**6. Case studies**

Although the use of liposomes for aerosol formulations is certainly encouraging, liposome nebulization still presents some problems, i.e. storage stability (mainly related to oxidation processes) and leakage of encapsulated drugs. In addition, it should be also considered that

Accordingly, IL-6 levels are increased in COPD patients (Bucchioni et al., 2003), and during experimental respiratory viral infections in humans and mice (Decramer & Janssens, 2010).

Mucus rheology plays a critical role in maintaining respiratory health. Mucins are large, highly glycosylated proteins. The polyanionic nature of mucin stems primarily from sialic acid, sulfate, and carboxyl groups present in these linked oligosaccharides. Beside physical entanglement, cationic calcium ions can act as crosslinkers that condense the mucin matrices inside mucin granules before exocytosis. Upon release, phase transition mainly driven by the Donnan effect triggers the massive decondensation of mucin networks. Hydrogen bonding, hydrophilic and hydrophobic interactions have also been proposed to contribute to the gel properties of mucin The gel characteristics and rheological properties of mucin are critical for the maintainance the integrity of epithelia by trapping bacteria and viruses and

Mucus is mainly composed of large and heavily glycosylated glycoproteins called mucins. The gel-forming mucins rapidly hydrate after exocytosis and, due to their tangle network properties, anneal with other mucins to form a protective barrier at the airway-surface liquid layer. The mucin gel layer lines the epithelial surface of various organs such as the vaginal tract, eyes, gastric wall and pulmonary lumen. Mucus in the airway of lungs serves as an innate immune defense against inhaled particulates, bacteria and viruses. Maintenance of the airway protection mechanism stems from the delicate balance between normal mucus production, transport and clearance. The mucin polymer network of mucus has a characteristic tangled topology. Since the rheological properties of mucus are governed mainly by the tangle density of mucin polymers, which decreases with the square of the volume of the mucin matrix, the mucin network hydration (degree of swelling) is the most critical factor in determining the rheological properties of mucus. The diffusivity of mucin matrices, which is closely related to mucin viscosity, can be calculated from polymer swelling kinetics. Based on the polymer network theory, polymer diffusivity is inversely proportional to its viscosity (Lodge, 1999). Thus, lower rate of mucin diffusivity is associated with higher viscosity, less dispersed and less transportable mucins that appear to characterize the clinical symptoms of thick mucus accumulation and obstruction commonly

The clinical manifestation of major respiratory diseases (Rogers & Barnes, 2006; Quinton,

The relationship between mucin dehydration and defective mucus clearance has been well established (Mall et al., 2004). As a result, the poorly hydrated, highly viscous and less transportable mucus appears to accumulate within airway passages (Randell et al., 2006). Obstruction of airway lumen with viscous mucus is usually accompanied by chronic

Although the use of liposomes for aerosol formulations is certainly encouraging, liposome nebulization still presents some problems, i.e. storage stability (mainly related to oxidation processes) and leakage of encapsulated drugs. In addition, it should be also considered that

bacterial infection, inflammation and impaired mucociliary transport.

for mucociliary clearance (Bansil & Stanley, 1995; Verdugo, 1990).

found in asthma, COPD and CF 44. (Rogers, 2007).

2008) are related to thick mucus.

**6. Case studies** 

**5.1 Mucus rheological properties** 

synthetic phospholipids are usually expensive and, on the other side, natural phospholipids show a variable degree of purity (Desai & Finlay, 2002).

An alternative approach to the liposomal approach is the use of liposome-like vesicles made up of non-ionic surfactants, the so-called niosomes. These carriers were proposed for both topical (Carafa et al., 2000; Carafa et al., 2004; Paolino et al., 2007; Paolino et al., 2008) and systemic administration (Cosco et al., 2009).

Here we report the evaluation of the possible advantages of a new type of non-phospholipid vesicle system for pulmonary drug delivery that can lead to an improved mucus permeation. Vesicles consisting of one or more surfactant bilayers enclosing aqueous spaces (non ionic surfactant vesicles NSVs), are of particular interest because they offer several advantages with regard to chemical stability, lower cost and availability of materials compared to conventional liposomes

In the formulation of inhaled drugs for the treatment of asthma and COPD, considerable attention has been devoted to new aerosol morphologies which can either enhance the local effect and/or increase the penetration through the mucus, secreted in bronchial inflammatory diseases. In diseases characterized by bronchial hypersecretion, lipophilic substances, such as corticosteroids, can be remarkably impeded in reaching their receptors, which are localized within the cytoplasm of bronchial epithelial cells.

In particular, alveolar macrophages are important target cells for the therapeutic action of glucocorticoids, because these cells are the major source of both proinflammatory and antiinflammatory cytokines. The action of glucocorticoids is mediated by an intracellular receptor belonging to the steroid thyroid/retinoic acid receptor superfamily (Oakley et al., 1999).

With the purpose of carrying out research leading to an innovative formulation for lung delivery capable of permeating the mucous layer and of an efficient delivery to alveolar macrophages, beclomethasone dipropionate (BDP), clinically used for the treatment of asthma and COPD, was entrapped in non-phospholipid vesicles.

BDP, as a reference model drug, was encapsulated in vesicular structures obtained with polysorbate 20. The aim of the study was to evaluate *in vitro* the effectiveness of such delivery system that should enhance permeation through mucosal barriers because of the presence of vesicles formed with a remarkably hydrophilic non-ionic surfactant usually considered as unsuitable for the formation of vesicular structures because of its high HLB value (HLB 16.7) (Santucci et al., 1996).

The intracellular availability of BDP and the safety of the delivery system are the two main issues to be addressed to propose these innovative non-ionic surfactant vesicles as carriers for the pulmonary delivery of this drug to be effectively used for the treatment of pulmonary inflammatory diseases. Therefore, the aim of this investigation was the evaluation of the interaction between our innovative non-ionic surfactant vesicles and human lung fibroblast (HLF) cells, the carrier tolerability, the vesicle localization within the cells and the amount of BDP actually internalized by the cells.

Unilamellar vesicles were obtained from a non-ionic surfactant/BDP aqueous dispersion (Hepes pH 7.4) by means of the "film" method as previously reported (Santucci et al., 1996), according to the compositions reported in Table 1.

A B

Fig. 5. Trasmission electron micrographs of BDP-loaded vesicles after freeze-fracture, before

et al., 1996; Darwis et al., 2001). For this reason, for permeation and nebulization

The possibility to use the novel vesicular dispersion in a conventional jet nebulizer, widely used in clinical applications, was also evaluated. For this purpose, samples were characterized also by means of rheological measurements and the aerodynamic diameter was determined (Table 3) as well as the nebulizer mass output (Figure 6) after completion of nebulization. Evaluation of Mass Median Aerodynamic Diameter (2.0±0.2 µm) and of geometric standard deviation (GSD) were also carried out; the GSD value (1.5) demonstrates the polydisperse nature of the distribution of the aerosolized droplets that, on the other side,

Aerodynamic diameter Percentage <10 100 <5 99.5 <2 65 Table 3. Percentage of particles with aerodynamic diameter <10, < 5, <2 µm, containing

Fig. 6. Deposition of vesicle-encapsulated BDP (BDP/ves) on filters upon nebulization, compared to a BDP/surfactant solution and to a BDP commercial product (n=3, ±SD).

experiments the formulation corresponding to sample 8 of Table 1 was used.

(A) and after (B) nebulization. The scale bar represents 0.5 m.

contained a monodisperse vesicular system.

non-ionic surfactant vesicles, delivered by a jet nebulizer


Table 1. Sample composition (expressed as % w/v)

All compositions are able to form nanovesicles with different size and zeta potential (ζpotential) according to the various formulations (Table 1). The size and the polydispersity index (PDI) obtained by dynamic light scattering measurements indicated those formulations with the smallest size and the most homogeneous nanovesicles population that can be obtained: samples 2 and 8, chosen to perform further experiments.

Size measurement experiments indicate that BDP-loaded vesicles are slightly larger than empty ones as reported in Table 2 for samples 2 and 8 of Table 1, there is an increase in diameter between 10 and 20%, and this expected effect can be related to drug partition between the bilayer and the aqueous core of the vesicles. Accordingly, the presence of BDP in the formulation may affects the ζ-potential values; as it is possible to observe from Table 2, the corresponding samples 2 and 8 show a significant decrease in ζ-potential that approaches the value obtained with BDP alone. This effect can be related to the chemical steroidic structure of the drug that is somehow similar to that of cholesterol, thus allowing it to fit well within the vesicular structure.


Table 2. Vesicle dimensions (nm) and ζ- potential (mV) of analyzed samples (n=3; ±SD)

Furthermore, it should be pointed out that electron microscopy carried out on numerous samples (10) indicated that nebulization does not influence drug-loaded vesicle dimensions (Figure 5A and B).

Analyzed samples showed a good stability in terms of possible changes in vesicle dimensions after aggregation.

Size measurement experiments indicated that after 1 month at 25 °C, no appreciable vesicle dimension variations could be detected.

The best entrapment efficiency (e.e.) was obtained for sample 8 and the calculated drug e.e. indicated that only about 20% of the overall amount of BDP is actually enclosed within the vesicles. This result is in agreement with the data reported by previous authors (Montenegro

**Sample Polysorbate 20 Cholesterol BDP**  1 1.84 0.58 0.5 2 1.84 0.58 1.0 3 1.84 0.58 3.0 4 1.84 0.58 5.0 5 3.68 1.16 0.5 6 3.68 1.16 1.0 7 3.68 1.16 3.0 8 3.68 1.16 5.0

All compositions are able to form nanovesicles with different size and zeta potential (ζpotential) according to the various formulations (Table 1). The size and the polydispersity index (PDI) obtained by dynamic light scattering measurements indicated those formulations with the smallest size and the most homogeneous nanovesicles population that

Size measurement experiments indicate that BDP-loaded vesicles are slightly larger than empty ones as reported in Table 2 for samples 2 and 8 of Table 1, there is an increase in diameter between 10 and 20%, and this expected effect can be related to drug partition between the bilayer and the aqueous core of the vesicles. Accordingly, the presence of BDP in the formulation may affects the ζ-potential values; as it is possible to observe from Table 2, the corresponding samples 2 and 8 show a significant decrease in ζ-potential that approaches the value obtained with BDP alone. This effect can be related to the chemical steroidic structure of the drug that is somehow similar to that of cholesterol, thus allowing it

can be obtained: samples 2 and 8, chosen to perform further experiments.

Sample Dimensions (nm) ζ -potential (mV)

Table 2. Vesicle dimensions (nm) and ζ- potential (mV) of analyzed samples (n=3; ±SD)

Furthermore, it should be pointed out that electron microscopy carried out on numerous samples (10) indicated that nebulization does not influence drug-loaded vesicle dimensions

Analyzed samples showed a good stability in terms of possible changes in vesicle

Size measurement experiments indicated that after 1 month at 25 °C, no appreciable vesicle

The best entrapment efficiency (e.e.) was obtained for sample 8 and the calculated drug e.e. indicated that only about 20% of the overall amount of BDP is actually enclosed within the vesicles. This result is in agreement with the data reported by previous authors (Montenegro

2 163±0.03 -32±0.2 8 174±0.02 -34±0.3 Empty vesicles 146±0.05 -40±0.2 BDP solution 0.05% w/v = -30±0.1

Table 1. Sample composition (expressed as % w/v)

to fit well within the vesicular structure.

(Figure 5A and B).

dimensions after aggregation.

dimension variations could be detected.

Fig. 5. Trasmission electron micrographs of BDP-loaded vesicles after freeze-fracture, before (A) and after (B) nebulization. The scale bar represents 0.5 m.

et al., 1996; Darwis et al., 2001). For this reason, for permeation and nebulization experiments the formulation corresponding to sample 8 of Table 1 was used.

The possibility to use the novel vesicular dispersion in a conventional jet nebulizer, widely used in clinical applications, was also evaluated. For this purpose, samples were characterized also by means of rheological measurements and the aerodynamic diameter was determined (Table 3) as well as the nebulizer mass output (Figure 6) after completion of nebulization. Evaluation of Mass Median Aerodynamic Diameter (2.0±0.2 µm) and of geometric standard deviation (GSD) were also carried out; the GSD value (1.5) demonstrates the polydisperse nature of the distribution of the aerosolized droplets that, on the other side, contained a monodisperse vesicular system.


Table 3. Percentage of particles with aerodynamic diameter <10, < 5, <2 µm, containing non-ionic surfactant vesicles, delivered by a jet nebulizer

Fig. 6. Deposition of vesicle-encapsulated BDP (BDP/ves) on filters upon nebulization, compared to a BDP/surfactant solution and to a BDP commercial product (n=3, ±SD).

The transmission signal remained close to the base line value and ΔT profiles close to 0% were observed for all the investigated NSV formulations during the entire time of analysis. Therefore, NSVs suspensions maintained a constant opalescent aspect along the height of various samples. At the same time, positive or negative variations of the backscattering profiles of the different formulations (Figure 7) were not correlated to destabilization processes under the sample height of 2 mm and over that of 20 mm, the values having been determined by enclosed air in the bottom and/or on the top of the cylindrical glass tube, respectively. Different NSVs formulations showed that backscattering emerged as the prevalent signal in the different measurements (Figure 7). ΔBS signals are close to ~1% during the time of analysis for the entire height of the samples of both purified BDP-loaded and unloaded non-ionic surfactant vesicles (Figure 7 panels B, D and F). It was interesting to observe that the different amounts of the entrapped drug do not influence the colloidal stability of non-ionic surfactant vesicles both in terms of vesicle migration and coalescence. Different stability behaviors, as shown by ΔBS profiles (Figure 7 panels A and C), were observed in the case of not purified BDP loaded non-ionic surfactant vesicles (i.e. before gel

In particular, vesicles prepared in the presence of the highest drug concentration (50 mg/ml) showed a high colloidal instability just after the beginning of the analysis. A moderate stability (ΔBS profile within the 10% during the 3 h of analysis) was observed for unpurified non-ionic surfactant vesicles prepared in the presence of 0.4 mg/ml of the drug, while an elevated stability (equal to purified formulations) was observed for unpurified unloaded vesicles (Figure 7 panel E). These findings can be due to the presence of high amount of free drug, that with time leads to the formation of aggregates. Therefore, the purification procedure is essential to achieve a stable vesicular colloidal carrier for the delivery of BDP. The stability findings by Turbiscan Lab® Expert measurement were also supported by light scattering size analysis during a storage period of 3 months, which showed no appreciable vesicle size variation for purified BDP-loaded non-ionic surfactant

According to the aim of this research, the capability of ensuring a better penetration through

In Figure 8, the permeation rate of BDP from the vesicular dispersion is reported and compared with that obtained using a BDP/polysorbate 20 (at the same concentration used for vesicle preparation) suspension as well as with that of the commercial preparation. The vesicular formulation (BDP-0.4) was used in its unpurified form, thus with the drug

This situation allows an appropriate comparison with the other preparations used in permeation experiments: since both micellar surfactant solutions and the commercial one contained free BDP and BDP included within aggregated structures (micelles). As it can be observed, the presence of NSVs in the formulation remarkably increases the permeation rate through the model mucosal barrier with respect to the other tested preparation thus indicating that the novel BDP formulation can be proposed for a better targeting of

An important aspect to evaluate an innovative drug delivery system is its safety. This aspect is much more relevant in the case of pulmonary delivery, since several side effects may

exclusion chromatography).

the mucus layer of vesicular formulation was tested.

partitioned inside and outside the vesicular structure.

corticosteroids in the treatment of COPD.

vesicles.

Furthermore, in all conditions of nebulization (TurboBoy nebulizer and Clenny nebulizer), the dispersion BDP/vesicles releases a greater amount of drug, dosed by HPLC, with respect to commercial formulations.

An important aspect to be taken into account for an actual application of these non-ionic surfactant vesicles as possible carriers to be aerosolized for the pulmonary delivery of drugs is their colloidal and storage stability.

In fact, the occurrence of aggregation phenomena can lead to a significant worsening of the biopharmaceutical features of nanosized colloidal suspensions, such as NSVs. Therefore, the colloidal stability of BDP-loaded NSVs was evaluated using the Turbiscan Lab® Expert (Celia et al., 2009) i.e. the optical transmission and the photon backscattering profiles of various samples were recorded. Any variation of the vesicle volume fraction (migration) or mean size (coalescence) triggers the variation of backscattering (BS) and transmission (T) signals, which are graphically reported as positive (backscattering/transmission increase) or negative (backscattering/transmission decrease) peaks. It can be assumed that no variation of particle size occurs when the ΔBS and ΔT profiles are within an interval of ±2% while variations greater than 10% either as a positive or a negative value are representative of an instable formulation.

Two different BDP concentrations, i.e. 50 mg/ml (sample BDP-50) and 0.4 mg/ml (BDP-0.4), were used in this stability investigations. The first concentration led to the maximum possible amount of drug entrapment within the NSVs, while the second concentration led to an amount of entrapped drug similar to that actually present in the most common commercial products.

The ΔBS and ΔT profiles of BDP -loaded and unloaded non-ionic surfactant vesicles are shown in Figure 7.

Fig. 7. Transmission and backscattering profiles of niosomes by using Turbiscan Lab® Expert. The image represents the analysis of different formulations: (1) unpurified BDP-50 niosomes; (2) purified BDP-50-niosomes; (3) unpurified BDP-0.4-niosomes; (4) purified BDP-0.4-niosomes; (5) unloaded niosomes. Data are reported as a function of time (0–3 h) and sample height (from 2 to 20 mm).

Furthermore, in all conditions of nebulization (TurboBoy nebulizer and Clenny nebulizer), the dispersion BDP/vesicles releases a greater amount of drug, dosed by HPLC, with

An important aspect to be taken into account for an actual application of these non-ionic surfactant vesicles as possible carriers to be aerosolized for the pulmonary delivery of drugs

In fact, the occurrence of aggregation phenomena can lead to a significant worsening of the biopharmaceutical features of nanosized colloidal suspensions, such as NSVs. Therefore, the colloidal stability of BDP-loaded NSVs was evaluated using the Turbiscan Lab® Expert (Celia et al., 2009) i.e. the optical transmission and the photon backscattering profiles of various samples were recorded. Any variation of the vesicle volume fraction (migration) or mean size (coalescence) triggers the variation of backscattering (BS) and transmission (T) signals, which are graphically reported as positive (backscattering/transmission increase) or negative (backscattering/transmission decrease) peaks. It can be assumed that no variation of particle size occurs when the ΔBS and ΔT profiles are within an interval of ±2% while variations greater than 10% either as a positive or a negative value are representative of an

Two different BDP concentrations, i.e. 50 mg/ml (sample BDP-50) and 0.4 mg/ml (BDP-0.4), were used in this stability investigations. The first concentration led to the maximum possible amount of drug entrapment within the NSVs, while the second concentration led to an amount of entrapped drug similar to that actually present in the most common

The ΔBS and ΔT profiles of BDP -loaded and unloaded non-ionic surfactant vesicles are

**1 2 3 4 5** 

Fig. 7. Transmission and backscattering profiles of niosomes by using Turbiscan Lab® Expert. The image represents the analysis of different formulations: (1) unpurified BDP-50 niosomes; (2) purified BDP-50-niosomes; (3) unpurified BDP-0.4-niosomes; (4) purified BDP-0.4-niosomes; (5) unloaded niosomes. Data are reported as a function of time (0–3 h) and

respect to commercial formulations.

is their colloidal and storage stability.

instable formulation.

commercial products.

sample height (from 2 to 20 mm).

shown in Figure 7.

The transmission signal remained close to the base line value and ΔT profiles close to 0% were observed for all the investigated NSV formulations during the entire time of analysis.

Therefore, NSVs suspensions maintained a constant opalescent aspect along the height of various samples. At the same time, positive or negative variations of the backscattering profiles of the different formulations (Figure 7) were not correlated to destabilization processes under the sample height of 2 mm and over that of 20 mm, the values having been determined by enclosed air in the bottom and/or on the top of the cylindrical glass tube, respectively. Different NSVs formulations showed that backscattering emerged as the prevalent signal in the different measurements (Figure 7). ΔBS signals are close to ~1% during the time of analysis for the entire height of the samples of both purified BDP-loaded and unloaded non-ionic surfactant vesicles (Figure 7 panels B, D and F). It was interesting to observe that the different amounts of the entrapped drug do not influence the colloidal stability of non-ionic surfactant vesicles both in terms of vesicle migration and coalescence. Different stability behaviors, as shown by ΔBS profiles (Figure 7 panels A and C), were observed in the case of not purified BDP loaded non-ionic surfactant vesicles (i.e. before gel exclusion chromatography).

In particular, vesicles prepared in the presence of the highest drug concentration (50 mg/ml) showed a high colloidal instability just after the beginning of the analysis. A moderate stability (ΔBS profile within the 10% during the 3 h of analysis) was observed for unpurified non-ionic surfactant vesicles prepared in the presence of 0.4 mg/ml of the drug, while an elevated stability (equal to purified formulations) was observed for unpurified unloaded vesicles (Figure 7 panel E). These findings can be due to the presence of high amount of free drug, that with time leads to the formation of aggregates. Therefore, the purification procedure is essential to achieve a stable vesicular colloidal carrier for the delivery of BDP. The stability findings by Turbiscan Lab® Expert measurement were also supported by light scattering size analysis during a storage period of 3 months, which showed no appreciable vesicle size variation for purified BDP-loaded non-ionic surfactant vesicles.

According to the aim of this research, the capability of ensuring a better penetration through the mucus layer of vesicular formulation was tested.

In Figure 8, the permeation rate of BDP from the vesicular dispersion is reported and compared with that obtained using a BDP/polysorbate 20 (at the same concentration used for vesicle preparation) suspension as well as with that of the commercial preparation. The vesicular formulation (BDP-0.4) was used in its unpurified form, thus with the drug partitioned inside and outside the vesicular structure.

This situation allows an appropriate comparison with the other preparations used in permeation experiments: since both micellar surfactant solutions and the commercial one contained free BDP and BDP included within aggregated structures (micelles). As it can be observed, the presence of NSVs in the formulation remarkably increases the permeation rate through the model mucosal barrier with respect to the other tested preparation thus indicating that the novel BDP formulation can be proposed for a better targeting of corticosteroids in the treatment of COPD.

An important aspect to evaluate an innovative drug delivery system is its safety. This aspect is much more relevant in the case of pulmonary delivery, since several side effects may

Another important feature of an innovative drug delivery system is to increase the amount of active compound in the target district, thus improving the therapeutic effect of the drug. Therefore, to evaluate the delivery ability and the mechanisms of interaction of fluoresceinlabelled non-ionic surfactant vesicles with HLF cells, CLSM experiments were carried out. Figure 9 shows how the fluorescein-labelled vesicles interacted with HLF cells at different incubation times. A green fluorescence distribution was observed in the cells just after 1 h incubation. After 3 h incubation the fluorescence of the cellular membrane and the cytoplasm became more intense and increased slightly up to 24 h of incubation. These findings prompted us to suppose that the main mechanism involved in the NSVs/cell interaction was the endocytosis of the carrier (Di Marzio et al., 2008), which enabled a rapid internalization in the cytoplasm. It is worthy of note that at all incubation times the localization of fluorescence was in the cytoplasm compartment and these results represent an important aspect because the glucocorticoid receptor is localized in the cytoplasm.

No fluorescence was detected in the untreated HLF cell line (control) and hence there was

no interfering auto-fluorescence phenomenon (Figure 9 panel 6).

1 h 3 h 6 h

12 h 24 h control

a greater intracellular delivery of the entrapped drug.

investigated as a function of time (Figure 10).

Fig. 9. CLSM micrographs of the interaction of fluoresceine-labelled NSVs with primary HLF cells as a function of the incubation times,. The reflectance CLSM micrographs of untreated cells were used as controls and no significant cellular fluorescence was observed

As a consequence, the improved interaction of NSVs with primary HLF cells should lead to

For this reason the intracellular uptake of BDP prompted by the various formulations was

Considering that surfactant molecules and unloaded non-ionic surfactant vesicles may act as drug cellular penetration enhancers and to evaluate the effective role of the vesicles in the promotion of the intracellular uptake of BDP, a mixture surfactant/BDP and a mixture empty vesicles/BDP was also assayed. As reported in Figure 10, BDP loaded non-ionic surfactant vesicles (BDP-0.4) showed a significant improvement of the intracellular uptake of the drug with respect to a mixture surfactant/BDP, a mixture empty non-ionic surfactant

Fig. 8. Comparison of the permeation patterns through a gel-like mucin solution (0.1%w/v), expressed as percentage of permeated drug as a function of time (n=3, ±SD) (BDP/surfactant ▲BDP/commercial product BDP/vesicles).

result from an unsafe material, i.e. fibrosis, pulmonary oedema, inflammation, as reported in section 4.

Safety of empty non-ionic surfactant vesicles was evaluated in vitro on HLF cells by using the trypan blue dye exclusion assay (cell mortality) and MTT viability test. Purified and unpurified empty NSVs were assayed in vitro at different surfactant concentrations (from 0.01 to 10 μM) and incubation-times (24, 48 and 72 h).

Purified non-ionic surfactant vesicles did not show a significant cytotoxic activity on HLF cells at all incubation times for concentrations ranging from 0.01 to 1 μM, i.e. the mortality values ranged from 1.2 to 5.81%, respectively. Only at the highest investigated concentration (10 μM) and after 48 h of incubation a slight cytotoxic effect was observed (mortality value of 16%).

It is interesting to point out that unpurified vesicles showed a significant (P<0.001) increase of cytotoxicity with respect to purified vesicles at all the investigated conditions (exposition times and surfactant concentrations). In this sense it was also observed that the increase of the cytotoxic effect was dependent on surfactant concentration but not on the exposition time. This finding was due to the fact that the cytotoxic action of unpurified NSVs was determined by the presence of the free molecules of surfactants, which were able to exert immediately their cytotoxic action during the incubation period by noticeably perturbing the cellular membranes (Dimitrijevic et al., 2000; Lin et al., 2007) and hence causing the cellular death. This hypothesis was strongly supported by the evidence that HLF cells treated with the free surfactants showed a greater (P<0.001) cytotoxic insult than those treated with unpurified non-ionic surfactant vesicles. Therefore, the assembling of surfactant into non-ionic surfactant vesicles determined a drastic reduction of cytotoxicity, due to a concomitant reduction of the free surfactant molecules in solution and/or surfactant micelles, which are able to alter the cellular permeability and homeostasis.

Fig. 8. Comparison of the permeation patterns through a gel-like mucin solution (0.1%w/v),

result from an unsafe material, i.e. fibrosis, pulmonary oedema, inflammation, as reported

Safety of empty non-ionic surfactant vesicles was evaluated in vitro on HLF cells by using the trypan blue dye exclusion assay (cell mortality) and MTT viability test. Purified and unpurified empty NSVs were assayed in vitro at different surfactant concentrations (from

Purified non-ionic surfactant vesicles did not show a significant cytotoxic activity on HLF cells at all incubation times for concentrations ranging from 0.01 to 1 μM, i.e. the mortality values ranged from 1.2 to 5.81%, respectively. Only at the highest investigated concentration (10 μM) and after 48 h of incubation a slight cytotoxic effect was observed (mortality value

It is interesting to point out that unpurified vesicles showed a significant (P<0.001) increase of cytotoxicity with respect to purified vesicles at all the investigated conditions (exposition times and surfactant concentrations). In this sense it was also observed that the increase of the cytotoxic effect was dependent on surfactant concentration but not on the exposition time. This finding was due to the fact that the cytotoxic action of unpurified NSVs was determined by the presence of the free molecules of surfactants, which were able to exert immediately their cytotoxic action during the incubation period by noticeably perturbing the cellular membranes (Dimitrijevic et al., 2000; Lin et al., 2007) and hence causing the cellular death. This hypothesis was strongly supported by the evidence that HLF cells treated with the free surfactants showed a greater (P<0.001) cytotoxic insult than those treated with unpurified non-ionic surfactant vesicles. Therefore, the assembling of surfactant into non-ionic surfactant vesicles determined a drastic reduction of cytotoxicity, due to a concomitant reduction of the free surfactant molecules in solution and/or surfactant

micelles, which are able to alter the cellular permeability and homeostasis.

expressed as percentage of permeated drug as a function of time (n=3, ±SD)

(BDP/surfactant ▲BDP/commercial product BDP/vesicles).

0.01 to 10 μM) and incubation-times (24, 48 and 72 h).

in section 4.

of 16%).

Another important feature of an innovative drug delivery system is to increase the amount of active compound in the target district, thus improving the therapeutic effect of the drug. Therefore, to evaluate the delivery ability and the mechanisms of interaction of fluoresceinlabelled non-ionic surfactant vesicles with HLF cells, CLSM experiments were carried out.

Figure 9 shows how the fluorescein-labelled vesicles interacted with HLF cells at different incubation times. A green fluorescence distribution was observed in the cells just after 1 h incubation. After 3 h incubation the fluorescence of the cellular membrane and the cytoplasm became more intense and increased slightly up to 24 h of incubation. These findings prompted us to suppose that the main mechanism involved in the NSVs/cell interaction was the endocytosis of the carrier (Di Marzio et al., 2008), which enabled a rapid internalization in the cytoplasm. It is worthy of note that at all incubation times the localization of fluorescence was in the cytoplasm compartment and these results represent an important aspect because the glucocorticoid receptor is localized in the cytoplasm.

No fluorescence was detected in the untreated HLF cell line (control) and hence there was no interfering auto-fluorescence phenomenon (Figure 9 panel 6).

Fig. 9. CLSM micrographs of the interaction of fluoresceine-labelled NSVs with primary HLF cells as a function of the incubation times,. The reflectance CLSM micrographs of untreated cells were used as controls and no significant cellular fluorescence was observed

As a consequence, the improved interaction of NSVs with primary HLF cells should lead to a greater intracellular delivery of the entrapped drug.

For this reason the intracellular uptake of BDP prompted by the various formulations was investigated as a function of time (Figure 10).

Considering that surfactant molecules and unloaded non-ionic surfactant vesicles may act as drug cellular penetration enhancers and to evaluate the effective role of the vesicles in the promotion of the intracellular uptake of BDP, a mixture surfactant/BDP and a mixture empty vesicles/BDP was also assayed. As reported in Figure 10, BDP loaded non-ionic surfactant vesicles (BDP-0.4) showed a significant improvement of the intracellular uptake of the drug with respect to a mixture surfactant/BDP, a mixture empty non-ionic surfactant

inhibited by anti-inflammatory glucocorticoids. Therefore, the in vitro determination of the levels of NGF secreted by HLF cells is a direct evidence of the pro- or anti-inflammatory

As shown in Figure 11, the stimulation of HLF cells with IL-1β led to an over-secretion of NGF up to 210% of basal values (control). The treatment with the free BDP (1 μM) significantly reduced both the constitutive and the IL-1β-stimulated secretion of NGF by 18.7% and 61.23%, respectively. BDP-loaded non-ionic surfactant vesicles (BDP-0.4) were much more effective than the free drug, i.e. a reduction of 68% and 85% with respect to the constitutive and IL-1β-stimulated NGF secretion were respectively observed. These findings

Fig. 11. Anti-inflammatory activity of various formulations containing BDP (1 μM) evaluated as inhibition of NGF secretion in primary HLF cells treated with IL-1β (as pro-inflammatory stimulating agent). Control was untreated HLF cells, which secrete the basal level of NGF.

The use of NSV formulations was investigated not only to increase intracellular uptake of BDP in HLF cells and to improve diffusion through mucus layer but also to design an innovative system able to increase therapeutic efficacy of BDP in pulmonary diseases thus

Despite the many promising proof - of - concepts of various delivery technologies, there is still a long way ahead that must be covered. This means there are still many challenges that

reducing the dosage and potential side effects of this drug.

**7. Conclusions** 

are in agreement with both CLSM and intracellular uptake experiments.

effect of a substance.

Fig. 10. HLF intracellular uptake (I.U. at 37 °C) of beclomethasone dipropionate (expressed as percentage of the applied dose) as a function of time by different formulations: BDP loaded NSVs (BDP-0.4), ●; surfactant/BDP mixture, ▲; empty non-ionic surfactant vesicle/BDP mixture, ■; free drug, ♦.

vesicle/BDP and a free drug solution. This result was correlated to the ability of NSVs to easily penetrate across cell membranes of primary HLF cells (in full agreement with CSLM experiments), thus achieving a noticeable cytoplasm accumulation of BDP. These results clearly evidenced that the improvement of the drug intracellular uptake was mainly mediated by the vesicular carrier and no positive influence was exerted by the surfactant molecules and/or empty vesicles, i.e. the drug has to be entrapped within the carrier. In fact, the mixtures surfactant/drug and empty vesicle/drug showed only a slight improvement of the intracellular uptake of BDP. The profiles of BDP intracellular uptake as a function of time showed (Figure 10) a Tmax (time at which the maximum drug concentration was reached) value of 1 h followed by a gradual reduction of the intracellular drug accumulation up to 6 h in the case of the drug-loaded non-ionic surfactant vesicles. On the other side, all other tested formulations showed Tmax values of 30 min.

The rapid internalization of BDP formulated in niosomes was not considered as a critical parameter.

The evidence of the improved intracellular entrance of NSVs and the noticeable increase of the intracellular uptake of BDP mediated by the vesicular carrier should match an improved pharmacological activity of the delivered drug. For this reason the anti-inflammatory activity of BDP-loaded non-ionic surfactant vesicles was evaluated in comparison with the free drug, as the capacity to inhibit the secretion of NGF.

NGF is an important inflammatory mediator, which contributes to the development of airway hyper-responsiveness (de Vries et al., 1999). The NGF production is stimulated by the presence of pro-inflammatory cytokines and asthma-associated cytokines, i.e. IL-1β, and

Fig. 10. HLF intracellular uptake (I.U. at 37 °C) of beclomethasone dipropionate (expressed as percentage of the applied dose) as a function of time by different formulations: BDP loaded NSVs (BDP-0.4), ●; surfactant/BDP mixture, ▲; empty non-ionic surfactant

vesicle/BDP and a free drug solution. This result was correlated to the ability of NSVs to easily penetrate across cell membranes of primary HLF cells (in full agreement with CSLM experiments), thus achieving a noticeable cytoplasm accumulation of BDP. These results clearly evidenced that the improvement of the drug intracellular uptake was mainly mediated by the vesicular carrier and no positive influence was exerted by the surfactant molecules and/or empty vesicles, i.e. the drug has to be entrapped within the carrier. In fact, the mixtures surfactant/drug and empty vesicle/drug showed only a slight improvement of the intracellular uptake of BDP. The profiles of BDP intracellular uptake as a function of time showed (Figure 10) a Tmax (time at which the maximum drug concentration was reached) value of 1 h followed by a gradual reduction of the intracellular drug accumulation up to 6 h in the case of the drug-loaded non-ionic surfactant vesicles. On the other side, all

The rapid internalization of BDP formulated in niosomes was not considered as a critical

The evidence of the improved intracellular entrance of NSVs and the noticeable increase of the intracellular uptake of BDP mediated by the vesicular carrier should match an improved pharmacological activity of the delivered drug. For this reason the anti-inflammatory activity of BDP-loaded non-ionic surfactant vesicles was evaluated in comparison with the

NGF is an important inflammatory mediator, which contributes to the development of airway hyper-responsiveness (de Vries et al., 1999). The NGF production is stimulated by the presence of pro-inflammatory cytokines and asthma-associated cytokines, i.e. IL-1β, and

vesicle/BDP mixture, ■; free drug, ♦.

I.U. (%)

parameter.

other tested formulations showed Tmax values of 30 min.

free drug, as the capacity to inhibit the secretion of NGF.

inhibited by anti-inflammatory glucocorticoids. Therefore, the in vitro determination of the levels of NGF secreted by HLF cells is a direct evidence of the pro- or anti-inflammatory effect of a substance.

As shown in Figure 11, the stimulation of HLF cells with IL-1β led to an over-secretion of NGF up to 210% of basal values (control). The treatment with the free BDP (1 μM) significantly reduced both the constitutive and the IL-1β-stimulated secretion of NGF by 18.7% and 61.23%, respectively. BDP-loaded non-ionic surfactant vesicles (BDP-0.4) were much more effective than the free drug, i.e. a reduction of 68% and 85% with respect to the constitutive and IL-1β-stimulated NGF secretion were respectively observed. These findings are in agreement with both CLSM and intracellular uptake experiments.

Fig. 11. Anti-inflammatory activity of various formulations containing BDP (1 μM) evaluated as inhibition of NGF secretion in primary HLF cells treated with IL-1β (as pro-inflammatory stimulating agent). Control was untreated HLF cells, which secrete the basal level of NGF.

The use of NSV formulations was investigated not only to increase intracellular uptake of BDP in HLF cells and to improve diffusion through mucus layer but also to design an innovative system able to increase therapeutic efficacy of BDP in pulmonary diseases thus reducing the dosage and potential side effects of this drug.

#### **7. Conclusions**

Despite the many promising proof - of - concepts of various delivery technologies, there is still a long way ahead that must be covered. This means there are still many challenges that

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are being faced, which, in turn, mean there are still many chances for the academic and industrial scientist to make a decisive impact.

Further research efforts are needed to ensure the safety of long-term in vivo applications. There is an urgent requirement for cautiously designed toxicology and toxicokinetic studies for each nanocarrier type; the protocols should be customized for an appropriate clinical use. Furthermore, it should be pointed out that scale up from laboratory to industry is still poorly investigated in this specific area, despite its obvious importance in the ultimate goal of the development a product that can reach the market and actually give benefits to the patients.

In spite of the above reported difficulties and challenges; hopefully, within a few years, the safety and large - scale production at affordable costs of the delivery technologies described in this book will be a dream that will become true.
