Chem. Pharm. Bull. 74, 28–36 (2026) Vol. 74, No. 1 https://doi.org/10.1248/cpb.c25-00532 Current Topics
Introduction to Various Inhaled Formulation Technologies Supporting Diverse Therapeutic Modalities Review
Advancements in Inhalation Technologies for Pulmonary Delivery of Protein Therapeutics Mayumi Ikeda-Imafuku,* Hiroko Fukuda, Tatsuya Fukuta, and Kazunori Kadota* Department of Physical Pharmaceutics, School of Pharmaceutical Sciences, Wakayama Medical University, 25–1 Shichibancho, Wakayama 640–8156, Japan. * Correspondence: imayu@wakayama-med.ac.jp; kazunori-kadota@wakayama-med.ac.jp Received July 31, 2025 Inhalation delivery of protein therapeutics has emerged as a promising non-invasive alternative to traditional injectable formulations that offers potential for both localized and systemic treatment of pulmonary diseases. This review comprehensively summarizes the current advances in inhalable protein formulations, with emphasis on design strategies, formulation technologies, barriers to effective delivery, and diseasespecific applications. Key aspects include the role of particle size, surface charge, and protein engineering in optimizing lung deposition and cellular uptake, as well as techniques such as spray freeze drying and PEGylation to enhance protein stability. The review also explores novel therapeutic approaches that target cystic fibrosis, asthma, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, lung infections, and cancer, including the use of antibodies, nanobodies, exosomes, and albumin-based carriers. Clinical translation remains limited, but ongoing innovation in delivery systems and molecular design is thought to hold significant promise for expanding the therapeutic landscape of inhaled protein drugs. Key words
der inhaler (DPI) delivery, including the use of PEGylation. Finally, we discuss the challenges and future of the clinical translation of protein-based nanoparticles.
The development of therapeutic agents utilizing proteins as active pharmaceutical ingredients has accelerated in recent years, driven by advances in biopharmaceuticals.1,2) Intravenous administration has traditionally been the primary route for protein-based formulations. However, as an efficient, non-invasive alternative, inhalation of proteins has gained increasing attention.3) The lungs provide an ideal site for drug absorption due to their large surface area, extensive vascularization, and the ability to bypass first-pass metabolism.4) Nonetheless, pulmonary administration of proteins presents several challenges, including enzymatic degradation, particle deposition, and potential immune responses. In practice, many protein-based inhalation therapies under development are designed to exert their effects locally within the lungs. Pulmonary delivery aimed at achieving systemic effects has been primarily investigated in the context of treatment for diabetes, particularly with insulin and other related peptides (e.g., Afrezza®).5) In this review, we summarize the latest research trends in protein inhalation formulations, focusing on design principles, formulation technologies, immunological safety, and diseasespecific applications. We also highlight recent advances in drying techniques and nanoparticle engineering for dry pow- 2.
Protein-based inhalation formulations include therapeutic antibodies, cytokines, enzymes, and other bioactive proteins, as well as protein-based drug carriers. Among U.S. Food and Drug Administration (FDA)-approved protein therapeutics across all routes of administration, monoclonal antibodies represent the largest proportion, accounting for approximately half of the total.6) Other approved protein drugs include coagulation factors, enzymes, fusion proteins, hormones, vehicles, and growth factors. When used as drug delivery carriers, proteins are valued for their high biocompatibility and biodegradability. Serum albumin as an endogenous component in human serum has been extensively studied as a drug carrier due to its high drug-binding capacity, favorable permeability, and low immunogenicity.7,8) Serum albumin has also been well studied in nanoformulations, which are used to improve uptake and drug loading.9–11) Other proteins from natural products, such as silk fibroin and gelatin, have also attracted attention as formulation materials capable of modulating particle rigidity and disintegration properties.12–14)
© 2026 The Author(s). This is an open access article distributed under the terms of Creative Commons Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/). Published by The Pharmaceutical Society of Japan. 28
Chem. Pharm. Bull. Vol. 74, No. 1 (2026) been comprehensively reviewed elsewhere.20) Unlike intravenously injectable formulations, there are currently no clear regulatory standards regarding protein aggregates in inhalable products. The establishment of new guidelines for both formulation design and analytical evaluation is therefore urgently required. 3.3. Interfacial Stress during Inhalation Interfacial stress in inhalation formulations refers to the physicochemical impact of drugs or nanoparticles on the pulmonary air–liquid interface, particularly the surfactant film lining the alveoli. To assess the ‘interfacial stress’ exerted by inhaled aerosols on lung surfactant function, Bäckman et al. developed a modified constrained drop surfactometer (CDS), which enabled quantification of drug or particle effects via surface tension.26) Micronized albumin was demonstrated to impair surfactant function, with minimum surface tension exceeding 10 mN/m, thereby compromising interfacial stability. In contrast, inhaled corticosteroids and lactose did not exhibit such effects. The surface activity of protein-based formulations is suggested by these findings to interfere with pulmonary surfactant function, which highlights the importance of CDS-based evaluation in toxicity assessment. Surface charge is also a critical parameter influencing interfacial stress. Positively charged nanoparticles reportedly electrostatically interact with pulmonary surfactant, forming microscale particle–vesicle complexes.27) Such aggregationinduced structural alterations are thought to impair the biophysical function of the surfactant layer. 3.4. Control of Particle Distribution In inhalation formulations, a particle size range of 1–5 µm is considered to be optimal for pulmonary deposition, and a narrow particle size distribution, reflected by a low geometric standard deviation, contributes to consistent inhalation performance.24) Particles that are too large tend to deposit in the oropharyngeal region, while those that are too small are likely to be exhaled before reaching the lungs. Precise control of the aerodynamic particle size is therefore critical. To investigate the impact of initial particle size, paclitaxel was encapsulated in albumin microparticles of 0.5, 1.0, and 3.0 µm, and formulated into dry powders with a consistent aerodynamic diameter (approx. 5 µm).28) Their intrapulmonary distribution and antitumor activity were assessed in a murine lung cancer model. The formulations prepared from particles with initial sizes of 1.0–3.0 µm demonstrated sustained drug release and prolonged pulmonary retention, which resulted in good therapeutic outcomes. These findings underscore the significance of the initial particle size as a critical factor in the therapeutic efficacy of inhalable formulations and they highlight the need to carefully consider this parameter during formulation design and quality control.
Modifications to aerodynamic particle size, surface charge, and hydrophobicity are well known to greatly influence adhesion to alveolar epithelial cells and the efficiency of endocytosis, primarily by affecting the interaction with and penetration through the mucus layer.15) These design parameters also affect the site of particle deposition (e.g., bronchi vs. alveoli) and the risk of clearance by alveolar macrophages. Additionally, proteins have been used as excipients in some formulations. For example, the inclusion of albumin as an excipient has been reported to reduce particle aggregation, to enhance spherical morphology and uniformity, and to improve respirable deposition.16)
3.1. Enzymes The lungs contain a variety of metabolic enzymes, including CYPs and esterases, which can significantly affect the local efficacy, systemic exposure, and safety of administered drugs.17) Neutrophil elastase, a serine protease stored in the primary granules of neutrophils, plays a critical role in host defense against bacterial infections.18) Neutrophil elastase transcriptionally and post-translationally activates mucin genes, thereby inducing excessive production and secretion of airway mucus. Its secretion is elevated in pulmonary diseases such as cystic fibrosis, chronic obstructive pulmonary disease (COPD), and in acute lung injury.18,19) Matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, are upregulated in acute respiratory distress syndrome and act as key mediators of tissue damage and inflammatory amplification.19) These proteolytic enzymes may impair the therapeutic efficacy of protein-based inhalation formulations. The expression of these enzymes can vary depending on the pathological condition, so it is crucial to perform disease-specific evaluations during formulation development. 3.2. Aggregation Protein aggregation is a major difficulty in the development of inhalable protein formulations. Physical stresses associated with spray drying or nebulization (i.e., shear forces, thermal exposure, air-liquid interfacial stress, and rapid dehydration) can induce structural denaturation and aggregation, potentially leading to local immune responses or toxicity in the lungs.20) Depending on the size and type of aggregates, clearance by alveolar macrophages may be hindered, so there are concerns about the induction of chronic inflammation. In the preparation of DPIs, thermal denaturation is a concern when drying is performed using an oven.21) Typical spray-drying processes involve drying temperatures ranging between 60 and 100 °C, which can cause irreversible heatinduced denaturation of proteins.22–24) To avoid such thermal degradation, there are some promising alternative technologies, such as lyophilization and freeze spray drying.21,25) Other stabilization strategies include the addition of excipients such as trehalose, mannitol, and amino acids (e.g., glycine) to preserve hydrogen bonding and suppress structural denaturation.24) Such additives are particularly effective due to their ability to replace water molecules through hydrogen bonding (the water replacement hypothesis) and to form a glassy matrix upon drying, thereby restricting molecular mobility and stabilizing protein structure. Alternatively, hydrophobic polymers and surfactants may be employed to prevent denaturation at the air–liquid interface. The risks and mechanisms of protein aggregation in inhalation formulations have
4.1. Drying Methods for Protein Drugs in DPI Formulations Several drying techniques have been explored for the development of protein-based dry powder inhalers, including lyophilization, spray drying, spray freeze drying (SFD), and supercritical fluid (SCF) drying (Fig. 1). Among them, SFD has been suggested to have considerable promise for particle engineering and inhalation applications because it has the advantage of preventing protein aggregation. In addition, emerging methods, such as supercritical drying, are gaining atten29
Difference in the Drying Process for DPI Formulation tion as potential alternatives.29) The choice of drying method should be optimized based on the physicochemical properties of the protein, the selected excipients, and the intended therapeutic application. In spray drying, atomized liquid droplets are dried by exposure to hot air, often yielding uniform, spherical particles with good aerodynamic properties.30,31) In protein formulation, parameters such as the protein concentration during atomization greatly influence the resulting particle morphology and aerosolization efficiency, as shown in studies that used serum albumin.32) SFD, in contrast, subjects the formulation to much lower thermal stress than conventional spray drying, and it tends to produce porous particles that are favorable for inhalation.33,34) Compared with conventional freeze drying, SFD has also been shown to reduce oxidation in lipid-based systems, which suggests additional potential advantages in preventing protein oxidation.35) As an example of SFD for inhalable protein formulation, Ito et al. successfully formulated a decoy protein as a DPI using SFD (while preserving antibody activity) by incorporating trehalose and leucine as stabilizing excipients.36) In comparison of SFD and spray drying using deoxyribonuclease, Maa et al. found that spray drying produced small, dense particles of approximately 3 µm, while SFD produced particles of approximately 8–10 µm, which were porous. SFD was therefore superior to spray drying in terms of aerosolization, and the aerodynamic particle size of the particles produced by SFD was smaller.37) SCFs, such as carbon dioxide, exist under conditions above their critical temperature and pressure, and they exhibit properties of both gases and liquids.38) In supercritical drying, a transition from liquid to SCF and then to the vapor phase is used to precipitate and dry the protein by exploiting the antisolvent effect of SCFs.29) This technique allows precise control over particle formation and it has gained attention as a viable drying method for protein inhalation formulations. The drying method was shown in studies on whey protein aerogels to significantly influence the structural, adsorptive, and mechanical properties.39) Compared with freeze drying, supercritical drying produced denser aerogels with higher oil retention capacity. Lyophilization remains the most widely used drying technique for injectable protein formulations. Although it typically results in solid cakes that are less amenable to particle design,
it can be suitable in certain nanoparticle-based formulations, such as lysozyme.40) Nebulizers mechanically generate aerosols from liquid formulations, making them suitable for patients with limited inspiratory capacity. In contrast, DPIs offer advantages in portability and storage stability, but their efficacy depends on the patient’s ability to generate sufficient inspiratory flow. With recent advancements in powder formulation technologies, DPIs have gained attention for delivering proteins and nanoparticle-based drugs, which has led to a decline in the use of nebulizers for formulation development. Nonetheless, nebulizers remain widely used in basic research due to their simplicity and the ease of administration.41)
5. Inhalable Protein-Based Therapies for Lung Local Diseases
5.1. Cystic Fibrosis Cystic fibrosis is a genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator protein, leading to the accumulation of viscous mucus in the lungs and resulting in chronic infection and inflammation.42) Effective management requires strategies to enhance mucus clearance, control infection, and restore cystic fibrosis transmembrane conductance regulator function. Alpha-1 antitrypsin (AAT) is a serine protease inhibitor that protects tissues from esterases derived from neutrophils and other inflammatory cells. AAT deficiency is associated with the development of pulmonary emphysema. Griese et al. investigated the pulmonary administration of AAT in patients with cystic fibrosis. No significant improvement was seen in lung function, but there were reductions in elastase activity, pro-inflammatory cytokines, and enhancements in bactericidal capacity.43) Two inhalation modes were tested via a SMART CARD-connected device (AKITA1 inhalation device with a Pari LC Plus, or a Pari LC Star nebulizer), and there were no differences in outcomes. Although subsequent clinical development of AAT for cystic fibrosis has slowed, its inhalational pre-treatment has shown promising anti-inflammatory and cytoprotective effects in models of acute lung injury induced by toxic gases such as phosgene, which suggests potential applications in other pulmonary diseases.44) Recombinant human deoxyribonuclease I (rhDNase, Pulmozyme®) is approved in many countries as an inhaled therapeutic for cystic fibrosis. Its efficacy in reducing mucus viscosity and improving lung function has been shown in clinical stud30
ies. Frederiksen et al. reported that 12 months of rhDNase inhalation significantly improved forced expiratory volume in 1 s (FEV1) and that it reduced the incidence of respiratory infections, including those caused by Staphylococcus aureus, potentially contributing to the slowing of disease progression.45) A biosimilar drug, Tigeras®, has also been developed, and has been shown to be both effective and safe.46) Lactoferrin is an approx. 83 kDa glycoprotein with antimicrobial, anti-inflammatory, and immunomodulatory activities, and it has attracted attention as a candidate for inhaled therapy in patients with cystic fibrosis.47) Marshall et al. developed spray-dried powder formulations containing lactoferrin or apolactoferrin combined with aminoglycoside antibiotics (tobramycin or gentamicin), and tested them in cystic fibrosis lung infection models.48) These formulations demonstrated multiple beneficial effects, including inhibition of biofilm formation, disruption of existing biofilms, and enhanced antibiotic penetration. The resulting particles measured between 1.5–1.9 µm, with 90% being under 5 µm, which indicates good aerosolization and suitability for inhalation. This combination is thought to represent a promising protein-based DPI candidate for the treatment of chronic infections in patients with cystic fibrosis. ALX-009, an inhalable formulation combining lactoferrin and hypothiocyanite, exhibited potent bactericidal activity against Pseudomonas aeruginosa and Burkholderia cepacia complex in sputum samples of patients with cystic fibrosis.49) ALX-009 has shown superior antibacterial activity to tobramycin, with enhanced effects observed upon twice-daily administration. ALX-009 is suggested by these results to be a promising therapeutic option for multidrug-resistant infections in cystic fibrosis. Additionally, lactoferrin has been demonstrated to have protective effects in models of acute respiratory distress syndrome and oxygen toxicity, so there is potential utility in severe respiratory diseases beyond CF.50) 5.2. Idiopathic Pulmonary Fibrosis Inhaled interferongamma (IFN-γ) is a cytokine with both antifibrotic properties and macrophage-activating functions, and it has attracted interest as a potential therapeutic agent for idiopathic pulmonary fibrosis. Nebulized administration of IFN-γ reportedly suppressed the transforming growth factor-beta (TGF-β) signaling pathway and inhibited fibrosis progression in the lungs.51,52) In a safety study involving patients with idiopathic pulmonary fibrosis over an 80-week period, bronchoalveolar lavage fluid IFN-γ concentrations were shown to have increased by 60-fold. This was accompanied by a reversal of the declining trends in diffusing capacity of the lungs for carbon monoxide and total lung capacity. Systemic exposure was minimal, and no adverse events were observed. Subcutaneous administration had previously shown no clinical benefit, and there was no detection of IFN-γ in bronchoalveolar lavage fluid, so these findings highlight the significance of direct pulmonary delivery via inhalation. PRS-220 is an inhaled Anticalin® protein that targets CCN2 (formerly known as connective tissue growth factor, or CTGF). In preclinical idiopathic pulmonary fibrosis models, PRS-220 demonstrated greater antifibrotic efficacy than systemically administered antibodies.53) Due to its selective pulmonary distribution and low systemic exposure, PRS-220 also exhibited a favorable safety profile. Notably, in ex vivo and human lung tissue slice models, PRS-220 showed stronger antifibrotic activity than other therapeutic agents such as nint-
edanib and pamrevlumab. These results underscore the potential of PRS-220 as a promising inhaled protein-based therapy for idiopathic pulmonary fibrosis, offering both efficient lung delivery and a potent therapeutic effect. 5.3. Asthma The therapeutic potential of inhaled interferon-λ2/3 (IFN-λ2/3) in an allergen-induced asthma model was investigated by Won et al.54) Administration of IFN-λ2/3 after asthma onset led to a marked reduction in airway inflammation and hyperresponsiveness. Inhalation of IFN-λ significantly suppressed pro-inflammatory cytokines associated with T-helper 2 (Th2) and Th17 responses (including interleukin (IL)-4, IL-5, IL-13, and IL-17A) and also promoted an anti-inflammatory response characterized by an increase in IL-10-producing CD4+ T cells. IFN-λ may therefore serve as a promising inhaled protein-based therapeutic candidate for asthma management. Nanobodies, which are small antibody fragments (approx. 15 kDa) composed of the variable region of antibodies, offer advantages for inhalation therapy due to their high pulmonary distribution and intrinsic stability.55) LQ036, a nanobody that targets the α1 chain of the IL-4 receptor (IL-4Rα1), significantly suppressed airway inflammation, goblet cell hyperplasia, and immunoglobulin E (IgE) production in a humanized asthma model.56) Inhaled administration of LQ036 resulted in high local concentration and prolonged retention within the lungs. Compared with full-length antibodies, fragmented antibodies such as nanobodies exhibit reduced aggregation tendencies, making them attractive candidates for inhaled delivery systems. Depemokimab (GSK3511294) is a long-acting monoclonal antibody with high affinity for IL-5, and it has been shown to significantly reduce exacerbations in patients with severe eosinophilic asthma when administered subcutaneously once every six months (Phase III, NCT04719832).57) Although Depemokimab itself has not been developed for inhalation, studies have demonstrated that inhaled anti–IL-5 monoclonal antibodies can significantly attenuate airway inflammation and hyperresponsiveness in allergen-induced asthma mouse models.58) Inhalation may therefore offer therapeutic efficacy comparable to that of intravenous administration. IL-33 is a key cytokine involved in mucosal inflammatory diseases, including asthma. A human-derived, inhalable single-domain antibody (UdAb A12) that selectively inhibits IL-33 signaling was developed by Huange et al. In an allergic airway inflammation model, UdAb A12 exhibited strong anti-inflammatory effects. Compared with the control immunoglobulin G (IgG) antibody itepekimab, UdAb A12 demonstrated superior pulmonary distribution and local efficacy, which indicates its potential as a novel platform for inhaled antibody therapeutics.59) 5.4. COPD DAS181 is an inhalable fusion protein composed of a human epithelial growth factor and a bacterial sialidase. It was designed to inhibit viral entry into host cells. Inhaled administration of DAS181 in a COPD mouse model (cigarette smoke exposure combined with influenza infection) effectively suppressed pneumonia progression, reduced viral load, inhibited pro-inflammatory cytokines (IL-6, IL-1β, and tumor necrosis factor (TNF)), and preserved lung function, as indicated by maintained pulmonary compliance.60) Furthermore, DAS181 modulated the expression of immunoregulatory Siglecs in lung macrophages and promoted memory T cell re31
sponses, which suggests enhanced protection against reinfection. Compared with oseltamivir, DAS181 exhibited superior efficacy in suppressing pneumonia. These findings highlight its dual function as an inhalable protein therapeutic that combines antiviral activity with immune modulation, offering a novel strategy for respiratory viral infections in COPD. In addition, inhaled IFN-γ, which was previously discussed in the context of asthma, and inhaled AAT, which has been described in CF treatment, have also gained attention for their potential application in COPD management.51,61) 5.5. Infections Inhaled antibody therapies for coronavirus disease 2019 (COVID-19) have been developed to achieve direct viral suppression at the pulmonary site of infection. Inhaled delivery of the neutralizing antibody 1212C2 has been shown to significantly reduce the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load in the lungs of hamsters and to improve histopathological scores.62) Other promising candidates include the nanobody PiN-21 (a multivalent single-domain antibody) and inhaled Regdanvimab (IN-006), both of which exhibited high pulmonary concentrations and potent antiviral efficacy.63) The achievement of high lung concentrations in inhaled antibody therapies results from a combination of factors, including molecular size optimization, tailored inhalation device design, and limited systemic absorption. PiN-21, due to its small nanobody structure, demonstrated enhanced diffusion and deep lung penetration. IN-006, on the other hand, achieved over 100-fold higher pulmonary concentrations compared to intravenous administration, which has been attributed to its muco-trapping capability via binding to mucin.62–64) 5.6. Lung Cancer Inhaled administration of cetuximab has been shown in lung cancer models to have antitumor efficacy.65) Pharmacokinetic studies using neonatal Fc receptor (FcRn)-knockout mice and non-human primate models suggest that the FcRn plays a greater role in local pulmonary retention than in systemic absorption from the lungs. Pulmonary delivery of monoclonal antibodies may therefore enable high local concentrations in the lungs while minimizing systemic exposure, which makes inhaled mAb therapy a promising approach for chronic respiratory diseases, particularly in outpatient or home-care settings. An inhalable liposomal formulation (CTX-OB-LPs) in which osimertinib, an epidermal growth factor (EGFR) tyrosine kinase inhibitor (EGFR-TKI), is encapsulated within cetuximab-conjugated liposomes, was developed by Daram et al.66) This formulation exhibited strong antitumor activity in a non-small cell lung cancer (NSCLC) model, as evidenced by enhanced cytotoxicity, suppression of colony formation, and reduced cell migration. In this context, cetuximab was utilized to confer tumor-targeting ability upon pulmonary delivery. Future studies are warranted to assess how cetuximab modification influences in vivo biodistribution and pharmacokinetics. Bevacizumab is an anti-vascular endothelial growth factor (VEGF) monoclonal antibody that is widely used in the treatment of NSCLC. However, systemic intravenous administration requires high doses and is associated with significant systemic toxicity. An inhalable dry powder formulation of bevacizumab using spray drying technology was developed by Shepard et al.67) In a rat NSCLC model, it demonstrated equivalent tumor suppression to intravenous administration at only one-tenth of the dose. The inhaled formulation retained
VEGF-inhibitory activity and it showed good physicochemical stability. Inhaled anti-VEGF antibody therapies may therefore represent a novel strategy for lung cancer treatment by reducing adverse effects and improving patient compliance. Given the growing number of antibody-based therapies in oncology (including molecular targeted agents and immune checkpoint inhibitors), there is thought to be considerable potential for expanding inhaled formulations of therapeutic antibodies in the future. 5.7. Other Lung Inflammation Diseases Granulocytemacrophage colony-stimulating factor (GM-CSF) is a cytokine secreted by various cell types, including macrophages, T cells, and endothelial cells. It plays a crucial role in immune regulation and alveolar macrophage function. GM-CSF has been applied in the treatment of autoimmune pulmonary alveolar proteinosis (aPAP), a rare disease characterized by the accumulation of surfactant proteins and lipids in the alveoli, leading to progressive respiratory insufficiency.68) A dry powder inhalation formulation of GM-CSF was approved in 2024 in Japan and is marketed under the brand name Sargmalin®. Favorable responses have been reported in patients, which demonstrates the therapeutic potential of inhaled GM-CSF in managing this otherwise difficult-to-treat condition.69)
6. Albumin and Other Proteins as a Drug Delivery Carrier
6.1. Albumin as a Carrier of Anti-cancer Agents Serum albumin is the most abundant protein in serum and has high biocompatibility and biodegradability. In addition, it is actively uptaken by cancer cells via a surface protein, secreted protein acidic and rich in cysteine. Albumin is also a ligand of glycoprotein 60 (gp60) that is overexpressed in cancer vascular endothelial cells, and gp60 supports the distribution of albumin in tumors by transcytosis. Serum albumin is therefore commonly used as a drug carrier for cancer treatment. Paclitaxel-loaded albumin microparticle dry powders with different sizes (0.5, 1, 3 µm) were developed by Chaurasiya et al. for lung cancer.28) Compared with the Taxol solution, the microparticles showed a higher antitumor effect in lung cancer orthotopic model mice. While 66% of paclitaxel accumulated in the liver in the Taxol solution group, more than 78% of paclitaxel was distributed in tumors in the microparticleadministrated group. Compared to the particle with a size of 0.5 µm, the particle with 1 and 3 µm accumulated tumor better, probably due the long retention time in the lungs. Other groups use albumin nanoparticles as a carrier for inhalation.10,70–72) Nanoparticle formation is expected to increase the penetration and cellular uptake, and to take advantage of controlled release.73) Albumin particles were sometimes converted into powder using spray-dryer with heat,28,70) SFD,74) or lyophilized,75) whereas sometimes they administrated as a solution.10,71,72) The negative surface charge of albumin is considered to be advantageous in inhalation formulations because it reduces electrostatic interactions with mucin, thereby enhancing mucus penetration. A novel formulation (PEG-pHSA@PMB) was developed by Li et al., in which polymyxin B was electrostatically loaded onto chemically modified, PEGylated human serum albumin with enhanced negative charge.76) This system was designed to exploit the acidic environment of infection 32
Mucus Targeting Strategies for Inhalable Particles is the predominant route, but rapid clearance by the liver and spleen limits systemic bioavailability. Inhalation has emerged as an alternative strategy to achieve localized delivery to the lungs, with reduced systemic exposure. Intratracheal administration of exosomes derived from canine stem cells (cST-Exo) demonstrated anti-inflammatory effects in an lipopolysaccharide-induced acute lung injury (ALI) mouse model, including the induction of M2 macrophages and regulatory T cells, suppression of pro-inflammatory cytokines (TNF-α, TGF-β), and upregulation of antiinflammatory cytokine IL-10.80) Compared with intravenous injection, airway-localized delivery exhibited greater therapeutic efficacy, supporting the potential of inhaled exosome-based therapies for pulmonary diseases. Inhalation of exosomes derived from lung spheroid cells reportedly improved cardiac function, inhibited fibrosis, and promoted cardiomyocyte proliferation in both mouse and pig models of myocardial infarction.81) Noninvasive inhaled administration could therefore represent a novel delivery route for cardiac regenerative therapies. In the context of infectious disease prevention, Wang et al. developed an inhalable hybrid nanovaccine (NVRBDMLipo) that was composed of SARS-CoV-2 receptor-binding domain (RBD)-expressing cell-derived nanovesicles fused with lung surfactant-mimicking liposomes (MLipo).82) Intratracheal administration of MLipo induced robust mucosal and systemic immune responses, which included high titers of RBD-specific secretory IgA (sIgA) and IgG, activation of both CD4+ and CD8+ T cells, and broad-spectrum neutralization against viral variants. The vaccine also activated pulmonary macrophages via the TLR4/NF-κB pathway and demonstrated a favorable safety profile, positioning it as a promising nextgeneration inhalable vaccine candidate. 6.4. PEGylation PEGylation, which is the covalent attachment of PEG chains, has been widely employed to enhance the stability and pharmacokinetics of protein therapeutics.83) The PEG moiety protects the protein from renal clearance and enzymatic degradation, while also reducing immunogenicity. For example, PEGylated interferons and granulocyte colonystimulating factor have enabled reduced dosing frequencies (ranging from once weekly to once monthly) thereby improv-
sites, where a pH-triggered surface charge shift facilitates drug release. The combination of mild negative charge and polyethylene glycol (PEG)-mediated hydrophilicity improved mucus permeability, while pH-responsiveness enabled sitespecific drug release. Such charge modulation strategies are thought to represent a key design principle in the development of inhalable particulate drug delivery systems. 6.2. Mucoadhesion/Mucodegradating Proteins To achieve site-specific therapy for lung cancer, Jeong et al. developed an inhalable nanomedicine termed thMAP@Cur nanoparticles, which was based on a mucoadhesive peptide derived from adhesion proteins.41) This formulation involved thiol modification of a dopamine-containing mucin-binding protein. The resulting redox-responsive nanoparticles (thMAP NPs) were loaded with curcumin, a compound with known antitumor activity, forming thMAP@Cur NPs. These particles exhibited redox-triggered drug release specifically within the reductive environment of cancer cells. When administered via nebulization, thMAP@Cur NPs demonstrated prolonged pulmonary retention and high tumor localization in a lung metastasis model. This study exemplifies a mucoadhesion-based strategy for enhancing site-specific drug delivery (Fig. 2). In contrast, other approaches aim to disrupt the mucus barrier rather than to interact with it. Sousa et al. incorporated mucolytic enzymes derived from pineapple, specifically papain and bromelain, into poly(acrylic acid) nanoparticles to degrade mucus and to enhance penetration.77) Local enzymatic degradation of the mucus structure significantly increased permeability. Notably, bromelain exhibited superior deep tissue penetration compared to papain in an ex vivo porcine intestinal mucus model. Such strategies to enhance penetration into the deep lung may be particularly advantageous for pulmonary administration aimed at achieving systemic circulation. 6.3. Extracellular Vesicles Exosomes are naturally derived membrane-bound vesicles (30–150 nm in diameter) that encapsulate proteins, nucleic acids, and lipids, and they play a critical role in intercellular communication. Due to their low immunogenicity, biocompatibility, and intrinsic targeting capability, exosomes have garnered attention as promising nanocarriers for drug delivery.78,79) Intravenous administration 33
ing patient adherence and treatment outcomes.84,85) However, challenges remain, such as that the PEGylation may mask the active site of the protein, potentially diminishing bioactivity, and the induction of anti-PEG antibodies has been reported.86) Maintaining a balance between preserving biological activity and selecting the most appropriate modification sites is therefore essential in formulation design. Although still in the experimental stage, several studies have explored PEGylation as a strategy to enhance the pharmacological performance of inhaled protein therapeutics. Dornase alfa (recombinant human DNase I, rhDNase) is a mucolytic enzyme that is widely used in CF treatment via inhalation, but its short pulmonary residence time remains a limitation. N-terminal PEGylation of rhDNase with 20–40 kDa PEG chains was shown in a mouse model to prolong lung retention time to over 15 d.87) This improvement is because PEGylation attenuated rapid clearance by alveolar macrophages, which enabled sustained therapeutic concentrations in the pulmonary environment. In addition, the PEGylated rhDNase retained enzymatic activity while reducing systemic absorption and hepatic accumulation, thereby improving both local efficacy and safety. These findings support the utility of PEGylation in optimizing inhaled protein therapeutics for clinical application. Inhalable protein formulations using PEG-based polymers have also been investigated. An inhalable dry powder formulation composed of a protein complexed with vitamin B12-modified PEG–poly(glutamic acid), targeting the vitamin B12 internalization receptor (CD320), was developed by Nieto-Orellana et al., and it achieved efficient delivery to the lungs.88) PEG–poly(glutamic acid) can form electrostatic interactions with proteins, allowing for the formulation of non-covalent complexes without requiring covalent linkage between the drug and PEG. The significance of PEGylation in inhalation formulations has been extensively reviewed by Guichard et al.89) PEGylation enhances the pulmonary retention and the stability of inhaled proteins through multiple mechanisms, including molecular size optimization, reduced interaction with mucus, protection against enzymatic degradation, and evasion of macrophage-mediated clearance. However, PEG chains can hinder systemic absorption, so careful design is required when systemic therapeutic effects are desired.