Intratracheal Delivery of Nano- and Microparticles and Hyperpolarized Gases

Accurate diagnosis is crucial to improve the treatment and prognosis of respiratory disease, especially lung cancer. Tumors and lesions located deep in the lung are directly accessible via dendritic tracheal bronchus, thereby opening a new way to tackle respiratory disease. Intratracheal delivery is an innovative, noninvasive approach for imaging and treating respiratory disease efficiently, when compared with other delivery methods. Intratracheal delivery of nano- and microparticles and hyperpolarized gases offers valuable clinical advantages, such as assessing lung function, monitoring ventilation and perfusion, controlling disease progression, and inhibiting tumor growth. Especially, versatile nanosized particles have enormous potential to benefit precision imaging and therapy at the molecular level. Here we discuss advances of intratracheal delivery of nano- and microparticles and hyperpolarized gases for respiratory disease imaging and treatment, with an emphasis on intratracheal nanoparticles delivery for pulmonary imaging, which has extremely valuable clinical applications in precise theranostics for respiratory disease. Accurate diagnosis is crucial to improve the treatment and prognosis of respiratory disease, especially lung cancer. Tumors and lesions located deep in the lung are directly accessible via dendritic tracheal bronchus, thereby opening a new way to tackle respiratory disease. Intratracheal delivery is an innovative, noninvasive approach for imaging and treating respiratory disease efficiently, when compared with other delivery methods. Intratracheal delivery of nano- and microparticles and hyperpolarized gases offers valuable clinical advantages, such as assessing lung function, monitoring ventilation and perfusion, controlling disease progression, and inhibiting tumor growth. Especially, versatile nanosized particles have enormous potential to benefit precision imaging and therapy at the molecular level. Here we discuss advances of intratracheal delivery of nano- and microparticles and hyperpolarized gases for respiratory disease imaging and treatment, with an emphasis on intratracheal nanoparticles delivery for pulmonary imaging, which has extremely valuable clinical applications in precise theranostics for respiratory disease. Respiratory diseases are prevalent and have increasing incidence globally, especially lung cancer, which remains the leading cause of cancer mortality.1Bray F.I. Ferlay J. Soerjomataram I. Siegel R.L. Torre L.A. Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.CA Cancer J Clin. 2018; 68: 394-424Crossref PubMed Scopus (51287) Google Scholar Traditional imaging diagnostic methods, such as CT scanning, provide imaging with higher resolution and finer slice thickness (< 1 mm), enabling discovery of 50 mm3 noncalcified nodules.2Mulshine J.L. Jablons D.M. Volume CT for diagnosis of nodules found in lung-cancer screening.N Engl J Med. 2009; 361: 2281-2282Crossref PubMed Scopus (14) Google Scholar However, insufficient molecular information about respiratory diseases limits precise diagnosis and subsequent treatment. Current treatment strategies for respiratory disease are based mostly on the systemic administration of antibiotics or antitumor drugs by oral or IV delivery, with low concentrations at the target site and high levels of systemic drug, risking toxicity and adverse effects. Thus, exploring innovative and noninvasive approaches to precisely diagnose and treat respiratory disease at a molecular level is of great significance. The unique anatomical structure and microenvironment of the lungs present particular challenges for molecular diagnosis and treatment. Most of the respiratory diseases occur deep in the gas-rich lung tissue, which is easily affected by breathing motions, limiting the resolution and efficacy of current imaging techniques. Furthermore, lung tissue has a powerful clearance mechanism and physiologic barriers, compounded in lung cancer by a complex tumor microenvironment and blood supply by the bronchial arteries. These physiologic features determine the local concentration of drugs and imaging agents in the lungs. Efficient, precise, and innovative theranostic strategies are urgently needed. Yet, the unique anatomical location of respiratory diseases also affords opportunities. The lungs contact the environment directly via tracheal and bronchial dendritic ramifications rich in blood vessels. In part, tumors and lesions located in deep lung tissue can be directly accessed via dendritic tracheal bronchus. In addition, the epithelium covering the trachea and bronchi varies in composition according to cellular functions. The alveolar epithelium consists of cuboidal type II and tabular type I alveolar epithelial cells, with the latter covering over 95% of the alveolar surface. This provides a thin air-blood barrier (about 0.1-0.2 μm) and large epithelial surface area (about 70-140 m2),3Gehr P. Bachofen M. Weibel E.R. The normal human lung: ultrastructure and morphometric estimation of diffusion capacity.Respir Physiol. 1978; 32: 121-140Crossref PubMed Scopus (436) Google Scholar thereby allowing homogeneous distribution and rapid absorption and uptake, with high bioavailability after intratracheal delivery of imaging agents and therapeutic drugs. Intratracheal delivery, or inhalation, has made great progress since it was first used to deliver medications to the lungs more than 2,000 years ago.4Sanders M. Inhalation therapy: an historical review.Prim Care Respir J. 2007; 16: 71-81Crossref PubMed Scopus (117) Google Scholar A number of nano- and microparticle formulations and hyperpolarized gases have been designed for intratracheal delivery as contrast agents or delivery carriers including dry powder, aerosol, and spray. In the wake of delivery strategy innovation, intratracheal delivery devices have also made tremendous progress, for instance, pressurized metered dose inhalers, dry powder inhalers, nebulizers, and soft mist inhalers, among others.5Yang W. Peters J.I. Williams III, R.O. Inhaled nanoparticles: a current review.Int J Pharm. 2008; 356: 239-247Crossref PubMed Scopus (502) Google Scholar Therefore, promising and exciting progress in intratracheal delivery for imaging and treatment of respiratory diseases, including lung cancer, has been reported. Among those delivery formulations, nanoparticle is an extraordinary actor in delivery systems. Nanoparticles with a size range from 1 to 1,000 nm, enabling unique medical effects,6Wagner V. Dullaart A. Bock A.K. Zweck A. The emerging nanomedicine landscape.Nat Biotechnol. 2006; 24: 1211-1217Crossref PubMed Scopus (1338) Google Scholar have been successfully employed for biomedical applications in the past several decades.7Petros R.A. Desimone J.M. Strategies in the design of nanoparticles for therapeutic applications.Nat Rev Drug Discov. 2010; 9: 615-627Crossref PubMed Scopus (2894) Google Scholar To achieve specific imaging and therapeutic effects, nanoparticles can enter the human body via ocular penetration,8Sah A.K. Suresh P.K. Verma V.K. PLGA nanoparticles for ocular delivery of loteprednol etabonate: a corneal penetration study.Artif Cells Nanomed Biotechnol. 2017; 45: 1-9Crossref PubMed Scopus (35) Google Scholar IV injection,9Xu X. Yan Y. Liu F. Wu L. Shen B. Folate receptor-targeted 19F MR molecular imaging and proliferation evaluation of lung cancer: 19F MR and proliferation evaluation.J Magn Reson Imaging. 2018; 48: 1617-1625Crossref PubMed Scopus (6) Google Scholar oral administration,10Song Q. Jia J. Niu X. et al.An oral drug delivery system with programmed drug release and imaging properties for orthotopic colon cancer therapy.Nanoscale. 2019; 11: 15958-15970Crossref PubMed Google Scholar and the subcutaneous route.11Reva G.V. Reva I.V. Yamamoto T. et al.Reaction of dermal structures to subcutaneous injection of gold nanoparticles to CBA mice.Bull Exp Biol Med. 2014; 156: 491-494Crossref PubMed Scopus (2) Google Scholar However, factors such as hepatic first-pass metabolism, rapid blood clearance, and high-dose exposure in off-target tissues limit the efficacy of these conventional delivery methods, especially for respiratory disease. Intratracheal nanoparticle delivery is a promising noninvasive strategy for respiratory disease. By controlling the size of nanoparticles, they can reach the alveolar region in deep lung tissue, enabling homogeneous distribution, improved deposition, and increased delivery efficacy. Tailorable nanosized particles offer additional advantages, as versatile surface modification and high surface area-to-volume ratios favor drug binding and loading, thereby improving efficacy and residence time. Multifunctional nanoparticles loaded with targeting ligands, imaging agents, and therapeutic drugs enable the synthesis of multimodal compounds that provide highly sensitive imaging and treatment at molecular and cellular levels.12Chow E.K. Ho D. Cancer nanomedicine: from drug delivery to imaging.Sci Transl Med. 2013; 5: 216rv214Crossref Scopus (378) Google Scholar These properties enable inhaled nanoparticles to reach specific targeted sites and bind biomarkers in the deep lung tissue, amplifying signals of molecular information, enhancing local drug concentrations, and protecting secondary organs from systematic exposure. Thus, nanoparticles are very attractive candidates for the intratracheal delivery imaging and treatment strategy of respiratory disease.13Aghebati-Maleki A. Dolati S. Ahmadi M. et al.Nanoparticles and cancer therapy: perspectives for application of nanoparticles in the treatment of cancers.J Cell Physiol. 2020; 235: 1962-1972Crossref PubMed Scopus (155) Google Scholar Figure 1 depicts typical intratracheal delivery strategies.14Siva S. Hardcastle N. Kron T. et al.Ventilation/perfusion positron emission tomography-based assessment of radiation injury to lung.Int J Radiat Oncol Biol Phys. 2015; 93: 408-417Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 15Wu L. Wen X. Wang X. et al.Local intratracheal delivery of perfluorocarbon nanoparticles to lung cancer demonstrated with magnetic resonance multimodal imaging.Theranostics. 2018; 8: 563-574Crossref PubMed Scopus (28) Google Scholar, 16Mizuno T. Mohri K. Nasu S. Danjo K. Okamoto H. Dual imaging of pulmonary delivery and gene expression of dry powder inhalant by fluorescence and bioluminescence.J Control Release. 2009; 134: 149-154Crossref PubMed Scopus (45) Google Scholar This promising and fascinating intratracheal delivery technique, combined with the advantages of nanotechnology, paves a new avenue for respiratory disease theranostics. This review summarizes essential developments and advances in the intratracheal delivery of nano- and microparticles and hyperpolarized gases, particularly for delivered nanoparticles imaging respiratory disease. This is a valuable resource with systematized information on intratracheal particle delivery systems and their advantages and prospects for clinical applications in precise imaging and treatment of respiratory disease. Significant research efforts have been devoted to the understanding and improvement of nano- and microparticle deposition in the lungs. Key factors including respiratory anatomy and particle size influence delivered particle deposition, as summarized in Figure 1. Given their greater gravitation and inertia, microparticles with diameters greater than 5 μm are transported to the upper airways and nasopharynx, and subsequently swallowed into the GI tract and coughed in sputum.17Costa A. Pinheiro M. Magalhães J. et al.The formulation of nanomedicines for treating tuberculosis.Adv Drug Deliv Rev. 2016; 102: 102-115Crossref PubMed Scopus (66) Google Scholar However, microparticles sized between 1 and 5 μm are prone to deposition in the bronchi and bronchioles by inertial impaction and sedimentation.18Heyder J. Gebhart J. Rudolf G. Schiller C.F. Stahlhofen W. Deposition of particles in the human respiratory tract in the size range 0.005-15 μm.J Aerosol Sci. 1986; 17: 811-825Crossref Scopus (830) Google Scholar Nanoparticles with a diameter ≤ 1,000 nm deposit in the alveolar region, due to sedimentation and Brownian diffusion, and could be optimized for intratracheal delivery.19Chow A.H. Tong H.H. Chattopadhyay P. Shekunov B.Y. Particle engineering for pulmonary drug delivery.Pharm Res. 2007; 24: 411-437Crossref PubMed Scopus (526) Google Scholar A large number of nanoparticles remaining in the lung are removed by slow clearance mechanisms, including alveolar macrophages and epithelial transcytosis, and these processes may last from days to several months.20Sutunkova M.P. Katsnelson B.A. Privalova L.I. et al.On the contribution of the phagocytosis and the solubilization to the iron oxide nanoparticles retention in and elimination from lungs under long-term inhalation exposure.Toxicology. 2016; 363-364: 19-28Crossref PubMed Scopus (33) Google Scholar Intratracheally delivered nanoparticles (< 6 nm) translocate to the lung capillaries and lymph nodes, and may reach other body regions via the bloodstream by crossing the air-blood barrier and entering the circulation.21Mohammad A.K. Amayreh L.K. Mazzara J.M. Reineke J.J. Rapid lymph accumulation of polystyrene nanoparticles following pulmonary administration.Pharm Res. 2013; 30: 424-434Crossref PubMed Scopus (32) Google Scholar,22Choi H.S. Ashitate Y. Lee J.H. et al.Rapid translocation of nanoparticles from the lung airspaces to the body.Nat Biotechnol. 2010; 28: 1300-1303Crossref PubMed Scopus (480) Google Scholar Nanoparticles deposited within the lungs have a greater chance of escaping from lung clearance mechanisms and reaching the epithelium directly, when compared with microparticles. The most commonly used nanoparticles measure between 50 and 500 nm, which is an optimal size for efficient endocytosis.23Oh N. Park J.H. Endocytosis and exocytosis of nanoparticles in mammalian cells.Int J Nanomedicine. 2014; 9: 51-63PubMed Google Scholar However, the lung mucus and surfactant barriers present a major obstacle to the translocation and deposition of intratracheally delivered nanoparticles. The modification of surface coating, such as with polyethylene glycol, minimizes nanoparticle adhesion to mucus and protein.24Schuster B.S. Suk J.S. Woodworth G.F. Hanes J. Nanoparticle diffusion in respiratory mucus from humans without lung disease.Biomaterials. 2013; 34: 3439-3446Crossref PubMed Scopus (268) Google Scholar Mucus-penetrating particles as large as 300 nm exhibit uniform distribution and markedly enhanced retention in the lungs after inhalation.25Schneider C.S. Xu Q. Boylan N.J. et al.Nanoparticles that do not adhere to mucus provide uniform and long-lasting drug delivery to airways following inhalation.Sci Adv. 2017; 3e1601556Crossref PubMed Scopus (159) Google Scholar Indeed, nanoparticles, with their versatile size scale and properties, provide tunable options for imaging and treating respiratory disease. PET/CT imaging is a comprehensive detection method that depends on the uptake of radiotracers; it provides incremental value because of its higher sensitivity and accuracy, and superior quantification of lung function. Indeed, a large number of studies have used radioaerosol PET/CT imaging to assess lung function and physiology in respiratory diseases. Administration of inhaled ultrasonically nebulized 99mTc-albumin colloid (diameter, 3.4 μm) is a noninvasive and easily performed method to test mucociliary function of the airways.26Munkholm M. Nielsen K.G. Mortensen J. Clinical value of measurement of pulmonary radioaerosol mucociliary clearance in the work up of primary ciliary dyskinesia.EJNMMI Res. 2015; 5: 118Crossref PubMed Scopus (12) Google Scholar Le Roux et al27Le Roux P.Y. Siva S. Callahan J. et al.Automatic delineation of functional lung volumes with 68Ga-ventilation/perfusion PET/CT.EJNMMI Res. 2017; 7: 82Crossref PubMed Scopus (16) Google Scholar presented an automated threshold-based approach to quantify pulmonary function, using 68Ga-V˙/Q˙ (ventilation/perfusion) PET/CT imaging. More recently, they demonstrated the feasibility and accuracy of 68Ga-V˙/Q˙ PET/CT imaging in patients with cancer with suspected pulmonary embolism, compared with CT pulmonary angiography.28Le Roux P.Y. Iravani A. Callahan J. et al.Independent and incremental value of ventilation/perfusion PET/CT and CT pulmonary angiography for pulmonary embolism diagnosis: results of the PECAN pilot study.Eur J Nucl Med Mol Imaging. 2019; 46: 1596-1604Crossref PubMed Scopus (11) Google Scholar Radiation-induced regional lung functional deficits in patients with lung cancer can be estimated by simple linear models and four-dimensional 68Ga-V˙/Q˙ PET/CT imaging (Fig 2).14Siva S. Hardcastle N. Kron T. et al.Ventilation/perfusion positron emission tomography-based assessment of radiation injury to lung.Int J Radiat Oncol Biol Phys. 2015; 93: 408-417Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar In addition, high-resolution PET/CT imaging of 13NH3-labeled isotonic saline (inhalable aerosol, 4.9 μm) was shown to accurately detect the aerosol distribution pattern in patients with asthma.29Greenblatt E.E. Winkler T. Harris R.S. Kelly V.J. Kone M. Venegas J. Analysis of three-dimensional aerosol deposition in pharmacologically relevant terms: beyond black or white ROIs.J Aerosol Med Pulm Drug Deliv. 2015; 28: 116-129Crossref PubMed Scopus (15) Google Scholar Cossío et al30Cossío U. Gómez-Vallejo V. Flores M. Gañán-Calvo B. Jurado G. Llop J. Preclinical evaluation of aerosol administration systems using positron emission tomography.Eur J Pharm Biopharm. 2018; 130: 59-65Crossref PubMed Scopus (12) Google Scholar reported three different methods for pulmonary administration of aerosol labeled with 2-deoxy-2-[18F]fluoro-d-glucose (18F-FDG; diameter range, 0.9-5.25 μm) for PET imaging and compared their regional distribution in rat lungs, providing paramount information for the optimization of aerosol delivery systems. By providing anatomical information and accurate estimation of diseased lung function, PET imaging of inhalable radioaerosols can benefit clinical practice, but motion artifacts, radiation exposure, and cost are persistent issues. With advances in MRI techniques and optimization of imaging sequences, good-quality MR images of the lungs can be obtained by the administration of potent contrast agents. For example, inhalable hyperpolarized 3He and 129Xe gases (atom size, < 1 nm) have been regarded as promising MRI agents for evaluation of lung function because of their longer longitudinal relaxation times (T1) resulting from a nuclear spin quantum number of 1/2.31Kern A.L. Vogel-Claussen J. Hyperpolarized gas MRI in pulmonology.Br J Radiol. 2018; 91: 20170647Crossref PubMed Scopus (32) Google Scholar As shown in Figure 3,32Washko G.R. Parraga G. Coxson H.O. Quantitative pulmonary imaging using computed tomography and magnetic resonance imaging.Respirology. 2012; 17: 432-444Crossref PubMed Scopus (47) Google Scholar hyperpolarized 3He lung functional MR imaging has been used to assess inhaled gas distribution and to quantify lung function in healthy young and older never smokers, and in patients with asthma, COPD, cystic fibrosis (CF), and radiation-induced lung injury. Reproducibility, feasibility, tolerability, and safety of lung function quantitative evaluation by 129Xe gas MRI were also demonstrated in patients and animal models with various respiratory diseases.33Stewart N.J. Horn F.C. Norquay G. et al.Reproducibility of quantitative indices of lung function and microstructure from 129Xe chemical shift saturation recovery (CSSR) MR spectroscopy.Magn Reson Med. 2017; 77: 2107-2113Crossref PubMed Scopus (28) Google Scholar, 34Walkup L.L. Thomen R.P. Akinyi T.G. et al.Feasibility, tolerability and safety of pediatric hyperpolarized 129Xe magnetic resonance imaging in healthy volunteers and children with cystic fibrosis.Pediatr Radiol. 2016; 46: 1651-1662Crossref PubMed Scopus (63) Google Scholar, 35Li H. Zhang Z. Zhao X. Sun X. Ye C. Zhou X. Quantitative evaluation of radiation-induced lung injury with hyperpolarized xenon magnetic resonance.Magn Reson Med. 2016; 76: 408-416Crossref PubMed Scopus (32) Google Scholar Oxygen-enhanced MRI has also been used to evaluate regional lung function, as paramagnetic oxygen molecules can shorten T1 and T2* values in the lungs.36Sasaki T. Takahashi K. Obara M. Viability of oxygen-enhanced ventilation imaging of the lungs using ultra-short echo time MRI.Magn Reson Med Sci. 2017; 16: 259-261Crossref PubMed Scopus (4) Google Scholar Hyperpolarized gas MRI is a highly specific, noninvasive, time-saving, and easy-to-operate methodology providing systematic and comprehensive information on lung function. However, the sophisticated production of polarized gases limits its clinical applications. Given the excellent advantages of 19F, such as its 100% natural abundance, high gyromagnetic ratio (∼95% of 1H), and high sensitivity of 83%,9Xu X. Yan Y. Liu F. Wu L. Shen B. Folate receptor-targeted 19F MR molecular imaging and proliferation evaluation of lung cancer: 19F MR and proliferation evaluation.J Magn Reson Imaging. 2018; 48: 1617-1625Crossref PubMed Scopus (6) Google Scholar it is opportune to develop 19F-MR imaging. In 1996, perfluorocarbon (PFC) partial liquid ventilation was shown to alleviate neonatal respiratory distress syndrome and improve pulmonary function, due to its high oxygen-dissolving and releasing capability, low surface tension, and excellent physical-chemical properties.37Leach C.L. Greenspan J.S. Rubenstein S.D. et al.Partial liquid ventilation with perflubron in premature infants with severe respiratory distress syndrome.N Engl J Med. 1996; 335: 761-767Crossref PubMed Scopus (409) Google Scholar The combination of these features makes PFC the best 19F-MR contrast agent candidate for pulmonary imaging by intratracheal delivery. For example, aerosol deposition and oxygenation patterns in rat lungs have been detected by 19F-MR imaging after inhalation of PFC aerosol (1.2 μm) produced by pneumatic generator.38Thomas S.R. Gradon L. Pratsinis S.E. et al.Perfluorocarbon compound aerosols for delivery to the lung as potential 19F magnetic resonance reporters of regional pulmonary pO2.Invest Radiol. 1997; 32: 29-38Crossref PubMed Scopus (21) Google Scholar In another study in rats, high-resolution three-dimensional 19F-MR imaging was able to assess airway pressures and strains with liquid PFC instilled into the lung.39Weigel J.K. Steinmann D. Emerich P. Stahl C.A. Elverfeldt D.V. Guttmann J. High-resolution three-dimensional 19F-magnetic resonance imaging of rat lung in situ: evaluation of airway strain in the perfluorocarbon-filled lung.Physiol Meas. 2011; 32: 251-262Crossref PubMed Scopus (6) Google Scholar 19F-MR imaging with nanosized PFC enables highly sensitive diagnostic imaging of lung cancer. PFC nanoparticles (approximately 150 nm in size) administered by intratracheal delivery were shown to diffuse and distribute densely and deeply into the lung tumors, producing excellent fluorescence and 1H- and 19F-MR imaging signals that persisted for at least 72 hours.15Wu L. Wen X. Wang X. et al.Local intratracheal delivery of perfluorocarbon nanoparticles to lung cancer demonstrated with magnetic resonance multimodal imaging.Theranostics. 2018; 8: 563-574Crossref PubMed Scopus (28) Google Scholar Furthermore, fluorine imaging, demonstrated for the first time in lung cancer, obviates the need for gadolinium and circumvents potential safety issues. Indeed, PFC nanoparticles are safe in vitro and in vivo and are constrained to the tumor.9Xu X. Yan Y. Liu F. Wu L. Shen B. Folate receptor-targeted 19F MR molecular imaging and proliferation evaluation of lung cancer: 19F MR and proliferation evaluation.J Magn Reson Imaging. 2018; 48: 1617-1625Crossref PubMed Scopus (6) Google Scholar,15Wu L. Wen X. Wang X. et al.Local intratracheal delivery of perfluorocarbon nanoparticles to lung cancer demonstrated with magnetic resonance multimodal imaging.Theranostics. 2018; 8: 563-574Crossref PubMed Scopus (28) Google Scholar As PFC has a long history of medical applications as an artificial blood substitute, in liquid ventilation, and as a drug delivery vehicle,37Leach C.L. Greenspan J.S. Rubenstein S.D. et al.Partial liquid ventilation with perflubron in premature infants with severe respiratory distress syndrome.N Engl J Med. 1996; 335: 761-767Crossref PubMed Scopus (409) Google Scholar,40Squires J.E. Artificial blood.Science. 2002; 295: 1002-1005Crossref PubMed Scopus (138) Google Scholar the logical extension of this work suggests that PFC nanoparticles may represent an important theranostic opportunity to address the formidable challenges in treating lung cancer. More importantly, active targeted PFC nanoparticles show great promise as quantitative biomarkers for lung cancer molecular imaging. Recent data have demonstrated that intratracheal administration of αvβ3-targeted PFC nanoparticles (about 170 nm) produced significantly higher 19F-MR signal in tumors and actively targeted tumor angiogenesis in orthotopic lung cancer models (Fig 4A).41Xu X. Zhang R. Liu F. et al.19F MRI in orthotopic cancer model via intratracheal administration of αvβ3-targeted perfluorocarbon nanoparticles.Nanomedicine (Lond). 2018; 13: 2551-2562Crossref PubMed Scopus (6) Google Scholar Of note, multimodal imaging methods, including optical, 1H-MR, and 19F-MR imaging, have also contributed to intratracheally delivered PFC-based preclinical and clinical tests in lung cancer imaging. In addition to PFC nanoparticles, iron oxide nanoparticles are suitable for intratracheal delivery pulmonary MR imaging. Intratracheally delivered antibody-conjugated superparamagnetic iron oxide nanoparticles (SPIONs, about 100 nm) have been shown by MRI to actively target and detect M1 and M2 macrophage subpopulations in a COPD mouse model, and to provide early diagnosis of pulmonary inflammatory diseases.42Al Faraj A. Shaik A.S. Afzal S. Al S.B. Halwani R. MR imaging and targeting of a specific alveolar macrophage subpopulation in LPS-induced COPD animal model using antibody-conjugated magnetic nanoparticles.Int J Nanomedicine. 2014; 9: 1491-1503Crossref PubMed Scopus (49) Google Scholar SPIONs (about 130 nm) have been nebulized and delivered to rat lungs, enabling lung imaging with high sensitivity and efficacy via magnetic particle imaging (Fig 4B).43Tay Z.W. Chandrasekharan P. Zhou X.Y. Yu E. Zheng B. Conolly S. In vivo tracking and quantification of inhaled aerosol using magnetic particle imaging towards inhaled therapeutic monitoring.Theranostics. 2018; 8: 3676-3687Crossref PubMed Scopus (65) Google Scholar Novel nanosized MRI contrast agents developed by Gao et al,44Gao J. Li L. Liu X. Guo R. Zhao B. Contrast-enhanced magnetic resonance imaging with a novel nano-size contrast agent for the clinical diagnosis of patients with lung cancer.Exp Ther Med. 2018; 15: 5415-5421PubMed Google Scholar chitosan/Fe3O4-enclosed bispecific antibodies, were shown to significantly enhance signal intensity for lung cancer after pulmonary inhalation, when compared with MRI alone. Fluorescence and bioluminescence imaging experienced rapid development with the nanotechnology revolution and innovation in nanomedicine. In particular, intratracheally delivered nano- and microparticles have shown tremendous value in pulmonary optical imaging. Mizuno et al16Mizuno T. Mohri K. Nasu S. Danjo K. Okamoto H. Dual imaging of pulmonary delivery and gene expression of dry powder inhalant by fluorescence and bioluminescence.J Control Release. 2009; 134: 149-154Crossref PubMed Scopus (45) Google Scholar succeeded in visualizing pulmonary delivery and gene expression simultaneously in vivo by fluorescence and bioluminescence of administered well-designed indocyanine green-plasmid DNA dry powder (about 20 μm in diameter) (Fig 5). Fluorescence images of live xenografted mice after inhalation of nebulized gelatin nanoparticles modified with biotinylated epidermal growth factor (size, 0.4-2 μm) revealed that the aerosol particles bound to epidermal growth factor receptor (EGFR) and targeted cancerous lung tissue, with lower accumulation in normal lungs.45Tseng C.L. Wu S.Y.H. Wang W.H. et al.Targeting efficiency and biodistribution of biotinylated-EGF-conjugated gelatin nanoparticles administered via aerosol delivery in nude mice with lung cancer.Biomaterials. 2008; 29: 3014-3022Crossref PubMed Scopus (128) Google Scholar In another study, polystyrene nanoparticles loaded with near-infrared fluorescent dye (100 nm) were demonstrated by fluorescence imaging to accumulate in alveolar M2 macrophages and alveolar areas after administration to allergic airway inflammation mice via the intranasal tract.46Markus M.A. Napp J. Behnke T. et al.Tracking of inhaled near-infrared fluorescent nanoparticles in lungs of SKH-1 mice with allergic airway inflammation.ACS Nano. 2015; 9: 11642-11657Crossref PubMed Scopus (17) Google Scholar Cy5.5-labeled dry small interfering RNA (siRNA)/chitosan powder was shown to exhibit effective and specific gene silencing and excellent fluorescence imaging in vivo, thereby integrating diagnosis and treatment.47Okuda T. Kito D. Oiwa A. Fukushima M. Hira D. Okamoto H. Gene silencing in a mouse lung metastasis model by an inhalable dry small interfering RNA powder prepared using the supercritical carbon dioxide technique.Biol Pharm Bull. 2013; 36: 1183-1191Crossref PubMed Scopus (51) Google Scholar Chronic respiratory infections such as asthma, COPD, CF, and TB, and other infectious lung diseases, are usually treated by systemic administration (orally or intravenously) of high doses of antibiotics, which may cause several adverse effect