Aerosolised micro and nanoparticle: formulation and delivery method for lung imaging.

Purpose: The application of contrast and tracing agents is essential for lung imaging, as indicated by the wide use in recent decades and the discovery of various new contrast and tracing agents. Different aerosol production and pulmonary administration methods have been developed to improve lung imaging quality. This review details and discusses the ideal characteristics of aerosol administered via pulmonary delivery for lung imaging and the methods for the production and pulmonary administration of dry or liquid aerosol.
Methods: We explored several databases, including PubMed, Scopus, and Google Scholar, while preparing this review to discover and obtain the abstracts, reports, review articles, and research papers related to aerosol delivery for lung imaging and the formulation and pulmonary delivery method of dry and liquid aerosol. The search terms used were "dry aerosol delivery", "liquid aerosol delivery", "MRI for lung imaging", "CT scan for lung imaging", "SPECT for lung imaging", "PET for lung imaging", "magnetic particle imaging", "dry powder inhalation", "nebuliser", and "pressurised metered-dose inhaler".
Results: Through the literature review, we found that the critical considerations in aerosol delivery for lung imaging are appropriate lung deposition of inhaled aerosol and avoiding toxicity. The important tracing agent was also found to be Technetium-99m (99mTc), Gallium-68 (68Ga) and superparamagnetic iron oxide nanoparticle (SPION), while the essential contrast agents are gold, iodine, silver gadolinium, iron and manganese-based particles. The pulmonary delivery of such tracing and contrast agents can be performed using dry formulation (graphite ablation, spark ignition and spray dried powder) and liquid aerosol (nebulisation, pressurised metered-dose inhalation and air spray).
Conclusion: A dual-imaging modality with the combination of different tracing or contrast agents is a future development of aerosolised micro and nanoparticles for lung imaging to improve diagnosis success.

Introduction

Lung disease is one of the major causes of illness-related death, accounting for approximately six million deaths worldwide in 2019 (12% of total deaths) [1]. This number can remain unchanged in the future, as predicted by The Global Burden of Disease study, in which chronic obstructive pulmonary disease (COPD) and lower respiratory infection will be the third and fourth cause of death globally in 2030, respectively [2]. Furthermore, lower respiratory infection is predicted to rise in the future, particularly in tropical countries, due to the increased infectious activity of non-tuberculous mycobacteria, which tends to infect people with either genetic or acquired structural typical lung diseases, such as COPD and cystic fibrosis [3]. This condition is worsened by the emergence of coronavirus disease 2019 (COVID-19), which has become a global pandemic and has caused more than 6.24 million deaths around the world, as reported by the World Health Organization (WHO) [4]. Aside from the high mortality rate, lung disease is also predicted to be one of the global burdens of severe health-related suffering until 2060 [5]

The economic burden is one of the global impacts of lung diseases, caused by either unnecessary drug overprescription or treatment failure due to over-or underdiagnosis, respectively [6]. For example, studies on COPD misdiagnosis revealed that the underdiagnosis and overdiagnosis prevalence of this disease was 56.7–81.4% and 29.0–65.0%, respectively [7], leading to 55.4% dissipation of treatment cost [8]. The misdiagnosis of lung disease not only aggravates the economic burden but also can increase mortality due to false medical treatment. For instance, lung cancer is frequently misdiagnosed as tuberculosis in a country with tuberculosis prevalence. This phenomenon highlights the importance of lung diagnosis to reduce the lung disease burden and its mortality rate [9].

Lung imaging, comprising of computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT), is a standard method in lung disease diagnosis, as presented in Table 1. Nevertheless, each modality for lung imaging has weaknesses, such as the limited spatial resolution in molecular imaging-based modality [10-12]. To increase the performance of such a diagnosis method, micro and nano-inhalable particles have been investigated and applied for decades as a tracing agent or contrast agent [13-15]. As a tracing agent, the lung deposition of an inhaled particle can be detected due to the nature of the particle; for example, Technetium-99m (99mTc) emits gamma-ray, which can be detected by SPECT, resulting in an image of the lung [13]. On the other side, the contrast agent can alter the result of existing imaging methods (CT and MRI), providing brighter or darker regions for better diagnosis or interpretation [16-18].

Table 1

Detection mechanism in lung imaging

Detection source		Imaging method	Advantages	Disadvantages
Element	Mechanism
Tracing agent
99mTc	Gamma energy (140 keV)	SPECT	Trace amount is required (nanomolar) [19]	Radiations, limited spatial resolution (~ 15 mm), no lung morphology image [12]
68Ga	Positron (Emax = 1899 keV)	PET	Trace amount is required (nanomolar) [19]	Radiations, limited spatial resolution (~ 6 mm), no lung morphology image [12]
AuNP	X-ray absorption	CT	Therapeutic effect (photothermal and radiosensitiser) [20, 21]	higher concentration required compared to other CAs [22]
AgNP	X-ray absorption	CT	Antimicrobial activity [23]	Easy to be deposited in other tissues after pulmonary delivery [24]
Contrast agent
FeNP	Shortening the T1 relaxation time of nearby water	MRI	Magnetic hyperthermia therapy [25]	Inflammatory response and extrapulmonary toxicity were observed upon inhalation [26]
	Imaging electronic magnetisation of SPIONs	MPI
GdNP	Shortening the T1 and T2 relaxation time of nearby water	MRI	Radiosensitiser [27]	Toxicity of free ion [14]
MnNP	Shortening the T1 relaxation time of nearby water	MRI	Enhancement of photo- and chemotherapy [28]	Neurotoxicity upon inhalation [29]
I-CA	X-ray absorption	CT	Low cost [30]	IODINE sensitivity and high osmolality [30]

AgNP, silver-based nanoparticle; AuNP, gold-based nanoparticle; CA, contrast agent; CT, computed Tomography; FeNP, iron-based nanoparticle; 68Ga, Gallium-68; I-CA, iodonated-based contrast agent; MnNP, manganese nanoparticle; MRI, magnetic resonance imaging; PET, positron emission tomography; SPECT, Single photon emission computed tomography; SPIONs, super paramagnetic iron oxide nanoparticles; T1, longitudinal; T2, transverse; 99mTc, Technetium-99m

To date, 38 tracing and contrast agents have been approved for commercial use [30-32], and the development of new agents is underway to improve the safety aspect and image quality [17, 18, 23, 33, 34]. This review aimed to address the challenges of pulmonary delivery for lung imaging, as well as the aerosol formulations that have been commercially approved or are still in development study. There have been reviews on the application of nano or microparticles for imaging diagnosis purposes (CT: [15, 22]; MRI: [14, 35, 36]; PET: [37]; SPECT: [38, 39]), but there is no review which discusses the application of aerosol for lung imaging. This review is focused on understanding the critical parameters of aerosol delivery into the lung, which are essential for formulating micro- or nanoparticles for pulmonary delivery. Furthermore, the fundamental concepts of different aerosol production methods that contribute to the development of aerosolised contrast and tracing agent for lung imaging quality improvement are also discussed. We explored several databases, including PubMed, Scopus, and Google Scholar, during preparing this review to discover and obtain the abstracts, reports, review articles, and research papers related to aerosol delivery for lung imaging and the formulation and pulmonary delivery method of dry or liquid aerosol. The following search terms were used: “dry aerosol delivery”, “liquid aerosol delivery”, “MRI for lung imaging”, “CT scan for lung imaging”, “SPECT for lung imaging”, “PET for lung imaging”, “magnetic particle imaging”, “dry powder inhalation”, “nebuliser”, and “pressurised metered-dose inhaler”.

Challenges of aerosol delivery for lung imaging

Pulmonary delivery for contrast or tracing agent administration route has provided an image for lung disease diagnosis for decades [13]. Furthermore, an attempt to discover new inhalable particles with increased image quality and safety profile has also been reported in recent years [17, 18, 23, 33, 34]. For instance, Silva et al. reported the development of a dual-modality contrast agent consisting of Fe and gold (Au) for detection using MRI and CT, respectively [20]. Despite its success in lung imaging and potential for future development, several challenges need to be overcome in designing inhalable particles for tracing or contrast agents.

Toxicity

The first challenge is attributed to the toxic nature of different tracing and contrast agents. The established tracing agents are typically an element with radioactive hazards due to ionising radiation or particle emission. Nevertheless, typical radionuclides used for diagnosis are considered safe due to the low radiation energy and short half-life [13]. 99mTc, Tc, is the most widely used diagnostic radionuclide, including in lung scintigraphy. It has a half-life of 6 h and a gamma energy of 140 keV, yet sufficient for diagnosis purposes. [40]. Another radionuclide used for lung scintigraphy, Gallium-68 (68Ga, a positron-emitting radionuclide), has a shorter half-life (67.71 min) with Emax of 1899 keV [41]. The amount of radioactive element used for the diagnostic purpose is also meagre; for instance, the typical radioactivity of 99mTc for clinical use is 185–925 MBq, equal to only 0.95–4.70 ng of Tc metal [19]. On the other hand, a significantly higher amount of element is required for the contrast agent, leading to potential toxicity in the human body. Another issue in the use of radioactive tracing agents is the possibility of free radionuclide release from radiolabelled compound or complex, leading to free radionuclide extrapulmonary distribution. For instance, free pertechnetate has been found in thyroid upon lung scintigraphy due to unstable radiolabelled compound or complex [42]. Although such extrapulmonary distribution will not harm the organs, free radionuclides in significant amounts can disturb lung scintigraphy [43, 44]. Therefore, standard quality control for radiopharmaceuticals, e.g., radiochemical purity, should also be performed in the lung scintigraphy.

As an endogenous element in the human body, the use of iron (Fe) as a contrast agent is considered safe since ionic Fe presents as a complex with binding protein and the metal one has a low bio-solubility. Different binding and regulatory proteins are available for Fe absorption in the human digestive system without toxic effects [45]. However, such absorption facilitation is significantly limited in the respiratory tract, which might induce local adverse effects, such as inflammatory response. Furthermore, the inhaled Fe can easily be transported to other organs and causes more severe effects, including pro-atherosclerotic effect and alteration of autonomic regulation [26]. Similar to Fe, the inhalation of different elements for contrast agents can potentially induce intra- or extrapulmonary side effects, where the result can be even worse for exogenous substances, i.e. Au [46], gadolinium (Gd), and silver (Ag) [47]. In general, although the toxicity of these elements is enhanced in the ionic form [32], the nanoparticle form can also induce cell death due to the easy cellular uptake, resulting in cellular toxicity [26]. The toxicity of the iodinated contrast agent relies on the molecular form, in which inorganic iodine and ionic compounds exhibit higher toxicity [30].

Continuous efforts have been made toward minimising the toxicity of contrast agents, including the formulation of the stable complex to avoid the release of free ionic elements and interaction with cellular membranes, proteins, and other biological structures [30, 48]. Another attempt to reduce the toxicity is minimising cellular uptake, and particle absorption into the systemic circulation, which can be achieved by preparing micron or sub-micron size particle since effective cellular membrane penetration can only effectively occurs in size of < 200 nm [49]. Although larger particles can still be engulfed by alveolar macrophage, this type of cell is able to generate protein complex to minimise the toxicity of quantitatively large metallic element absorption, such as Fe [50].

Radio-protection of radioactive aerosol or gas

Radioactive contamination is one of the concerns during the utilisation of radiopharmaceuticals in the medical field. Common liquid radiopharmaceuticals typically pose a low contamination risk, requiring simple handling during preparation and administration to the patients. On the other hand, radioactive aerosol poses a relatively higher risk due to its nature that easily diffuses into the air. Brudecki et al. reported that the activity of 99mTc aerosol in a typical ventilation-perfusion SPECT facility ranged from 99 ± 11 to 6.1 ± 0.5 kBq m−3, resulting in daily intake by male technicians, female technicians, male nurses and female nurses of 5.4 kBq, 4.4 kBq, 3.0 kBq and 2.5 kBq, respectively [51]. Although such internal contaminations are considered safe for the medical staff (over three orders of magnitude lower than the dose limit), the contamination level should be kept as low as possible to avoid the reduction of gamma camera performance [52].

Lung deposition

Aside from toxicity and radio-protection concern, designing the inhalable particle size should also consider lung deposition since particle larger than 5 µm is most likely deposited in upper airways, followed by rapid pulmonary clearance and resulting in poor lung image in the healthy lung [39]. The presence of disease in the lung is most likely to alter the particle deposition due to the decrease in airway cross-section. Several studies, as presented in Table 2, revealed that most lung diseases enhanced lung deposition, although the deposition pattern has become more heterogenous as some areas are poorly ventilated and less penetrated by inhaled particles [53]. Such a condition will lead to great disadvantages in lung disease therapy since the drug bioavailability will be under or over the therapeutic window in some areas, resulting in underdose or toxic effects, respectively [54, 55]. For diagnostic purposes, the heterogenous deposition of inhaled particles might not be disadvantageous since less penetrated lung area can be an indication of a certain disease. Nevertheless, a particular aerodynamic size range is still required to ensure that the lung deposition is adequate for generating a good diagnostic image [53].

Table 2

The inhaled particle deposition in different clinical lung conditions

Clinical indication	Inhaled particles	Aerodynamic size	Key findings	References
(AAT) deficiency	99mTc-AAT (nebulisation into human airways)	4 μm	No significant difference in lung deposition in the healthy and diseased lung (C/P ratio 1.48–1.66)	[61]
CF	99mTc-AAT (nebulisation into human airways)	4 μm	No significant difference in lung deposition between the healthy and diseased lung (C/P ratio 1.37–1.48)	[61]
	99mTc-iron oxide (nebulisation into human airways)	4.6–4.9 μm	No significant difference in lung deposition between the healthy and diseased lung (C/P ratio 1.02–1.78)	[56]
	Levofloxacin (nebulisation into lung model)	3.65 μm	Lung deposition in CF was higher (30–50%) than in healthy lung (15–30%)	[62]
	99mTc-DTPA-Tobramycin (nebulisation into human airways)	eFlow: 3.95 μm	Lung deposition in CF was lower (26.6%) than in healthy lung (40.0%)	[63]
		LC: 3.54 μm	No significant difference in lung deposition between the healthy and diseased lung (44.3–45.2%)
COPD	Diesel exhaust particle	10–500 nm	The deposition fraction increased with increasing the severity of the disease. The deposition fraction of particles of < 100 nm was decreased in COPD patients	[58]
	Polystyrene latex nanospheres (Electrospray aerosol generation into human airways)	50 and 100 nm	The deposition fraction was significantly lower in the lung with emphysema compared to healthy ones (p = 0.001–0.01)	[64]
	Polystyrene latex nanospheres (Electrospray aerosol generation into human airways)	50 nm	Exhaled particle in COPD was significantly higher (0.128) compared to the healthy one (0.074) ( p = 0.01)	[59]
	99mTc-carbon/Technegas (aerosolisation into human airways)	100 nm	No significant difference in lung deposition, retention, and clearance in COPD and healthy lung	[65]
	Particulate matter in the air of industrial area	82–5960 nm	Lung deposition was significantly higher in the diseased lung compared to healthy ones	[57]
Asthma	Nanocarbon (spark generator)	23 nm	The efficient respiratory deposition was higher in subjects with asthma	[66]
	Aerosolisation into lung model	man: 8.1 μm chi: 6.1 μm	The deposition is significantly higher in the upper airways of the asthmatic model compared to healthy ones	[67]
	99mTc-BDP/FF combination (nebulisation into human airways)	BDP: 1.5 μm FF: 1.4 μm	No significant difference in lung deposition in the healthy and diseased lung (54.9–56.2%)	[68]
OLD, RLD	DEHS (aerosolised into human airways)	0.02–0.24 μm	Lung deposition was increased in OLD and unchanged in RLD compared to healthy ones	[69]

AAT, a1-antitrypsin; CF, cystic fibrosis; COPD, chronic obstructive pulmonary disease; DEHS, di-2-ethylhexyl sebacate; C/P ratio, central to peripheral ratio; OLD, obstructive lung disease; RLD, restrictive lung disease

As seen in Table 2, the lung deposition of inhaled particles with an aerodynamic size of 1–5 μm was typically not affected or increased in the presence of lung diseases. Brown et al. suggested that although obstructed airways in diseased lungs should have reduced lung deposition, mucociliary clearance impairment and slow-deep breathing patterns can enhance the deposited particles and make them similar to those of the healthy lung [56]. Higher particle deposition in the diseased lung is also possible, as reported by Furi et al., since narrower airways induce airflow turbulence resulting in higher inertial impaction. Such a higher deposition might also be induced by the use of polydisperse particles (82–5960 nm), where larger particles will be retained in the upper airways, while smaller ones penetrate deeper lung [57]. In contrast, monodisperse particles are most likely to give more heterogenous lung deposition since large particles will only be deposited in the upper area, and ultrafine particles tend to be exhaled [58-60]. To ensure the size of inhalable particles falls within the desirable range, especially for underdeveloped products, in vitro aerosolisation devices, e.g., cascade impactor, can be utilised for quality control [54].

It can be concluded that the inhalable powder should be designed in a size of 200 nm–5 µm to minimise systemic absorption and to reach deep lung deposition for the best quality image and to minimise systemic absorption, respectively, although the presence of a particular disease inducing airways obstruction requires smaller inhaled particles to reach deep lung effectively. Different formulations and aerosol delivery methods have been developed to achieve this purpose, as discussed in the next section (Fig. 1).

Fig. 1

The fate of inhaled particles depends on their size [26, 54]

Formulation and aerosol delivery method into respiratory airways

Dry aerosol

Dry formulation provides a stable form, which can be stored for a long time. However, the main challenge of this form is the preparation method, which might require a sophisticated instrument to produce an ideal dry aerosol for pulmonary delivery [54].

Graphite ablation

Graphite ablation method is used in Technegas to produce 99mTc-radiolabelled carbon aerosol with a size of < 100 nm for lung ventilation scintigraphy [70]. 99mTc-pertechnetate in saline solution yielded from 99Mo/99mTc generator is used as 99mTc source in this method. The 99mTc-radiolabelled carbon aerosol generation method has been detailed in a review by Wiebe et al. as illustrated in Fig. 2 [71]. In brief, 99mTc solution is added to a crucible, followed by evaporation at 70ºC for 6 min in an ultrapure argon environment. Subsequently, graphite and 99mTc are ablated by an alternating-current arc generated between the terminals holding the crucible. Aerosolised carbon nanoparticles are produced by heating at 2750 °C for 15 s in an ultrapure argon environment [13].

Fig. 2

Production of 99mTc-carbon aerosol by graphite ablation method.

Adapted from Wiebe et al. [71]

Carbon nanoparticle generated from the graphite ablation method has a size of < 140 nm with a gas-like characteristic upon aerosolisation, which provides an ideal lung deposition for imaging purpose [13, 71]. Many studies reported different sizes of carbon aerosol from Technegas, depending on the particle collection and characterisation methods [72]. For instance, the use of electrostatic precipitation and transmission electron microscopy (TEM) by Senden et al. reported size of 30–60 nm, while a 400-mesh filter coupled with glass-fibre filter paper and photo correlation spectroscopy resulted in a size of 97–140 nm [71, 73]. A size of ~ 100 nm observed from Technegas is most likely due to the aggregation upon particle collection [71].

As an inhalable particle, carbon aerosol with a size of < 140 nm will follow Brownian diffusion upon inhalation, leading to deposition in the alveolar region and lower respiratory tracts [54]. Such lung deposition is considered an ideal particle distribution for lung imaging [74]. Such particle size for inhalable particles theoretically can easily penetrate the cellular membrane and induce toxicity [49]. Nevertheless, the clinical use of Technegas for more than three decades proves the safety of the 99mTc-labelled carbon aerosol [13, 71]. This safety profile can be explained by the fact that the amount of carbon nanoparticles inhaled is very low, which means the toxicity effect can be neglected [75]. Furthermore, a study by Zhang et al. revealed that carbon nanoparticles with a size of < 100 nm showed no toxicity in RAWS264.7 cells, and a toxic effect was observed in the nanoparticle in size of > 250 nm [76]. A review by Pacurari et al. also demonstrated that multi-walled carbon nanotubes with smaller sizes were less potent in inducing lung fibrosis upon inhalation [77].

Due to an ideal lung distribution and no toxicity, 99mTc-labelled carbon aerosol has been widely used in lung imaging to diagnose different lung-related diseases, such as COPD, pulmonary embolism (PE), and tracheobronchitis [38, 78, 79]. Nasr et al. reported that lung scintigraphy by 99mTc-labelled carbon aerosol in COVID-19 patients has successfully diagnosed COPD and PE [74]. The same method has also found the presence of tracheobronchitis in COVID-19 patients by observing an increase in tracheobronchial uptake of 99mTc-labelled carbon aerosol, as reported by Verger et al. [70]. In conclusion, graphite ablation method generates radiolabelled carbon aerosol ideal for lung ventilation scintigraphy. Nevertheless, graphite ablation is a sophisticated method and can only be performed using a commercial aerosol generator called Technegas.

Spark ignition

Spark ignition method has been utilised to produce different nanoparticles, including alloy, composite, metal, oxide, and semiconductor [80]. This method generates nanoparticles as an aerosol via spark discharge from two heated conducting electrodes with gas flow, as illustrated in Fig. 3 [81]. Similar to graphite ablation, this method spontaneously produces nanoparticles directly from large and solid material, resulting in spherical particle with a size of < 10 nm that tends to agglomerate into a larger particle (~ 20 nm) [80].

Fig. 3

Aerosolisation devices for liquid formulation: A nebuliser, B pressurised-metered dose inhaler (pMDI), C air sprayer [114–116]

Gold nanoparticle (AuNP) has been formulated into nano aerosol by spark ignition method as a CT contrast agent. Kreyling et al. successfully produced 20 nm-sized AuNP by this method and used radioactive gold (195Au) as a material for tracing purposes. An intratracheal inhalation into Wistar-Kyoto rats demonstrated that approximately 30% of AuNP reached the respiratory tract, followed by rapid mucociliary clearance, and about 80% of AuNP deposited in alveoli was moved from the inner surface into the interstitium within 24 h. Although Kreyling et al. did not perform a CT scan to evaluate the contrast agent performance, the primary deposition of inhaled AuNP in the alveolar region indicates its potential for lung imaging. Nevertheless, an extrapulmonary deposition was also found, mainly in the liver, which increased up to 28 days [33]. Therefore, the toxicity in other organs should be carefully observed to ensure the safety of the AuNP administration.

Another CT contrast agent that has been formulated into nano aerosol by spark ignition is AgNP, which was investigated for its potential toxicity upon inhalation. Campagnolo et al. delivered ~ 25 nm AgNP, generated by spark ignition, into pregnant C57BL/6 female mice via nose-only inhalation, resulting in AgNP accumulation in placentas, albeit very low, and increasing the expression of pregnancy-relevant inflammatory cytokines in the placentas [82]. A biokinetic study of spark-ignited AgNP in Wistar-Kyoto rats administered via tracheal inhalation was also reported by Kreyling et al. Tracing method using radioactive 105AgNP showed that 105AgNP was mainly excreted via larynx into the gastrointestinal tract due to poor solubility in the lung fluid [83]. These findings imply that the extrapulmonary toxic effect of inhaled AgNP can be minimised by lowering the solubility of the nanoparticle in lung fluid to enhance the mucociliary clearance and reduce the epithelium penetration.

Spray drying

Spray drying method has been widely investigated for preparing dry powder for drug administration via pulmonary delivery. The ability to incorporate different nanoparticles into size-controllable powder makes spray drying a promising method for developing inhalable powder for lung imaging purposes [54]. In general, spray dryer is classified based on droplet generation and powder collection system. A conventional instrument utilises a gas atomiser and cyclone or bag filter for droplet generation and powder collection, respectively [54]. In a gas atomiser system, the liquid formulation is atomised by pressurised nitrogen gas, in which droplet diameter is affected by the liquid feed and gas flow rate. Droplet diameter can be increased by the increase of feed rate and reduced by increasing the gas flow rate. The cyclone separation system utilises the gravitational force of rotating flow to collect the powder with high inertia [84]. The new instrument, the so-called nano spray dryer, uses a nebuliser to generate droplets and an electrostatic separation system to collect powder [85].

Despite the better particle engineering offered by a nano-spray dryer, only conventional spray dryer type has been utilised for the preparation of an inhalable powder containing 99mTc-diethylenetriamine pentaacetic acid (DTPA) for lung imaging, as reported by Yang et al. in this study, a clinical trial in nine healthy subjects demonstrated that inhaled powder reached lung at 50–70 L/min peak inhalation flow rate. Yang et al. concluded that total and regional lung depositions were not significantly affected by inhaler resistance. Instead, such lung depositions are significantly influenced by inhalation rate and particle size [86]. Despite the success in a clinical trial, the spray drying of radioactive material is a sophisticated process, particularly in avoiding the leakage of radioactive dust from the instrument. It requires tight containment and radioactive material entrapment to ensure the safety of the personnel from radioactive contamination [87]. In contrast, a non-radioactive contrast agent formulation can be carried out using a typical spray dryer without any additional equipment. Furthermore, a combination of different nanoparticles or elements for dual-imaging or theranostic purpose can simply be performed by pre-mixing prior to spray drying, as reported by Julián-López et al. who developed an iron-silica hybrid using a home-made conventional spray dryer [88].

Aside from conventional and nano-spray dryer, supercritical CO2-assisted spray dryer has also been utilised to produce a dry powder with complex structures, such as aerogel. Such a technique allows the material to pass through the supercritical region with the assistance of supercritical fluid, resulting in the absence of interfacial stress [89]. The application of supercritical CO2-assisted spray drying (SASD) for the preparation of inhalable powder containing contrast agents has been reported in the literature.

However, the SASD method poses challenges, including sophisticated instrumentation, high costs, and the presence of organic solvent residuals in the end product [89], which hampers further development in inhalable powder technology [20, 90–92]. For instance, a nanohybrid for dual-imaging consisting of strawberry-like gold-coated magnetite nanocomposites was developed by Silva et al. by SASD method. Using chitosan as a filler, the spray drying process resulted in powder with an aerodynamic diameter of 2.6–2.8 μm and a fine particle fraction (FPF) of 48–55%, which is sufficient for deep lung deposition [20]. Nevertheless, supercritical CO2-assisted spray drying poses challenges, namely high costs, complex instrumentation, and the presence of organic solvent residuals in the end product [89].

Although gold and iron NP are the only investigated contrast agent as inhalable powder via spray drying [90-92], this method is suitable for formulating other contrast or tracing agent into dry powder. The ability to design particle size, structure, and composition makes spray drying a promising method to produce inhalable powder for lung imaging.

Liquid aerosol

Liquid aerosol for pulmonary delivery is a simpler and cheaper formulation than the dry one. The drug formulation can be dissolved in a suitable solvent, followed by aerosolisation using relatively unsophisticated devices compared to those used for dry aerosol. Another characteristic of liquid aerosol is the immediate solubility upon deposition in the lung airways, avoiding a mucus trap and mucociliary clearance [54]. Therefore, the pulmonary clearance profile of inhaled aerosol is merely affected by the cargo’s characteristics, i.e. size and solubility, simplifying the designing process of inhalable particles [93]. Aside from the aforementioned advantages, the application of liquid aerosol in pulmonary delivery also poses challenges, namely the difficulty in aerosol size adjustment and the lower drug stability in liquid formulation compared to that in the dry one [94]. Standard devices used to produce liquid aerosol are nebuliser, pressurised metered-dose inhaler (pMDI), and air spray [55].

Nebulisation

Nebuliser is the oldest aerosol device and the most widely used for pulmonary drug administration. This device can produce aerosol from liquid formulation using different instrument types, including ultrasonic, jet and vibrating mesh (Fig. 3a). Ultrasonic nebuliser applies high-frequency vibration of the piezoelectric element that transforms liquid formulation into tiny droplets due to sound wave effect. This device type might generate heat upon aerosolisation, which may impact the drug cargo's stability. [93]. A jet nebuliser uses compressed gas that passes through a narrow gap to generate lower pressure and faster gas flow. Subsequently, such a gas flow draws and leads the liquid formulation to hit the baffle for the generation of tiny droplets. Jet nebuliser has two types of valves: the first valve opens during inhalation to increase the aerosol flow, while the second one opens during exhalation for gas release to the atmosphere, avoiding a reverse gas flow. A vibrating mesh nebuliser applies mesh vibration to generate tiny droplets from liquid formulation [55].

To date, nebulisation is the most widely investigated method to deliver contrast or tracing agent via pulmonary delivery for lung imaging, with 31 publications in the past decade (Table 3). Such an extensive investigation of the nebulisation method is most likely due to the simple operating technique during aerosolisation from liquid formulation [93]. This simple method can also be used to aerosolise radioactive material since a commercial nebuliser equipped with the lead container, e.g. SmartVent™, is available for this purpose [95]. Furthermore, different types of nebulisers lead to dissimilar performance, providing device options to adapt to various drug cargo types [96].

Table 3

Application and development of nebulisation method for lung imaging in the last decade

Contrast/tracing agent	Nebuliser type	Research objective	Key finding	References
99mTc-sestamibi	Vibrating mesh	Evaluating MRP1 expression in the lung as a result of smoking	Smoking upregulates MRP1 and delays clearance of inhaled Tc-99m-MIB	[95]
99mTc-DTPA	–	Investigating aerosol deposition in ferrets’ lung	The respiratory tract deposition was ~ 9% for liquid formulation and 5–6% for the dry one. The lung deposition in both formulations were similar	[98]
99mTc-DTPA	–	Evaluation the use of V/Q scintigraphy as an alternative modality for diagnosis in patients with bronchopleural fistula	bronchoscopy or the methylene blue test failed to identify residual air leak detected by V/Q scintigraphy	[109]
99mTc-sestamibi/DTPA/MDP	–	Comparing the use of 99mTc-sestamibi, 99mTc-DTPA, and 99mTc-MDP in P/Q scintigraphy to asses pulmonary thromboembolism	99mTc-sestamibi demonstrated the highest count rate, the slowest alveolar clearance, and the best quality image	[117]
99mTc-nanocarbon or 99mTc-DTPA	–	Evaluating the occurrence of COPD in patients suspected PE using V/Q scintigraphy and investigating the performance of V/Q scintigraphy to diagnose PE in patients with COPD	PE was diagnosed in 353 (28%) patients. Mild, moderate or severe airway obstruction were shown in 697 patients (55%). Among patients with PE, 90 patients (25%) were also diagnosed as COPD	[74]
99mTc-DTPA	–	examination alveolar fluid clearance in CHF patients compared against healthy controls	A significantly faster alveolar fluid clearance was observed in CHF patients than controls	[108]
99mTc-sulfur colloid	Jet	Evaluating the aerosol deposition and mucociliary clearance following Ivacaftor treatment in Cystic Fibrosis	The homogeneity improvement was observed, indicated by a decrease in number ratio of pixels with higher and lower deposition. Mucociliary clearance also increased	[118]
99mTc-arginine	Jet	Evaluation the deposition of 99mTc-arginine in the lung for COPD therapy	15 ± 2% of aerosolised 99mTc-arginine was deposited in the lung	[119]
99mTc-sestamibi	Vibrating mesh	Investigating the correlation between 99mTc-sestamibi clearance and MRP1 expression	99mTc- sestamibi clearance was faster in patients with low MRP1 expression compared with those with higher MRP1 expression	[120]
99mTc-DTPA	Vibrating	Comparing lung deposition in two invasive mechanical ventilation: pressure support ventilation and volume‐controlled ventilation	The lung deposition for pressure support and volume-controlled ventilation was 10.5 ± 3.0 and 15.1 ± 5.0%, respectively. Pressure support ventilation demonstrated higher endotracheal tube and tracheal deposition	[101]
99mTc-DTPA	–	Evaluating the effect of positive expiratory pressure on 99mTc-DTPA clearance	99mTc-DTPA clearance was significantly faster with the presence of positive expiratory pressure	[102]
99mTc-sulfur colloid	Vibrating mesh	Evaluating the effect of posture (supine and seated) on lung deposition	99mTc-sulfur colloid deposition in the alveolar region for supine was significantly lower than for seated. The deposition in the intermediate region and small airways for supine was significantly higher than for seated	[121]
99mTc-DTPA	Jet	Comparing lung deposition after central and peripheral administration	There was no significant difference in lung deposition after central and peripheral administration	[103]
99mTc-DTPA	Vibrating mesh	Evaluating whether 99mTc-DTPA can distinguish asthma and COPD	The T1⁄2 values of 99mTc-DTP of the non-smoking COPD patients were significantly lower than non-smoking asthma patients. The cap value was significantly higher in non-smoking COPD patients	[106]
99mTc-tin colloid	–	Comparing the effect of Laerdal and Mapleson-C circuits for positive end-expiratory pressure setting on the lung deposition	Aerosol deposition was higher in the right lung region than in the left one, and the lowest deposition was observed in the left lower region	[122]
99mTc-DTPA	Ultrasound	Evaluating the application of low dose CT to distinguish between COPD and PE during V/Q SPECT/CT	The mean lung tissue density in healthy areas was 758 HU, in V/Q mismatch defects (PE) 695 HU, and in V/Q match defects (COPD) 900 HU	[107]
99mTc-DTPA	–	Investigating the influence of continuous positive airway pressure and body position on the lung clearance	20 cmH2O of air pressure significantly reduced the lung clearance in the supine and seated positions. However, 10 cmH2O air pressure did not affect the lung clearance in both positions	[100]
99mTc-DTPA	Jet and vibrating mesh	Comparing the performance jet and vibrating mesh nebuliser in healthy subjects	Vibrating mesh nebulisers delivered more than twofold 99mTc-DTPA into the lung compared to jet nebulisers	[96]
99mTc-DTPA	Jet	Investigating the effect of Acapella positive expiratory pressure device and nebuliser configuration on the lung deposition	Administration without Acapella and with nebuliser in between Acapella and patient resulted in higher lung deposition than setting the nebuliser distal to the Acapella	[104]
99mTc-DTPA	Jet and vibrating mesh	Comparing the performance jet and vibrating mesh nebuliser coupled with NIV in COPD patients	Vibrating mesh nebulisers delivered more than threefold 99mTc-DTPA into the lung compared to jet nebulisers	[97]
99mTc-DTPA	Jet	Investigating the effect of NIV coupling on the lung deposition in the asthma patients	The application of NIV upon nebulisation did not improve the lung deposition of 99mTc-DTPA	[105]
NH2-(PVA/PEG) AuNPs	Vibrating mesh	Investigation of aerosolised AuNPs functionalised with NH2PVA and NH2-PEG	Accumulation in macrophages (20%), epithelial (60%), and dendritic cells (20%) was observed after repeated aerosolisation. No cytotoxicity and proinflammation were shown but significant apoptosis was observed after 96 h	[110]
AgNP	Jet	Documenting the kinetic time course of inhaled Ag NPs with size of 20 nm	Only small percentage of aerosol reached the lung (1–2.5%) since most of the aerosol were retained in the upper airways. AgNP was also found in gastrointestinal tract, liver, lymphatic node, OB, kidney, and spleen, 6 h following the onset of inhalation	[23]
AgNP with a size of 20 or 110 nm	Jet collision	Evaluating: the deposition and retention of AgNP in the nasal cavity; the translocation and retention time of AgNP in the OB; and the effect of AgNP in the OB on the activity of microglial cells	The highest nasal deposition was found immediately after AgNP deposition in the nose, translocation rate to the brain, and subsequent microglial activation in the OB was affected by AgNP size and time after inhalation	[24]
SPIONs	Jet	Pysicochemical characterisation of SPIONs aerosol	The aerosol size measured by TEM, PCS, NTA, impinger, and SMPS was 9 ± 2, 27 ± 7, 56 ± 10, 159 ± 46, and 75 nm, respectively	[113]
SPIONs	Vibrating mesh	Development of MPI for inhaled aerosol tracking and quantification	SPIONs have potential as highly sensitive tracer for lung imaging and theranostic agent via magnetic actuation or hyperthermia effect	[17]
Gd-DOTA	Jet	evaluating the performance of UTE sequences for imaging and quantifying the deposition of inhaled Gd-DOTA	3D acquisitions with isotropic millimetric resolution were obtained rapidly. Images demonstrated homogeneous and significant signal enhancement (> 200%). The visualisation of deposited Gd-DOTA in the level of the bronchi of secondary lobules was obtained	[16]
Gd-DOTA	–	aerosol deposition and to characterize signal enhancement in asthmatic rat lungs	A lower signal enhancement and deposited aerosol were observed in asthmatic model rats. Approximately 1 μmol/kg body weight in the standard clinical MRI has potential application for asthma diagnosis	[34]
Gd-DOTA (gadoterate meglumine)	Jet	Evaluating the performance of UTE sequences for quantifying the deposition of inhaled Gd-DOTA	In the lung parenchyma, 50% signal enhancement was observed. Gd-DOTA clearance was 14% per h and Gd-DOTA deposition was homogeneous in the lung	[111]
MnCl2	–	Investigating the effectiveness of MnCl2 on targeting adenocarcinoma and its toxicity	Tumours of submillimeter size can be identified by pulmonary delivery of MnCl2 with dose of 20 times lower than that of systemic. No Mn2+ was detected in the brain and liver	[112]
Iodine-based liquid (Ultravist-370)	Vibrating mesh	Exploring the use of dynamic X-ray phase-contrast imaging to observe pulmonary drug delivery	Only 8% of liquid reached the lung. Temporal resolution was up to 1.5 fps	[18]

AgNP, silver nanoparticle; AuNP, gold nanoparticle; CHF, chronic heart failure; COPD, chronic obstructive pulmonary disease; DOTA, dodecane tetraacetic acid; DTPA, diethylenetriaminepentaacetic acid; HU, Hounsfield units; MDP, methylene diphosphonate, MPI, magnetic particle imaging; MRP1, multidrug resistance protein-1; NIV, non-invasive ventilation; OB, olfactory bulb; PE, pulmonary embolism; PEG, polyethylene glycol; PVA, polyvinyl alcohol; SPIONs, super paramagnetic iron oxide nanoparticles; 99mTc, Technetium-99m

Among different types of nebulisers, a vibrating mesh is the most widely used in clinical or laboratory practice, as seen in Table 3. The reason behind the wide use of vibrating mesh nebulisers has been investigated by Galindo-Filho, et al., where the comparative study of jet and vibrating mesh nebulisers is carried out either in healthy subjects or COPD patients. In healthy subjects, vibrating mesh nebuliser delivered more than twofold 99mTc-DTPA into the lung compared to jet nebulisers [96], while in COPD patients, vibrating mesh nebuliser delivered more than threefold 99mTc-DTPA into the lung [97]. Galindo-Filho et al. suggested that such a difference is caused by different droplet sizes generated by both nebulisers, where jet and vibrating mesh nebuliser generated droplets with a size of 5 and 3.4 µm [96]. As discussed earlier, a particle size of < 5 µm is essential for deep lung deposition [54]. Aside from comparing different types of nebuliser, a comparative study between 99mTc-sulfur colloid powder prepared by Palas rotating brush generator and 99mTc-DTPA aerosolised with nebuliser has also been reported in the literature. Kuehl et al. reported that there is no significant difference in both administration methods (6.81–9.08%) since the particle sizes were similar (2.5–2.8 µm) [98].

Table 3 also shows that most of the literature was clinical studies with 99mTc-DTPA. It is understandable since 99mTc-DTPA is one of the radiopharmaceuticals recommended by the European Association of Nuclear Medicine (EANM) guideline and can easily be administered into the lung [99]. As seen in Table 3, nebulised 99mTc-DTPA is mainly used to investigate the effect of particular pulmonary delivery equipment, e.g. positive expiratory pressure device, on the aerosol performance of lung deposition [97, 100–105]. Other studies with 99mTc-DTPA focus on distinguishing different lung diseases [74, 106, 107], understanding the physiology of lung disease [108], and investigating the new application of ventilation scintigraphy [109].

Besides the clinical application, nebuliser can also be used for development study of a new contrast agent administration via pulmonary delivery, including AuNP [110], gadolinium nanoparticle (GdNP) [16, 34, 111], Iodine-based contrast agent (I-CA) [18], MnCl2 [112], silver nanoparticle (AgNP) [23, 24], and superparamagnetic iron oxide nanoparticle (SPION) [17, 113]. Such a study aimed to determine lung deposition and clearance [111], investigate biokinetics and toxicity [110, 112], and evaluate the lung image quality generated after inhalation [16, 18].

Despite the simple use of a nebuliser to produce aerosol for pulmonary delivery, this device also has challenges that need to be solved, namely stability issues in liquid formulation and difficulty in particle engineering. The first challenge can be overcome by preparing the drug solution into a lyophilised formulation that can be rehydrated prior to use. Such a lyophilisation method has been applied in radiopharmaceutical kits formulation that can be rehydrated and radiolabelled by 99mTc-pertechnetate in saline solution [94]. Therefore, this method can also be applied in the development of other contrast and tracing agents. Another challenge is the limitation of the nebuliser in particle engineering, unlike spray drying, which can easily design the inhalable powder, e.g. size and porosity [54].

Pressurised metered-dose inhaler (pMDI)

Pressurised metered-dose inhaler (pMDI) releases liquid formulation mixed with propellant from a pressurised canister, allowing droplets generation for aerosol inhalation (Fig. 3b) [123]. Due to the portable size and simplicity of use, patients can use pMDIs for inhalation treatment without any help from a medical worker [115]. The main drawback of pMDI is the incompatibility of particular drug cargo with the propellant mixture or pressurised storage. To date, 99mTc-labelled radiopharmaceutical is the only substance administered by pMDI for investigating the lung deposition of particular drugs and the performance of additional equipment for pulmonary delivery [123-126].

Radiotracing studies of pMDI-delivered aerosol revealed that lung deposition is influenced by the aerosol size, liquid formulation, and additional inhalation equipment. Clinical study of pMDI-aerosolised Beclomethasone and Fluticasone with hydrofluoroalkane (HFA) as a propellant in patients with asthma has been reported by Leach et al. This study demonstrated that the lung deposition of 99mTc-Beclomethasone was significantly higher than that of 99mTc-Fluticasone (55 vs. 24%), which can be explained by the difference of aerosol size (0.7 vs. 2.0 µm) [126]. A study reported by Ditcham et al. demonstrated that the deposition of inhaled 99mTc-Albuterol aerosol with a facemask was higher (18.1%) than that with a spacer mouthpiece (22.5%) [123]. A study comparing the lung deposition of radiolabelled drug-aerosol in healthy subjects and patients with asthma reported that the deposition patterns were similar in healthy and people with asthma (22–25%) [124]. Nevertheless, the lung deposition of pMDI-aerosol might be different in other lung diseases, as observed in a study using aerosol generated by other devices [13, 74].

Although the development of a pMDI-aerosolised contrast agent has not been reported, the delivery of nano-structured particles via pMDI is feasible, as reported by Taylor et al., where glycopyrronium/formoterol fumarate or phospholipid porous particles was 99mTc-radiolabelled and administered via pMDI into five healthy male subjects. A gamma scintigraphy assessment revealed that 38.4% of emitted dose reached the lung, 61.4% was detected in the oropharyngeal and stomach, and < 0.25% of the emitted dose was detected in the exhalation filter [125].

Air spray

The last method that has been investigated for the delivery of an inhalable tracing agent is air spraying method, which is similar to the atomisation method used in spray dryer for droplet generation. In brief, pressurised medical air and pumped liquid formulation are applied to generate an atomising flow of 0.5 L min−1 with a median particle size distribution of 40–60 μm. Although such a distribution range is theoretically too large for desirable lung deposition, the preclinical study of 99mTc-labelled porcine surfactant in six sedated 1-day-old piglets nasal-mask continuous positive pressure airway demonstrated 40% lung deposition after 28 min inhalation [116]. Such relatively high lung deposition compared to other aerosolisation methods, such as nebulisation [101, 119], can be due to the difference in an animal model or the presence of a continuous positive pressure airway [104]. Therefore, a comparative study should be performed between air spray and other nebulisation methods with identical experimental conditions to compare better.

Conclusion and future perspective

The use of contrast and tracing agent in lung imaging has been applied in the last decades, with 38 commercially available products. A high surface area, non-invasive administration, the potential to avoid extrapulmonary toxicity, and the availability of various administration devices make pulmonary delivery the best option for administering contrast and tracing agents to obtain optimal image quality. The key performance parameter of inhalable aerosol is the particle size affecting the lung deposition and clearance, eventually determining the image quality and toxicity profile, respectively. Another critical parameter of inhaled contrast and tracing agents is the elemental form, such as ionic or nanoparticle form, affecting the lung clearance profile. The research outputs reviewed here show that different aerosolisation methods are suitable depending on the characteristics of contrast and tracing agents, such as the graphite ablation method for a radionuclide-based tracing agent. Nevertheless, graphite ablation and nebulisation are the most widely used aerosolisation method for dry and liquid aerosol production, respectively.

Further development of aerosol delivery for lung imaging should also consider the combination of more than one element to improve the image quality or provide a dual-imaging modality. For instance, the combination of Fe and Au provides a dual contrast agent using MRI and CT scans. Such a combination can be prepared upon preparing nanoparticles, resulting in dual elements nanoparticle, or combined upon aerosol preparation, such as during spray drying. The latter method can be performed easily since the combination of available formulas is feasible as long as the formulas are compatible. Another further development of new aerosol for lung imaging is using an animal model, which mainly focuses on small animals, such as a mouse. Due to the distant physiological and anatomical gap between the animal model and human lung, the use of small animals in pre-clinical study typically has low translational success. Therefore, the use of relatively larger animals, such as piglets or ferrets, should be considered in future studies.