeric systems, polyplexes, liposomes, lipid nanoparticles, and polymer-based nanocarriers. Moreover, it delves into a thorough analysis of the most recent progress and the challenges faced in the evolution of inhalation-based RNA therapeutic delivery platforms. It underscores the critical necessity for these systems to offer augmented delivery efficiency, therapeutic effectiveness, and safety. The discussion accentuates the importance of optimizing these vectors to meet stringent requirements of respiratory drug delivery and to harness the full potential of RNA-based therapeutics in treating various pulmonary conditions.
4.1　 Adeno-associated virus (AAV)
AAV belongs to the parvovirus family, harboring a single-stranded DNA genome of approximately 4.7 kb. The working principle of AAV vaccines involves using non-pathogenic AAV viruses for genetic delivery. The target antigen's gene sequence is inserted into the genome of the AAV virus. Once inside the body, the AAV virus releases the antigen gene sequence, enabling cells to express the antigen and induce an immune response, thereby protecting the body from infection. AAVs are widely used as gene manipulation tools in biology due to their safety, durability, high efficiency, and specificity. Chen et al.107 have publicly released the world's first published clinical trial results on mucosal immunity against COVID-19. The study assessed the safety and immunogenicity of the Ad5-nCoV vaccine by aerosol inhalation in adults. The research findings indicated that inhaled Ad5-nCoV was well-tolerated and did not cause any vaccine-related serious adverse events108. Recently, the Information Office of Shanghai Municipal People's Government announced on its official WeChat account “Shanghai Release” that the city had opened reservation registrations for booster immunizations with the CanSinoBIO recombinant COVID-19 vaccine (adenovirus type 5 vector) and stated that this inhaled COVID-19 vaccine was officially being used for immunizations. CanSinoBIO developed this adenovirus vector vaccine for COVID-19 and has been certified by the World Health Organization for emergency use109. Although AAV are DNA virus vector and not the primary focus of this review, the COVID-19 pandemic has prompted the proposal of utilizing inhaled delivery of AAV vaccines. The objective was to employ AAV viruses as vectors for converting COVID-19 vaccines into inhalable forms, enabling direct administration to respiratory epithelial cells. This concept offers valuable insights and translational possibilities for developing inhaled RNA therapeutics.
4.2　 Extracellular vesicles (EVs)
EVs with suitable size, lipophilicity, and surface proteins have garnered extensive interest as delivery systems because they can package diverse contents, penetrate through cell membranes, and release their contents intracellularly. At the same time, EVs of homogeneous origin acting as delivery systems negligibly induce the innate immune responses that are always elicited by artificial delivery vectors110. A recent study showed that a vibrating mesh nebulizer could deliver serum-derived EVs to murine lungs. In vivo EV tracking revealed that inhaled EVs were distributed exclusively in the lungs and localized mainly in lung macrophages and airway epithelial cells. Furthermore, EVs loaded siRNAs through inhalation could attenuate lipopolysaccharide-induced lung injury in mice, supporting the use of this inhalation method to deliver functional small RNAs54. EVs come in several types, including micro-vesicles, exosome-like vesicles, exosomes, and membrane particles. There have been studies on exosomes as delivery carriers of nucleic acid drugs111,112. Liu et al.53 selected lung-derived exosomes (lung-exo) with natural lung homing ability for the inhalation delivery of mRNA. The lung-exo maintained its original function after one month of storage at room temperature, indicating high stability. This work has greatly promoted the delivery of nucleic acid therapeutics using functional exosomes as platforms for inhaled drug delivery. However, Lipo does not represent LNPs containing other lipid molecules, including ionizable, cholesterol, helper, and PEG conjugated-lipid. Therefore, it is still unclear whether exosomes have a betterinhaled delivery effect than LNP (Fig. 3)113.
4.3　 Polymers
As early as 2000, Densmore et al.114 researched DNA inhalation delivery. Researchers compared PEI-DNA formulations and cationic lipid-DNA formulations in the inhalation system. For example, DC-cholesterol (3(-[N-[(N,N-dimethyl-amino)ethane]carbamoyl]cholesterol), guanidinium cholesterol (BGTC), GL-67 (N4-spermine hydrayl carbamate), 1,2-dilauroyl-sn-glycero-3-ethylphos-phocholine (DL-EPC), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecytoxy)-1-propanaminium bromide (DMRIE), and dioleoyl trimethylammonium propane (DOTAP) were used to prepare the cationic lipid-DNA formulations, which exhibited obvious advantages in delivery efficiency over PEI formulations. PEI produces efficient expression of delivered DNA encoding human growth hormone and produces specific antibodies that are several times higher than those of cationic lipid-DNA formulations. In another polymer-based inhalation delivery system, Kumari Patel et al.8 synthesized hyperbranched poly(beta amino esters) (hPBAEs) to enable nanoformulations of stable and concentrated polyplexes for inhalation. Importantly, repeat dosing of inhaled hPBAEs-mRNA generated consistent protein production in the lung without local or systemic toxicity. The efficiency of this delivery system based on hPBAEs was much better than that based on PEI. Another study on hyperbranched polymers also demonstrated their advantages in inhaled mRNA delivery. Rotolo et al. synthesized a poly-β-amino-thioester (PBATE) (P76), which enabled effective delivery of mRNA regardless of cargo size and complexity to hamsters, ferrets, cows, and rhesus macaques. P76 was safe and well tolerated, with greater expression than previous inhaled PBAE candidates. It allowed for fourfold dose sparing compared with previous inhaled PBAEs in a Casl3a efficacy study against SARS-CoV-2 in the Syrian hamster model. This approach was also shown to be competitive with high doses of intraperitoneal-delivered neutralizing antibodies, COV2-2381, which is a gold-standard control115.
Polymer materials also have a wide range of applications in the inhalation delivery of siRNA. For example, Conti et al.64,116 developed a siGFP–polymer conjugate that preserved silencing efficiency for up to 40% dispersed in HFA using a 4th generation dendrimer poly(amidoamine) and a mannitol or CSLA co-oligomer shell to achieve microparticle formation upon inhalation. The polyplex also showed promising aerosol performance, with 46%–49% (w/w) of inhaled siRNA reaching the lower respiratory tract to be therapeutically beneficial. McCarroll et al.52 developed a polymer-based star-siRNA nanoparticle. By aerosolizing the nanoparticle, the researchers observed that they could accumulate in the lungs and silence the expression of beta III-tubulin and Polo-Like Kinase 1 (PLK1) in lung tumors in mice, which delays tumor growth.
4.4　 Polycomplex
Ternary complex containing polymer, peptide, and nucleic acid has also been used for mRNA inhalation. Qiu et al.51 attached cationic KL4 peptide to a monodisperse linear PEG of 12-mer to synthesize PEG12KL4, which formed nanosized complexes with mRNA at 10:1 ratio (w/w). In vivo studies demonstrated PEG12KL4 enhanced mRNA uptake in the lungs of BALB/c mice compared to naked mRNA or mRNA-lipoplexes, resulting in effective transfection in human lung epithelial cells. Besides, Guan et al.117 employed poloxamine-based copolymers, peptides, DNA, or mRNA to build a ternary complex for drug inhalation. The delivery efficiency of this ternary complex was much higher than that of the cationic lipid DOTAP. The peptides developed by modular design approaches could spontaneously form compact and monodisperse nanoparticles with poloxamine and nucleic acids via self-assembly. Both mRNA and plasmid DNA expression mediated by peptide-poloxamine nanoparticles is greatly boosted in vitro and the lungs of cystic fibrosis mice. Notably, negligible toxicity was monitored during therapy, indicating promising potential of this strategy in clinic translation. More recently, an inhaled ribosomal protein-based mRNA nanoformulation was reported to clear the intrapulmonary extracellular matrix and re-epithelialize the disrupted alveolar epithelium, thereby reversing established fibrotic foci in idiopathic pulmonary fibrosis. A ribosomal protein-condensed mRNA core, a bifunctional peptide-modified corona and keratinocyte growth factor with a PEGylated shielding shell sequentially assembled the nanoformulation. In this polymer nanoparticle, the P–N3 terminal presented on the carrier loaded nucleic acid with DBCO via a click reaction50.
4.5　 Lipid nanoparticles (LNPs)
LNP-based delivery systems are predominantly utilized for intravenous, subcutaneous, and intramuscular injections. In the case of LNPs-enabled RNA vaccines, a tightly regulated cold chain infrastructure is necessary118—123. Parenteral formulations face further hurdles, such as the instability of liquid formulations at ambient temperatures, the need for skilled healthcare personnel, patient reluctance towards this delivery method (due to needle phobia, injection site pain, and risks of unintended local or systemic immune responses), and the potential for contamination (related to needle or injection site). These challenges have been thoroughly examined in previous studies124. Some of these challenges could be overcome by changing to a dry powder formulation suitable for inhalation. Friis et al.125 described a proof-of-concept study on engineering an mRNA-LNPs formulation suitable for spray drying. This process produced a dry powder formulation that maintained stability and preserved mRNA functionality with increased performance compared to liquid formulations stored for two weeks at 4 °C. The spray-dried LNPs may be used in future research for inhalation or intratracheal delivery systems. The process is illustrated in Fig. 4A. As current clinical formulations are not optimized for lung inhalation, effectively addressing the complex pulmonary vaccine landscape necessitates a meticulous reassessment of mRNA formulation strategies that can enhance stability and prolong shelf life. Among other methods, we summarized the most recently published studies on the use of mRNA-LNPs in inhaled delivery systems. They have made a series of optimizations to improve LNPs inhalation delivery. These were then categorized into the following five aspects: LNPs formulation, excipient addition, inhalation buffer, electrostatic repulsions, PEG concentration, and cholesterol analog. The properties of inhaled LNP, both structural and biological, can be attributed to a single factor and the optimal combination(Fig. 4B)34,39. Both Dahlman and Anderson analyzed prescription screening for the components of inhaled LNPs. Fortunately, Lokugamage and coworkers34 revealed the development strategy for inhalation therapy in their research. The employment of 7Cl lipid caused the 28-fold difference in delivery between the best- and worst-performing LNPs simply by changing the formulation ratio. In addition, the team proposed three principles for inhaled LNPs delivery systems: (1) PEG-lipids were essential for forming stable 7C1-based LNPs structures. (2) Combining cation-assisted lipids and a high molar percentage of PEG resulted in increased mRNA delivery after inhalation. (3) LNPs formulated with neutral phospholipid lipids required less PEG than cation-assisted lipids. Based on these three principles, the research team constructed an LNP called Nebulized Lung Delivery 1 (NLD1) for further analysis. By comparing the relative size of different dynamic light scattering peaks, the researchers found that NLD1 was more stable than cKK-E12 and MC3. Finally, NLD1 was stable and well tolerated after inhalation, and the lung transfection efficiency was higher. The lung intensively expressed the delivered mRNA and all six mice infected with the virus survived the therapeutic period. After the initial screening of inhaled LNPs prescription, Anderson observed that LNPs formulations could be stabilized to resist inhalation-induced aggregation by altering the inhalation buffer to increase the LNPs charge during inhalation and by the addition of a branched polymeric excipient (Fig. 4C and D)39. This will greatly improve the inhalation effect of mRNA-LNPs. In addition, a dense PEG layer helps LNPs achieve pulmonary delivery of mRNA following inhalation126,127. The density of PEG has an impact on the random motion of nanoparticles, which subsequently alters their movement within mucus128,129. Studies have demonstrated that an increase in PEG content leads to higher efficiency in encapsulating mRNA and a reduction in the size of LNPs. However, an excess amount of the PEG layer inhibited receptor-mediated endocytosis by decreasing the adsorption of serum proteins and hindering the escape of LNPs from endosomes, thereby significantly limiting the intracellular delivery of mRNA130—132. Therefore, Kim et al.38 exploited β-sitosterol, a phytosterol facilitating LNP’ endosomal escape, to meet such criteria. They adopted that combinations of β-sitosterol and high PEG contents would permit nebulization, mucus penetration, and endosomal escape of LNPs. This inhaled LNPs retained its physicochemical properties and efficiently delivered mRNA after inhalation (Fig. 4E). Interestingly, in a recent study, Liu et al.40 developed a charge-assisted stabilization (CAS) strategy aimed at inducing electrostatic repulsions among LNPs to enhance their colloidal stability. By optimizing the surface charges using a peptide–lipid conjugate, the leading CAS-LNPs demonstrated exceptional stability during inhalation (Fig. 4F). These factors affect the effectiveness and controllability of the inhaled mRNA-LNP delivery system.
In general, LNPs-based inhalation delivery systems should be the most advanced delivery systems in development. At least four inhaled mRNA nanocarriers have been used in clinical trials worldwide. MRT5005, developed by Translate Bio, an mRNA therapy research and development company in the United States, was approved to enter clinical trials for cystic fibrosis in 2018 and was the world's first LNPs-mRNA drug to enter clinical trials in an aerosol preparation133. Inhaled LNPs-mRNA drugs (ARCT-032, VX-522, and RCT1100) were approved for clinical trials in 2023 to treat cystic fibrosis lung disease and primary ciliary dyskinesia (PCD). Unfortunately, developing inhalation gene therapies can be more complex than other delivery methods. Despite their significant impact, inhaled gene therapies for CF have yet to enter the market due to disappointing results in human trials despite promising preclinical research.
4.6　 Cationic liposomes
Cationic liposomes were the earliest nucleic acid carriers and have an extensive research basis. A majority of studies have also explored inhaled delivery of cationic liposomes. DOTAP and DOTMA are the most representative ionizable lipids due to their extensive research foundation, relatively low cost, and ease of synthesis. Pei et al.134 synthesized functional analogs of DOTAP and successfully incorporated them into mRNA-wrapping cLNPs. These cLNPs have similar physicochemical properties to cLNPs using DOTAP, but the delivery effect was not significantly better than DOTAP. Although a large number of studies have shown that delivery systems based on ionizable lipids are significantly superior to cationic delivery systems, the use of cationic carriers as auxiliary lipids has been extensively studied and proved to have some unique advantages. From this perspective, it may provide a meaningful reference for future research. We present the related carriers for inhaled delivery of RNAs in Fig. 5.
4.7　 Nanoparticles (NPs)
The advancements in nanotechnology have facilitated the development of nanoparticle-based drug delivery systems for cancer immunotherapy135. Researchers have demonstrated the efficacy of utilizing nanoparticle carriers with a precise size of 100 nm to enhance drug retention duration and concentration within solid tumors through the augmented permeability and retention effect following intravenous administration136. However, there remains considerable scope for enhancing the efficiency of drug accumulation, specifically at targeted tumor sites137. Simultaneously, the intricate fabrication process and high-cost is barriers to the widespread clinical implementation of these nanoparticle drug carriers138. Due to the distinctive physiological state of the lungs within the respiratory system, the noninvasive administration of drugs through aerosol nebulization has demonstrated exceptional benefits in managing respiratory ailments139, 140. For mRNA, Zhang et al.50 sought to develop inhaled nanoparticles co-delivering the messenger RNA of MMP13 and KGF to fibrotic lung tissues to reverse established pulmonary fibrosis in a bleomycin-induced murine model. Tang et al.43,47 recently also reported a novel approach involving the development of dual-targeted mRNA NPs using cationic lipid and hyaluronic acid. The designed NPs exhibited excellent stability and demonstrated efficient transfection of targeted proteins into lung tissues. Importantly, the optimized dual-targeted mRNA NPs exhibited a dual capacity: They primarily accumulated in lung tumor cells and inflammatory macrophages following inhalation delivery, enabling effective expression of desired proteins (Fig. 6A–C). Regarding siRNA delivery, Zhao et al.45,75 prepared an inhalable and mucus-penetrative NP system incorporating siRNA against IL11 and KRAS-mutant NSCLC based on ionizable lipid compound G0-C14. This work presented a versatile NP platform for the locally inhaled delivery of siRNA therapeutics. It exhibited promising clinical potential in treating numerous respiratory diseases, including IPF and KRAS-mutant NSCLC (Fig. 6D and E). In addition, Ma et al.52 investigated the potential of inhaled star-siRNA NPs to accumulate in orthotopic mouse lung tumors to inhibit gene expression of βIII-tubulin and Polo-Like Kinase 1, which were upregulated in lung cancer cells and promoted tumor growth (Fig. 6F). These results indicated a proof-of-concept for inhaled delivery of RNA nanoparticles as a novel therapeutic strategy to treat respiratory-related disease and tumor.
5.　The applications of inhaled RNA delivery systems
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5.1　 SARS-CoV-2
5.1.1　 Systematic and mucosal immune responses
The SARS-CoV-2 virus, responsible for the COVID-19 pandemic, continues to evolve through mutations and genetic recombination, resulting in new variants121. These variants pose challenges to existing management strategies and vaccine efficacy122. Currently, mRNA vaccines like mRNA1273 (Moderna) and BNT162b2 (Pfizer) are widely used to provide systemic immunization against SARS-CoV-2141—143. However, these vaccines require injection and frozen storage, making accessibility and delivery difficult, particularly in lower-income-countries. To address these challenges, there is a need to explore alternative formulation and delivery methods, such as inhalation, for mRNA vaccines, which could offer easier storage requirements and enhanced mucosal protection144—146. Working from the principle of SARS-CoV-2 virus infection, this virus migrates towards the posterior region of the nasal passageways, where it binds to and penetrates host cells via the ACE2 present on the membrane of bronchial epithelial cells (Fig. 7A)147—149. After entering the dendritic cells (DCs), The two vaccine formulations-LNP or adenovirus (AdV) vectors encoding the S protein, could produce high levels of S protein, which were produced by the host cell and could induce an immune response. Meanwhile, the mRNA vaccines also shortened time using the body's molecular mechanisms (Fig. 7B). However, more than 90% of pathogens enter the body through the mucosal site. The primary site of SARS-Cov-2 replication is the upper respiratory tract mucosa. More importantly, type I mucosa tends to focus on the areas of the respiratory (the upper respiratory tract (URT) and the lower respiratory tract (LRT) tract mucosa. Mucosal DCs can migrate and transport antigens to systemic inductive sites such as the lymph nodes and spleen. It has often been demonstrated that mucosal vaccination induces robust systemic humoral immunity, eliminating virus particle that evades the main immune response at the mucosal site150,151. Equally important, effective protection against pathogens necessitates the coordinated engagement of both the systemic and mucosal immune responses, employing the production of both immunoglobulin G (IgG) and IgA antibodies. Therefore, it is essential to examine the persistence of vaccine efficacy and the vaccine-induced mucosal immunity for SARS-CoV-2 prevention (Fig. 7C–E)145,152,153. Recent results showed that SARS-CoV-2 virus was delivered to the respiratory tract through inhalation and attaches first to airway multi-cilia via the ACE2 receptor (Fig. 7F). Normally, viruses are impenetrable to the pericililiary layer. However, they can use motile cilia as tracks to access the cell body and achieve infection of ciliated epithelial cells. Moreover, upon initial SARS-CoV-2 virus contagion, the SARS-CoV-2 hijacks the host cell machinery to induce elongated and highly branched microvilli, and this microvillus enables the virus to exit across the PCL layer before lateral spread to other regions (Fig. 7G). Importantly, an anti-spike glycoproteinmonoclonal antibody that neutralizes SARS-CoV-2 inhibited attachment of SARS-CoV-2 to cilia and decreased infected cell numbers (Fig. 7H)147. Therefore, this mode of virus transmission indicates that the respiratory mucosae explicitly act as the immune system's frontline in response to viruses. Consequently, mucosal immunity has the potential to provide a robust defense to prevent initial infection and subsequent transmission. This represents a significant opportunity for the development of inhaled mRNA vaccines.
5.1.2　 Case studies
Mucosal immunization could stimulate mucosal IgA antibodies, which can capture and neutralize respiratory pathogens on the mucosal surfaces, thus providing the first line of defense against infection156—158. Recent scientific studies have utilized animal models and clinical trials to investigate the efficacy of mucosal vaccines against SARS-CoV-2. These studies have indicated that the combination of mucosal and systemic immunity can provide comprehensive protection against SARS-CoV-2155,154. For example, CanSino Biologics Inc. and Feng et al.159 for the first time, reported the safety, mucosal and systematic immunogenicity of inhaled adenovirus type-5 vector-based COVID-19 vaccine employing inhalation that was firstly utilized for SARS-CoV-2 vaccine delivery. The first inhaled COVID-19 vaccine induced robust humoral, cellular, and mucosal immunization responses, including IgG and IgA, and were elicited in all vaccinated people. Meanwhile, Ye et al.160 report a vaccine formulation that cannot only induce respiratory mucosal immunity after local lung delivery but is also inhaled as a dry powder, avoiding the need for cold chains and the use of needles. This confirmed that the developed inhalation delivery strategy was to have shots deliverable through the airways to generate mucosal immunity that today's injectable vaccines cannot provide.
Pulmonary drug delivery can be achieved via three different routes of administration: intranasal, intratracheal, and inhalation. Although inhaled vaccines can be delivered nasally160, can spread to part of the brain (the olfactory bulb) and are limited mainly to the URT rather than the LRT, thus posing a potential safety concern and efficacy if used in humans. In the second case, intratracheal delivery is an invasive procedure unsuitable for the pulmonary delivery of therapeutics in humans. Inhalation is the most common route and relies on normal breathing to administer aerosolized therapeutics through the airway. Liquid or dry powder vaccines can be inhaled through the mouth using an aerosol-generating nebulizer. The inhalation of the vaccine to the LRT aims to induce a range of immune responses, including the production of antibodies, activation of T cells, and stimulation of the innate immune system. Simultaneously, the vaccine also maintains immune responses in the URT and bloodstream (Fig. 8A). The carriers of actives in inhaled vaccines remain difficult with optimized physiochemical parameters. Most COVID-19 vaccines under clinical development are based on a viral-vector system and are delivered through the nose. Except for inhaled adenoviral delivery systems, exosomes are a class of naturally derived extracellular vesicles (approximately 100 nm in diameter) secreted from most cells. These have raised remarkable attention in drug delivery and immunization areas. Recently, Wang et al.161 developed room-temperature-stable inhalable lung-derived extracellular vesicles or exosomes (Lung-Exos) as mRNA and protein drug carriers. Compared with standard synthetic nanoparticle Lipos, Lung-Exos exhibited superior distribution to the bronchioles and parenchyma. They were deliverable to the lungs of rodents and nonhuman primates by dry powder inhalation. In a vaccine application, the SARS-CoV-2 spike (S) protein-encoding mRNA-loaded Lung-Exos (S-Exos) elicited greater IgG and secretory IgA responses than its loaded liposome (S-Lipo) counterpart. Importantly, S-Exos remained functional at room-temperature storage for one month. Their results suggested that extracellular vesicles could serve as an inhaled mRNA drug-delivery system superior to synthetic liposomes (Fig. 8B–E).
Meanwhile, Cheng and coworkers also designed a novel inhalable COVID-19 vaccine based on exosomes, with effective mucosal immune stimulation and long-term stability. Such vaccine consisted of a recombinant SARS-CoV-2 RBD conjugated to lung-derived exosomes, which, concerning liposomes, created the virus-like particles imitating the morphology of authentic virus and enhanced the retention of the RBD in both the mucus-lined respiratory airway and in lung parenchyma (Fig. 8F). In mice, the inhalation vaccine of RBD-Exo VLPs produced the highest amount of SIgA antibodies in nasopharyngeal lavage fluid (NPLF) and bronchoalveolar lavage fluid (BALF) (Fig. 8G). Furthermore, researchers estimated the effect of inhalable RBD-Exo VLPs on preventing high-dose live SARS-CoV-2 infection in the hamster model that could replicate serious diseases in the clinic. Compared with others, RBD-Exo VLPs groups showed lower virus detection amounts and the highest RBD-specific serum antibody concentration in every tested timepoint (Fig. 8H). Therefore, the inhalation vaccine was considered the most promising of all kinds of vaccine delivery strategies. It was expected to improve the people's willingness to vaccinate and further achieve global COVID-19 vaccine popularization. Their study provided a direction for the development of COVID-19 therapy and a reference for the treatment of other respiratory illnesses161.
5.2　 Influenza
Influenza virus is classified into four types: A, B, C, and D. Influenza virus can cause human influenza, avian influenza, swine influenza, equine influenza, and other diseases in humans and animals164—166. Although more than 130 influenza A subtype combinations have been detected in nature, mainly from wild birds, there are potentially many more influenza A subtype combinations due to the virus's “reassortment” ability167. As the virus has zoonotic potential and is capable of an antigenic shift, the gene reassortment of influenza A viruses of different origins results in highly contagious hybrid strains resistant to the existing therapy. Vaccination is the most common prevention strategy for influenza A virus infections. As mentioned in this review, inhaled vaccines stimulate mucosal immunity primarily, effectively preventing pathogen invasion and enhancing the immunoprophylactic effect of vaccination. Therefore, several studies have been conducted on inhalation vaccines for influenza virus prevention and control. Lokugamage et al.34 designed an LNP-mRNA named inhaled NLD1 to immunize mice against the influenza virus by optimizing the LNP formulation. They optimized the composition, molar ratios, and structure of LNPs made of lipids, neutral or cationic helper lipids, and PEG by using cluster-based workflows and then allowed for maximizing in vivo screening and delivery efficiency (Fig. 9A and B). It was also demonstrated that the mRNA carried by NLD1 could be transferred to ciliated bronchial epithelial cell, bronchial club cells, and alveolar type I and II in the lung epithelial cells and could continue to be translated in the body for up to a week (Fig. 9C and D). As expected with the inhaled viral dose, five of the six control mice died after progressively losing weight. In contrast, all of the mice treated with NLD1 survived, suggesting that this cluster-based screening system provided a novel approach for rapidly treating high-risk infectious diseases (Fig. 9E and F)34. Many studies have focused on applying the CRISPR/Cas13a system, which could target RNA for gene cutting.168, 169 Compared with traditional RNAi technology, Cas13A-mediated gene silencing has advantages, such as high efficiency and low off-targeting rate, thus theoretically having higher safety in disease treatment. Compared with Cas9-mediated gene knockout technology, Cas13D-mediated gene silencing does not alter genomic DNA, indicating that this gene silencing is reversible. Treating some acquired diseases, such as acquired metabolic diseases, is more advantageous. Santangelo et al. designed CRISPR RNAs specific for PB1 and highly conserved regions of PB2 of the influenza virus and selected the crRNAs that reduced viral RNA levels most efficiently in cell culture. A nebulizer delivered the polymer-formulated Cas13a mRNA and the validated guides to the respiratory tract. In mice, Cas13a degraded influenza RNA in lung tissue efficiently after inhalation. This finding suggested that Cas13a-mediated targeting of pathogenic viruses could mitigate respiratory infections (Fig. 9G–J)48. In general, regarding influenza virus prevention and control, most studies mainly focus on optimizing nucleic acid delivery vectors and screening targets. Although this 7C1 vector based on PEI improved the inhaled delivery efficiency of LNP through prescription optimization, only a few vectors have been reported so far. Therefore, further research on nucleic acid inhalation carriers is imperative to provide insights for future efficiency development. At the same time, further exploration of the broad spectrum of ways to combat influenza viruses should be conducted, and new technologies and new antiviral targets should also be discovered.
5.3　 Idiopathic pulmonary fibrosis (IPF)
IPF, a lethal respiratory disease with few treatment options, occurs due to repetitive micro-injuries to alveolar epithelial cells and progresses with an overwhelming deposition of extracellular matrix, ultimately resulting in fibrotic scars and destroying the alveolar architecture. IPF can cause lung infection, respiratory failure, pulmonary hypertension, pulmonary heart disease, heart failure, and other symptoms. Nowadays, inhaled delivery of nucleic acid drugs to treat IPF has been reported in many cases. Zhang et al.50 constructed a nanoparticle assembled by a ribosomal protein-condensed mRNA core, a bifunctional peptide-modified corona, and a keratinocyte growth factor (KGF) with a PEGylated shielding shell. When inhaled via a nebulizer, the nanoformulations carried by microdrops were deposited in the alveoli and penetrated fibrotic foci, where the outer KGFs were detached after matrix metalloproteinase 2 triggering. The core of the RGD-based graft then exposed and specifically targeted cells with elevated integrins for intracellular delivery of mRNA. The results suggested that repeated nanomaterial inhalation could synergically improve bleomycin-induced lung function in mouse models by promoting intralesional expression of MMP13 and KGFS-mediated alveolar re-epithelialization to accelerate local collagen clearance. In addition, Sahay et al. constructed an LNP based on ionizable lipids of DLin-MC3-DMA and substituted β-sitosterol for cholesterol to deliver mRNA encoding the cystic fibrosis transmembrane conductivity regulator (CFTR). The formulation was delivered to an animal model of CFTR deficiency after inhalation, resulting in lung expression of this therapeutic protein, significantly improving IPF (Fig. 10A–E)50. Kim et al.38 utilized PEG lipids to enhance the surficial stability of LNPs by including a cholesterol analogue, β-sitosterol, to improve endosomal escape. Increased PEG concentrations in LNPs enhanced the shear resistance and mucus penetration, while β-sitosterol provided LNPs with a polyhedral shape, facilitating endosomal escape. This study demonstrated the rational design approach for cystic fibrosis of inhalable LNP-based mRNA therapies (Fig. 10F and G).
Furthermore, Bai et al.75 obtained siIL11@PPGC NPs using nanoparticles prepared by lipid compounds coated with siRNA for IL11, which has the following outstanding advantages: 1) It has good stability, can withstand the shear force generated in the process of inhalation and keeps the particle size, morphology, cell uptake and encapsulation rate unchanged before and after inhalation; 2) could be condensed into a small volume; 3) After inhalation, it passed through the lung mucus layer and reached deep into the lung tissue. In vitro experiments showed that siIL11@PPGC NPs could significantly inhibit fibroblast conversion to myofibroblast, extracellular matrix deposition, and migration. In bleomycin-induced pulmonary fibrosis in mice, aerosol inhalation of siIL11@PPGC NPs effectively reduced the expressions of ACTA2 and COL1A1, significantly reduced hydroxyproline content, and improved fibrosis area and collagen content (Fig. 10H–J). The inhaled RNA drugs have a significant anti-fibrosis effect and significantly improve lung function in mice with pulmonary fibrosis, providing a new therapeutic method for repairing lung tissue damage and intervening in functional recovery after pulmonary fibrosis.
5.4　 Asthma
Asthma is a chronic, noncommunicable disease characterized by various degrees of airway inflammation, obstruction, mucus hypersecretion, and hyperresponsiveness. It represents the most prevalent and intractable disease due to its complex pathophysiology and multifactorial aetiology163. The global prevalence of asthma in adults is 4.3% and causes death in approximately 0.4 million people annually worldwide162,171. Traditional treatment methods, such as use of bronchodilators, anti-inflammatory drugs and Reducing mucolytic agent, could provide only symptomatic relief but hardly prevent the progressive deterioration of lung function in asthmatic patients172. It has recently been reported that inhaled delivery systems are fruitful in treating asthma37,173. For example, Zhang et al.37 exploited novel inhaled LNP targeting intercellular adhesion molecule-1 (ICAM-1) receptors on the apical side of AECs. A cyclic peptide that resembled part of the capsid protein of rhinovirus and bound to ICAM-1 receptor was initially conjugated with cholesterol and subsequently assembled with ionizable cationic lipids to form the LNP (Pep-LNP) loaded with siRNA against thymic stromal lymphopoietin (TSLP siRNA). After inhalation of the Pep-LNP-siTSLP, it was engulfed by AECs by ICAM-1 receptor-mediated endocytosis and remarkably downregulated the expression of TSLP in AECs and effectively alleviated inflammatory cell infiltration, and reduced the secretion of other proinflammatory cytokines (Fig. 11A–E). These results suggested that inhaled Pep-LNP-siTSLP could be a promising therapy to alleviate epithelium-mediated inflammatory responses in asthmatic conditions. Another recent study confirmed the feasibility of efficient inhaled lung drug delivery for asthma treatment. Keil et al.170 first identified a suitable transferrin receptor-mediated uptake pathway to target efficiently and specifically activated TH2 cells with a melittin-PEI conjugate, forming polyplexes with siRNA. The new formulation showed improved endosomal escape and gene silencing efficacy. Additionally, to develop a clinically relevant dosage form for pulmonary delivery of siRNA, they have lately focused on a dry powder formulation by spray drying to produce inhalable nano-in-microparticles. Their efforts were devoted to the development of a novel treatment for asthma that could be translated from bench to bedside. In this work, the inhalation of multifunctional nanogels was developed for asthma treatment (Fig. 11F). Therefore, the application of inhaled RNA therapy in asthma disease will provide new ideas for the treatment of asthma and other related diseases.
5.5　 Lung cancer
Lung carcinoma is one of the most common cancers and has one of the lowest survival rates in the world. RNA-based therapies have gained much attention as biomedicines due to their remarkable therapeutic effects and high specificity and potency. Inhaled RNA drugs for the treatment of lung disease may have a good potential for application. This chapter summarized the use of inhaled delivery systems for different types of RNA in anti-tumor research for lung cancer. Liu's group53 developed an inhalable exosome loaded with IL-12 mRNA for inhaled mRNA therapy. Inhalation administration leads to targeted administration and fewer systemic side effects, with IL-12 mRNA producing interferon-gamma in both the primary and adaptive immune cell populations. Activation of the tumor microenvironment increases immunogenicity, resulting in a more robust immune response. This enhanced response facilitates the expansion of cytotoxic immune cells, immune memory formation, improved antigen presentation, and activation of tumor-specific T cells. This approach shows promise in treating both situ and metastatic lung tumors (Fig. 12A–C). Similarly, Patel et al.8 synthesized hPBAEs to enable the nanoformulation of stable and concentrated polyplexes suitable for inhalation. This strategy achieves uniform distribution of luciferase mRNA throughout all five lung lobes and produces stable luciferase protein expression 24 h after inhalation of hPBAE polyplexes. Delivery is localized to the lung; no luminescence is observed in other tissues. Furthermore. Repeated dosing of inhaled hPBAE-mRNA generates consistent protein production in the lung without local or systemic toxicity. The results indicate that inhaled delivery of IVT-mRNA facilitated by hPBAE vectors may provide a clinically relevant delivery system for lung cancer (Fig. 12D and E). In the inhaled miRNA study, Zhang et al.56 investigated the efficacy of inhaled let-7b miRNA treatment in lung cancer prevention. Inhaled let-7b mimic showed significant inhibition of lung adenoma by immune-promoting effects via downregulating PD-L1 in tumors or PD-1 on CD8+ T cells. These changes potentiated antitumor CD8+ T cell immune responses. The results suggested that this inhaled let-7b mimic was a promising approach for lung cancer prevention (Fig. 12F–H). Finally, research on the inhaled delivery of siRNA for lung cancer has also demonstrated its tremendous potential. Zhao et al.45 investigated the antitumor efficacy of inhaled siKRAS@GCLPP NPs for KRAS-mutant non-small-cell lung cancer utilizing a murine orthotopic lung cancer model. Their findings suggested that inhaled siKRAS@GCLPP NPs could deliver equivalent effectiveness to intravenously injected NPs while reducing the adverse effects associated with systemic administration. Inhalable siKRAS@GCLPP NPs showed significant tumor-targeting capability and enhanced antitumor activity in an orthotopic mouse model of human KRAS-mutant NSCLC. This work presented a new avenue for noninvasive inhaled siRNA delivery that holed tremendous potential for treating KRAS-mutant NSCL (Fig. 12I and J). The above studies indicated that RNA delivered through the respiratory tract had good research value for lung cancer treatment. However, there are still few relevant studies and more attempts are needed to develop new delivery systems, select new targets, and explore more clinical translational approaches.
5.6　 Obstructive pulmonary disease (COPD) and tuberculosis (TB)
COPD is a common severe chronic pulmonary disease characterized by airway inflammation, airflow obstruction, as well as damage to the lung parenchyma. COPD is a complex and multifactorial respiratory disease that is caused by environmental factors, for example, tobacco smoking, air pollution, allergens, genetic factors, or occupational risks. Major pathological features of COPD are obstructive bronchiolitis, emphysema, pulmonary hypersecretion, and small airway obstruction (Fig. 13A)176. Current therapeutics for COPD are largely borrowed from the drug armamentarium for the treatment of asthma, which has different pathophysiological mechanisms from COPD. COPD shows a predominant involvement of peripheral airways (bronchioles) and lung parenchyma, which COPD has been linked to dysregulated expression of mRNAs and noncoding RNAs, including miRNAs, PIWI-interacting RNAs, long noncoding RNAs, and circular RNAs (Fig. 13B)177. Identifications of disease-triggering pathways and gene targets for COPD have opened an opportunity for inhaled RNA therapeutics. However, clinical translation has remained limited due to insufficient understanding of the intricate disease mechanism and a lack of robust and predictive animal models. TPI 1100, which consists of ASO targetingphosphodiesterases (PDE4 and PDE7), is the only reported RNAi-based product launched into phase I clinical trial to treat COPD. However, the pipeline was soon terminated. Recent pre-clinical experiments have used non-RNA drug inhalation therapy178 or RNA drug pulmonary administration (non-inhalation therapy)179 and have been mainly dedicated to targeting inflammatory cytokines. Of note, more and more research on inhaled RNA therapy may shift the current paradigm of COPD management. Tuberculosis is the infectious disease that causes the most deaths per year worldwide. TB is caused by Mycobacterium tuberculosis (Mtb), which most frequently enters the body through the respiratory route. Despite the potential therapeutic regimens comprising both the conventional drugs (isoniazid and rifampicin) and the newly developed chemotherapy approaches that are now undergoing various stages of clinical trials, the threatening prevalence of multidrug-resistant and extensive-drug resistant-TB keeps calling for safer and more efficient treatment methods. Moreover, exposure of the mycobacteria to sub-therapeutic levels of anti-TB drugs during treatment is a driver for the emergence of drug-resistant microbial strains180. One effective approach to increase the bactericidal effect of antibiotics is to achieve a high local concentration of the drug by localized drug delivery, such as inhalation therapy. Although the COVID-19 pandemic has impacted TB diagnosis and treatment caused by the SARS-CoV-2, the RNA-based may present a golden opportunity to accelerate the development and approval of new potentially effective TB vaccine candidates. RNA interference (RNAi) therapy targeting TB is being investigated to enhance host antibacterial capacity or improve drug efficacy on drug resistance strains while minimizing the associated adverse effects. One of the key challenges of RNAi therapeutics arises from the delivery of the RNAi molecules into the target cells. Inhalation could be a direct administration route for treating pulmonary TB non-invasively (Fig. 13C)174. The clinical pipeline includes an mRNA-based candidate (BNT164a1/BNT164b1, BioNTech) recently entering phase I clinical trials in Germany (NCT05547464) and South Africa175. Recently, this pioneering project, “RNA inhaled vaccines against Tuberculosis”, led by Certest Biotec in collaboration with the University of Zaragoza and the University of the Basque Country, aims to develop a new inhaled RNA vaccine against tuberculosis as a booster for current and future vaccines against the disease. All of the above studies indicate the potential of inhaled RNA therapy in treating obstructive pulmonary disease and tuberculosis.
6.　Conclusions and perspective
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In the past decade, RNA therapies have had an exciting potential to greatly improve our understanding of disease and treatment. Inhalation delivery of therapeutic aerosols in treating respiratory tract diseases allows for targeted delivery, minimizing off-target dosing and associated side effects. This review systematically expounds on the principle, RNA classifications, and application of inhalation delivery systems. We first mentioned the nebulizers used in the inhalation systems and compared the characteristics of different inhalers. Different nebulizers have their advantages and disadvantages, and they have a diverse application range. Of these devices, nebulizers and DPIs are of particular interest since they are distributed easily and the administration is simple. In terms of inhaled vaccines, we hope it has been deposited in the lower respiratory tract. Liquid or dry powder vaccines can also be inhaled through the mouth using an aerosol-generating nebulizer or dry powder inhaler. Meanwhile, we hope to make nebulizers more portable and simplified, which could greatly optimize patient use. Different patients need to deposit the drug in different places, and the smaller the particle, the deeper the drug can go into the respiratory tract. Moderate inhalation power is suitable for ordinary use, but for acute attack patients, it is more suitable for immediate high-power inhaled treatment. Therefore, the power of inhalers and aerosol particle size can also become controllable, which will become the development trend That is convenient for carrying and cleaning. In addition, Early method development work can ascertain the maintenance of desired integrity for a gene therapy formulation–device combination. For particularly sensitive formulations, optimization of nebulizers and other delivery devices is crucial to mitigate aerosolization stresses in inhalation therapy. Despite promising preclinical findings on candidate inhaled RNA therapies, the outcomes of human trials have been disappointing. The reason is that the challenges encountered in inhalation therapy pertain to numerous biological and otherwise barriers that impede the effective treatment of diseases through RNA therapy aerosols. The distribution of inhaled therapeutics in the lungs is determined by the aerodynamic diameter of RNA loaded. Such complex anatomical patterns will further increase the barriers to the clinical translation of RNA therapeutics to human patients. The other reason lies in using mice as the animal model in most in vivo investigations. Due to major differences in lung physio-anatomy between mice and humans, the pre-clinical data cannot be easily extrapolated to human patients. We therefore recommend using lung on a chip, in vitro cell models, larger animal models (e.g., non-human primates, ferrets and pigs), and developing more inhalation-based RNAi therapeutics to increase the probability of future use in clinics. Next, we summarized the research progress on siRNA, ASO, and other types of RNA inhaled delivery. A related advance was the need to understand how chemical modifications to the RNA payload influence RNA stability avoidance of intracellular off-target effects. Much of the clinical and preclinical research has focused on siRNA and mRNA for the inhaled delivery of RNA. With the vast growth of RNA-based therapies, Various other types and functions of RNA will also be attempted for inhalation delivery to treat respiratory infections and other pulmonary diseases.
To better accelerate the clinical translation of non-viral platforms, rigorous material screening, and optimization should be conducted to maximize RNA delivery efficiency. Although various types of inhaled delivery carriers have been reported, such as chemical delivery carriers, including polymer, polycomplex, cationic lipid, and ionizable lipids, these delivery systems all have their specific advantages. Polymer and polycomplex have the advantages of stable delivery and high efficiency, but quality control is difficult. However, the traditional cationic lipid has the advantages of a high encapsulation rate and stable transfection, but its safety and delivery efficiency are not as good as ionizing lipids. Currently, two commercially available mRNA vaccines are based on ionizable lipid delivery systems, which seem to have obvious advantages. However, more studies are still needed on optimizing lipid structure and delivery system prescription. Recently, with the rapid development of more ionizable lipid materials and formulation optimization of LNPs, such as CKK-E12, DLin-MC3-DMA, G0-C14, SM102, IR-117-17, IR-19-Py and C12-200, the field of nebulized mRNA delivery have attracted increasing attention. Additional research should be pursued, and more innovations should be made in the structure of ionizable lipids and prescription optimization of delivery systems. Another kind of biomaterial delivery system is also used for inhaled delivery of RNA. Such carriers include AAV and EVs. Although these biological delivery carriers can successfully deliver nucleic acid drugs to the lungs, it is still unclear which biological delivery system is suitable for inhaled delivery of nucleic acid drugs—inhaled delivery carriers, whether viral or nonviral vectors have advantages and disadvantages. In addition, few studies have compared these biomaterial delivery vectors with chemical delivery vectors. Therefore, we are not sure which type of delivery nonviral vectors are more suitable for inhaled delivery of nucleic acid drugs, and much relevant research is still needed.
RNA inhalation delivery can be used to treat various diseases, and it is reported in SARS-CoV-2, Influenza, pulmonary fibrosis, Asthma, lung cancer, and other pulmonary diseases. Future research should focus on an efficient and safe delivery system, selecting potential disease targets for RNA therapies, designing specific drugs for specific viral infections, and broad-spectrum antiviral drugs for multiple viral infections to address the challenges of viral mutations. In cancer biology, the researchers found that using genetically engineered mice more accurately recreated clinical results from human trials carried out in parallel. Suppose we can expand our understanding of how the RNA drug, drug delivery system, and body interact. In that case, patients will benefit from effective next-generation inhaled RNA therapies. Regrettably, preclinical research for inhalation RNA delivery remains somewhat under-described in the literature, perhaps due to the nascent nature of the field. We encourage researchers and sponsors to publish methods and techniques used during preclinical evaluations of inhalation RNA therapy products to aid in refining experimental setups and analysis for future inhaled RNA therapy programs. We expect to see accelerating interest in gene therapy products targeting inhalation delivery. We may seek application innovations in RNA inhalation delivery to develop atomized drugs suitable for various diseases in the coming years.
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Year 2025 volume 15 Issue 5
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doi: 10.1016/j.apsb.2025.03.033
  • Receive Date：2024-09-18
  • Online Date：2026-09-17
Article Data
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  • Received：2024-09-18
  • Revised：2024-12-28
  • Accepted：2025-01-15
Affiliations
    aKey Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Department of Chemistry, Tsinghua University, Beijing 100084, China
    bState Key Laboratory of Biotherapy and Cancer Center, Research Unit of Gene and Immunotherapy, Chinese Academy of Medical Sciences, Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China
    cInstitute for Immunology, School of Basic Medical Sciences, Tsinghua University, Beijing 100084, China
    dVascular Surgery Center, the Third Hospital of Jilin University, Changchun 130031, China

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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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