The lung parenchyma consists of airways and finally the alveoli. The alveoli are constructed from a thin tissue layer covered with microvessels. It has been observed that particles $\le$5 $\mu$m go through the oropharynx to the trachea, then the smaller airways and finally the alveoli where they are absorbed to the systemic circulation [1]. The absorption and safety of an aerosol compound depends on the PH, particle size, salt of particles, residual cup loading, residual cup design, patient respiratory function, aerosol production source and drug formulation [1, 2]. Antibiotics have been used for several years for cystic fibrosis. Inhaled insulin has been investigated as aerosol administration versus subcutaneous [3, 4]. Inhaled antibiotics are also in use for cystic fibrosis [5]. Moreover, we can use inhaled nitric oxide for idiopathic pulmonary fibrosis [6]. Finally, several drugs have been used or investigated as aerosol administration of pulmonary hypertension. Pulmonary hypertension is observed when the mean pulmonary arterial pressure (PAP) is $\ge$25 mmHg at rest [7]. It is considered a fatal disease due to vascular proliferation, small vessel obstruction and remodeling. Moreover; these patients have increased pulmonary vascular resistance (PVR). As a result right-sided heart failure (HF) is observed and death occurs if not treated properly [8]. According to current guidelines the disease has been divided into: (a) Pulmonary arterial hypertension, (b) Pulmonary hypertension due to left heart disease, (c) Pulmonary hypertension due to lung disease and/or hypoxia, (d) Pulmonary hypertension due to pulmonary artery obstruction and (e) Pulmonary hypertension with unclear and/or multifactorial mechanisms [9, 10, 11]. The major diagnostic examination is right-heart catheterization, afterwards an optical coherence tomography follows. The major treatment options for pre-capillary pulmonary arterial hypertension are: General measures, pregnancy avoidance, Influenza and pneumococcal vaccination, Psychosocial support, supervised exercise training and avoid excessive physical activity that leads to distressing symptoms, Inflight oxygen (O${}_2$) therapy for partial pressure of oxygen (PO${}_2$) less than 60 mmHg or class III–IV, avoidance of general anesthesia and use epidural instead, if feasible. Supportive therapy with: (a) Diuretics for RV failure and fluid overload. Long-term O${}_2$ if PO${}_2$ is consistently less than 60 mmHg, (b) Correct anemia and/or iron deficiency, (c) Calcium channel blockers (CCBs). In case of inadequate clinical response to initial combination or monotherapy, sequential double or triple combination therapy is recommended (riociguat and phosphodiesterase type 5 inhibitor (PDE-5i) are contraindicated). In case of inadequate clinical response with sequential double combination therapy, triple combination should be attempted. Refer to lung transplant if there is inadequate response to combination therapy. The following dual combination therapies are recommended on the basis of evidence: (1) Macitentan and sildenafil. (2) Riociguat and bosentan. (3) Selexipag and endothelin receptor antagonist or PDE5i, or both. Other agents: (a) Endothelin receptor antagonists (ERA), (b) Ambrisentan, (c) Bosentan, (d) Macitentan, (e) PDE-5i and guanylate cyclase stimulators, (f) Sildenafil, (g) Tadalafil, (h) Vardenafil, (i) Riociguat, (j) Prostacyclin analogs and (k) prostacyclin receptor agonists, (l) Epoprostenol, (m) Iloprost [12], (o) Treprostinil. Novel agents are currently being investigated such as; Rho-Kinase inhibitors, riociquat and imatinib. Rho-Kinase inhibitors, sildenafil, tadalafil, riociquat and macitentan. Other drugs such as; angiotensin-receptor blockers, diuretics, nitrates, $\beta$-blockers and angiotensin-converting enzyme inhibitors. In the case of chronic thromboembolic pulmonary hypertension angioplasty is depended on a case by case evaluation [13]. Almost 12 years ago inhaled treprostinil was been approved by the FDA (2009) for patients with pulmonary arterial hypertension with NYHA III. Prostacyclin is a drug that induces vasodilation in large and small pulmonary vessels. Moreover, it has observed that inhibits the smooth muscle cell growth. Prostcyclin-2 is working through cycloxygenase-2 pathway activation which leads to vasodilation and platelet aggregation [14]. Inhaled prostanoids are administered in patients who can be treated with parenteral treatment. In the TRIUMPH trial treprostinil provided data where the 6-minute walking test was improved when compared to stable doses of bonsentan or sildenafil alone [15]. It has been previously investigated that different types and models of nebulisers in combination with different residual cup design and fillings affect the size of the aerosol production size [4, 16, 17]. The aim of this experiment was to investigate the possible administration of these drugs as aerosols and which would be the best equipment combination and residual cup filling.

Underlying disease is a serious issue that has to be evaluated in every patient. There has been an extensive research initially for inhaled insulin for patients with asthma and chronic obstructive pulmonary disease (COPD) [3]. The mucus, airway clearance mechanisms and local genes/transporters play a crucial role in the local absorption of an inhaled drug [23]. The viscosity of the mucus does not let the drug to be released to the airway surface [1]. Patients with COPD and cystic fibrosis have thick mucus and the clearance mechanisms do not work properly [24, 25]. There are different transporters in different sites of the airways. In the airways there is also 90% humidity, therefore molecules with high salt concentration will expand by almost 50% as they reach the alveoli from the upper respiratory system [1]. Exacerbations of COPD and asthma or another underlying disease is a contraindication for inhalational drugs [26]. Moreover, a careful respiratory evaluation has to be performed for every patient not only with spirometry, but also with diffusing capacity of the lung (D${}_{LCO}$) which evaluates the capacity of the smaller airways. Six-minute walking test is another test that is also necessary for pulmonary hypertension where the respiratory evaluation is combined with the cardiologic evaluation [27]. Inhaled drugs for pulmonary hypertension are available however; certainly we need more, since different drugs are effective for each patient and disease stage. The necessary technology is available to transform the drugs and also several aerosol and production systems are already on the market [4, 16, 28, 29, 30, 31]. The major advantage of inhaled delivery of the drug directly to the lungs are less systemic side effects and large drug concentration in a small period of time [32, 33, 34]. There is indication for patients with pulmonary hypertension that need a treatment combination and therefore rapid absorption is feasible with aerosol administration. The aerosol characteristics does not depend only on the nebulizer design but from the combination of nebulizer, nebulizer cup design, drug characteristics and quantity of the drug within the residual cup. All these factors interact between them. In our study we observed that Tadalafil produced the smallest aerosol droplets with residual cup B 2.43 $\mu$m with a filling of 6 mL followed by residual cup C and F. The smallest aerosol droplets were produced with the jet-nebulisers. Regarding Macitentan the smallest droplet size was 3.14 $\mu$m again with residual cup B with a 6ml filling followed by C and F. Again the smallest droplet sizes were produced with jet-nebulisers. The residual cup design and filling probably affects the impact of the droplets within the walls of the residual cup along with the air pressure inserted from the jet nebulizer through the connection cable. The multiple impacts assist in the production of smallest droplets. In a previous study the inlet in front of the nebulizer cup further reduced the mass median aerodynamic diameter of the droplets and in a recent study novel nozzle designs were able to further reduce the aerosol droplets [4, 35]. In another study other factors such as the electrostatic precipitation was investigated as a factor affecting the aerosol distribution within the peritoneal environment and a new methodology was proposed to overcome this issue [36]. The underlying pulmonary disease plays a crucial role in the absorption of aerosols, as with other drugs, we should avoid administering aerosol drugs if we have pulmonary infection or COPD and asthma exacerbation. Based on our results the drugs tadalafil and macitentan can be administered as aerosol with current nebulisers, both jet- and ultrasound. We more experiments to evaluate several aspects of the produced aerosol such as the drug concentration and the absorption. If possible if the future we could have a sustain release aerosol formulation. The aerosol administration is easier to use than subcutaneous pumps and maybe a portable system such as a metered dose pressurized inhaler could be manufactured (mdpi) in the future. Aerosol administration has certainly the advantage of fast absorption as previously observed with other drugs. In any case every patient should have a variety of choices for his medication. Tablets have the disadvantage of non-linear sustain release, and in some patients with gastric issues the absorption is not complete. The aerosol administration can be performed in any patients even in those with a Levin or gastrostomy and they the aerosol administration is fast acting. In our manuscript we investigated whether the drugs macitetan and tadalafil were possible to be converted firstly to powder and secondly aerosol mist. We used a simple technique which has been previously used in other publications [28]. Major limitations were that we were not able to measure the concentration of the drugs within the aerosol mist and several other aspects of the aerosol such as the z potential which are important for a future drug design. We concluded that the residual cup design and residual cup filling play a crucial role in the formation of mmad $\le$5 $\mu$m which is the threshold for optimal distribution and absorption within the airways. The larger residual cups produce the smallest aerosol droplets. The three different residual cups with the jet-nebulisers produce the smallest aerosol droplets. Both ultrasound nebulisers are capable of producing optimal size aerosol droplets $\le$5 $\mu$m mmad. Additionally, targeting multiple pathways in this disease is necessary [37, 38, 39, 40]. We need long term studies to evaluate the safety of these drugs to the lung parenchyma, in in vitro study would elucidate this aspect. Our next steps will be to make the appropriate measurements with HPLC and then agree on the appropriate dosage that has to be delivered according to each disease.

PZ, DP, CS, CK, KT, DM, HH, CB, WHS, GP, IK, GG, SP and CK wrote the manuscript and performed the experiments. DP and PZ performed the statistics. IK, GP and GG provided useful insights.

Not applicable.

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This research received no external funding.

The authors declare no conflict of interest.