Aerosol particles are deposited in the airways are removed by mucociliary clearance mechanisms. The airway surface consists of epithelial goblet cells and submucosal glands secreting mucus. These two cell types produce a two-layer mucus blanket over the ciliated epithelium: (a) the low-viscosity sol layer and (b) a high-viscosity gel layer. All insoluble particles are trapped in the gel layer and with the help of the beating cilia are moved towards the pharynx or the gastrointestinal tract or even removed through coughing. The activity of the mucus varies depending on the airway area and the clearance is also synergistically determined by the number of ciliated cells and their beat frequency which differs in different parts of the airways. For normal mucociliary clearance there must be no underlying lung disease. Furthermore, the chemical composition of the sol layer should be optimal along with the rheology of the mucus. Mucociliary clearance is dysfunctional in lung diseases such as immotile cilia syndrome, chronic obstructive pulmonary disease (COPD), cystic fibrosis, bronchiectasis, and asthma [14]. It has been observed that lipophilic molecules pass easily through the airway epithelium via passive transport, while the hydrophilic molecules cross via extracellular pathways and exocytosis [15]. Particles deposited on the bronchial epithelium are absorbed into the systemic bronchial circulation and then into the lymphatic system. Aerosol particles that are deposited in the alveolar region may either be phagocytosed and cleared by alveolar macrophages, or they are absorbed into the pulmonary circulation. Alveolar macrophages situated in the alveoli are phagocytic cells and they are the first defense layer against inhaled microorganisms and particles [16]. Macrophages phagocytose insoluble particles that are deposited in the alveoli, and are either cleared by the lymphatic system or moved with the help of beating cilia covered in alveolar fluid in the upper airways to the gastrointestinal tract [17]. However; depending on the particle’s chemical structure, this process can take weeks to months to complete [18]. There are cases where particles can be enzymatically degraded intracellularly with the help of local enzymes [19]. The bacterial biofilm of the mucus also plays a crucial role in the rate of drug absorption of the bronchial epithelium and alveoli [20]. An extensive investigation of these factors has been done in cystic fibrosis patients and therefore all factors have been more or less elucidated [21]. Pre-treatment with aerosolized corticosteroids or saline in other cases provides enhanced local drug absorption, in dosages of 1 mg/2 mL $\times$ 3 budesonide or saline 2mL $\times$ 3 per day.

The lungs receive the largest part of the cardiac output through the pulmonary arteries and are considered to be the highest perfused organs in the body. The alveoli are the parts of the lungs that are the most highly supplied. Only 1% of the cardiac output circulates to the trachea and bronchi. In bronchiectasis the bronchial blood flow is augmented from 1% to as much as 30% of the cardiac output [22]. It has been observed that inhaled drugs that are deposited in tracheobronchial regions can be locally absorbed by the local vessels [23].

It has been observed in previous studies that tumor size affects the distribution and deposition of an inhaled compound and therefore the maximum diameter of the tumors had to be up to 5 cm upon diagnosis, otherwise patients were excluded from trials [24, 25, 26, 27, 28, 29]. Anticancer drugs have been observed to penetrate normal tissues by both diffusion and convection and finally reabsorption into the lymphatic circulation. However; a major issue is the unstructured neo-angiogenesis and lack of functional lymphatics in several tumors [30, 31]. This tumor microenvironment increases the levels of interstitial fluid pressure in tumors [32, 33, 34], reduces convection and inhibits distribution of macromolecules [35, 36]. The penetration of a drug particle depends on its: (a) charge, (b) shape, (c) molecular weight, and (d) aqueous solubility. Water-soluble drugs are distributed in the extracellular matrix and diffuse efficiently around and between the cells. Lipid-soluble drugs, on the other hand, penetrate lipid membranes, and so can be transported through cells. One can use 22G, 21G and 19G needles for local drug administration. Indeed, 19G needles can deliver larger quantities, however; depending on the stiffness of the tissue, the drug penetration will differ. Needles with pores on their sides exist to facilitate absorption of more cells during biopsy. Such needles can also be used to deliver more drug in different parts of a lesion when they are used for drug delivery [12, 37].

The most important physical properties that have to be considered when creating an aerosolized drug are: (a) viscosity ionic strength, (b) pH and (c) osmolarity. It has been observed that if pH and osmolarity are not in the normal range, then bronchoconstriction and coughing will occur due to mucosal irritation [38, 39].

It has been observed when studying aerosolized drugs that lung disease plays a crucial role in local drug absorption. This knowledge comes from studies of large patient populations with diabetes and aerosolized insulin administration, either as liquid or powder form [24]. However; all these drugs are deposited to a large percentage of the surface of the airway epithelium. In the case of local intratumoral administration only the extracellular matrix (ECM) and the parameters contained in this environment play a key role. However, one should keep in mind that chronic obstructive pulmonary disease (COPD) or heart failure might be contraindications to performing an endoscopy for the administration of intratumoral therapy. In cases of underlying disease exacerbation, one should increase the dosage and administrations. Additional issues such as the oxygenation of a patient need to be resolved and aerosol pretreatment as previously described must be increased up to double. In the worst case scenario, one should switch to intravenous administration until the exacerbation is adequately managed. All this information can be found in a previously published article by Zarogoulidis et al. [24].

Liposomes are drug carriers that help secure sustained release, reduced toxicity and reduced irritation to the lung parenchyma. They can be easily manipulated and have stability [40]. The dose of the drug carried by the liposomes, its release rate and the deposition in the alveoli depends on: (a) lipid composition, (b) particle size, (c) charge, (d) drug/lipid ratio, and (e) method of delivery [41, 42, 43]. Liposomes are produced from phospholipids, and they may or may not be electrically charged [44, 45]. Their structure consists of an aqueous part, which is entrapped by a synthetic lipid single layer or bilayer, with or without the addition of cholesterol. They can encapsulate either hydrophilic or lipophilic chemical entities [46]. However, drugs with intermediate solubility are poorly retained by liposomes, so they have to be manipulated to achieve a higher degree of retention [47]. Liposomes are prepared for inhalation either in liquid or dry powder form [48]. Unfortunately, it has been observed that during nebulization, an amount of the formulation is lost and hence the operating conditions need to be optimized in order to minimize loss [49, 50, 51, 52]. The dry powder liposome formulations are produced by lyophilization, usually followed by milling or by spray-drying [53, 54]. The sustained release capability of liposomes has been observed in examples of aerosolized treatments for lung diseases in several studies [24, 55, 56]. In order to enhance the sustained-release properties of liposomes, a polymer surface coating called polyethylene glycol (PEG) has been developed recently. PEG coating provides a “stealth” shield to the molecule to bypass the body’s defense mechanisms [47, 57, 58].

Microparticles are produced from naturally occurring or synthetic polymers. Their size ranges between 0.1 and 500 $\mu$m. Their physical and chemical properties allow them to be more stable than liposomes, and so are capable of higher drug loading. Microparticles are an ideal carrier system for proteins and peptides [59, 60]. The main factors affecting them are: (a) pH, (b) heat, (c) moisture, (d) solvents, (d) oxygen, and (e) mechanical stresses. The preparation for aerosolized delivery of microparticles can be undertaken using: (a) emulsion-solvent evaporation, (b) spray-drying, (c) phase separation, (d) emulsion-solvent diffusion and e) supercritical fluid technology [61, 62, 63, 64, 65, 66, 67]. The following parameters influence drug release: (a) porosity, (b) size, (c) solubility, (d) molecular weight, (e) nature of micromolecular drug, (f) concentration, g) tortuosity and h) uniformity of the polymer. A coating can be added to improve the time release characteristics, and also 1,2-dipalmitoylphosphatidylcholine is also added to poly(DL-lactide-co-glycolide) microspheres to decrease the uptake by macrophages [64]. When chitosan and hydroxypropylcellulose are added to the particles, their local absorption over time is increased [67]. It was observed in previous studies that particles of 1–3 $\mu$m, tend to aggregate [68] and are cleared by alveolar macrophages [69]. Therefore it was necessary to develop large porous particles of a geometric diameter of 0.5 $\mu$m, an aerodiameter of 0.5 $\mu$m, and a low density of 0.1 mg/mL [65, 70].

Further development of this molecules led to the development of “Trojan” particles [69], these have the ability to escape both phagocytic and mucociliary clearance of the respiratory system. They are prepared from nanoparticles, of low density (0.1 mg/mL). These particles can be aerosolized from dry powder [61]. A single cancer cell can ingest one or multiple microparticles and these particles can be arranged in such a way so as to reduce the space occupied inside the cell [71, 72].

There are currently three carbohydrates used as used drug carriers: (a) lactose (a-lactose monohydrate), (b) glucose, and (c) mannitol. These carriers act also as stability enhancers. In the last decade several carriers were assessed and it was observed that mannitol is the best candidate for dry powder inhaler formulations [73]. Current techniques that are used to produce a inhalable formulation are: (a) supercritical fluid technology, (b) lyophilizing followed by milling/jet milling or spray-drying, (c) freeze-drying, and (d) spray-freeze drying [74, 75, 76, 77, 78]. Lactose enhances the uptake of poly-D-lysine into airway cells, increasing intracellular localization of proteins and peptides [79]. Lactose can be mixed with fine lactose particles (about 5 $\mu$m in diameter) with coarse lactose to improve disaggregation, as well as the fine particle fraction [80]. Lactose can have a ternary component, such as L-leucine, to the formulation to increase dispersibility [45].

Cyclodextrins (cyclic oligosaccharides), have also been proven to be useful excipients in the respiratory distribution of small molecules [81]. In a recent study, the use of dimethyl-$\beta$-cyclodextrin resulted in increased bioavailability of carbohydrates by increasing concentrations of cyclodextrin [73].

Polyethylene glycol (PEG) added to proteins enables a sustained release at the site of deposition, due to a “stealth” effect, by bypassing the defense mechanisms of the respiratory tract (macrophages), and by decreasing degradation of the formulation in the lungs [47, 82]. PEG has been shown to be a safe carrier for inhalable agents [83].

Polylactic acid has properties that favor sustained release, but it is not suitable for lung delivery due to its prolonged biological half-life. However, an oligomer of lactic acid, with a shorter half-life (6–8 days), can be used instead for drug delivery. Hydroxypropyl cellulose, is another option absorbed over approximately 24 hours that bypasses mucociliary clearance. However, not enough data exist about its the toxicity profile [45, 84].

Bioadhesives have been used to prolong the attachment of the carrier-drug complex to the bronchial cells of the airways [85, 86]. Several multivalent binding agents have been incorporated in drug-carrier systems, such as; lectins, heparin, octa-arginine, peptides, heparin sulfate, and antibodies [87, 88].

Intracellular targeting is an additional strategy enabling a drug to reach the proper surface area [93, 94]. Most chemotherapy regimens interact within the reproductive cell cycle, so this delivery strategy should be further pursued [95]. Three are the most important parameters concerning the cell microenvironment and drug-formulation interactions: (a) endosomal release, (b) intracellular trafficking, and (c) nuclear localization.

ABC transporters are a large family (50 members) of transmembrane proteins, consisting of seven subfamilies. ABC transporters act as a defense mechanism in lung epithelial tissue [96]. P-glycoprotein, also known as multidrug resistant proteins, has been shown to play a role in multidrug resistance, especially in chemotherapy resistance [97]. P-glycoprotein and its properties have been extensively studied in the lung. It has been observed that its presence decreases oral drug absorption, prevents drug entry in the central nervous system, and it is responsible for many drug to drug interactions [98]. The transporter is found on the apical membrane of the bronchial and bronchiolar epithelium [99, 100, 101, 102], in the endothelial cells of the bronchial capillaries [103], and in alveolar macrophages [100, 101]. Unfortunately the expression of P-glycoprotein in smokers with lung disease versus people with normal lungs has not been adequately investigated. It has been observed that P-glycoprotein and immunohistochemical multidrug-resistant protein analyses can predict a patient’s response to chemotherapy [104]. However; in one study, the mRNA levels in the lung tissue of smokers, non-smokers, and ex-smokers were not found to be significantly different [99]. Several studies have previously demonstrated a direct or indirect connection between the P-glycoprotein transporter expression and underlying lung disease. In cystic fibrosis, for example, due to changes induced by the disease, it has been shown that the P-glycoprotein transporter is upregulated [105, 106]. Furthermore, it has been observed in another study that toxins released from microorganisms infecting patients with cystic fibrosis also inhibit P-glycoprotein [107]. On the other hand, there are no significant data indicating modification of P-glycoprotein expression between different disease stages in chronic obstructive pulmonary disease patients [108, 109] and no relevant data exist for asthma patients. It has been observed that corticosteroids administered by inhalation, oral, and intraperitoneal routes upregulate the P-glycoprotein transporter [110, 111]. In a previous study, inhibition of P-glycoprotein was observed by lipid nanocapsules, which is a crucial mechanism of resistance for paclitaxel [112]. Nine multidrug resistance proteins (MRPs) exist. In normal lung tissue, MRP 1 and MRP 5 have been found to be highly expressed, MRP 6 and MRP 7 are moderately expressed, and MRPs 2, 3, 4, 8, and 9 are either low or undetectable [113, 114]. MRP-1 and MRP-2 are found in the bronchial and bronchiolar epithelium [100, 115, 116]. MRP-1 is also found in alveolar macrophages [100, 115]. MRP-1 expression levels are altered in patients with chronic obstructive pulmonary disease [108, 115]. It has been shown that smoking downregulates these transporters, and these transporters play a protective role against cell damage [108, 117]. Ipratropium, N-acetylcysteine, and budesonide stimulate MRP-1 efflux and activity [118]. Formoterol on its own does not have an effect on the transporter, however, the co-administration of inhaled corticosteroids reduces the transporter expressions [118]. In a previous study on the use of inhaled doxorubicin, MRP-1 and MRP-2 were overexpressed [119]. This information is important to keep in mind, because most lung cancer patients are also diagnosed with chronic obstructive disease and because most are or have been smokers, the transporter expression has been found to be increased after doxorubicin use.

Organic cation transporters comprise of five types of carriers: (a) electrogenic OCT 1-3, (b) electroneutral OCT N1 and (c) OCT N2. OCT 1–3 can be found in the trachea, smooth muscles of the airway, and ciliated bronchial cells. OCT N1 is expressed in the tracheal epithelium and alveolar macrophages. OCT N2 is expressed in the alveolar epithelium and airway epithelium [120, 121, 122, 123]. Previously published data from animal and in vitro cell lines studies implicate upregulation or downregulation of OCT transporters upon induced inflammation or drug interactions related to asthma and/or chronic obstructive pulmonary disease. Nevertheless, these data have not been clearly demonstrated in humans [122, 123, 124, 125].

Peptide transporters belong to the proton-coupled oligopeptide transporter group. PEPT 1 and PEPT 2 are the two main transporters, which contribute to the high bioavailability of peptide-like molecules. These two peptides can affect the absorption and distribution of inhaled antibiotics and antiviral drugs [126]. PEPT 2 and PEPT 1 have been detected in the airway epithelium and bronchial epithelium [127, 128]. PEPT 1 and PEPT 2 have been found in animal tissues and various cell lines [129, 130]. Their mechanism of action, however, has not been fully investigated [131].

There are six members identified: (a) OAT 1–4, (b) URAT 1, and (c) OAT 5, mostly found in the kidneys [132]. Gene microarrays have confirmed their absence in human and murine lungs, but OAT 2 is highly expressed at these sites [120]. Moreover; OAT 4 mRNA was highly expressed in the bronchial cell lines Calu-3 and 16HBE14o- [129].

There are 11 human organic anion transporting polypeptides (OATPs), which are divided into six families [133]. Their actual tissue distribution has not been fully investigated [133]. OATP 2B1, OATP 3A1, OATP 4C1, and OATP 4A1 expression has been observed in: (a) cell lines, (b) human lungs, and (c) animals [120, 129, 134].

Several studies have been performed, both in humans and in animals, with inhaled platinum analogs and instillation day 1 and cisplatin (50 mg/m${}^2$ daily) on days 1 and 2. The administration was performed with jet-nebulizers as a production system and under strict protection measures such as the high efficiency particulate air (HEPA) filter or a protective chamber [27, 135, 136, 137, 138, 139, 140]. Safety and efficacy assessment was performed with: radiographic examinations (x-rays, computed tomography scans), blood and urine analysis, ki-67 cell proliferation, pathological findings, bronchoalveolar lavage, high-performance liquid chromatography, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay and recording of Eastern Cooperative Oncology Group common toxicity criteria. The main adverse effects observed were dose-dependent: (a) cough, (b) fatigue, (c) nausea and (d) weight loss.

5-FU was the first chemotherapy drug to be used as local chemotherapy treatment almost 30 years ago [141]. A high-efficiency particulate arrestance filter and a plexi-glass chamber were used for the administration. Safety and efficacy were evaluated via blood samples, high performance liquid chromatography and imaging techniques [142, 143, 144, 145]. The drug was administered intrabronchially and regression of the tumor was observed with endoscopy in all their treatment sessions. The drug has not been delivered intratumorally as in other studies 100 mg/m${}^2$ on day [146]. It was observed that the aerosol drug was absorbed via the alveoli to the systematic circulation. This has not been reported in other studies [135]. This was an early study, conducted before intratumoral administration began [37], where the aerosol as a local therapy interacted with the surface of the tumor and was absorbed by the extracellular matrix of the tumor and alveoli [147]. The adverse effects observed were cough, weight loss, dizziness and bronchospasm, and were dose dependent. 5-FU has been administered either alone or coated with lipids or liposome nanoparticles [144, 145].

Taxanes (paclitaxel and docetaxel) have been administered locally in the respiratory system alone or in combination with other drugs [148, 149]. In one of the first published studies cyclosporin A was co-administered as a method to augment the efficiency of aerosolized taxanes paclitaxel (175 mg/m${}^2$) [150]. The safety and efficacy was evaluated with blood samples, pathology findings, high performance liquid chromatography and V/Q scans. All previously published studies were performed in animals and the administration was performed in sealed cages. The drug formulation administered was either coated with liposomes or a lipid base formulation. Unfortunately, severe adverse effects, such as weight loss and neurological toxicity were observed.

This chemotherapy drug has been administered as local treatment. Safety and efficacy assessments were performed with respiratory capacity tests (spirometry, diffusion capacity and 6 minute walking test), high performance liquid chromatography, V/Q display, imaging techniques, pathology findings, blood samples, confocal laser scanning microscopy and colorimetric assay for cell proliferation. Adverse effects observed included (a) acute chest pain, (b) wheezing, alveolar hemorrhage, (c) weight loss, (d) cardiac toxicity, (e) hypoxia and $>$20% reduction of pulmonary function tests values were observed. In order to cope with the adverse effects intravenous corticosteroids were administered. Consequently, novel compounds were developed to engulf doxorubicin as loaded microparticles or liposomes to coat the drug. The previously observed side effects were subsequently reduced [26, 119, 151, 152]. The administration of the aerosolized drug was performed in specially designed cages, chambers or HEPA with hood 60 mg/m${}^2$ q14D.

Gemcitabine has less severe systemic side effects when administered intravenously [153]. In previous studies gemcitabine was administered as aerosol in specially designed chambers and cages 2362 mg/m${}^2$/week for 3 weeks. The evaluation of the drug distribution, safety and efficacy were performed with: (a) V/Q scan, (b) high performance liquid chromatography, (c) bronchoalveolar lavage, (d) blood samples, (e) pathology findings and (f) cell proliferation. The most severe adverse effects observed were emesis, bronchospasm and excessive cough and were dose depended. Moreover; acute pulmonary toxicity was observed in the form of pulmonary edema, neovascularization, and connective tissue formation [154, 155, 156].

9-NC has been previously investigated in animals and humans as aerosol and via instillation. The safety and efficiency profile has been investigated with blood and urine samples, imaging techniques, Ki-67 proliferation, pathology samples, platelet-endothelial cell adhesion and performance liquid chromatography. The only adverse effects observed included; (a) cough, (b) pharyngitis and (c) bronchitis and these were dose-dependent. The 9-NC has been administered only as a liposome form in doses of 26.6 $\mu$g/kg/day [55, 157].

Bevacisumab has not been administered as a local treatment for NSCLC, however, it is a strong anti-neovascularization drug used to treat NSCLC [158]. It has been used for hereditary hemorrhagic telangiectasia-associated epistaxis as anasal spray and submucosal injections [159, 160]. It has also been used as local instillation treatment in bronchial HPV patients [3]. The concept for its usage in general is to block the local anarchic proliferation of the vessels. The adverse effects observed were mainly ageusia, anosmia, headache, hemorrhage, hypertension, septal perforation and nasal obstruction (15 mg/kg on day 1Assessment of safety and efficacy was performed via blood samples, epistaxis severity score and phone administered custom-made questionnaires.

In a recently published multicenter study immunotherapy was administered locally with the help of 19G needles under the guidance of endobronchial ultrasound with a convex probe. The patients were stage four NSCLC and the safety and efficiency of their treatment was evaluated with positron emission tomography (PET-CT) and next generation sequencing (NGS) [12]. Indeed, the combination of intratumoral chemotherapy and immunotherapy improved survival rates in patients with $\le$50% PD-L1 expression and in total to all patients that received intratumoral treatment versus only intravenous treatment (Nivolumab 10 mg/mL was purchased from Bristol-Myers-Squibb with the dosage being administered at 3 mg/kg, and calculated according to the weight). The reason for this positive effect was probably due to the local transformation of the tumor matrix were initial local application of chemotherapy initiated a production of proteins that enhanced the synergistic local effect of immunotherapy administration. The same concept as i.v chemotherapy plus immunotherapy, however, the major local adverse effect was the formation of abscess. Again abscess formation has been observed in a previous case series due to high PD-L1 expression and it is common in large NSCLC lesions [161]. There are two more studies that support that local aerosol administration of immunotherapy has a favorable effect and could be used alternatively in NSCLC for selected patients [162, 163].