European Journal of Pharmaceutics and Biopharmaceutics 88 (2014) 1076–1085
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Research paper
Impact of atomization technique on the stability and transport efficiency of nebulized liposomes harboring different surface characteristics Bernhard Lehofer a,b,1, Florian Bloder a,1, Pritesh P. Jain a, Leigh M. Marsh a, Gerd Leitinger c,d, Horst Olschewski a,e, Regina Leber a,f, Andrea Olschewski a, Ruth Prassl a,b,⇑ a
Ludwig Boltzmann Institute for Lung Vascular Research, Graz, Austria Institute of Biophysics, Medical University of Graz, BioTechMed-Graz, Graz, Austria Institute of Cell Biology, Histology and Embryology, Research Unit Electron Microscopic Techniques, Medical University of Graz, Graz, Austria d Center for Medical Research, Medical University of Graz, Graz, Austria e Division of Pulmonology, Medical University of Graz, Graz, Austria f Institute of Molecular Biosciences, University of Graz, Graz, Austria b c
a r t i c l e
i n f o
Article history: Received 16 June 2014 Accepted in revised form 15 October 2014 Available online 23 October 2014 Keywords: Liposome Nebulization Aerosol Pulmonary drug delivery Inhalation technique Vibrating-mesh nebulizer
a b s t r a c t The objective of this study was to evaluate the impact of nebulization on liposomes with specific surface characteristics by applying three commercially available inhaler systems (air-jet, ultrasonic and vibrating-mesh). Conventional liposome formulations composed of phosphatidylcholine and cholesterol were compared to sterically stabilized PEGylated liposomes and cationic polymer coated liposomes. Liposomes of similar size (between 140 and 165 nm in diameter with polydispersity indices 0.1 a more detailed size distribution analysis of the PCS data based on intensity, volume and number calculations was performed. The zeta potential was determined by Laser Doppler Micro-electrophoresis using a Zetasizer Nano ZS (Malvern Instruments, Herrenberg, Germany). For the zeta potential measurements the liposomes were diluted with 2 mM HEPES containing 2 mM CsCl to a final concentration of 0.3 mg/ml of lipids. All size and zeta potential measurements were taken in triplicate at room temperature with each measurement consisting of 10 runs. 2.4. Negative staining transmission electron microscopy (TEM) The three liposomal formulations each before and after nebulization were diluted to a concentration of 0.6 mg/ml and pipetted on carbon coated copper grids. After 1 min incubation time, the excess sample was blotted off and replaced by 1% aqueous uranyl acetate as staining reagent. This treatment was repeated twice each with 30 s incubation times. Samples were visualized using an FEI TECNAI 20 Transmission Electron Microscope at 120 kV as acceleration voltage with the help of a GATAN Ultrascan 1000 CCD camera at 2 k 2 k resolution.
(Fig. S2). The calculations are based on fluorescence intensity measurements; F0 is the initial quenched fluorescence intensity of ANTS before nebulization and Fmax is the fluorescence intensity corresponding to 100% leakage, which was measured after lysis of the liposomes by the addition of 10 ll of 10% Triton X-100. Ft represents the fluorescence intensity of ANTS measured after nebulization. The correction term (F max =F max ) accounts for changes in sample concentration during nebulization with F max being the fluorescence intensity after lysis of nebulized liposomes with Triton X-100. Encapsulation efficiency (EE%) was calculated as follows
EE% ¼
F max F 0 100 F max
ð1Þ
and liposome leakage (%) corresponding to drug release as
leakage ¼
Ft
F max F max
F0
F max F 0
100
ð2Þ
2.6. Stability of liposomes in artificial lung surfactant ANTS/DPX loaded liposomes were diluted to a lipid concentration of 1 mg/ml with HEPES buffer and 5 ll thereof was incubated with 200 ll of diluted AlveofactÒ (0.5 mg/ml). The measurements were taken in a 10 2 mm quartz cuvette at a temperature of 37 °C. Leakage was recorded for 60 min at the same instrumental settings as described in Section 2.5. At the end of the experiment 10 ll of 10 % Triton X-100 was added and the increase in intensity was recorded for other 60 s. The background autofluorescence of AlveofactÒ was subtracted from the overall fluorescence intensity of the liposomes in AlveofactÒ. The measured fluorescence intensity is expressed in kilocounts per second (kcps). 2.7. Nebulization of liposomes All liposomal formulations were diluted with HEPES buffer to a final lipid concentration of 1 mg/ml. Each nebulizer reservoir was filled with 3 ml of liposomal solution and 2 ml thereof was nebulized, while the time for nebulization was recorded. The weight of the nebulizer was determined before and after nebulization to calculate the total aerosol output rate. Generated aerosol was
2.5. Determination of encapsulation efficiency and release by fluorescence spectroscopy The fluorophore/quencher (ANTS/DPX) system was used as model drug system to assess the encapsulation efficiency and leakage tendency of liposomal formulations [21,22]. The fluorescence of ANTS was recorded on a SPEX FLUOROMAX-3 fluorescence spectrophotometer (HORIBA Jobin–Yvon, Longjumeau, France). Measurements were taken using 10 10 mm quartz cuvettes (Hellma Analytics, Germany), an excitation wavelength of 360 nm and an emission wavelength of 530 nm. The slit width was 5 nm for both excitation and emission monochromators. Samples were diluted with HEPES buffer and incubated at 37 °C without stirring during the measurements. Details on the mode of operation of the ANTS/DPX system are provided in Supporting Information
Fig. 1. Volume median diameter (VMD) of aerosol droplets depends on the sodium chloride concentration of the nebulized solution. Three nebulizer devices are compared, each one based on a different principle. The aerosol solutions are as follows: 10 mM HEPES (pH 7.4) without NaCl (h), with 70 mM NaCl ( ) and 140 mM NaCl ( ). Bars are the mean ± SD, n = 3. The different levels of significance are indicated as ⁄p < 0.05, ⁄⁄p < 0.016.
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to the droplet size at which 50% of the volume is in droplets smaller than the VMD. 2.9. Statistical analysis All measurements were taken in triplicate and the values are presented as the mean ± S.D. A one-way ANOVA statistical test with a Bonferroni post-t-test correction was made. Probability values of p < 0.05 and p0 < 0.016 for Bonferroni correction (comparison among three groups) were considered significant. Statistical analysis was done with Microsoft Excel 2010. 3. Results and discussion Fig. 2. Influence of liposomes on the aerosol droplet size distribution. Liposomal solutions in HEPES (pH 7.4) containing 140 mM NaCl nebulized with the vibratingmesh nebulizer (eFlowÒ rapid). HEPES buffer (continuous line) L1, L2 and L3 (dotted lines).
collected at 4 °C with the disposable exhaled condensate collecting system TURBO-DECCS purchased from Ital-chill, Parma, Italy, for further analysis. Since the nebulizers have different airflow rates, a compressor (DeVO/MC 29, DeVILBISS GmbH Medical Products, Germany) to enhance airflow (for low flow rate in case of the OptinebÒ-ir) or an additional collecting vessel (for high flow rate in case of MicroDrop MasterJetÒ) was applied following the condensation chamber. As a reference, a 10 mM HEPES buffer (pH 7.4) containing different salt concentrations (0, 70 and 140 mM NaCl) was nebulized. 2.8. Analysis of the aerodynamic diameter of the aerosol The aerosol droplet size distribution of nebulized samples was measured by laser diffraction technique using a Mastersizer 2000 (Malvern Instruments, Herrenberg, Germany). Measurements were taken at a laser obscuration between 4% and 6% and taken in triplicates. The lipid concentration of all liposomal formulations was set to 1 mg/ml by dilution with HEPES buffer before nebulization. The Malvern Mastersizer 2000 software Version 5.60 was used to characterize the volume median diameter (VMD) corresponding
3.1. Physicochemical properties of liposomes used for nebulization To evaluate the applicability of each nebulizer device for atomization of liposomes, three types of liposomal formulations of similar particle sizes but with different chemical composition and surface properties were investigated. Namely, a conventional pharmaceutical formulation composed of zwitterionic phosphatidylcholines (L1) was compared to a polymer-coated (PEGylated) sterically stabilized formulation (L2), representative for the second generation of liposomal drug carriers. Such PEGylated liposomes are stabilized by steric repulsion caused by the PEG chains in addition to a hydration shell of a non-diffusible water layer around the liposomes [23,24]. Hence, PEGylated liposomes have a longer life time in vivo since they are not readily recognized by the immune system [25]. The third formulation (L3) was additionally enriched with a cationic lipid to exert a net positive surface charge to the liposomes. Cationic liposomes are becoming more and more interesting for pharmaceutical applications, especially as delivery systems for oligonucleotides, RNA or DNA [26]. The main lipid component of the three formulations was DPPC, a phosphatidylcholine with saturated acyl chains. Additionally, the liposomes were enriched with 30 mol% of cholesterol. With this lipid (DPPC/cholesterol) composition the bilayer membrane is in an ordered liquid phase [27], being more rigid and less permeable for water soluble, non-bilayer interacting molecules. The average particle sizes ranged from 140 to 165 nm. For L2 and L3 the size
Fig. 3. Aerosol output efficiencies of nebulized liposomes for different nebulizer devices. (A) Amount of liposomes (mg total lipid concentration) transported per ml of aerosol and (B) transport efficiency for liposomes (L1–L3) per minute of nebulization time (mg lipid/min). Equal volumes of liposomal solutions with 1 mg/ml phospholipid concentration were aerosolized with three different nebulizers. Bars are the mean ± SD, n = 3. The different levels of significance are indicated as ⁄p < 0.05, ⁄⁄p < 0.016.
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Fig. 4. Impact of nebulizers on the integrity of liposomal formulations. (A) Size of liposomes determined before (pre) and after (post) nebulization for three nebulizer devices in aerosol and remainder. (B) Polydispersity index (PDI) of nebulized liposomes measured by PCS. Bars are the mean ± SD, n = 3.
values were very similar, while L1, the conventional formulation without PEG-coating, was slightly, but significantly (p0 < 0.016) larger. The PDI-values were below 0.08 for all three formulations indicating highly homogeneous populations. The surface zeta potential, which is related to the surface charge of the particles and the ionic strength of the medium, was markedly positive for the cationic formulation L3 and slightly negative for L1 and L2. The chemical compositions and physicochemical properties of the three formulations are listed in Table 1. For all formulations the encapsulation efficiency of ANTS/DPX was high (>70 %EE). These values were taken as 100%EE for the subsequent nebulization studies. To address the stability of liposomes under more physiological conditions the liposomes were incubated with artificial lung surfactant (AlveofactÒ) at physiological temperature.
We found that none of the three formulations showed any leakage of the encapsulated fluorophore (Fig. S3) during the observation period of 60 min. 3.2. Aerosol droplet size Aerosols for deep lung penetration require the generation of droplets with an aerodynamic diameter of 1–3 lm [9,28]. Since it is known that the addition of electrolytes can influence aerosol droplet size [29,30], we aimed to assess the impact of sodium chloride (70 and 140 mM NaCl) on the VMD of droplets generated by the different nebulizer devices. Fig. 1 shows that the aerosol droplet size of nebulized 10 mM HEPES buffer (pH 7.4) decreased with increasing concentrations of sodium chloride. While for the
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3.3. Aerosol output and nebulizer efficiency
Fig. 5. Negative staining transmission electron micrographs of the three liposomal formulations L1, L2 and L3 before nebulization (left panel) and after nebulization (right panel) with the eFlowÒ rapid nebulizer. The bars represent lengths of 100 nm.
vibrating-mesh and the ultrasonic device the addition of 70 mM NaCl was sufficient to generate adequate droplet sizes below 3 microns, higher concentrations of salt were required for the airjet device. By increasing the concentration of NaCl to 140 mM, the droplet sizes for the vibrating-mesh and the air-jet nebulizer were significantly (p0 < 0.016) reduced. These data indicate that optimal droplet sizes for deep lung deposition of droplets can be reliably achieved by addition of a moderate, close to physiological, concentration of sodium chloride. Details on the size analysis of aerosol droplets containing different sodium chloride concentrations are provided in Supporting Information Fig. S4. Based on these initial results revealing the effects of electrolytes on droplet size, all further experiments with liposomes were performed in HEPES buffer containing 140 mM NaCl. The liposomes were prepared and nebulized at room temperature at a concentration of 1 mg/ml phospholipid. We found that at these experimental conditions the liposomes had little influence on the aerodynamic diameter and the aerosol droplet size distribution regardless of the nebulizer type. Likewise, very similar volume distribution profiles were obtained for all three formulations showing similar characteristics as those obtained with HEPES buffer. Fig. 2 shows the data obtained for the vibrating-mesh nebulizer (eFlowÒ rapid) as a representative example. About 90% of the aerosolized volume contained droplets less than 5.5 lm in size; approximately 75% contained droplets below 3 lm. No marked differences were observed for the three liposomal formulations.
Effective transport of liposomes within aerosol droplets is a prerequisite for therapeutic inhalative applications of liposomal drug formulations. However, the actual amount of aerosolized liposomes transferred with the spray mist might depend on the employed nebulizer device and the characteristics of the formulation itself [10]. To address this issue the aerosol output for nebulized liposomes, corresponding to the transport efficiency of the nebulizer, was calculated based on the lipid concentration determined in the collected aerosol and correlated with the initial lipid concentration before nebulization (1 mg/ml phospholipid). Fig. 3A reveals that the transport efficiency was very much dependent on the working principle of the inhaler device. The lowest atomization efficiency was observed with the ultrasonic device, ranging between 21% and 39% of liposomes transported in the spray mist. No significant differences could be detected when comparing the three formulations. In contrast to the ultrasonic device, both the air-jet and the vibrating-mesh nebulizer generated aerosols containing the same amount of liposomes as compared to the non-nebulized solution. Uncharged PEGylated liposomes were as efficiently transported in the spray mist as conventional ones (almost 100% are transferred). A lower output of about 60–70% lipid was achieved for the positively charged formulation L3. These data suggest that surface coating of liposomes with the polymeric stabilizer PEG2000 had no impact on the aerosol output, at least at the PEG concentrations used in this study, which are typical for sterically stabilized pharmaceutical formulations [31]. To determine the output rate of the different nebulizer devices the absolute amount of liposomes in the aerosol was normalized to the time required to nebulize the corresponding solution (Fig. 3B). The ultrasonic nebulizer was the least efficient transferring less than 10% of the liposomes per minute into the aerosol independent of the formulation used. These data suggest that large parts of liposomes are excluded from the emitted aerosol droplets produced by the ultrasonic nebulizers. This agrees with the findings of O’Riordan [32] indicating that the suitability of ultrasonic nebulizers for liposomal formulations is limited. The vibratingmesh nebulizer was the most efficient device being three times more effective than the air-jet nebulizer. Approximately 10 min was required for nebulizing 2 ml of liposomal formulations with the air-jet nebulizer, whereas the actively vibrating-mesh nebulizer required only 3 min for the same volume. The output rate was in general lower for the positively charged liposomes (L3) compared to L1 and L2. One possible explanation could be that for the cationic formulation the concentration of ions that is present at the air–liquid interface is lower [33] and that this effect influences the transport efficiency for cationic liposomes. 3.4. Liposome stability upon nebulization Next we have determined the stability of the three liposomal formulations upon nebulization (Fig. 4A). None of the formulations exhibited any substantial changes in the average hydrodynamic diameter, as would be expected from bilayer disruption, aggregation or fusion of liposomes during nebulization. Likewise, the mean particle size of liposomes remaining in the reservoir of the nebulizer devices remained essentially the same. The same is true for the PDI values, which remained below 0.1 during nebulization for L1 and L2 (Fig. 4B). A strong increase in PDI was observed for the positively charged formulation (L3) for all three nebulizer devices and being most pronounced for the vibrating-mesh nebulizer. A direct comparison of the PDI values revealed that for none of the liposomal formulations the PDI increased above a mean value of 0.22; such PDI values still indicate a narrow size distribution. By performing a detailed size distribution analysis it
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Fig. 6. Drug release upon nebulization. The leakage of the model drug system ANTS/DPX in relation to the initially encapsulated amount was determined fluorimetrically for the three liposomal formulations L1 (h), L2 ( ) and L3 ( ). The values are expressed as %leakage. Bars are the mean ± SD, n = 3.
becomes obvious that in case of the vibrating-mesh nebulizer more than 90% of the liposomes kept their original size during nebulization and only a very small fraction of liposomes aggregate to larger particles with diameters between 1 and 2 lm. For clarity, some original data obtained for the positively charged formulation nebulized with the eFlowÒ rapid nebulizer are shown in Fig. S5. Overall, these data stress that especially for bi- or multimodal particle size distributions, e.g. small particles in presence of some large aggregates, the mean values of hydrodynamic diameters and the PDI values typically provided in publications, have to be challenged. To obtain more detailed information on the morphology of the liposomes and on the structural stability upon nebulization we have performed negative staining TEM. The TEM images displayed in Fig. 5 show predominantly single spherical particles for all three formulations before nebulization. Very similar images were observed for the liposomes after nebulization with the eFlowÒ rapid nebulizer, where almost no aggregates were visible. These data are in support of our size analysis, which revealed that only a few liposomes lose their stability during nebulization. 3.5. Liposome drug release profile upon nebulization To address the release properties of the three formulations upon nebulization we determined the release of an encapsulated hydrophilic model drug system by fluorescence spectroscopy. The initial %EE of ANTS/DPX ranged between 70% and 85% for the three formulations. The highest %EE was achieved for the PEGylated formulation. The fluorescence intensity values prior to nebulization were taken as reference corresponding to 0% leakage. During nebulization some drug release was observed for all nebulizer devices (Fig. 6). We found that the release profile was especially reliant on the composition of the formulation. Less than 10% leakage was observed for the conventional DPPC/cholesterol formulation (L1); for the PEGylated formulation (L2) the leakage increased to a maximum of 25% for the vibrating-mesh nebulizer.
For all nebulizers the most pronounced leakage was observed for the cationic formulation (L3). For the vibrating-mesh nebulizer the drug release from the cationic formulation exceeded 40%. A considerable loss of drug was also observed in the residual fluid of the nebulizer reservoir, which has not passed through the mesh. This suggests that the energetics of the vibrating-mesh may directly affect the integrity of liposomes [34]. Even more pronounced was the drug release from the cationic formulation in the remainder of the air-jet and the ultrasonic nebulizer. In ultrasonic nebulizers liposomes are exposed to high frequency waves and heat, which might change lipid fluidity and membrane rigidity to favor drug release. Both air-jet and vibrating-mesh nebulization procedures expose the liposomes to shear stress and transient air– liquid interfaces known to be destabilizing for liposomes. Moreover, in the air-jet nebulizer the formulation is repeatedly exposed to the nebulization process as >98% of the droplets are re-deposited within the nebulizer after each pass through the jet nozzle [35]. This recurrent exposure to air could compromise stability and induce drug release [35,36]. Moreover, traditional air-jet nebulizers are relatively inefficient, with low amounts of active drugs reaching the airways [37]. To meet these shortcomings, more recent studies concentrate on the use of actively vibrating mesh technologies for nebulization. Following this trend, we have directly compared the performance of two different vibratingmesh nebulizers to investigate whether transport and release characteristics of the two advanced nebulization devices working on the same technical principle are comparable. As previously mentioned, the VMD values determined as a function of salt concentration were not significantly different between the two vibratingmesh inhalers (Fig. 7A). Likewise, the transport efficiencies, where 100% efficiency corresponds to a complete transport of liposomes to the spray mist, were very similar (Fig. 7B). Aggregation propensity of liposomes was apparently the same for both vibrating-mesh nebulizers (Fig. 7C). Again, the cationic formulation was less efficiently transported and less stable than the other two formulations (Fig. 7D). The conventional zwitterionic formulation composed of
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Fig. 7. Comparison between the two vibrating-mesh nebulizers. eFlowÒ rapid and M-nebÒ nebulizers were compared regarding droplet size (volume median diameter) of solutions with different NaCl concentration (A), nebulizer output efficiency of liposomes in percent of the start concentration (B), size integrity (polydispersity index) pre- and post-nebulization (C) and leakage of the ANTS/DPX model drug system in percent of the initially encapsulated amount (D). Bars are the mean ± SD, n = 3.
rigid phospholipids (DPPC) and cholesterol was most stable and had the highest drug retention rate exceeding more than 90%, regardless of which vibrating-mesh device was used. Also the polymer coated PEGylated formulation was remarkably stable in size with a tolerable increase in the polydispersity index. In comparison with the conventional formulation the drug release rate for L2 significantly increased to about 20%. For the cationic formulation the propensity for aggregation and drug release further increased. Both vibrating-mesh nebulizers caused 50% release of the encapsulated drug, which demonstrates no significant differences between these two nebulizers. To conclude, although the vibrating-mesh devices were most efficient in transporting liposomes with the highest output rate, the liposomes were slightly more prone to aggregation and loss of the encapsulated model drug. These findings are in agreement with previous reports showing that air-jet and ultrasonic nebulizer devices are largely ineffective in nebulizing liposomal formulations [15,38–41]. By contrast, the vibrating-mesh technology is reported to be more gentle in aerosol generation and in general drug retention should be improved [34,38,42]. As shown above this was not the case for all the liposomal formulations tested in this study. A fast release kinetic for part of the active drug may be desired when rapid absorption and fast onset of drug action are required. The remaining part of encapsulated drug will probably persist in the lungs for a longer period of time for prolonged action.
Many different physicochemical parameters of the liposomes and drug may interfere with the nebulization behavior of the respective formulation. According to the literature, some limitations may be overcome by changing the liposome composition to make the bilayers more rigid e.g. by the incorporation of longchain saturated lipids [43], or by addition of high concentrations of cholesterol to stabilize the formulation in the liquid ordered phase [44–46]. Another important parameter is particle size, and this means that smaller unilamellar liposomes, typically below 200 nm in size, are less susceptible to shear forces during nebulization [35]. All these recommendations have been taken into account for the design of the surface modified formulations used in this study with the goal to provide optimal preconditions for nebulization. Currently, no polymer coated liposomes are evaluated for nebulization in human clinical setting [10], and only a few systematic nebulization studies using PEGylated liposomes are reported in the literature. Kleemann et al. [41], for example, observed a high drug loss for PEG-coated liposomes during nebulization using two distinct different drug systems. Therefore they concluded that PEGylated liposomes are not suitable for inhalation therapy. By contrast Anabousi et al. [39] reported that PEGylation had no effect on the stability and integrity of transferrin conjugated liposomal formulations during nebulization using air-jet and ultrasonic nebulizers. Notably, a significantly increased stability of PEGylated liposomes in lung surfactant was observed, and this has to be
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considered as a crucial point for successful delivery [47]. These differences suggest that the performance of liposomes and the actual drug release profile will not only depend on the physicochemical characteristics of the liposomal formulation (size, lipid composition, surface charge, bilayer membrane rigidity), but also depend on the nature and the physical characteristics of the encapsulated drug [48]. For instance, negatively charged drugs will be more readily stabilized and captured in positively charged liposomes than non-charged ones. Likewise, liposomal membranes have a lower permeability to hydrophilic drugs than to amphiphilic or hydrophobic agents [2]. In any case drug release rates from liposomes are inevitably critical to successful lung delivery and therapeutic outcome [43]. This leads to the conclusion that the chemical characteristics of the liposomal formulations have to be specifically adjusted to the therapeutics to be delivered. 4. Conclusion In the current study the aerodynamic properties of three liposomal drug formulations with distinct different surface characteristics were compared to each other by applying four different nebulizer devices. None of the liposomal formulations had an impact on the aerosol droplet size distribution, which was appropriately adjusted for deep lung deposition of drugs. The most efficient transport and output rates were achieved using the vibratingmesh nebulizers. However, for these devices the highest release rate of the encapsulated model drug system was observed regardless of the surface characteristics of the liposomes. In all nebulization experiments the conventional liposomes (L1) were most stable, with their size and size distribution almost unaffected by the forces generated during nebulization. By contrast, polymer coated (L2) and positively charged liposomes (L3) were more prone to aggregation and revealed a considerable loss of the encapsulated hydrophilic agent during nebulization. This tendency was observed for all considered atomization techniques. This study reveals the vibrating-mesh nebulizers as being the most efficient devices for the nebulization of liposomes and herewith the nebulizer of choice for liposomal solutions. Moreover the conventional liposomes, containing just DPPC and cholesterol showed the best performance throughout all different techniques. Overall our data emphasize that controlled pulmonary drug delivery via inhalation necessitates the finding of a compromise between nebulizer efficiency, formulation stability and drug release profiles. Our data suggest that only with the appropriate choice of the liposome composition being specifically tailored to the physicochemical characteristics of the drugs in combination with an efficiently operating nebulizer device, it will become possible to employ the emerging new generation of surface modified liposomes for advanced inhalation therapy. Conflict of interest The authors report no conflicts of interest in this work. Acknowledgements This work was co-financed by the Medical University of Graz (PhD Program Molecular Medicine to P.P. Jain). We would like to thank NEBU-TEC (Elsenfeld, Germany) for providing us with the M-nebÒ nebulizer. The authors wish to thank Gebhard Schratter and Elisabeth Pritz for excellent technical assistance. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ejpb.2014.10.009.
References [1] H.I. Chang, M.K. Yeh, Clinical development of liposome-based drugs: formulation, characterization, and therapeutic efficacy, Int. J. Nanomed. 7 (2012) 49–60. [2] T.M. Allen, P.R. Cullis, Liposomal drug delivery systems: from concept to clinical applications, Adv. Drug Deliv. Rev. 65 (2013) 36–48. [3] T.M. Allen, P.R. Cullis, Drug delivery systems: entering the mainstream, Science 303 (2004) 1818–1822. [4] O. Meyer, D. Kirpotin, K. Hong, B. Sternberg, J.W. Park, M.C. Woodle, D. Papahadjopoulos, Cationic liposomes coated with polyethylene glycol as carriers for oligonucleotides, J. Biol. Chem. 273 (1998) 15621–15627. [5] D.D. Stuart, S.C. Semple, T.M. Allen, High efficiency entrapment of antisense oligonucleotides in liposomes, Methods Enzymol. 387 (2004) 171–188. [6] T.S. Zimmermann, A.C. Lee, A. Akinc, B. Bramlage, D. Bumcrot, M.N. Fedoruk, J. Harborth, J.A. Heyes, L.B. Jeffs, M. John, A.D. Judge, K. Lam, K. McClintock, L.V. Nechev, L.R. Palmer, T. Racie, I. Rohl, S. Seiffert, S. Shanmugam, V. Sood, J. Soutschek, I. Toudjarska, A.J. Wheat, E. Yaworski, W. Zedalis, V. Koteliansky, M. Manoharan, H.P. Vornlocher, I. Maclachlan, RNAi-mediated gene silencing in non-human primates, Nature 441 (2006) 111–114. [7] C. Jaafar-Maalej, A. Elaissari, H. Fessi, Lipid-based carriers: manufacturing and applications for pulmonary route, Expert. Opin. Drug Deliv. 9 (2012) 1111– 1127. [8] S.P. Vyas, M.E. Kannan, S. Jain, V. Mishra, P. Singh, Design of liposomal aerosols for improved delivery of rifampicin to alveolar macrophages, Int. J. Pharm. 269 (2004) 37–49. [9] Y.Y. Huang, C.H. Wang, Pulmonary delivery of insulin by liposomal carriers, J. Control. Release 113 (2006) 9–14. [10] D. Cipolla, I. Gonda, H.K. Chan, Liposomal formulations for inhalation, Ther. Deliv. 4 (2013) 1047–1072. [11] M.M. Gaspar, U. Bakowsky, C. Ehrhardt, Inhaled liposomes-current strategies and future challenges, J. Biomed. Nanotechnol. 4 (2008) 245–257. [12] C. O’Callaghan, P.W. Barry, The science of nebulised drug delivery, Thorax 52 (Suppl. 2) (1997) S31–S44. [13] C.F. Lange, W.H. Finlay, Liquid atomizing: nebulizing and other methods of producing aerosols, J. Aerosol Med.-Deposition Clear. Effects Lung 19 (2006) 28–35. [14] L.Y. Yeo, J.R. Friend, M.P. McIntosh, E.N. Meeusen, D.A. Morton, Ultrasonic nebulization platforms for pulmonary drug delivery, Expert. Opin. Drug Deliv. 7 (2010) 663–679. [15] A.M. Elhissi, W. Ahmed, K.M. Taylor, Laser diffraction and electron microscopy studies on inhalable liposomes generated from particulate-based proliposomes within a medical nebulizer, J. Nanosci. Nanotechnol. 12 (2012) 6693–6699. [16] J.C. Waldrep, R. Dhand, Advanced nebulizer designs employing vibrating mesh/aperture plate technologies for aerosol generation, Curr. Drug Deliv. 5 (2008) 114–119. [17] R. Dhand, Nebulizers that use a vibrating mesh or plate with multiple apertures to generate aerosol, Respir. Care 47 (2002) 1406–1416. [18] T. Ghazanfari, A.M. Elhissi, Z. Ding, K.M. Taylor, The influence of fluid physicochemical properties on vibrating-mesh nebulization, Int. J. Pharm. 339 (2007) 103–111. [19] B. Stark, P. Debbage, F. Andreae, W. Mosgoeller, R. Prassl, Association of vasoactive intestinal peptide with polymer-grafted liposomes: structural aspects for pulmonary delivery, Biochim. et Biophys. Acta – Biomembranes 1768 (2007) 705–714. [20] P.P. Jain, R. Leber, C. Nagaraj, G. Leitinger, B. Lehofer, H. Olschewski, A. Olschewski, R. Prassl, L.M. Marsh, Liposomal nanoparticles encapsulating iloprost exhibit enhanced vasodilation in pulmonary arteries, Int. J. Nanomed. 9 (2014) 3249–3261. [21] H. Ellens, J. Bentz, F.C. Szoka, PH-induced destabilization of phosphatidylethanolamine-containing liposomes: role of bilayer contact, Biochemistry 23 (1984) 1532–1538. [22] A.S. Ladokhin, P.W. Holloway, Fluorescence of membrane-bound tryptophan octyl ester: a model for studying intrinsic fluorescence of protein-membrane interactions, Biophys. J. 69 (1995) 506–517. [23] O. Tirosh, Y. Barenholz, J. Katzhendler, A. Priev, Hydration of polyethylene glycol-grafted liposomes, Biophys. J. 74 (1998) 1371–1379. [24] M.L. Immordino, F. Dosio, L. Cattel, Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential, Int. J. Nanomed. 1 (2006) 297–315. [25] T.M. Allen, Ligand-targeted therapeutics in anticancer therapy, Nat. Rev. Cancer 2 (2002) 750–763. [26] J.M. Ramsey, A. Hibbitts, J. Barlow, C. Kelly, N. Sivadas, S.A. Cryan, ‘Smart’ nonviral delivery systems for targeted delivery of RNAi to the lungs, Ther. Deliv. 4 (2013) 59–76. [27] T.T. Mills, J. Huang, G.W. Feigenson, J.F. Nagle, Effects of cholesterol and unsaturated DOPC lipid on chain packing of saturated gel-phase DPPC bilayers, Gen. Physiol. Biophys. 28 (2009) 126–139. [28] J.S. Patton, P.R. Byron, Inhaling medicines: delivering drugs to the body through the lungs, Nat. Rev. Drug. Discov. 6 (2007) 67–74. [29] M. Najlah, A. Vali, M. Taylor, B.T. Arafat, W. Ahmed, D.A. Phoenix, K.M. Taylor, A. Elhissi, A study of the effects of sodium halides on the performance of air-jet and vibrating-mesh nebulizers, Int. J. Pharm. 456 (2013) 520–527. [30] M. Beck-Broichsitter, N. Oesterheld, M.C. Knuedeler, W. Seeger, T. Schmehl, On the correlation of output rate and aerodynamic characteristics in vibratingmesh-based aqueous aerosol delivery, Int. J. Pharm. 461 (2014) 34–37.
B. Lehofer et al. / European Journal of Pharmaceutics and Biopharmaceutics 88 (2014) 1076–1085 [31] Y. Barenholz, Liposome application: problems and prospects, Curr. Opin. Colloid Interface Sci. 6 (2001) 66–77. [32] T.G. O’Riordan, Formulations and nebulizer performance, Respir. Care 47 (2002) 1305–1312. [33] M. Najlah, I. Parveen, M.A. Alhnan, W. Ahmed, A. Faheem, D.A. Phoenix, K.M. Taylor, A. Elhissi, The effects of suspension particle size on the performance of air-jet, ultrasonic and vibrating-mesh nebulisers, Int. J. Pharm. 461 (2014) 234–241. [34] M.M. Gaspar, O. Gobbo, C. Ehrhardt, Generation of liposome aerosols with the Aeroneb Pro and the AeroProbe nebulizers, J. Liposome Res. 20 (2010) 55–61. [35] R.W. Niven, H. Schreier, Nebulization of liposomes. I. Effects of lipid composition, Pharm. Res. 7 (1990) 1127–1133. [36] R.W. Niven, M. Speer, H. Schreier, Nebulization of liposomes. II. The effects of size and modeling of solute release profiles, Pharm. Res. 8 (1991) 217–221. [37] D. Cipolla, H.K. Chan, J. Schuster, D. Farina, Personalizing aerosol medicine: development of delivery systems tailored to the individual, Ther. Deliv. 1 (2010) 667–682. [38] A.M. Elhissi, M. Faizi, W.F. Naji, H.S. Gill, K.M. Taylor, Physical stability and aerosol properties of liposomes delivered using an air-jet nebulizer and a novel micropump device with large mesh apertures, Int. J. Pharm. 334 (2007) 62–70. [39] S. Anabousi, E. Kleemann, U. Bakowsky, T. Kissel, T. Schmehl, T. Gessler, W. Seeger, C.M. Lehr, C. Ehrhardt, Effect of PEGylation on the stability of liposomes during nebulisation and in lung surfactant, J. Nanosci. Nanotechnol. 6 (2006) 3010–3016.
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[40] M. Zaru, S. Mourtas, P. Klepetsanis, A.M. Fadda, S.G. Antimisiaris, Liposomes for drug delivery to the lungs by nebulization, Eur. J. Pharm. Biopharm. (2007) 655–666. [41] E. Kleemann, T. Schmehl, T. Gessler, U. Bakowsky, T. Kissel, W. Seeger, Iloprostcontaining liposomes for aerosol application in pulmonary arterial hypertension: formulation aspects and stability, Pharm. Res. 24 (2007) 277– 287. [42] V. Wagner, A. Dullaart, A.K. Bock, A. Zweck, The emerging nanomedicine landscape, Nat. Biotechnol. 24 (2006) 1211–1217. [43] M. Beck-Broichsitter, M. Rieger, R. Reul, T. Gessler, W. Seeger, T. Schmehl, Correlation of drug release with pulmonary drug absorption profiles for nebulizable liposomal formulations, Eur. J. Pharm. Biopharm. 84 (2013) 106– 114. [44] D.D. Lasic, Doxorubicin in sterically stabilized liposomes, Nature 380 (1996) 561–562. [45] G.W. Feigenson, Phase behavior of lipid mixtures, Nat. Chem. Biol. 2 (2006) 560–563. [46] P.J. Quinn, C. Wolf, Thermotropic and structural evaluation of the interaction of natural sphingomyelins with cholesterol, Biochim. Biophys. Acta 1788 (2009) 1877–1889. [47] R. Abu-Dahab, U.F. Schäfer, C.M. Lehr, Lectin-functionalized liposomes for pulmonary drug delivery: effect of nebulization on stability and bioadhesion, Eur. J. Pharm. Sci. 14 (2001) 37–46. [48] A.S. Ulrich, Biophysical aspects of using liposomes as delivery vehicles, Biosci. Rep. 22 (2002) 129–150.
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European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb
Research paper
Impact of atomization technique on the stability and transport efficiency of nebulized liposomes harboring different surface characteristics Bernhard Lehofer a,b,1, Florian Bloder a,1, Pritesh P. Jain a, Leigh M. Marsh a, Gerd Leitinger c,d, Horst Olschewski a,e, Regina Leber a,f, Andrea Olschewski a, Ruth Prassl a,b,⇑ a
Ludwig Boltzmann Institute for Lung Vascular Research, Graz, Austria Institute of Biophysics, Medical University of Graz, BioTechMed-Graz, Graz, Austria Institute of Cell Biology, Histology and Embryology, Research Unit Electron Microscopic Techniques, Medical University of Graz, Graz, Austria d Center for Medical Research, Medical University of Graz, Graz, Austria e Division of Pulmonology, Medical University of Graz, Graz, Austria f Institute of Molecular Biosciences, University of Graz, Graz, Austria b c
a r t i c l e
i n f o
Article history: Received 16 June 2014 Accepted in revised form 15 October 2014 Available online 23 October 2014 Keywords: Liposome Nebulization Aerosol Pulmonary drug delivery Inhalation technique Vibrating-mesh nebulizer
a b s t r a c t The objective of this study was to evaluate the impact of nebulization on liposomes with specific surface characteristics by applying three commercially available inhaler systems (air-jet, ultrasonic and vibrating-mesh). Conventional liposome formulations composed of phosphatidylcholine and cholesterol were compared to sterically stabilized PEGylated liposomes and cationic polymer coated liposomes. Liposomes of similar size (between 140 and 165 nm in diameter with polydispersity indices 0.1 a more detailed size distribution analysis of the PCS data based on intensity, volume and number calculations was performed. The zeta potential was determined by Laser Doppler Micro-electrophoresis using a Zetasizer Nano ZS (Malvern Instruments, Herrenberg, Germany). For the zeta potential measurements the liposomes were diluted with 2 mM HEPES containing 2 mM CsCl to a final concentration of 0.3 mg/ml of lipids. All size and zeta potential measurements were taken in triplicate at room temperature with each measurement consisting of 10 runs. 2.4. Negative staining transmission electron microscopy (TEM) The three liposomal formulations each before and after nebulization were diluted to a concentration of 0.6 mg/ml and pipetted on carbon coated copper grids. After 1 min incubation time, the excess sample was blotted off and replaced by 1% aqueous uranyl acetate as staining reagent. This treatment was repeated twice each with 30 s incubation times. Samples were visualized using an FEI TECNAI 20 Transmission Electron Microscope at 120 kV as acceleration voltage with the help of a GATAN Ultrascan 1000 CCD camera at 2 k 2 k resolution.
(Fig. S2). The calculations are based on fluorescence intensity measurements; F0 is the initial quenched fluorescence intensity of ANTS before nebulization and Fmax is the fluorescence intensity corresponding to 100% leakage, which was measured after lysis of the liposomes by the addition of 10 ll of 10% Triton X-100. Ft represents the fluorescence intensity of ANTS measured after nebulization. The correction term (F max =F max ) accounts for changes in sample concentration during nebulization with F max being the fluorescence intensity after lysis of nebulized liposomes with Triton X-100. Encapsulation efficiency (EE%) was calculated as follows
EE% ¼
F max F 0 100 F max
ð1Þ
and liposome leakage (%) corresponding to drug release as
leakage ¼
Ft
F max F max
F0
F max F 0
100
ð2Þ
2.6. Stability of liposomes in artificial lung surfactant ANTS/DPX loaded liposomes were diluted to a lipid concentration of 1 mg/ml with HEPES buffer and 5 ll thereof was incubated with 200 ll of diluted AlveofactÒ (0.5 mg/ml). The measurements were taken in a 10 2 mm quartz cuvette at a temperature of 37 °C. Leakage was recorded for 60 min at the same instrumental settings as described in Section 2.5. At the end of the experiment 10 ll of 10 % Triton X-100 was added and the increase in intensity was recorded for other 60 s. The background autofluorescence of AlveofactÒ was subtracted from the overall fluorescence intensity of the liposomes in AlveofactÒ. The measured fluorescence intensity is expressed in kilocounts per second (kcps). 2.7. Nebulization of liposomes All liposomal formulations were diluted with HEPES buffer to a final lipid concentration of 1 mg/ml. Each nebulizer reservoir was filled with 3 ml of liposomal solution and 2 ml thereof was nebulized, while the time for nebulization was recorded. The weight of the nebulizer was determined before and after nebulization to calculate the total aerosol output rate. Generated aerosol was
2.5. Determination of encapsulation efficiency and release by fluorescence spectroscopy The fluorophore/quencher (ANTS/DPX) system was used as model drug system to assess the encapsulation efficiency and leakage tendency of liposomal formulations [21,22]. The fluorescence of ANTS was recorded on a SPEX FLUOROMAX-3 fluorescence spectrophotometer (HORIBA Jobin–Yvon, Longjumeau, France). Measurements were taken using 10 10 mm quartz cuvettes (Hellma Analytics, Germany), an excitation wavelength of 360 nm and an emission wavelength of 530 nm. The slit width was 5 nm for both excitation and emission monochromators. Samples were diluted with HEPES buffer and incubated at 37 °C without stirring during the measurements. Details on the mode of operation of the ANTS/DPX system are provided in Supporting Information
Fig. 1. Volume median diameter (VMD) of aerosol droplets depends on the sodium chloride concentration of the nebulized solution. Three nebulizer devices are compared, each one based on a different principle. The aerosol solutions are as follows: 10 mM HEPES (pH 7.4) without NaCl (h), with 70 mM NaCl ( ) and 140 mM NaCl ( ). Bars are the mean ± SD, n = 3. The different levels of significance are indicated as ⁄p < 0.05, ⁄⁄p < 0.016.
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to the droplet size at which 50% of the volume is in droplets smaller than the VMD. 2.9. Statistical analysis All measurements were taken in triplicate and the values are presented as the mean ± S.D. A one-way ANOVA statistical test with a Bonferroni post-t-test correction was made. Probability values of p < 0.05 and p0 < 0.016 for Bonferroni correction (comparison among three groups) were considered significant. Statistical analysis was done with Microsoft Excel 2010. 3. Results and discussion Fig. 2. Influence of liposomes on the aerosol droplet size distribution. Liposomal solutions in HEPES (pH 7.4) containing 140 mM NaCl nebulized with the vibratingmesh nebulizer (eFlowÒ rapid). HEPES buffer (continuous line) L1, L2 and L3 (dotted lines).
collected at 4 °C with the disposable exhaled condensate collecting system TURBO-DECCS purchased from Ital-chill, Parma, Italy, for further analysis. Since the nebulizers have different airflow rates, a compressor (DeVO/MC 29, DeVILBISS GmbH Medical Products, Germany) to enhance airflow (for low flow rate in case of the OptinebÒ-ir) or an additional collecting vessel (for high flow rate in case of MicroDrop MasterJetÒ) was applied following the condensation chamber. As a reference, a 10 mM HEPES buffer (pH 7.4) containing different salt concentrations (0, 70 and 140 mM NaCl) was nebulized. 2.8. Analysis of the aerodynamic diameter of the aerosol The aerosol droplet size distribution of nebulized samples was measured by laser diffraction technique using a Mastersizer 2000 (Malvern Instruments, Herrenberg, Germany). Measurements were taken at a laser obscuration between 4% and 6% and taken in triplicates. The lipid concentration of all liposomal formulations was set to 1 mg/ml by dilution with HEPES buffer before nebulization. The Malvern Mastersizer 2000 software Version 5.60 was used to characterize the volume median diameter (VMD) corresponding
3.1. Physicochemical properties of liposomes used for nebulization To evaluate the applicability of each nebulizer device for atomization of liposomes, three types of liposomal formulations of similar particle sizes but with different chemical composition and surface properties were investigated. Namely, a conventional pharmaceutical formulation composed of zwitterionic phosphatidylcholines (L1) was compared to a polymer-coated (PEGylated) sterically stabilized formulation (L2), representative for the second generation of liposomal drug carriers. Such PEGylated liposomes are stabilized by steric repulsion caused by the PEG chains in addition to a hydration shell of a non-diffusible water layer around the liposomes [23,24]. Hence, PEGylated liposomes have a longer life time in vivo since they are not readily recognized by the immune system [25]. The third formulation (L3) was additionally enriched with a cationic lipid to exert a net positive surface charge to the liposomes. Cationic liposomes are becoming more and more interesting for pharmaceutical applications, especially as delivery systems for oligonucleotides, RNA or DNA [26]. The main lipid component of the three formulations was DPPC, a phosphatidylcholine with saturated acyl chains. Additionally, the liposomes were enriched with 30 mol% of cholesterol. With this lipid (DPPC/cholesterol) composition the bilayer membrane is in an ordered liquid phase [27], being more rigid and less permeable for water soluble, non-bilayer interacting molecules. The average particle sizes ranged from 140 to 165 nm. For L2 and L3 the size
Fig. 3. Aerosol output efficiencies of nebulized liposomes for different nebulizer devices. (A) Amount of liposomes (mg total lipid concentration) transported per ml of aerosol and (B) transport efficiency for liposomes (L1–L3) per minute of nebulization time (mg lipid/min). Equal volumes of liposomal solutions with 1 mg/ml phospholipid concentration were aerosolized with three different nebulizers. Bars are the mean ± SD, n = 3. The different levels of significance are indicated as ⁄p < 0.05, ⁄⁄p < 0.016.
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Fig. 4. Impact of nebulizers on the integrity of liposomal formulations. (A) Size of liposomes determined before (pre) and after (post) nebulization for three nebulizer devices in aerosol and remainder. (B) Polydispersity index (PDI) of nebulized liposomes measured by PCS. Bars are the mean ± SD, n = 3.
values were very similar, while L1, the conventional formulation without PEG-coating, was slightly, but significantly (p0 < 0.016) larger. The PDI-values were below 0.08 for all three formulations indicating highly homogeneous populations. The surface zeta potential, which is related to the surface charge of the particles and the ionic strength of the medium, was markedly positive for the cationic formulation L3 and slightly negative for L1 and L2. The chemical compositions and physicochemical properties of the three formulations are listed in Table 1. For all formulations the encapsulation efficiency of ANTS/DPX was high (>70 %EE). These values were taken as 100%EE for the subsequent nebulization studies. To address the stability of liposomes under more physiological conditions the liposomes were incubated with artificial lung surfactant (AlveofactÒ) at physiological temperature.
We found that none of the three formulations showed any leakage of the encapsulated fluorophore (Fig. S3) during the observation period of 60 min. 3.2. Aerosol droplet size Aerosols for deep lung penetration require the generation of droplets with an aerodynamic diameter of 1–3 lm [9,28]. Since it is known that the addition of electrolytes can influence aerosol droplet size [29,30], we aimed to assess the impact of sodium chloride (70 and 140 mM NaCl) on the VMD of droplets generated by the different nebulizer devices. Fig. 1 shows that the aerosol droplet size of nebulized 10 mM HEPES buffer (pH 7.4) decreased with increasing concentrations of sodium chloride. While for the
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3.3. Aerosol output and nebulizer efficiency
Fig. 5. Negative staining transmission electron micrographs of the three liposomal formulations L1, L2 and L3 before nebulization (left panel) and after nebulization (right panel) with the eFlowÒ rapid nebulizer. The bars represent lengths of 100 nm.
vibrating-mesh and the ultrasonic device the addition of 70 mM NaCl was sufficient to generate adequate droplet sizes below 3 microns, higher concentrations of salt were required for the airjet device. By increasing the concentration of NaCl to 140 mM, the droplet sizes for the vibrating-mesh and the air-jet nebulizer were significantly (p0 < 0.016) reduced. These data indicate that optimal droplet sizes for deep lung deposition of droplets can be reliably achieved by addition of a moderate, close to physiological, concentration of sodium chloride. Details on the size analysis of aerosol droplets containing different sodium chloride concentrations are provided in Supporting Information Fig. S4. Based on these initial results revealing the effects of electrolytes on droplet size, all further experiments with liposomes were performed in HEPES buffer containing 140 mM NaCl. The liposomes were prepared and nebulized at room temperature at a concentration of 1 mg/ml phospholipid. We found that at these experimental conditions the liposomes had little influence on the aerodynamic diameter and the aerosol droplet size distribution regardless of the nebulizer type. Likewise, very similar volume distribution profiles were obtained for all three formulations showing similar characteristics as those obtained with HEPES buffer. Fig. 2 shows the data obtained for the vibrating-mesh nebulizer (eFlowÒ rapid) as a representative example. About 90% of the aerosolized volume contained droplets less than 5.5 lm in size; approximately 75% contained droplets below 3 lm. No marked differences were observed for the three liposomal formulations.
Effective transport of liposomes within aerosol droplets is a prerequisite for therapeutic inhalative applications of liposomal drug formulations. However, the actual amount of aerosolized liposomes transferred with the spray mist might depend on the employed nebulizer device and the characteristics of the formulation itself [10]. To address this issue the aerosol output for nebulized liposomes, corresponding to the transport efficiency of the nebulizer, was calculated based on the lipid concentration determined in the collected aerosol and correlated with the initial lipid concentration before nebulization (1 mg/ml phospholipid). Fig. 3A reveals that the transport efficiency was very much dependent on the working principle of the inhaler device. The lowest atomization efficiency was observed with the ultrasonic device, ranging between 21% and 39% of liposomes transported in the spray mist. No significant differences could be detected when comparing the three formulations. In contrast to the ultrasonic device, both the air-jet and the vibrating-mesh nebulizer generated aerosols containing the same amount of liposomes as compared to the non-nebulized solution. Uncharged PEGylated liposomes were as efficiently transported in the spray mist as conventional ones (almost 100% are transferred). A lower output of about 60–70% lipid was achieved for the positively charged formulation L3. These data suggest that surface coating of liposomes with the polymeric stabilizer PEG2000 had no impact on the aerosol output, at least at the PEG concentrations used in this study, which are typical for sterically stabilized pharmaceutical formulations [31]. To determine the output rate of the different nebulizer devices the absolute amount of liposomes in the aerosol was normalized to the time required to nebulize the corresponding solution (Fig. 3B). The ultrasonic nebulizer was the least efficient transferring less than 10% of the liposomes per minute into the aerosol independent of the formulation used. These data suggest that large parts of liposomes are excluded from the emitted aerosol droplets produced by the ultrasonic nebulizers. This agrees with the findings of O’Riordan [32] indicating that the suitability of ultrasonic nebulizers for liposomal formulations is limited. The vibratingmesh nebulizer was the most efficient device being three times more effective than the air-jet nebulizer. Approximately 10 min was required for nebulizing 2 ml of liposomal formulations with the air-jet nebulizer, whereas the actively vibrating-mesh nebulizer required only 3 min for the same volume. The output rate was in general lower for the positively charged liposomes (L3) compared to L1 and L2. One possible explanation could be that for the cationic formulation the concentration of ions that is present at the air–liquid interface is lower [33] and that this effect influences the transport efficiency for cationic liposomes. 3.4. Liposome stability upon nebulization Next we have determined the stability of the three liposomal formulations upon nebulization (Fig. 4A). None of the formulations exhibited any substantial changes in the average hydrodynamic diameter, as would be expected from bilayer disruption, aggregation or fusion of liposomes during nebulization. Likewise, the mean particle size of liposomes remaining in the reservoir of the nebulizer devices remained essentially the same. The same is true for the PDI values, which remained below 0.1 during nebulization for L1 and L2 (Fig. 4B). A strong increase in PDI was observed for the positively charged formulation (L3) for all three nebulizer devices and being most pronounced for the vibrating-mesh nebulizer. A direct comparison of the PDI values revealed that for none of the liposomal formulations the PDI increased above a mean value of 0.22; such PDI values still indicate a narrow size distribution. By performing a detailed size distribution analysis it
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Fig. 6. Drug release upon nebulization. The leakage of the model drug system ANTS/DPX in relation to the initially encapsulated amount was determined fluorimetrically for the three liposomal formulations L1 (h), L2 ( ) and L3 ( ). The values are expressed as %leakage. Bars are the mean ± SD, n = 3.
becomes obvious that in case of the vibrating-mesh nebulizer more than 90% of the liposomes kept their original size during nebulization and only a very small fraction of liposomes aggregate to larger particles with diameters between 1 and 2 lm. For clarity, some original data obtained for the positively charged formulation nebulized with the eFlowÒ rapid nebulizer are shown in Fig. S5. Overall, these data stress that especially for bi- or multimodal particle size distributions, e.g. small particles in presence of some large aggregates, the mean values of hydrodynamic diameters and the PDI values typically provided in publications, have to be challenged. To obtain more detailed information on the morphology of the liposomes and on the structural stability upon nebulization we have performed negative staining TEM. The TEM images displayed in Fig. 5 show predominantly single spherical particles for all three formulations before nebulization. Very similar images were observed for the liposomes after nebulization with the eFlowÒ rapid nebulizer, where almost no aggregates were visible. These data are in support of our size analysis, which revealed that only a few liposomes lose their stability during nebulization. 3.5. Liposome drug release profile upon nebulization To address the release properties of the three formulations upon nebulization we determined the release of an encapsulated hydrophilic model drug system by fluorescence spectroscopy. The initial %EE of ANTS/DPX ranged between 70% and 85% for the three formulations. The highest %EE was achieved for the PEGylated formulation. The fluorescence intensity values prior to nebulization were taken as reference corresponding to 0% leakage. During nebulization some drug release was observed for all nebulizer devices (Fig. 6). We found that the release profile was especially reliant on the composition of the formulation. Less than 10% leakage was observed for the conventional DPPC/cholesterol formulation (L1); for the PEGylated formulation (L2) the leakage increased to a maximum of 25% for the vibrating-mesh nebulizer.
For all nebulizers the most pronounced leakage was observed for the cationic formulation (L3). For the vibrating-mesh nebulizer the drug release from the cationic formulation exceeded 40%. A considerable loss of drug was also observed in the residual fluid of the nebulizer reservoir, which has not passed through the mesh. This suggests that the energetics of the vibrating-mesh may directly affect the integrity of liposomes [34]. Even more pronounced was the drug release from the cationic formulation in the remainder of the air-jet and the ultrasonic nebulizer. In ultrasonic nebulizers liposomes are exposed to high frequency waves and heat, which might change lipid fluidity and membrane rigidity to favor drug release. Both air-jet and vibrating-mesh nebulization procedures expose the liposomes to shear stress and transient air– liquid interfaces known to be destabilizing for liposomes. Moreover, in the air-jet nebulizer the formulation is repeatedly exposed to the nebulization process as >98% of the droplets are re-deposited within the nebulizer after each pass through the jet nozzle [35]. This recurrent exposure to air could compromise stability and induce drug release [35,36]. Moreover, traditional air-jet nebulizers are relatively inefficient, with low amounts of active drugs reaching the airways [37]. To meet these shortcomings, more recent studies concentrate on the use of actively vibrating mesh technologies for nebulization. Following this trend, we have directly compared the performance of two different vibratingmesh nebulizers to investigate whether transport and release characteristics of the two advanced nebulization devices working on the same technical principle are comparable. As previously mentioned, the VMD values determined as a function of salt concentration were not significantly different between the two vibratingmesh inhalers (Fig. 7A). Likewise, the transport efficiencies, where 100% efficiency corresponds to a complete transport of liposomes to the spray mist, were very similar (Fig. 7B). Aggregation propensity of liposomes was apparently the same for both vibrating-mesh nebulizers (Fig. 7C). Again, the cationic formulation was less efficiently transported and less stable than the other two formulations (Fig. 7D). The conventional zwitterionic formulation composed of
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Fig. 7. Comparison between the two vibrating-mesh nebulizers. eFlowÒ rapid and M-nebÒ nebulizers were compared regarding droplet size (volume median diameter) of solutions with different NaCl concentration (A), nebulizer output efficiency of liposomes in percent of the start concentration (B), size integrity (polydispersity index) pre- and post-nebulization (C) and leakage of the ANTS/DPX model drug system in percent of the initially encapsulated amount (D). Bars are the mean ± SD, n = 3.
rigid phospholipids (DPPC) and cholesterol was most stable and had the highest drug retention rate exceeding more than 90%, regardless of which vibrating-mesh device was used. Also the polymer coated PEGylated formulation was remarkably stable in size with a tolerable increase in the polydispersity index. In comparison with the conventional formulation the drug release rate for L2 significantly increased to about 20%. For the cationic formulation the propensity for aggregation and drug release further increased. Both vibrating-mesh nebulizers caused 50% release of the encapsulated drug, which demonstrates no significant differences between these two nebulizers. To conclude, although the vibrating-mesh devices were most efficient in transporting liposomes with the highest output rate, the liposomes were slightly more prone to aggregation and loss of the encapsulated model drug. These findings are in agreement with previous reports showing that air-jet and ultrasonic nebulizer devices are largely ineffective in nebulizing liposomal formulations [15,38–41]. By contrast, the vibrating-mesh technology is reported to be more gentle in aerosol generation and in general drug retention should be improved [34,38,42]. As shown above this was not the case for all the liposomal formulations tested in this study. A fast release kinetic for part of the active drug may be desired when rapid absorption and fast onset of drug action are required. The remaining part of encapsulated drug will probably persist in the lungs for a longer period of time for prolonged action.
Many different physicochemical parameters of the liposomes and drug may interfere with the nebulization behavior of the respective formulation. According to the literature, some limitations may be overcome by changing the liposome composition to make the bilayers more rigid e.g. by the incorporation of longchain saturated lipids [43], or by addition of high concentrations of cholesterol to stabilize the formulation in the liquid ordered phase [44–46]. Another important parameter is particle size, and this means that smaller unilamellar liposomes, typically below 200 nm in size, are less susceptible to shear forces during nebulization [35]. All these recommendations have been taken into account for the design of the surface modified formulations used in this study with the goal to provide optimal preconditions for nebulization. Currently, no polymer coated liposomes are evaluated for nebulization in human clinical setting [10], and only a few systematic nebulization studies using PEGylated liposomes are reported in the literature. Kleemann et al. [41], for example, observed a high drug loss for PEG-coated liposomes during nebulization using two distinct different drug systems. Therefore they concluded that PEGylated liposomes are not suitable for inhalation therapy. By contrast Anabousi et al. [39] reported that PEGylation had no effect on the stability and integrity of transferrin conjugated liposomal formulations during nebulization using air-jet and ultrasonic nebulizers. Notably, a significantly increased stability of PEGylated liposomes in lung surfactant was observed, and this has to be
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considered as a crucial point for successful delivery [47]. These differences suggest that the performance of liposomes and the actual drug release profile will not only depend on the physicochemical characteristics of the liposomal formulation (size, lipid composition, surface charge, bilayer membrane rigidity), but also depend on the nature and the physical characteristics of the encapsulated drug [48]. For instance, negatively charged drugs will be more readily stabilized and captured in positively charged liposomes than non-charged ones. Likewise, liposomal membranes have a lower permeability to hydrophilic drugs than to amphiphilic or hydrophobic agents [2]. In any case drug release rates from liposomes are inevitably critical to successful lung delivery and therapeutic outcome [43]. This leads to the conclusion that the chemical characteristics of the liposomal formulations have to be specifically adjusted to the therapeutics to be delivered. 4. Conclusion In the current study the aerodynamic properties of three liposomal drug formulations with distinct different surface characteristics were compared to each other by applying four different nebulizer devices. None of the liposomal formulations had an impact on the aerosol droplet size distribution, which was appropriately adjusted for deep lung deposition of drugs. The most efficient transport and output rates were achieved using the vibratingmesh nebulizers. However, for these devices the highest release rate of the encapsulated model drug system was observed regardless of the surface characteristics of the liposomes. In all nebulization experiments the conventional liposomes (L1) were most stable, with their size and size distribution almost unaffected by the forces generated during nebulization. By contrast, polymer coated (L2) and positively charged liposomes (L3) were more prone to aggregation and revealed a considerable loss of the encapsulated hydrophilic agent during nebulization. This tendency was observed for all considered atomization techniques. This study reveals the vibrating-mesh nebulizers as being the most efficient devices for the nebulization of liposomes and herewith the nebulizer of choice for liposomal solutions. Moreover the conventional liposomes, containing just DPPC and cholesterol showed the best performance throughout all different techniques. Overall our data emphasize that controlled pulmonary drug delivery via inhalation necessitates the finding of a compromise between nebulizer efficiency, formulation stability and drug release profiles. Our data suggest that only with the appropriate choice of the liposome composition being specifically tailored to the physicochemical characteristics of the drugs in combination with an efficiently operating nebulizer device, it will become possible to employ the emerging new generation of surface modified liposomes for advanced inhalation therapy. Conflict of interest The authors report no conflicts of interest in this work. Acknowledgements This work was co-financed by the Medical University of Graz (PhD Program Molecular Medicine to P.P. Jain). We would like to thank NEBU-TEC (Elsenfeld, Germany) for providing us with the M-nebÒ nebulizer. The authors wish to thank Gebhard Schratter and Elisabeth Pritz for excellent technical assistance. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ejpb.2014.10.009.
References [1] H.I. Chang, M.K. Yeh, Clinical development of liposome-based drugs: formulation, characterization, and therapeutic efficacy, Int. J. Nanomed. 7 (2012) 49–60. [2] T.M. Allen, P.R. Cullis, Liposomal drug delivery systems: from concept to clinical applications, Adv. Drug Deliv. Rev. 65 (2013) 36–48. [3] T.M. Allen, P.R. Cullis, Drug delivery systems: entering the mainstream, Science 303 (2004) 1818–1822. [4] O. Meyer, D. Kirpotin, K. Hong, B. Sternberg, J.W. Park, M.C. Woodle, D. Papahadjopoulos, Cationic liposomes coated with polyethylene glycol as carriers for oligonucleotides, J. Biol. Chem. 273 (1998) 15621–15627. [5] D.D. Stuart, S.C. Semple, T.M. Allen, High efficiency entrapment of antisense oligonucleotides in liposomes, Methods Enzymol. 387 (2004) 171–188. [6] T.S. Zimmermann, A.C. Lee, A. Akinc, B. Bramlage, D. Bumcrot, M.N. Fedoruk, J. Harborth, J.A. Heyes, L.B. Jeffs, M. John, A.D. Judge, K. Lam, K. McClintock, L.V. Nechev, L.R. Palmer, T. Racie, I. Rohl, S. Seiffert, S. Shanmugam, V. Sood, J. Soutschek, I. Toudjarska, A.J. Wheat, E. Yaworski, W. Zedalis, V. Koteliansky, M. Manoharan, H.P. Vornlocher, I. Maclachlan, RNAi-mediated gene silencing in non-human primates, Nature 441 (2006) 111–114. [7] C. Jaafar-Maalej, A. Elaissari, H. Fessi, Lipid-based carriers: manufacturing and applications for pulmonary route, Expert. Opin. Drug Deliv. 9 (2012) 1111– 1127. [8] S.P. Vyas, M.E. Kannan, S. Jain, V. Mishra, P. Singh, Design of liposomal aerosols for improved delivery of rifampicin to alveolar macrophages, Int. J. Pharm. 269 (2004) 37–49. [9] Y.Y. Huang, C.H. Wang, Pulmonary delivery of insulin by liposomal carriers, J. Control. Release 113 (2006) 9–14. [10] D. Cipolla, I. Gonda, H.K. Chan, Liposomal formulations for inhalation, Ther. Deliv. 4 (2013) 1047–1072. [11] M.M. Gaspar, U. Bakowsky, C. Ehrhardt, Inhaled liposomes-current strategies and future challenges, J. Biomed. Nanotechnol. 4 (2008) 245–257. [12] C. O’Callaghan, P.W. Barry, The science of nebulised drug delivery, Thorax 52 (Suppl. 2) (1997) S31–S44. [13] C.F. Lange, W.H. Finlay, Liquid atomizing: nebulizing and other methods of producing aerosols, J. Aerosol Med.-Deposition Clear. Effects Lung 19 (2006) 28–35. [14] L.Y. Yeo, J.R. Friend, M.P. McIntosh, E.N. Meeusen, D.A. Morton, Ultrasonic nebulization platforms for pulmonary drug delivery, Expert. Opin. Drug Deliv. 7 (2010) 663–679. [15] A.M. Elhissi, W. Ahmed, K.M. Taylor, Laser diffraction and electron microscopy studies on inhalable liposomes generated from particulate-based proliposomes within a medical nebulizer, J. Nanosci. Nanotechnol. 12 (2012) 6693–6699. [16] J.C. Waldrep, R. Dhand, Advanced nebulizer designs employing vibrating mesh/aperture plate technologies for aerosol generation, Curr. Drug Deliv. 5 (2008) 114–119. [17] R. Dhand, Nebulizers that use a vibrating mesh or plate with multiple apertures to generate aerosol, Respir. Care 47 (2002) 1406–1416. [18] T. Ghazanfari, A.M. Elhissi, Z. Ding, K.M. Taylor, The influence of fluid physicochemical properties on vibrating-mesh nebulization, Int. J. Pharm. 339 (2007) 103–111. [19] B. Stark, P. Debbage, F. Andreae, W. Mosgoeller, R. Prassl, Association of vasoactive intestinal peptide with polymer-grafted liposomes: structural aspects for pulmonary delivery, Biochim. et Biophys. Acta – Biomembranes 1768 (2007) 705–714. [20] P.P. Jain, R. Leber, C. Nagaraj, G. Leitinger, B. Lehofer, H. Olschewski, A. Olschewski, R. Prassl, L.M. Marsh, Liposomal nanoparticles encapsulating iloprost exhibit enhanced vasodilation in pulmonary arteries, Int. J. Nanomed. 9 (2014) 3249–3261. [21] H. Ellens, J. Bentz, F.C. Szoka, PH-induced destabilization of phosphatidylethanolamine-containing liposomes: role of bilayer contact, Biochemistry 23 (1984) 1532–1538. [22] A.S. Ladokhin, P.W. Holloway, Fluorescence of membrane-bound tryptophan octyl ester: a model for studying intrinsic fluorescence of protein-membrane interactions, Biophys. J. 69 (1995) 506–517. [23] O. Tirosh, Y. Barenholz, J. Katzhendler, A. Priev, Hydration of polyethylene glycol-grafted liposomes, Biophys. J. 74 (1998) 1371–1379. [24] M.L. Immordino, F. Dosio, L. Cattel, Stealth liposomes: review of the basic science, rationale, and clinical applications, existing and potential, Int. J. Nanomed. 1 (2006) 297–315. [25] T.M. Allen, Ligand-targeted therapeutics in anticancer therapy, Nat. Rev. Cancer 2 (2002) 750–763. [26] J.M. Ramsey, A. Hibbitts, J. Barlow, C. Kelly, N. Sivadas, S.A. Cryan, ‘Smart’ nonviral delivery systems for targeted delivery of RNAi to the lungs, Ther. Deliv. 4 (2013) 59–76. [27] T.T. Mills, J. Huang, G.W. Feigenson, J.F. Nagle, Effects of cholesterol and unsaturated DOPC lipid on chain packing of saturated gel-phase DPPC bilayers, Gen. Physiol. Biophys. 28 (2009) 126–139. [28] J.S. Patton, P.R. Byron, Inhaling medicines: delivering drugs to the body through the lungs, Nat. Rev. Drug. Discov. 6 (2007) 67–74. [29] M. Najlah, A. Vali, M. Taylor, B.T. Arafat, W. Ahmed, D.A. Phoenix, K.M. Taylor, A. Elhissi, A study of the effects of sodium halides on the performance of air-jet and vibrating-mesh nebulizers, Int. J. Pharm. 456 (2013) 520–527. [30] M. Beck-Broichsitter, N. Oesterheld, M.C. Knuedeler, W. Seeger, T. Schmehl, On the correlation of output rate and aerodynamic characteristics in vibratingmesh-based aqueous aerosol delivery, Int. J. Pharm. 461 (2014) 34–37.
B. Lehofer et al. / European Journal of Pharmaceutics and Biopharmaceutics 88 (2014) 1076–1085 [31] Y. Barenholz, Liposome application: problems and prospects, Curr. Opin. Colloid Interface Sci. 6 (2001) 66–77. [32] T.G. O’Riordan, Formulations and nebulizer performance, Respir. Care 47 (2002) 1305–1312. [33] M. Najlah, I. Parveen, M.A. Alhnan, W. Ahmed, A. Faheem, D.A. Phoenix, K.M. Taylor, A. Elhissi, The effects of suspension particle size on the performance of air-jet, ultrasonic and vibrating-mesh nebulisers, Int. J. Pharm. 461 (2014) 234–241. [34] M.M. Gaspar, O. Gobbo, C. Ehrhardt, Generation of liposome aerosols with the Aeroneb Pro and the AeroProbe nebulizers, J. Liposome Res. 20 (2010) 55–61. [35] R.W. Niven, H. Schreier, Nebulization of liposomes. I. Effects of lipid composition, Pharm. Res. 7 (1990) 1127–1133. [36] R.W. Niven, M. Speer, H. Schreier, Nebulization of liposomes. II. The effects of size and modeling of solute release profiles, Pharm. Res. 8 (1991) 217–221. [37] D. Cipolla, H.K. Chan, J. Schuster, D. Farina, Personalizing aerosol medicine: development of delivery systems tailored to the individual, Ther. Deliv. 1 (2010) 667–682. [38] A.M. Elhissi, M. Faizi, W.F. Naji, H.S. Gill, K.M. Taylor, Physical stability and aerosol properties of liposomes delivered using an air-jet nebulizer and a novel micropump device with large mesh apertures, Int. J. Pharm. 334 (2007) 62–70. [39] S. Anabousi, E. Kleemann, U. Bakowsky, T. Kissel, T. Schmehl, T. Gessler, W. Seeger, C.M. Lehr, C. Ehrhardt, Effect of PEGylation on the stability of liposomes during nebulisation and in lung surfactant, J. Nanosci. Nanotechnol. 6 (2006) 3010–3016.
1085
[40] M. Zaru, S. Mourtas, P. Klepetsanis, A.M. Fadda, S.G. Antimisiaris, Liposomes for drug delivery to the lungs by nebulization, Eur. J. Pharm. Biopharm. (2007) 655–666. [41] E. Kleemann, T. Schmehl, T. Gessler, U. Bakowsky, T. Kissel, W. Seeger, Iloprostcontaining liposomes for aerosol application in pulmonary arterial hypertension: formulation aspects and stability, Pharm. Res. 24 (2007) 277– 287. [42] V. Wagner, A. Dullaart, A.K. Bock, A. Zweck, The emerging nanomedicine landscape, Nat. Biotechnol. 24 (2006) 1211–1217. [43] M. Beck-Broichsitter, M. Rieger, R. Reul, T. Gessler, W. Seeger, T. Schmehl, Correlation of drug release with pulmonary drug absorption profiles for nebulizable liposomal formulations, Eur. J. Pharm. Biopharm. 84 (2013) 106– 114. [44] D.D. Lasic, Doxorubicin in sterically stabilized liposomes, Nature 380 (1996) 561–562. [45] G.W. Feigenson, Phase behavior of lipid mixtures, Nat. Chem. Biol. 2 (2006) 560–563. [46] P.J. Quinn, C. Wolf, Thermotropic and structural evaluation of the interaction of natural sphingomyelins with cholesterol, Biochim. Biophys. Acta 1788 (2009) 1877–1889. [47] R. Abu-Dahab, U.F. Schäfer, C.M. Lehr, Lectin-functionalized liposomes for pulmonary drug delivery: effect of nebulization on stability and bioadhesion, Eur. J. Pharm. Sci. 14 (2001) 37–46. [48] A.S. Ulrich, Biophysical aspects of using liposomes as delivery vehicles, Biosci. Rep. 22 (2002) 129–150.
