The dense deep lymphatic network in the visceral pleural connective tissue, both in peribronchial and perivascular connective sheets, of all lung lobes, and in the juxta-alveolar regions plays a crucial role in removing foreign materials and in lung tumour dissemination.1–5

Liposomes have been proposed as promising anti-tumoral drug carriers, due to their encapsulating ability.6,7 They are also suitable for imaging the deep lymphatic network, as they will act as foreign particles to be drained. They can be administered by several routes such as aerosols. Based upon the pathophysiology of Bacillus tuberculosis pulmonary infection, specific liposomes can be modulated. They can be made to mimick the membrane composition of the Bacillus subtilis spores (a respiratory tract saprophyte microorganism) in order to be captured by pulmonary lymphatics.8,9

The used methodology enabled production of unilamellar vesicles (mean diameter 50–100nm) with a small polydispersity index (0.17, n = 3), and a surface charge of –45.8mV (n = 3), confirmed by zeta potential and by photon correlation spectroscopy determination.

The labelling efficiency of the liposome formulation was tested by ascendant instant thin layer microchromatography (ITLC-SG) with saline. The average labelling efficiency obtained for 99mTc-exametazime was 74.1 ± 13.9 % (n = 12). The previously referred in vitro stability studies are shown in Figure 1Af. Labelling efficiency of 99mTc-exametazime-liposomes was good, but the most important feature was that in vitro stability in the presence of human serum, human plasma and albumin solution was higher than in the presence of blood fraction.

Figure 1.

Stability of liposomes. A) Labelling efficiency (%) after incubation with different fluids (saline, human serum, human plasma, human serum albumin solution with a concentration of 4mg/mL), before and after warming at 56°C for 30min, in order to inactivate the complement of blood fractions. B) Temporal evaluation of the liposomes' aqueous core loss, using ascendant ITLC-SG with saline.

(0.07MB).

The liposomal formulation was administered as an aerosol, and its labelling efficiency (after 3min of US action) was not significantly different from the values determined pre- and post-US exposure. Study of liposomal aqueous core content loss, during 5.5 hours after the second ITLC, showed a good stability before and after ultrasonication (Fig. 1B).

Dynamic scintigraphic studies, both of 99mTc-exametazime and 99mTc-exametazime-liposomes aerosol inhalation, showed a good deposition in the lung, confirming that the produced liposomes reached the small airways and, hence, the alveolar surface.

Axillary lymph nodes were visualized 30min post-inhalation. 1 hour post-inhalation they became more evident, and abdominal aortic and inguinal lymph nodes were also observed. Later images did not give additional information, although activity is observed in abdominal organs (Fig. 2).

Figure 2.

Image sequence obtained at 30, 60, 90 and 120min after 99mTc-exametazime-liposome inhalation. Besides the high lung deposition, an high uptake by the axillary nodes should be noticed. In abdominal projections at 60, 90 and 120min abdominal aortic and inguinal lymph nodes can be observed.

(0.07MB).

The mean activity/pixel in each ROI, after background and decay correction, was used to plot regional time-activity curves, showing the radiotracer biokinetics variations in certain target areas during the study (Fig. 3). Total activity was measured in the lungs post-inhalation, as well as clearance rate (t1/2 = 77min) (Fig. 3A). An increase of activity was simultaneously observed in axillary lymph nodes, reaching maximum activity values for axillary and inguinal nodes ± 60min (Fig. 3B-D).

Figure 3.

Time-activity curves representing the kinetics of the 99mTc-Exametazime-liposomes in the lungs (A), axillary (B), periaortic (C) and inguinal (D) nodes. Graphics C and D begin only at 60min, since the collimator's dimension impairs to perform, simultaneously, a dynamic acquisition at thorax and abdominal levels.

(0.52MB).

Promoting passive movement of the feet, after 99mTc-Re2S7 injection, the left inguinal node was visualized at 45min, corresponding to the area visualized with 99mTc-exametazime-liposomes (Fig. 4A). In a control animal, 99mTc-exametazime biodistribution showed a lung fast clearance through alveolar-capillary permeability, which enabled a quick visualization of several organs (liver, gallbladder, spleen, kidneys and the ascending and transverse colon (Product datasheet, Ceretec®, 2001). Nevertheless, lymph nodes were not evident; as abdominal activity masks the lymphatic abdominal chains (Fig. 4B).

Figure 4.

Animal controls. A) Dynamic image sequence of the indirect lymphoscintigraphy obtained during the first 30min after interdigital feet 99mTc-Re2S7 injection, as well as static images at 60, 90 and 120min. The observed lymphatic abdominal drainage corresponds to the visualized areas with liposomes labelled with 99mTc-exametazime. B) 99mTc-exametazime biodistribution is a control and does not visualize the lymph nodes or lymphatic abdominal chains, only thoracic and abdominal activity uptake.

(0.07MB).

Following MIRD4 rules and applying the absorbed fraction method to calculate the absorbed dose, the obtained values are similar to those used in conventional nuclear medicine routine studies, to evaluate alveolar-capillary permeability with aerosols.39,40

Pulmonary lymphatic network is crucial for alveolar and interstitial clearance, being responsible for removal of many substances, particles, dusts, or pathogenic agents. Preferential lymphatic drainage correlates with certain specific surface components, such as those present in microorganisms.

This paper describes the obtained results of the in vivo animal studies in normal baboons, using the referred formulation (99mTc-exametazime-liposomes), administered as an aerosol as a diagnostic imaging agent to visualize the deep lung lymphatic drainage.

This carrier could be used both for visualization and therapy, whether it encapsulates an imaging agent or a therapeutic drug, respectively. Since anionic compounds are quickly removed by the lymphatic network negatively charged small calibrated unilamellar liposomes were produced by extrusion through polycarbonate membranes of 100nm pore size.41

Since 99mTc has 6 hours of half-life, labelling the liposome aqueous phase, after their formation, was done immediately prior to administration.7,11,18,23,24,34,42 Extrusion under moderate pressures was done, as liposome inhalation implied absence of organic solvents or detergents in the solution, to avoid possible allergic reactions.11

In vivo stability of liposomes may change and be altered by complement aggression, depending on the lipidic composition of the membrane.10 To evaluate these disruptive effects, 99mTc-exametazime-liposomes stability was tested in vitro, by incubation with saline, fresh human: serum, plasma and serum albumin solution (4mg/mL), as previously described. Results showed a statistically significant increase of labelling efficiency in presence of human serum and plasma for the studied formulation (t = 3.2; p = .01). This shows that biological fluids do not induce liposome aqueous content leakage. Stability to ultrasonication was determined analysing the labelling efficiency after 2.7MHz US action. Results do not show statistically significant differences for labelling efficiencies pre- and post-US action, as previously mentioned by other authors.37,41–45

Liposomal stability was also studied in terms of entrapped content loss (Fig. 1B), during 5.5 hours. Results showed a progressive increase of aqueous content loss, probably depending on the lipidic composition.

The inhaled thin liposomal aerosol duly reached the lung alveolar-capillary membrane. The vesicles could either be cleared by crossing the alveolar surface to lung interstitium after phagocytosis by alveolar macrophages, or directly through intercellular spaces or mucociliary escalator. Those, which crossed the interstitium, were eliminated by lymphatic drainage to the lymph nodes and subsequently to blood capillaries.46–48

Anatomically, pulmonary lymphatics can be grouped into two interconnected networks: the superficial pleural one, running in the connective tissue of the visceral pleura, and a deep intrapulmonary network forming the peribronchovascular lymphatics, located in the connective tissue sheets of the pulmonary bronchial and vascular trees. Several lymphatic capillaries can be seen in juxta-alveolar areas, in contiguity with the alveolar wall and separated from the alveolar lumen only by the alveolar epithelium and supporting connective tissue (usually very thin and richly vascularized). Pulmonary lymphatics can also be found in the loose connective tissue supporting more peripheral pleural cells and covering the pulmonary lobes, interalveolar septa and perivascular sheets.3–5,41,49

The clearance mechanism also depends on physicochemical characteristics and particle size. Only submicronic sizes (< 50nm Ø) can be deposited on the alveolar surface, being drained afterwards by the lymphatic system.50 After reaching the alveolar surface, the 99mTc-exametazime-liposomes cross into the lymphatic capillaries of juxta-alveolar areas through intercellular gaps and are engulfed by alveolar macrophages, migrating then to the hilar lymph nodes.3–5,41,49,51 In our animal model, the rather high radioactive dose deposited in the lungs, did not help to identify these nodes. Nevertheless, the main lymphatic drainage route of the whole organ is linked to the mediastinal and abdominal periaortic lymph nodes, therefore their visualization in the images agrees with the anatomical data (Fig. 2).1–5,41,49,51 There is a fast infra-abdominal lymphatic drainage, post-inhalation, confirmed by indirect lymphoscintigraphy. This clearance can possibly explain distant metastasis of lung cancer appearing in unexpected sites.

Aerosols of specially tailored 99mTc-exametazime-liposomes proved to be an interesting approach to study deep lung lymphatic drainage. The physiological behaviour of these drug carriers, mimicking some properties of micro-organisms, allowed visualization of a descendant lymphatic pathway to the abdominal aortic chain nodes, confirmed by indirect lymphoscintigraphy. Images of these chains could give highly relevant information for staging lung tumours, as well as to evaluate other pathologies with important pulmonary lymphatic contribution. In addition, this methodology may play an important role in targeted lung delivery of other pharmaceuticals, e.g. cytotoxic drugs.

Taking into account the promising obtained results in the tested animal models, the production of a tracer, for inhaled administration, providing information on the degree of pulmonary invasion and metastization through functional images, is in perspective.

Authors declare that they don't have any conflict of interest.