mRNA therapeutics have emerged as a promising strategy for the treatment of various diseases, especially respiratory diseases, such as cystic fibrosis, pulmonary hypertension, and COVID-19. However, the delivery of mRNA to the target cells and tissues poses significant challenges, such as low stability, poor cellular uptake, and undesired immune responses. Therefore, the development of effective and safe mRNA delivery systems is essential for the clinical translation and application of mRNA therapeutics. Pulmonary delivery of mRNA is a non-invasive and direct route of administration that offers several advantages over other routes, such as intravenous, intramuscular, or subcutaneous injection. Pulmonary delivery can enhance the local and systemic delivery of mRNA to the respiratory tract, where many diseases originate or manifest, and also reduce the dose requirement and the systemic toxicity of mRNA therapeutics, as well as the potential risk of infection or inflammation associated with invasive routes. Moreover, pulmonary delivery can facilitate the expression of therapeutic proteins in the lung epithelium, endothelium, or alveolar macrophages, which are critical for the modulation of immune responses and the restoration of lung function.
To protect mRNA from degradation and enhance its cellular uptake, various materials have been used to formulate mRNA delivery systems. These materials can be classified into four main categories: lipid nanoparticles, polymers, peptides, and hybrid systems.
Lipid nanoparticles (LNPs) are one of the most widely used and successful delivery systems for mRNA. LNPs are composed of four main components—cationic or ionizable lipids, helper lipids, cholesterol, and polyethylene glycol (PEG) lipids. The cationic or ionizable lipids can form electrostatic interactions with the negatively charged mRNA and also facilitate the endosomal escape of mRNA by proton sponge effect or pH-dependent membrane destabilization. The helper lipids and cholesterol can improve the stability and fluidity of the lipid bilayer, and the PEG lipids can provide steric stabilization and prolong the circulation time of LNPs.
LNPs have several advantages as mRNA delivery systems, such as high encapsulation efficiency, high transfection efficiency, low immunogenicity, and easy scalability. LNPs can also be modified with targeting ligands, such as antibodies, aptamers, or peptides, to enhance the specificity and selectivity of mRNA delivery to the desired cells or tissues.
However, LNPs also have some limitations, such as potential toxicity, accumulation in the liver and spleen, and instability under physiological conditions. Moreover, LNPs may induce innate immune responses, such as the activation of toll-like receptors (TLRs) or inflammasomes, which may affect the expression and function of the mRNA-encoded proteins.
LNPs have been extensively explored for pulmonary mRNA delivery, especially for the prevention and treatment of respiratory infections such as influenza, respiratory syncytial virus (RSV), and coronavirus. For example, Pardi et al. demonstrated that intranasal or intratracheal administration of LNPs encapsulating mRNA encoding the hemagglutinin (HA) protein of influenza virus induced robust and durable antibody and T cell responses in mice and protected them from lethal challenge with different strains of influenza virus. Similarly, Petsch et al. showed that intranasal delivery of LNPs containing mRNA encoding the fusion (F) protein of RSV elicited potent and long-lasting immune responses in mice and non-human primates and conferred protection against RSV infection. More recently, Sahin et al. reported that intramuscular injection of LNPs carrying mRNA encoding the spike (S) protein of SARS-CoV-2 induced high levels of neutralizing antibodies and T cell responses in humans and showed high efficacy and safety in phase 3 clinical trials.
Polymers are another common type of material for mRNA delivery systems. Polymers can be either natural or synthetic, form complexes with mRNA through electrostatic interactions, hydrogen bonding, or covalent bonding, provide protection, stability, and biodegradability for mRNA, and can be modified with various functional groups to improve the delivery efficiency and specificity.
Some examples of natural polymers used for mRNA delivery are chitosan, hyaluronic acid, and protamine. Chitosan is a cationic polysaccharide derived from chitin that can bind to mRNA and form nanoparticles with mucoadhesive properties. Chitosan nanoparticles can enhance the uptake and expression of mRNA in lung epithelial cells and can also modulate immune responses by activating macrophages and dendritic cells. Hyaluronic acid is an anionic glycosaminoglycan that can interact with mRNA through hydrogen bonding and can also target the CD44 receptor expressed in many cell types, including lung cancer cells. Hyaluronic acid nanoparticles can deliver mRNA to the lung tissue and induce the expression of therapeutic proteins, such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Protamine is a cationic peptide that can condense mRNA into compact and stable nanoparticles and can also facilitate the endosomal escape of mRNA by membrane disruption. Protamine nanoparticles can deliver mRNA to the lung, induce the expression of antigens such as ovalbumin (OVA) or HA, and elicit strong immune responses.
Some examples of synthetic polymers used for mRNA delivery are polyethylenimine (PEI), poly(lactic-co-glycolic acid) (PLGA), and poly(beta-amino ester) (PBAE). PEI is a cationic polymer that can bind to mRNA and form nanoparticles with high transfection efficiency and endosomal escape ability. However, PEI also has high toxicity and immunogenicity, which limit its clinical application. PEI nanoparticles can deliver mRNA to the lung and induce the expression of therapeutic proteins, such as erythropoietin (EPO) or interferon beta (IFN-β). PLGA is a biodegradable and biocompatible polymer that can encapsulate mRNA and form nanoparticles with sustained release and low toxicity. PLGA nanoparticles can deliver mRNA to the lung, induce the expression of antigens, such as OVA or S, and generate potent immune responses. PBAE is a cationic polymer that can bind to mRNA and form nanoparticles with high transfection efficiency and low toxicity. PBAE nanoparticles can deliver mRNA to the lung and induce the expression of therapeutic proteins, such as luciferase or vascular endothelial growth factor (VEGF).
Peptides are short sequences of amino acids that can form complexes with mRNA through electrostatic interactions, hydrophobic interactions, or covalent bonding. Peptides can also mediate the cellular uptake, endosomal escape, and intracellular trafficking of mRNA and can be modified with targeting ligands, such as cell-penetrating peptides (CPPs), to enhance the delivery specificity and efficiency.
Some examples of peptides used for mRNA delivery are histidine-lysine (HK), arginine-rich peptides, and elastin-like polypeptides (ELPs). HK is a cationic peptide that can bind to mRNA and form nanoparticles with high stability and transfection efficiency. HK nanoparticles can deliver mRNA to the lung and induce the expression of therapeutic proteins, such as erythropoietin (EPO) or superoxide dismutase (SOD). Arginine-rich peptides are a class of CPPs that can bind to mRNA and form nanoparticles with high cellular uptake and endosomal escape ability. Arginine-rich peptides can also target lung tissue by binding to the heparan sulfate proteoglycans (HSPGs) expressed on the lung epithelial cells. Arginine-rich peptides can deliver mRNA to the lung, induce the expression of antigens, such as OVA or S, and elicit strong immune responses. ELPs are a class of thermoresponsive peptides that can bind to mRNA and form nanoparticles with high stability and biocompatibility. ELPs can also target lung tissue by accumulating in the lung capillaries due to their phase transition behavior. ELPs can deliver mRNA to the lung and induce the expression of therapeutic proteins, such as luciferase or TRAIL.
Hybrid systems are composed of different classes of materials, such as lipids, polymers, and peptides, to combine the advantageous aspects of each material and overcome their limitations. Hybrid systems can provide enhanced protection, stability, efficiency, specificity, and biodegradability for mRNA delivery and can also modulate the immune responses and biodistribution of mRNA.
Some examples of hybrid systems used for mRNA delivery are lipid-polymer hybrid nanoparticles, lipid-peptide hybrid nanoparticles, and polymer-peptide hybrid nanoparticles. Lipid-polymer hybrid nanoparticles are composed of a polymer core and a lipid shell, which can encapsulate mRNA and form nanoparticles with high stability, transfection efficiency, and biocompatibility. Lipid-polymer hybrid nanoparticles can deliver mRNA to the lung and induce the expression of therapeutic proteins, such as luciferase or TRAIL. Lipid-peptide hybrid nanoparticles are composed of a lipid core and a peptide shell, which can bind to mRNA and form nanoparticles with high cellular uptake, endosomal escape, and targeting ability. Lipid-peptide hybrid nanoparticles can deliver mRNA to the lung, induce the expression of antigens, such as OVA or S, and generate potent immune responses. Polymer-peptide hybrid nanoparticles are composed of a polymer core and a peptide shell, which can bind to mRNA and form nanoparticles with high stability, transfection efficiency, and biodegradability. Polymer-peptide hybrid nanoparticles can deliver mRNA to the lung and induce the expression of therapeutic proteins, such as luciferase or VEGF.
Pulmonary administration of mRNA is a non-invasive and direct route of delivery that can target the respiratory tract, where many diseases originate or manifest. Pulmonary delivery can also reduce the dose requirement and the systemic toxicity of mRNA therapeutics, as well as the potential risk of infection or inflammation associated with invasive routes. Moreover, pulmonary delivery can facilitate the expression of therapeutic proteins in the lung epithelium, endothelium, or alveolar macrophages, which are critical for the modulation of immune responses and the restoration of lung function.
However, pulmonary delivery of mRNA also faces several challenges, such as the physical and biological barriers of the lung, the clearance and degradation of mRNA, and the variability and reproducibility of the delivery process. Therefore, the selection of appropriate routes and devices for pulmonary administration of mRNA is crucial for the optimization and standardization of delivery efficiency and safety.
There are three main routes for pulmonary administration of mRNA: inhalation, intratracheal injection, and aerosolization. Each route has its advantages and disadvantages, depending on the target site, the delivery device, and the formulation of mRNA.
Inhalation is the most common and convenient route for pulmonary administration of mRNA, which involves the delivery of mRNA through the mouth or nose into the lungs. Inhalation can target the upper or lower airways, depending on the particle size, shape, and density of the mRNA formulation. Inhalation can also achieve a rapid onset of action, as the lung has a large surface area and a high blood flow.
Intratracheal injection is an invasive route for pulmonary administration of mRNA, which involves the delivery of mRNA through a catheter or a syringe into the trachea. Intratracheal injections can target the lower airways and the alveoli and achieve a high and uniform distribution of mRNA in the lung. Intratracheal injection can also avoid the loss or degradation of mRNA in the upper airways or the environment.
Aerosolization is an emerging route for pulmonary administration of mRNA, which involves the delivery of mRNA through a spray or a mist into the lungs. Aerosolization can target the entire respiratory tract and achieve a large and deep delivery of mRNA in the lung. Aerosolization can also avoid the need for anesthesia or intubation and reduce the invasiveness and side effects of mRNA delivery.
| Route | Description | Advantages | Disadvantages |
|---|---|---|---|
| Inhalation | Breathing in mRNA-containing particles or droplets through the nose or mouth | Non-invasive, easy to use, target upper or lower respiratory tract | Low efficiency, variable deposition, potential immune response |
| Intratracheal injection | Injecting mRNA directly into the lung tissue through a needle or catheter inserted into the trachea | High efficiency, accurate delivery, target specific regions of the lung | Invasive, requires anesthesia and surgery, potential tissue damage |
| Aerosolization | Spraying mRNA-containing aerosols into the airways through a bronchoscope or endotracheal tube | Semi-invasive, target alveoli and bronchioles, where gas exchange occurs | Requires anesthesia and specialized equipment, potential airway irritation |
Table 1. Pulmonary Administration of mRNA: A Comparison of Three Routes
In conclusion, pulmonary mRNA delivery systems represent a promising and emerging field of research and development, with significant implications for the advancement of respiratory medicine and biotechnology. Pulmonary mRNA delivery systems have shown great potential for the prevention and treatment of various respiratory diseases, such as cystic fibrosis, asthma, and COVID-19. Pulmonary delivery can enhance the local and systemic delivery of mRNA to the respiratory tract, where many diseases originate or manifest. Pulmonary delivery can also reduce the dose requirement and the systemic toxicity of mRNA therapeutics, as well as the potential risk of infection or inflammation associated with invasive routes. Moreover, pulmonary delivery can facilitate the expression of therapeutic proteins in the lung epithelium, endothelium, or alveolar macrophages, which are critical for the modulation of immune responses and the restoration of lung function.
