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PAMAM dendrimers as aerosol drug nanocarriers for pulmonary delivery via nebulization
International Journal of Pharmaceutics 461 (2014) 242–250
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
PAMAM dendrimers as aerosol drug nanocarriers for pulmonary delivery via nebulization Maha Nasr a,b , Mohammad Najlah c , Antony D’Emanuele b , Abdelbary Elhissi b,∗ a b c
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Egypt Institute of Nanotechnology and Bioengineering, School of Pharmacy and Biomedical Sciences, University of Central Lancashire, United Kingdom Faculty of Pharmacy, Albaath University, Homs, Syria
a r t i c l e
i n f o
Article history: Received 20 September 2013 Received in revised form 11 November 2013 Accepted 15 November 2013 Available online 22 November 2013 Keywords: PAMAM dendrimers Pulmonary Beclometasone dipropionate Nebulizer Solubility
a b s t r a c t Polyamidoamine (PAMAM) dendrimers were evaluated as nanocarriers for pulmonary delivery of the model poorly soluble anti-asthma drug beclometasone dipropionate (BDP) using G3, G4 and G4(12) dendrimers. BDP-loaded dendrimers were characterized for drug solubility, in vitro drug release and aerosolization properties using three nebulizers: Pari LC Sprint (air-jet), Aeroneb Pro (actively vibratingmesh) and Omron MicroAir (passively vibrating-mesh) nebulizers. Solubilization of BDP using dendrimers was increased by increasing the dendrimer generation and by using higher pH media. In vitro release studies showed that BDP when complexed with dendrimers exhibited a sustained release, and for all dendrimer formulations less than 35% of the drug was released after 8 h. Nebulization studies revealed that aerosol performance was dependent on nebulizer rather than dendrimer generation. Nebulization output values for the Pari (air-jet) and Aeroneb Pro (active mesh) nebulizers were in the range of 90–92% and 85–89% respectively compared to 57–63% for the Omron (passive mesh) nebulizer. The size of the droplets generated from the jet nebulizer was slightly smaller and aerosol polydispersity was lower compared to both mesh devices. The “fine particle fraction (FPF)” of the aerosols was in the following order: Pari (air-jet) > Aeroneb Pro (active mesh) > Omron (passive mesh). This study demonstrates that BDP-dendrimers have potential for pulmonary inhalation using air-jet and vibrating-mesh nebulizers. Moreover, the aerosol characteristics are influenced by nebulizer design rather than dendrimer generation. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Dendrimers are hyperbranched polymers possessing welldefined structures with high degree of molecular uniformity and low polydispersity (Tomailia et al., 1985; D’Emanuele and Attwood, 2005). A dendrimer consists of a central core from which polymeric branches evolve, repeated units determining the solubilizing capacity of the dendrimer, and terminal groups responsible for the behaviour of the dendrimer in solutions (Tomailia et al., 1985). Polyamidoamine (PAMAM) dendrimers were the first commercialized family of dendrimers; their macromolecular architecture originates from an initiator ethylenediamine core, followed by consecutive branches whose number determines the generation of dendrimers (Fig. 1). Full and half generation PAMAM dendrimers are terminated with amine and carboxylate surface groups
∗ Corresponding author at: Institute of Nanotechnology and Bioengineering, School of Pharmacy and Biomedical Sciences, University of Central Lancashire, Preston PR1 2HE, United Kingdom. Tel.: +44 1772 89 5807. E-mail addresses: [email protected], [email protected] (A. Elhissi). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.11.023
respectively. PAMAM dendrimers may serve as carriers for a diversity of biomolecules including nucleic acids and proteins (Svenson and Tomalia, 2005; Bai et al., 2007; Venuganti and Perumal, 2008; Svenson, 2009; Navarro et al., 2010). Due to their high aqueous solubility and amenable surface functional groups, PAMAM dendrimers have been used for the solubilization of hydrophobic drugs (Milhem et al., 2000; Devarakonda et al., 2004; Najlah et al., 2007). Beclometasone dipropionate (BDP) is a poorly-water soluble steroid that is highly effective for prophylaxis against asthma via inhalation (Bakhbakhi et al., 2006; Xu et al., 2012). Attempts to nebulize BDP has involved the use of liposome formulations which may offer the ability to solubilize the drug and localize its action in the lung for prolonged periods (Saari et al., 1998; Saari et al., 1999; Darwis and Kellaway, 2001). Air-jet nebulizers are the most commonly used type of nebulizer for pulmonary delivery of aqueous solutions and dispersions (O’Callaghan and Barry, 1997). An air-jet nebulizer operates by employing a narrow “venturi” nozzle through which gas is forced using a compressor, resulting in formation of liquid films within the nebulizer reservoir. The resultant films convert into aerosol droplets as a result of surface tension (McCallion et al., 1996; Hess,
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Fig. 1. Structure of G3 PAMAM dendrimer.
2000). Air-jet nebulizers have become established for aerosol generation of novel drug delivery systems such as liposomes (Taylor et al., 1990; Bridges and Taylor, 1998; Saari et al., 1999; Darwis and Kellaway, 2001; Elhissi et al., 2012), solid lipid nanoparticles (Liu et al., 2008), ultradeformable vesicles (Elhissi et al., 2012), lipid nanoemulsions (Amani et al., 2010; Nasr et al., 2012a) and niosomes (Desai and Finlay, 2002; Abd-Elbary et al., 2008; Elhissi et al., 2013). As an alternative to jet nebulizers, vibrating-mesh nebulizers have been introduced and are classified into passively vibrating-mesh and actively vibrating-mesh nebulizers (Dhand, 2002; Newman and Gee-Turner, 2005). Passively vibrating-mesh devices (e.g. Omron MicroAir) operate by employing a piezoelectric crystal that transmits the vibrations via a transducer horn to a perforated plate having around 6000 tapered holes (each has a size of 3 m), resulting in “passive” vibrations of the plate, extrusion of the fluid through the holes and generation of aerosol droplets (Newman and Gee-Turner, 2005). By contrast, actively vibrating-mesh nebulizers such as the Aeroneb Pro device employ a perforated plate with more than 1000 dome-shaped apertures surrounded by a ceramic vibrational element which induces a “micropump” action to extrude the liquid and generate the aerosol (Dhand, 2002). Vibrating-mesh nebulizers have been demonstrated to be suitable for the delivery of novel drug delivery systems such as liposomes (Elhissi and Taylor, 2005; Elhissi et al., 2006; Wagner et al., 2006; Elhissi et al., 2007; Kleemann et al., 2007; Gaspar et al., 2010; Elhissi et al., 2011), nanoemulsions (Amani et al., 2010) and niosomes (Elhissi et al., 2013).
Dendrimers may constitute an alternative to all aforementioned nanocarrier systems for pulmonary delivery. The safety of dendrimer formulations on respiratory tissues and enhanced drug uptake by lung cells have been reported, suggesting potential applicability of dendrimers for pulmonary delivery (Bai et al., 2007; Bai and Ahsan, 2009; Inapagolla et al., 2010; Dong et al., 2011; Ryan et al., 2013). These studies, however, assessed the potential of pulmonary delivery of dendrimers using techniques such as direct instillation into anesthetized animals. For practical purposes, means of generating dendrimer aerosols for targeting the peripheral respiratory airways are required. In the present study, PAMAM dendrimer-BDP complexes were prepared and characterized in terms of complexation efficiency, solubility and release profile of the drug. The potential of nebulizers for generating inhalable aerosols from the dendrimer solutions was evaluated using Pari LC Sprint (air-jet), Aeroneb Pro (actively vibrating-mesh) and Omron MicroAir NE-U22 (passively vibratingmesh) nebulizers.
2. Materials and methods 2.1. Materials BDP, sodium dodecyl sulphate (SDS), phosphate buffered saline (PBS) and methanolic solutions of PAMAM dendrimers (ethylenediamine core; G3, G4 and G4.5) and PAMAM dendrimers
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(1,12-diamino dodecane core; G4(12)) were all purchased from Sigma–Aldrich, UK. Methanol and water used for high performance liquid chromatography (HPLC) were of HPLC grade and supplied by Fisher Scientific Ltd., UK. Pari LC Sprint nebulizer and its Pari TurboBoy S compressor (Pari GmBH, Germany) were purchased from Pari Medical Ltd., UK, and the Omron MicroAir NE-U22 nebulizer (Omron Healthcare, Japan) was supplied by EverGreen Nebulisers Ltd., UK. The Aeroneb Pro nebulizer was supplied by Aerogen Ltd., Ireland. 2.2. Methods
2.2.5. In vitro release of BDP from dendrimer complexes The release of BDP from dendrimer complexes through a dialysis bag was determined using HPLC (Section 2.2.3). A BDP-dendrimer complex equivalent to 500 g BDP was sealed in a dialysis bag (cut off MW 3500 Da) (Jin et al., 2011) and dialyzed against 15 ml PBS solution containing 20% methanol while stirring at 100 rpm and 25 ◦ C. Samples (100 l) were withdrawn at pre-determined intervals and replaced with fresh medium (Ma et al., 2007) and the released drug was monitored for up to 8 h using HPLC. For comparative purposes, the same amount of BDP in methanolic solution was tested for its release as control.
2.2.1. Preparation of BDP-dendrimer complexes Methanolic dendrimer solution (as supplied) was placed in a round bottomed flask and the solvent was evaporated at 35 ◦ C using a rotary evaporator (Büchi R-114, Büchi, Switzerland) at a pressure below 100 mbar for 1 h to obtain a dry residue. The weight of the dry dendrimers was determined by calculating the difference between the empty and dendrimer-loaded flasks after completed solvent evaporation (Vandamme and Brobeck, 2005). BDP was added at a concentration of 10% w/w of the dendrimers. Methanol (2 ml) was then added followed by solvent evaporation using the aforementioned conditions for 1.5 h. The residue was reconstituted in PBS (pH 7.4) followed by overnight stirring at 25 ◦ C. Separation of the unentrapped (i.e. uncomplexed) drug was performed by filtration using 0.45 m cellulose acetate membrane filters (Devarakonda et al., 2004).
2.2.6. Determination of aerosol droplet size, output and “fine particle fraction” (FPF) The size of aerosol droplets of BDP-dendrimer complexes was analyzed using Malvern’s Spraytec laser diffraction size analyzer (Malvern Instruments Ltd., UK). Pari LC Sprint (air-jet), Aeroneb Pro (actively vibrating-mesh) and Omron MicroAir (passively vibrating-mesh) nebulizers were used in this study. The nebulizer was positioned with its mouthpiece being directed perpendicular to the laser beam at a distance of 3 cm (Nasr et al., 2012a). BDP dendrimer solution (3 ml; G3, G4 or G4(12)) was loaded into the nebulizer and a vacuum line with a flow rate of 60 l/min was applied to draw the aerosol through the beam for droplet size analysis. The span and volume median diameter (VMD; 50% undersize) were recorded to present the size distribution (i.e. polydispersity) and size of the aerosol droplets respectively:
2.2.2. Phase solubility studies Phase solubility diagrams were determined by plotting the molar concentration of solubilized BDP versus the increased molar concentration of the dendrimers. The stability constants for complexation of BDP with the dendrimers were calculated using the following equation (Devarakonda et al., 2004):
Span =
K1:1 =
Slope So (1 − slope)
(1)
(90% undersize − 10% undersize) VMD
(3)
In different experiments, the same BDP-dendrimer formulations were used for nebulization. The nebulizer was operated to “dryness” (i.e. when the aerosol generation ceased completely). The aerosol output from the nebulizer was calculated as shown in Eq. (4): Output (%) =
weight of liquid nebulized weight of liquid before nebulization
× 100
(4)
where So is the equilibrium solubility of BDP in absence of the dendrimers. Complexation efficiency was calculated by the following equation (Devarakonda et al., 2005):
Furthermore, the “fine particle fraction (FPF)” of the aerosols was calculated according to the following equation:
Complexationefficiency = intrinsicmolarsolubilityofBDP
FPF (%) =
× equilibriumstabilityconstant.
Fraction of droplets ≤ 5.4 m Output
× 100
(5)
(2)
2.2.3. Determination of BDP solubility using high performance liquid chromatography (HPLC) The inherent molar solubility of BDP was determined in PBS using HPLC (Agilent 1200, USA). The drug solubility was also determined following complexation with dendrimers of different generations. The mobile phase utilized in HPLC consisted of methanol and water (75:25 v/v) at a flow rate of 1.7 ml/min. Separation was performed using an Agilent Eclipse column XDB-C18 (5 m, 4.6 × 150 mm) at a temperature of 40 ◦ C and the detection wavelength was 238 nm (Craparo et al., 2011). Samples (20 l) were injected via an autosampler. The molar solubility of BDP in 2% SDS was determined as a reference for comparison to that complexed in 2% dendrimers of different generations (Milhem et al., 2000). 2.2.4. Effect of pH on BDP solubility The molar solubility of BDP uncomplexed and when complexed with G3 dendrimers of molar concentration 0.00585 M was measured at three different pH values (9.8, 7.4 and 5). G3 was used as model dendrimers in this study. The pH of the medium was checked using a pH meter (Corning 220, Cole-Palmer, UK).
2.2.7. Statistical analysis All experiments were repeated in triplicate and the results were expressed as the mean ±standard deviation. Statistical analysis was undertaken using one way analysis of variance (ANOVA) followed by Tukey Kramer test using GraphPad InStat® software to check the statistical significance of differences. A difference was considered statistically significant if the p-value was ≤0.05. 3. Results and discussion 3.1. BDP-dendrimer phase solubility Fig. 2(a–d) shows the phase solubility diagrams of BDP in the presence of increasing concentrations of dendrimers at pH 9.8. For all formulations, the slopes were less than unity, suggesting that the complexes between BDP and dendrimers exhibited 1:1 stoichiometries (Devarakonda et al., 2004; Devarakonda et al., 2005). It has been reported that the interaction between dendrimer and drug molecules occurs either through encapsulation of the drug into the void spaces of dendrimer or by interaction of the drug with the dendrimer’s functional groups. The driving forces for the drug-dendrimer interactions are hydrogen bonding,
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Table 1 Stability constants and complexation efficiency for BDP-PAMAM complexes. Dendrimers generation
G3
G4
G4(12)
G4.5
K1:1 (M−1 ) Complexation efficiency (×103 )
3139 4.1
88,487 115.56
185,117 241.76
58.84 0.08
to the weak acidic nature of this drug (pKa = 13), thus, it existed in an unionized form at the pH range investigated. There was a significant increase in molar solubility of BDP upon increasing the concentration of all full generation dendrimers as shown in Fig. 2(a–c) (p < 0.05), which could be ascribed to the increased number of amine groups and internal cavities that are available to interact with or encapsulate BDP molecules (Yiyun et al., 2005; Cheng et al., 2007; Venkataraman et al., 2011). By contrast, increasing the concentration of the half generation dendrimer (G4.5) caused only a very slight trend of enhanced solubility of BDP (p > 0.05; Fig. 2d). This came in agreement with Devarakonda et al. (2005) upon loading niclosamide into half generation dendrimers, and in accordance with findings of Chauhan et al. (2003) by encapsulation of indometacin into G4.5 dendrimers. As shown in Fig. 2a and b, BDP exhibited a generation-dependent solubility, since increasing the dendrimer generation form G3 to G4 increased the molar solubility of the drug. This again may due to the increased number of interior amine groups in the dendrimer with generation size at a given pH, leading to the entrapment of more BDP molecules in the hydrophobic cores of the dendrimers (Yiyun et al., 2005; Cheng et al., 2007; Devarakonda et al., 2007; Ma et al., 2007). Results also showed that solubilization of the drug using G4(12) dendrimer was superior compared to solubilization using G4 dendrimers (Fig. 2b,c); this clearly emphasizes that the hydrophobicity of dendrimer core plays an important role in the solubilization of BDP molecules owing to enhanced hydrophobic interaction with the drug (Watkins et al., 1997; Cheng et al., 2008). Upon calculation of stability constants and complexation efficiency (Table 1), it was evident that amine terminated dendrimers (G3, G4 and G4(12)) formed very stable complexes with BDP while ester terminated (half-generation) dendrimers (G4.5) exhibited the lowest values. In a previous study, G4 and G4(12) showed more stable complexation with BDP than cyclodextrins (CD), which was reported by Worth et al. (1996) to be a 1:1 complexation having stability constant values of 33,313 and 10,428 M−1 for DM--CD and -CD respectively. This is also in agreement with Devarakonda et al. (2005) who reported dendrimers to be superior to CD for complexation with niclosamide. 3.2. Effect of pH on BDP solubility
Fig. 2. Solubility profiles of BDP in the presence of increasing concentrations of (a) G3, (b) G4, (c) G4(12) and (d) G4.5 PAMAM dendrimers (n = 3).
Van der Waals interactions and/or electrostatic attraction between opposite charged dendrimer and drug molecules (D’Emanuele and Attwood, 2005; Cheng et al., 2008; Svenson, 2009). BDP contains both hydrogen bond acceptor and donor groups, which could allow interaction with the dendrimer based on hydrogen bonding, in addition to the physical encapsulation within the dendrimer based on drug hydrophobicity. No marked electrostatic charges are expected to occur between dendrimers and BDP molecules owing
The inherent solubility of BDP was very low and did not change significantly (p > 0.05) by changing the pH of the medium (data not shown), due to the fact that BDP is a weak acid (pKa = 13.05). However, using G3 as a model dendrimer, the amount of BDP complexed with the dendrimers was found to increase upon increasing the pH from 5 to 7.4 with a maximum solubility found at pH 9.8 (Fig. 3). This might be attributed to the fact that PAMAM dendrimers have primary amines on their surfaces and tertiary amines within their internal cavities, which could act as hydrogen bond donors or acceptors, owing to the alteration of the protonation level of the amine groups in relation to pH (Cheng et al., 2007; Devarakonda et al., 2007). BDP can act as a hydrogen bond donor, promoting the formation of hydrogen bonds between the tertiary amines within the dendrimer cavity and BDP molecule at high pH media. In addition, the higher pH is expected to decrease the polarity of the dendrimer microenvironment and hence, promoting solubilization of the hydrophobic BDP molecules. This agrees with findings of other investigators using different hydrophobic drug molecules
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Fig. 3. Molar solubility of BDP complexed with G3 PAMAM dendrimers dispersed in media having pH values of 5, 7.4 or 9.8 (n = 3).
(Devarakonda et al., 2004; Cheng et al., 2007; Ma et al., 2007). For instance, nifedipine has a similar pKa to that of BDP and exhibited similar solubility behaviour in dendrimer solutions with a range of pH values (Devarakonda et al., 2004). These results suggest the pH-dependent solubility of BDP resulted from the pH-dependent hydrogen bond formation between BDP and dendrimer. As shown in Fig. 4, a comparison between the solubilization offered by dendrimers and a model micellar system using SDS at a concentration of 2% demonstrated that the solubilization capacity of both G4 and G4(12) dendrimers was significantly higher than that of SDS (p < 0.05), while G3 and G4.5 were significantly lower than SDS in their solubilizing capacity. This concurred with the results obtained by Milhem et al. (2000) who reported the superiority of G4 PAMAM dendrimers for solubilization of ibuprofen compared to the aqueous solution of the drug as well as 2% of SDS solution. The highest solubility of BDP was found with G4(12) dendrimers (Fig. 4), emphasizing that solubility of this hydrophobic drug directly correlates with the hydrophobicity of the dendrimer core. Since molar solubility studies revealed that full-generation dendrimers (G3, G4 and G4(12)) are superior to the halfgeneration dendrimer (G4.5) at solubilizing BDP (Fig. 2; Fig. 4), the half-generation dendrimer was excluded from the release and nebulization studies in subsequent studies.
Fig. 5. In vitro release of BDP from the complexes of G3, G4 and G4(12) dendrimers (n = 3).
release of the drug when complexed with dendrimers. These results are relatively consistent with those presented by Kolhe et al. (2003) who reported that drug–dendrimer complex is stable in methanol and water mixtures. Also, the release of ibuprofen from drug–dendrimer complex was found to be slower than that of pure ibuprofen (Milhem et al., 2000). Similarly, Ma et al. (2007) have reported that the release of sulfamethoxazole from the drug dendrimer solution was significantly slower compared to pure drug. After 8 h, 35.8 and 34.6% release was obtained for PAMAMG4(12)-DBP and PAMAM-G4-DBP complexes respectively, whilst 25% release was obtained for PAMAM-G3-DBP. The slower release of the drug from G3 complex might be attributed to the lower drug encapsulating capacity of G3 compared to that of G4 and G4(12) (Gupta et al., 2006). The slower release of BDP may indicate the stronger complex it forms upon encapsulation in the dendrimer. Controlled release of physically entrapped drugs within dendrimers has been previously reported (Liu and Fréchet, 1999).
3.3. In vitro release of BDP from dendrimers
3.4. Nebulizer output studies
Using dendrimer-free medium, 100% of BDP was released through the dialysis bag within 2 h (data not shown). By contrast, after 8 h less than 35% of BDP was released from dendrimer structures (Fig. 5), indicating the markedly slower and sustained
In this study, three nebulizers employing different operation mechanisms were used: Pari LC Sprint (air-jet), Aeroneb Pro (actively vibrating-mesh) and Omron MicroAir (passively vibrating-mesh) nebulizers. All medical nebulizers rely on the
Fig. 4. Comparison of inherent BDP molar aqueous solubility to 2% BDP-dendrimer complexes and SDS micellar system (n = 3).
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Fig. 6. Nebulization output of G3-BDP, G4-BDP and G4(12)-BDP dendrimers using Pari LC Sprint (air-jet), Aeroneb Pro (actively vibrating-mesh) and Omron MicroAir (passively vibrating-mesh) nebulizers (n = 3).
employment of energy to convert the liquid into inhalable aerosol droplets, and it is extremely important that the residual fluid left is minimal following completed nebulization (i.e. the output of the nebulizer is maximized) (Hess, 2000). Dispersed particles (especially particles in the micrometres size range) may not be efficiently nebulized, resulting in their concentration within the residual fluid of nebulizers (McCallion et al., 1996; O’Callaghan and Barry, 1997; Elhissi and Taylor, 2005). Our preliminary experiments have shown that all nebulizers employed in this study minimally concentrate nanoparticles such as dendrimers and nano-liposomes (data not shown), indicating that the measurement of output as aerosol output rather than dispersed phase output is valid in the present study. Thus, for convenience, the output of the nebulizers was measured by gravimetric determination of the aerosol output rather than drug output. Moreover, our preliminary investigations have confirmed that dendrimers are stable to nebulization, which is attributed to the small particle size of the dendrimers (Nasr et al., 2012b). All nebulizers generated high aerosol output regardless of dendrimer generation (Fig. 6). The aerosol output is arranged in the following order: Pari (air-jet) = Aeroneb Pro (active mesh) > Omron MicroAir (passive mesh) (Fig. 6). The Pari nebulizer shows a slightly higher output than the Aeroneb Pro device, but that was not statistically significant (p > 0.05). Thus, it seems that the energy provided by the passive vibrations of the Omron nebulizer was less efficient at converting the liquid into aerosols when compared to the energy provided by jet-nebulization of the Pari nebulizer or the active vibrations provided by the Aeroneb Pro device. This agrees with our recent findings using BDP niosomes (Elhissi et al., 2013). For each nebulizer, there was no effect of formulation (i.e. dendrimer generation) on the aerosol output (p > 0.05), being approximately 90–92% for the Pari (jet) nebulizer, 85–89% for the Aeroneb Pro (active mesh) nebulizer and 57–63% for the Omron (passive mesh) nebulizer (Fig. 6). It has been demonstrated that nebulization performance is influenced by fluid physicochemical properties such as viscosity, surface tension (McCallion et al., 1995; Ghazanfari et al., 2007) and the presence of electrolytes in the solution (Ghazanfari et al., 2007; Najlah et al., 2013). Thus, the absence of formulation effects on aerosol output suggests that the physicochemical properties of the different dendrimer formulations are highly similar; however, further studies are needed to investigate whether this assumption is valid. Overall, the output performance of dendrimers was influenced by nebulizer type rather than dendrimer generation (Fig. 6).
3.5. Aerosol droplet size and FPF Principally, for aerosols to be considered “respirable” (i.e. in FPF) droplet size should be smaller than 5–6 m (Stahlhofen et al., 1980; O’Callaghan and Barry, 1997). Patient-related factors such as lung pathophysiology, patient age and inhalation mode are additional factors that are known to affect the pulmonary deposition of inhaled aerosols; however, these factors are beyond the scope of this work. In this study, laser diffraction was used to determine droplet size, size distribution and FPF of dendrimer aerosols generated via nebulization. Laser diffraction has been shown to be highly reliable for size analysis of nebulized droplets, and has been reported to correlate with in vivo pulmonary deposition findings for non-volatile aerosols. Additionally, VMD measured by laser diffraction may be equivalent to the median aerodynamic diameter of nebulized droplets (Clark, 1995). It is worth noting that cascade impactors are more commonly used to analyze particle size of inhalable aerosols and attempts have been made to correlate the findings of cascade impaction with those of laser diffraction. A drawback of cascade impaction is solvent evaporation from liquid aerosols due to the air flow applied through the impactors; hence, correlation of cascade impaction with laser diffraction was possible when solvent evaporation was minimized either by cooling the cascade impactor before operation (Kwong et al., 2000) or by using low flow rate through the apparatus (None et al., 2001). Thus, the high reliability and convenience of laser diffraction justifies its use for the determination of size and size distribution of dendrimer aerosols. Size and size distribution of the aerosol droplets were expressed as VMD and span respectively (Table 2). The effect of formulation on the VMD and span was slight, and both vibrating-mesh devices generated droplets with larger VMD, higher span values and greater variability compared with the jet nebulizer (Table 2). These findings indicate that compressed gas forced through the “venturi” nozzle of the Pari nebulizer has provided more reliable means of generating small droplets with narrow size distribution as compared to the micro-perforated vibrating plates of the Aeroneb Pro and the Omron MicroAir nebulizers. In contrast to the findings of this study, other reports using liposomes have demonstrated narrower size distribution of vibrating-mesh nebulized aerosols (Elhissi and Taylor, 2005; Elhissi et al., 2006). Also, we reported that the vibrating-mesh devices failed at generating aerosols from amphotericin B nanoemulsions (Nasr et al., 2012a), whilst Amani et al. (2010) using steroid nanoemulsion formulations,
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Table 2 Size and size distribution of aerosol droplets generated from BDP-loaded PAMAM dendrimers of varying generations using air-jet and vibrating-mesh nebulizers. Dendrimer formulations VMD (m) Pari LC Sprint (air-jet) nebulizer Aeroneb Pro (active mesh) nebulizer Omron (passive mesh) nebulizer Span Pari LC Sprint (air-jet) nebulizer Aeroneb Pro (active mesh) nebulizer Omron (passive mesh) nebulizer
G3-BDP Complex
G4-BDP Complex
G4(12)-BDP Complex
4.05 ± 0.19 5.44 ± 1.64 5.30 ± 0.95
3.76 ± 0.23 5.21 ± 0.81 5.73 ± 2.41
4.25 ± 0.08 5.38 ± 1.47 5.82 ± 1.21
1.61 ± 0.17 2.21 ± 0.15 3.78 ± 1.67
1.68 ± 0.09 2.88 ± 0.75 2.83 ± 1.52
1.58 ± 0.21 2.89 ± 0.96 3.35 ± 0.49
Fig. 7. FPF of G3-BDP, G4-BDP and G4(12)-BDP dendrimers aerosols using Pari LC Sprint (air-jet), Aeroneb Pro (actively vibrating-mesh) and Omron MicroAir (passively vibrating-mesh) nebulizers (n = 3).
have reported that the Omron MicroAir (passive mesh) device exhibited superior performance compared with a standard jet nebulizer. The performance of vibrating-mesh nebulizers is highly dependent on formulation properties such as viscosity, surface tension (Ghazanfari et al., 2007) and the presence of electrolytes (Ghazanfari et al., 2007; Najlah et al., 2013). Thus, the conflicting findings amongst the various studies employing mesh nebulizers could be attributed to the marked effects formulation may have on the performance of aerosols generated using these devices. The size and size distribution (Table 2) both suggest that the aerosols of the three nebulizers are potentially suitable for generating “respirable” dendrimers. Aerosols of different dendrimer generations had different VMD and span values (p < 0.05) only when the Pari (air-jet) nebulizer was employed (Table 2). When the two vibrating-mesh devices were compared, VMD of the droplets was highly similar but the Aeroneb Pro (active mesh) nebulizer tended to generate aerosols with narrower size distribution (i.e. small span values) (Table 2), indicating that active mesh vibrations of the Aeroneb Pro may provide more control on the polydispersity of the aerosols compared with the passive vibrations of the Omron nebulizer. This contrasts with previous reports using liposomes (Elhissi et al., 2006) and niosomes (Elhissi et al., 2013) where the Omron MicroAir generated droplets with lower polydispersity, suggesting that this device behaves differently when different delivery systems are used. Nebulizers should generate small droplets with high output to ensure that a high proportion of the drug can potentially reach the peripheral airways. Thus, FPF of the aerosol was calculated by multiplying the aerosol output by the aerosol fraction ≤5.4 m. This figure was selected because it is the closest value to 5 m as presented by the Spraytec software. For each nebulizer, dendrimer generation did not affect the FPF (Fig. 7) which was consistent
with the findings of VMD and span (Table 2). However, the type of nebulizer significantly influenced FPF of the aerosols (p < 0.05) and the values were in the following order: Pari (air-jet) > Aeroneb Pro (active mesh) > Omron MicroAir (passive mesh) (Fig. 7). Despite the fact that passive and active vibrating mesh nebulizers both involve the presence of vibrating plates with multiple apertures, it is evident that since they operate according to different technologies they have significant differences in aerosol output (Fig. 6) and FPF (Fig. 7). 4. Conclusions BDP was successfully complexed with PAMAM dendrimers, and complexation was found to be dependent on generation and concentration of dendrimers and the pH of the dispersion medium. This work confirms the potential use of full generation PAMAM dendrimers as carriers for poorly soluble drugs such as BDP. The amine terminated dendrimers (G3, G4 and G4(12)) formed more stable complexes with BDP when compared with the ester terminated (half-generation; G4.5) dendrimers. The generation of dendrimer was also of great importance at enhancing the solubility of BDP. The highest solubility of BDP was found with G4(12) dendrimers, emphasizing that solubility of this hydrophobic drug directly correlates with the hydrophobicity of the dendrimer core. The sustained release profile and the high aerosol output and FPF suggests that dendrimers are a promising nanocarrier system for pulmonary delivery of BDP using air-jet or vibrating-mesh nebulizers. Pari LC Sprint (air-jet) and Aeroneb Pro (active mesh) nebulizers were more suitable for delivery of PAMAM dendrimers, producing high output and superior FPF. Overall, nebulization studies revealed that the aerosol properties of the dendrimer-BDP complexes were dependent on nebulizer design rather than dendrimer generation.
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Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
PAMAM dendrimers as aerosol drug nanocarriers for pulmonary delivery via nebulization Maha Nasr a,b , Mohammad Najlah c , Antony D’Emanuele b , Abdelbary Elhissi b,∗ a b c
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Egypt Institute of Nanotechnology and Bioengineering, School of Pharmacy and Biomedical Sciences, University of Central Lancashire, United Kingdom Faculty of Pharmacy, Albaath University, Homs, Syria
a r t i c l e
i n f o
Article history: Received 20 September 2013 Received in revised form 11 November 2013 Accepted 15 November 2013 Available online 22 November 2013 Keywords: PAMAM dendrimers Pulmonary Beclometasone dipropionate Nebulizer Solubility
a b s t r a c t Polyamidoamine (PAMAM) dendrimers were evaluated as nanocarriers for pulmonary delivery of the model poorly soluble anti-asthma drug beclometasone dipropionate (BDP) using G3, G4 and G4(12) dendrimers. BDP-loaded dendrimers were characterized for drug solubility, in vitro drug release and aerosolization properties using three nebulizers: Pari LC Sprint (air-jet), Aeroneb Pro (actively vibratingmesh) and Omron MicroAir (passively vibrating-mesh) nebulizers. Solubilization of BDP using dendrimers was increased by increasing the dendrimer generation and by using higher pH media. In vitro release studies showed that BDP when complexed with dendrimers exhibited a sustained release, and for all dendrimer formulations less than 35% of the drug was released after 8 h. Nebulization studies revealed that aerosol performance was dependent on nebulizer rather than dendrimer generation. Nebulization output values for the Pari (air-jet) and Aeroneb Pro (active mesh) nebulizers were in the range of 90–92% and 85–89% respectively compared to 57–63% for the Omron (passive mesh) nebulizer. The size of the droplets generated from the jet nebulizer was slightly smaller and aerosol polydispersity was lower compared to both mesh devices. The “fine particle fraction (FPF)” of the aerosols was in the following order: Pari (air-jet) > Aeroneb Pro (active mesh) > Omron (passive mesh). This study demonstrates that BDP-dendrimers have potential for pulmonary inhalation using air-jet and vibrating-mesh nebulizers. Moreover, the aerosol characteristics are influenced by nebulizer design rather than dendrimer generation. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Dendrimers are hyperbranched polymers possessing welldefined structures with high degree of molecular uniformity and low polydispersity (Tomailia et al., 1985; D’Emanuele and Attwood, 2005). A dendrimer consists of a central core from which polymeric branches evolve, repeated units determining the solubilizing capacity of the dendrimer, and terminal groups responsible for the behaviour of the dendrimer in solutions (Tomailia et al., 1985). Polyamidoamine (PAMAM) dendrimers were the first commercialized family of dendrimers; their macromolecular architecture originates from an initiator ethylenediamine core, followed by consecutive branches whose number determines the generation of dendrimers (Fig. 1). Full and half generation PAMAM dendrimers are terminated with amine and carboxylate surface groups
∗ Corresponding author at: Institute of Nanotechnology and Bioengineering, School of Pharmacy and Biomedical Sciences, University of Central Lancashire, Preston PR1 2HE, United Kingdom. Tel.: +44 1772 89 5807. E-mail addresses: [email protected], [email protected] (A. Elhissi). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.11.023
respectively. PAMAM dendrimers may serve as carriers for a diversity of biomolecules including nucleic acids and proteins (Svenson and Tomalia, 2005; Bai et al., 2007; Venuganti and Perumal, 2008; Svenson, 2009; Navarro et al., 2010). Due to their high aqueous solubility and amenable surface functional groups, PAMAM dendrimers have been used for the solubilization of hydrophobic drugs (Milhem et al., 2000; Devarakonda et al., 2004; Najlah et al., 2007). Beclometasone dipropionate (BDP) is a poorly-water soluble steroid that is highly effective for prophylaxis against asthma via inhalation (Bakhbakhi et al., 2006; Xu et al., 2012). Attempts to nebulize BDP has involved the use of liposome formulations which may offer the ability to solubilize the drug and localize its action in the lung for prolonged periods (Saari et al., 1998; Saari et al., 1999; Darwis and Kellaway, 2001). Air-jet nebulizers are the most commonly used type of nebulizer for pulmonary delivery of aqueous solutions and dispersions (O’Callaghan and Barry, 1997). An air-jet nebulizer operates by employing a narrow “venturi” nozzle through which gas is forced using a compressor, resulting in formation of liquid films within the nebulizer reservoir. The resultant films convert into aerosol droplets as a result of surface tension (McCallion et al., 1996; Hess,
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Fig. 1. Structure of G3 PAMAM dendrimer.
2000). Air-jet nebulizers have become established for aerosol generation of novel drug delivery systems such as liposomes (Taylor et al., 1990; Bridges and Taylor, 1998; Saari et al., 1999; Darwis and Kellaway, 2001; Elhissi et al., 2012), solid lipid nanoparticles (Liu et al., 2008), ultradeformable vesicles (Elhissi et al., 2012), lipid nanoemulsions (Amani et al., 2010; Nasr et al., 2012a) and niosomes (Desai and Finlay, 2002; Abd-Elbary et al., 2008; Elhissi et al., 2013). As an alternative to jet nebulizers, vibrating-mesh nebulizers have been introduced and are classified into passively vibrating-mesh and actively vibrating-mesh nebulizers (Dhand, 2002; Newman and Gee-Turner, 2005). Passively vibrating-mesh devices (e.g. Omron MicroAir) operate by employing a piezoelectric crystal that transmits the vibrations via a transducer horn to a perforated plate having around 6000 tapered holes (each has a size of 3 m), resulting in “passive” vibrations of the plate, extrusion of the fluid through the holes and generation of aerosol droplets (Newman and Gee-Turner, 2005). By contrast, actively vibrating-mesh nebulizers such as the Aeroneb Pro device employ a perforated plate with more than 1000 dome-shaped apertures surrounded by a ceramic vibrational element which induces a “micropump” action to extrude the liquid and generate the aerosol (Dhand, 2002). Vibrating-mesh nebulizers have been demonstrated to be suitable for the delivery of novel drug delivery systems such as liposomes (Elhissi and Taylor, 2005; Elhissi et al., 2006; Wagner et al., 2006; Elhissi et al., 2007; Kleemann et al., 2007; Gaspar et al., 2010; Elhissi et al., 2011), nanoemulsions (Amani et al., 2010) and niosomes (Elhissi et al., 2013).
Dendrimers may constitute an alternative to all aforementioned nanocarrier systems for pulmonary delivery. The safety of dendrimer formulations on respiratory tissues and enhanced drug uptake by lung cells have been reported, suggesting potential applicability of dendrimers for pulmonary delivery (Bai et al., 2007; Bai and Ahsan, 2009; Inapagolla et al., 2010; Dong et al., 2011; Ryan et al., 2013). These studies, however, assessed the potential of pulmonary delivery of dendrimers using techniques such as direct instillation into anesthetized animals. For practical purposes, means of generating dendrimer aerosols for targeting the peripheral respiratory airways are required. In the present study, PAMAM dendrimer-BDP complexes were prepared and characterized in terms of complexation efficiency, solubility and release profile of the drug. The potential of nebulizers for generating inhalable aerosols from the dendrimer solutions was evaluated using Pari LC Sprint (air-jet), Aeroneb Pro (actively vibrating-mesh) and Omron MicroAir NE-U22 (passively vibratingmesh) nebulizers.
2. Materials and methods 2.1. Materials BDP, sodium dodecyl sulphate (SDS), phosphate buffered saline (PBS) and methanolic solutions of PAMAM dendrimers (ethylenediamine core; G3, G4 and G4.5) and PAMAM dendrimers
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(1,12-diamino dodecane core; G4(12)) were all purchased from Sigma–Aldrich, UK. Methanol and water used for high performance liquid chromatography (HPLC) were of HPLC grade and supplied by Fisher Scientific Ltd., UK. Pari LC Sprint nebulizer and its Pari TurboBoy S compressor (Pari GmBH, Germany) were purchased from Pari Medical Ltd., UK, and the Omron MicroAir NE-U22 nebulizer (Omron Healthcare, Japan) was supplied by EverGreen Nebulisers Ltd., UK. The Aeroneb Pro nebulizer was supplied by Aerogen Ltd., Ireland. 2.2. Methods
2.2.5. In vitro release of BDP from dendrimer complexes The release of BDP from dendrimer complexes through a dialysis bag was determined using HPLC (Section 2.2.3). A BDP-dendrimer complex equivalent to 500 g BDP was sealed in a dialysis bag (cut off MW 3500 Da) (Jin et al., 2011) and dialyzed against 15 ml PBS solution containing 20% methanol while stirring at 100 rpm and 25 ◦ C. Samples (100 l) were withdrawn at pre-determined intervals and replaced with fresh medium (Ma et al., 2007) and the released drug was monitored for up to 8 h using HPLC. For comparative purposes, the same amount of BDP in methanolic solution was tested for its release as control.
2.2.1. Preparation of BDP-dendrimer complexes Methanolic dendrimer solution (as supplied) was placed in a round bottomed flask and the solvent was evaporated at 35 ◦ C using a rotary evaporator (Büchi R-114, Büchi, Switzerland) at a pressure below 100 mbar for 1 h to obtain a dry residue. The weight of the dry dendrimers was determined by calculating the difference between the empty and dendrimer-loaded flasks after completed solvent evaporation (Vandamme and Brobeck, 2005). BDP was added at a concentration of 10% w/w of the dendrimers. Methanol (2 ml) was then added followed by solvent evaporation using the aforementioned conditions for 1.5 h. The residue was reconstituted in PBS (pH 7.4) followed by overnight stirring at 25 ◦ C. Separation of the unentrapped (i.e. uncomplexed) drug was performed by filtration using 0.45 m cellulose acetate membrane filters (Devarakonda et al., 2004).
2.2.6. Determination of aerosol droplet size, output and “fine particle fraction” (FPF) The size of aerosol droplets of BDP-dendrimer complexes was analyzed using Malvern’s Spraytec laser diffraction size analyzer (Malvern Instruments Ltd., UK). Pari LC Sprint (air-jet), Aeroneb Pro (actively vibrating-mesh) and Omron MicroAir (passively vibrating-mesh) nebulizers were used in this study. The nebulizer was positioned with its mouthpiece being directed perpendicular to the laser beam at a distance of 3 cm (Nasr et al., 2012a). BDP dendrimer solution (3 ml; G3, G4 or G4(12)) was loaded into the nebulizer and a vacuum line with a flow rate of 60 l/min was applied to draw the aerosol through the beam for droplet size analysis. The span and volume median diameter (VMD; 50% undersize) were recorded to present the size distribution (i.e. polydispersity) and size of the aerosol droplets respectively:
2.2.2. Phase solubility studies Phase solubility diagrams were determined by plotting the molar concentration of solubilized BDP versus the increased molar concentration of the dendrimers. The stability constants for complexation of BDP with the dendrimers were calculated using the following equation (Devarakonda et al., 2004):
Span =
K1:1 =
Slope So (1 − slope)
(1)
(90% undersize − 10% undersize) VMD
(3)
In different experiments, the same BDP-dendrimer formulations were used for nebulization. The nebulizer was operated to “dryness” (i.e. when the aerosol generation ceased completely). The aerosol output from the nebulizer was calculated as shown in Eq. (4): Output (%) =
weight of liquid nebulized weight of liquid before nebulization
× 100
(4)
where So is the equilibrium solubility of BDP in absence of the dendrimers. Complexation efficiency was calculated by the following equation (Devarakonda et al., 2005):
Furthermore, the “fine particle fraction (FPF)” of the aerosols was calculated according to the following equation:
Complexationefficiency = intrinsicmolarsolubilityofBDP
FPF (%) =
× equilibriumstabilityconstant.
Fraction of droplets ≤ 5.4 m Output
× 100
(5)
(2)
2.2.3. Determination of BDP solubility using high performance liquid chromatography (HPLC) The inherent molar solubility of BDP was determined in PBS using HPLC (Agilent 1200, USA). The drug solubility was also determined following complexation with dendrimers of different generations. The mobile phase utilized in HPLC consisted of methanol and water (75:25 v/v) at a flow rate of 1.7 ml/min. Separation was performed using an Agilent Eclipse column XDB-C18 (5 m, 4.6 × 150 mm) at a temperature of 40 ◦ C and the detection wavelength was 238 nm (Craparo et al., 2011). Samples (20 l) were injected via an autosampler. The molar solubility of BDP in 2% SDS was determined as a reference for comparison to that complexed in 2% dendrimers of different generations (Milhem et al., 2000). 2.2.4. Effect of pH on BDP solubility The molar solubility of BDP uncomplexed and when complexed with G3 dendrimers of molar concentration 0.00585 M was measured at three different pH values (9.8, 7.4 and 5). G3 was used as model dendrimers in this study. The pH of the medium was checked using a pH meter (Corning 220, Cole-Palmer, UK).
2.2.7. Statistical analysis All experiments were repeated in triplicate and the results were expressed as the mean ±standard deviation. Statistical analysis was undertaken using one way analysis of variance (ANOVA) followed by Tukey Kramer test using GraphPad InStat® software to check the statistical significance of differences. A difference was considered statistically significant if the p-value was ≤0.05. 3. Results and discussion 3.1. BDP-dendrimer phase solubility Fig. 2(a–d) shows the phase solubility diagrams of BDP in the presence of increasing concentrations of dendrimers at pH 9.8. For all formulations, the slopes were less than unity, suggesting that the complexes between BDP and dendrimers exhibited 1:1 stoichiometries (Devarakonda et al., 2004; Devarakonda et al., 2005). It has been reported that the interaction between dendrimer and drug molecules occurs either through encapsulation of the drug into the void spaces of dendrimer or by interaction of the drug with the dendrimer’s functional groups. The driving forces for the drug-dendrimer interactions are hydrogen bonding,
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Table 1 Stability constants and complexation efficiency for BDP-PAMAM complexes. Dendrimers generation
G3
G4
G4(12)
G4.5
K1:1 (M−1 ) Complexation efficiency (×103 )
3139 4.1
88,487 115.56
185,117 241.76
58.84 0.08
to the weak acidic nature of this drug (pKa = 13), thus, it existed in an unionized form at the pH range investigated. There was a significant increase in molar solubility of BDP upon increasing the concentration of all full generation dendrimers as shown in Fig. 2(a–c) (p < 0.05), which could be ascribed to the increased number of amine groups and internal cavities that are available to interact with or encapsulate BDP molecules (Yiyun et al., 2005; Cheng et al., 2007; Venkataraman et al., 2011). By contrast, increasing the concentration of the half generation dendrimer (G4.5) caused only a very slight trend of enhanced solubility of BDP (p > 0.05; Fig. 2d). This came in agreement with Devarakonda et al. (2005) upon loading niclosamide into half generation dendrimers, and in accordance with findings of Chauhan et al. (2003) by encapsulation of indometacin into G4.5 dendrimers. As shown in Fig. 2a and b, BDP exhibited a generation-dependent solubility, since increasing the dendrimer generation form G3 to G4 increased the molar solubility of the drug. This again may due to the increased number of interior amine groups in the dendrimer with generation size at a given pH, leading to the entrapment of more BDP molecules in the hydrophobic cores of the dendrimers (Yiyun et al., 2005; Cheng et al., 2007; Devarakonda et al., 2007; Ma et al., 2007). Results also showed that solubilization of the drug using G4(12) dendrimer was superior compared to solubilization using G4 dendrimers (Fig. 2b,c); this clearly emphasizes that the hydrophobicity of dendrimer core plays an important role in the solubilization of BDP molecules owing to enhanced hydrophobic interaction with the drug (Watkins et al., 1997; Cheng et al., 2008). Upon calculation of stability constants and complexation efficiency (Table 1), it was evident that amine terminated dendrimers (G3, G4 and G4(12)) formed very stable complexes with BDP while ester terminated (half-generation) dendrimers (G4.5) exhibited the lowest values. In a previous study, G4 and G4(12) showed more stable complexation with BDP than cyclodextrins (CD), which was reported by Worth et al. (1996) to be a 1:1 complexation having stability constant values of 33,313 and 10,428 M−1 for DM--CD and -CD respectively. This is also in agreement with Devarakonda et al. (2005) who reported dendrimers to be superior to CD for complexation with niclosamide. 3.2. Effect of pH on BDP solubility
Fig. 2. Solubility profiles of BDP in the presence of increasing concentrations of (a) G3, (b) G4, (c) G4(12) and (d) G4.5 PAMAM dendrimers (n = 3).
Van der Waals interactions and/or electrostatic attraction between opposite charged dendrimer and drug molecules (D’Emanuele and Attwood, 2005; Cheng et al., 2008; Svenson, 2009). BDP contains both hydrogen bond acceptor and donor groups, which could allow interaction with the dendrimer based on hydrogen bonding, in addition to the physical encapsulation within the dendrimer based on drug hydrophobicity. No marked electrostatic charges are expected to occur between dendrimers and BDP molecules owing
The inherent solubility of BDP was very low and did not change significantly (p > 0.05) by changing the pH of the medium (data not shown), due to the fact that BDP is a weak acid (pKa = 13.05). However, using G3 as a model dendrimer, the amount of BDP complexed with the dendrimers was found to increase upon increasing the pH from 5 to 7.4 with a maximum solubility found at pH 9.8 (Fig. 3). This might be attributed to the fact that PAMAM dendrimers have primary amines on their surfaces and tertiary amines within their internal cavities, which could act as hydrogen bond donors or acceptors, owing to the alteration of the protonation level of the amine groups in relation to pH (Cheng et al., 2007; Devarakonda et al., 2007). BDP can act as a hydrogen bond donor, promoting the formation of hydrogen bonds between the tertiary amines within the dendrimer cavity and BDP molecule at high pH media. In addition, the higher pH is expected to decrease the polarity of the dendrimer microenvironment and hence, promoting solubilization of the hydrophobic BDP molecules. This agrees with findings of other investigators using different hydrophobic drug molecules
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Fig. 3. Molar solubility of BDP complexed with G3 PAMAM dendrimers dispersed in media having pH values of 5, 7.4 or 9.8 (n = 3).
(Devarakonda et al., 2004; Cheng et al., 2007; Ma et al., 2007). For instance, nifedipine has a similar pKa to that of BDP and exhibited similar solubility behaviour in dendrimer solutions with a range of pH values (Devarakonda et al., 2004). These results suggest the pH-dependent solubility of BDP resulted from the pH-dependent hydrogen bond formation between BDP and dendrimer. As shown in Fig. 4, a comparison between the solubilization offered by dendrimers and a model micellar system using SDS at a concentration of 2% demonstrated that the solubilization capacity of both G4 and G4(12) dendrimers was significantly higher than that of SDS (p < 0.05), while G3 and G4.5 were significantly lower than SDS in their solubilizing capacity. This concurred with the results obtained by Milhem et al. (2000) who reported the superiority of G4 PAMAM dendrimers for solubilization of ibuprofen compared to the aqueous solution of the drug as well as 2% of SDS solution. The highest solubility of BDP was found with G4(12) dendrimers (Fig. 4), emphasizing that solubility of this hydrophobic drug directly correlates with the hydrophobicity of the dendrimer core. Since molar solubility studies revealed that full-generation dendrimers (G3, G4 and G4(12)) are superior to the halfgeneration dendrimer (G4.5) at solubilizing BDP (Fig. 2; Fig. 4), the half-generation dendrimer was excluded from the release and nebulization studies in subsequent studies.
Fig. 5. In vitro release of BDP from the complexes of G3, G4 and G4(12) dendrimers (n = 3).
release of the drug when complexed with dendrimers. These results are relatively consistent with those presented by Kolhe et al. (2003) who reported that drug–dendrimer complex is stable in methanol and water mixtures. Also, the release of ibuprofen from drug–dendrimer complex was found to be slower than that of pure ibuprofen (Milhem et al., 2000). Similarly, Ma et al. (2007) have reported that the release of sulfamethoxazole from the drug dendrimer solution was significantly slower compared to pure drug. After 8 h, 35.8 and 34.6% release was obtained for PAMAMG4(12)-DBP and PAMAM-G4-DBP complexes respectively, whilst 25% release was obtained for PAMAM-G3-DBP. The slower release of the drug from G3 complex might be attributed to the lower drug encapsulating capacity of G3 compared to that of G4 and G4(12) (Gupta et al., 2006). The slower release of BDP may indicate the stronger complex it forms upon encapsulation in the dendrimer. Controlled release of physically entrapped drugs within dendrimers has been previously reported (Liu and Fréchet, 1999).
3.3. In vitro release of BDP from dendrimers
3.4. Nebulizer output studies
Using dendrimer-free medium, 100% of BDP was released through the dialysis bag within 2 h (data not shown). By contrast, after 8 h less than 35% of BDP was released from dendrimer structures (Fig. 5), indicating the markedly slower and sustained
In this study, three nebulizers employing different operation mechanisms were used: Pari LC Sprint (air-jet), Aeroneb Pro (actively vibrating-mesh) and Omron MicroAir (passively vibrating-mesh) nebulizers. All medical nebulizers rely on the
Fig. 4. Comparison of inherent BDP molar aqueous solubility to 2% BDP-dendrimer complexes and SDS micellar system (n = 3).
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Fig. 6. Nebulization output of G3-BDP, G4-BDP and G4(12)-BDP dendrimers using Pari LC Sprint (air-jet), Aeroneb Pro (actively vibrating-mesh) and Omron MicroAir (passively vibrating-mesh) nebulizers (n = 3).
employment of energy to convert the liquid into inhalable aerosol droplets, and it is extremely important that the residual fluid left is minimal following completed nebulization (i.e. the output of the nebulizer is maximized) (Hess, 2000). Dispersed particles (especially particles in the micrometres size range) may not be efficiently nebulized, resulting in their concentration within the residual fluid of nebulizers (McCallion et al., 1996; O’Callaghan and Barry, 1997; Elhissi and Taylor, 2005). Our preliminary experiments have shown that all nebulizers employed in this study minimally concentrate nanoparticles such as dendrimers and nano-liposomes (data not shown), indicating that the measurement of output as aerosol output rather than dispersed phase output is valid in the present study. Thus, for convenience, the output of the nebulizers was measured by gravimetric determination of the aerosol output rather than drug output. Moreover, our preliminary investigations have confirmed that dendrimers are stable to nebulization, which is attributed to the small particle size of the dendrimers (Nasr et al., 2012b). All nebulizers generated high aerosol output regardless of dendrimer generation (Fig. 6). The aerosol output is arranged in the following order: Pari (air-jet) = Aeroneb Pro (active mesh) > Omron MicroAir (passive mesh) (Fig. 6). The Pari nebulizer shows a slightly higher output than the Aeroneb Pro device, but that was not statistically significant (p > 0.05). Thus, it seems that the energy provided by the passive vibrations of the Omron nebulizer was less efficient at converting the liquid into aerosols when compared to the energy provided by jet-nebulization of the Pari nebulizer or the active vibrations provided by the Aeroneb Pro device. This agrees with our recent findings using BDP niosomes (Elhissi et al., 2013). For each nebulizer, there was no effect of formulation (i.e. dendrimer generation) on the aerosol output (p > 0.05), being approximately 90–92% for the Pari (jet) nebulizer, 85–89% for the Aeroneb Pro (active mesh) nebulizer and 57–63% for the Omron (passive mesh) nebulizer (Fig. 6). It has been demonstrated that nebulization performance is influenced by fluid physicochemical properties such as viscosity, surface tension (McCallion et al., 1995; Ghazanfari et al., 2007) and the presence of electrolytes in the solution (Ghazanfari et al., 2007; Najlah et al., 2013). Thus, the absence of formulation effects on aerosol output suggests that the physicochemical properties of the different dendrimer formulations are highly similar; however, further studies are needed to investigate whether this assumption is valid. Overall, the output performance of dendrimers was influenced by nebulizer type rather than dendrimer generation (Fig. 6).
3.5. Aerosol droplet size and FPF Principally, for aerosols to be considered “respirable” (i.e. in FPF) droplet size should be smaller than 5–6 m (Stahlhofen et al., 1980; O’Callaghan and Barry, 1997). Patient-related factors such as lung pathophysiology, patient age and inhalation mode are additional factors that are known to affect the pulmonary deposition of inhaled aerosols; however, these factors are beyond the scope of this work. In this study, laser diffraction was used to determine droplet size, size distribution and FPF of dendrimer aerosols generated via nebulization. Laser diffraction has been shown to be highly reliable for size analysis of nebulized droplets, and has been reported to correlate with in vivo pulmonary deposition findings for non-volatile aerosols. Additionally, VMD measured by laser diffraction may be equivalent to the median aerodynamic diameter of nebulized droplets (Clark, 1995). It is worth noting that cascade impactors are more commonly used to analyze particle size of inhalable aerosols and attempts have been made to correlate the findings of cascade impaction with those of laser diffraction. A drawback of cascade impaction is solvent evaporation from liquid aerosols due to the air flow applied through the impactors; hence, correlation of cascade impaction with laser diffraction was possible when solvent evaporation was minimized either by cooling the cascade impactor before operation (Kwong et al., 2000) or by using low flow rate through the apparatus (None et al., 2001). Thus, the high reliability and convenience of laser diffraction justifies its use for the determination of size and size distribution of dendrimer aerosols. Size and size distribution of the aerosol droplets were expressed as VMD and span respectively (Table 2). The effect of formulation on the VMD and span was slight, and both vibrating-mesh devices generated droplets with larger VMD, higher span values and greater variability compared with the jet nebulizer (Table 2). These findings indicate that compressed gas forced through the “venturi” nozzle of the Pari nebulizer has provided more reliable means of generating small droplets with narrow size distribution as compared to the micro-perforated vibrating plates of the Aeroneb Pro and the Omron MicroAir nebulizers. In contrast to the findings of this study, other reports using liposomes have demonstrated narrower size distribution of vibrating-mesh nebulized aerosols (Elhissi and Taylor, 2005; Elhissi et al., 2006). Also, we reported that the vibrating-mesh devices failed at generating aerosols from amphotericin B nanoemulsions (Nasr et al., 2012a), whilst Amani et al. (2010) using steroid nanoemulsion formulations,
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Table 2 Size and size distribution of aerosol droplets generated from BDP-loaded PAMAM dendrimers of varying generations using air-jet and vibrating-mesh nebulizers. Dendrimer formulations VMD (m) Pari LC Sprint (air-jet) nebulizer Aeroneb Pro (active mesh) nebulizer Omron (passive mesh) nebulizer Span Pari LC Sprint (air-jet) nebulizer Aeroneb Pro (active mesh) nebulizer Omron (passive mesh) nebulizer
G3-BDP Complex
G4-BDP Complex
G4(12)-BDP Complex
4.05 ± 0.19 5.44 ± 1.64 5.30 ± 0.95
3.76 ± 0.23 5.21 ± 0.81 5.73 ± 2.41
4.25 ± 0.08 5.38 ± 1.47 5.82 ± 1.21
1.61 ± 0.17 2.21 ± 0.15 3.78 ± 1.67
1.68 ± 0.09 2.88 ± 0.75 2.83 ± 1.52
1.58 ± 0.21 2.89 ± 0.96 3.35 ± 0.49
Fig. 7. FPF of G3-BDP, G4-BDP and G4(12)-BDP dendrimers aerosols using Pari LC Sprint (air-jet), Aeroneb Pro (actively vibrating-mesh) and Omron MicroAir (passively vibrating-mesh) nebulizers (n = 3).
have reported that the Omron MicroAir (passive mesh) device exhibited superior performance compared with a standard jet nebulizer. The performance of vibrating-mesh nebulizers is highly dependent on formulation properties such as viscosity, surface tension (Ghazanfari et al., 2007) and the presence of electrolytes (Ghazanfari et al., 2007; Najlah et al., 2013). Thus, the conflicting findings amongst the various studies employing mesh nebulizers could be attributed to the marked effects formulation may have on the performance of aerosols generated using these devices. The size and size distribution (Table 2) both suggest that the aerosols of the three nebulizers are potentially suitable for generating “respirable” dendrimers. Aerosols of different dendrimer generations had different VMD and span values (p < 0.05) only when the Pari (air-jet) nebulizer was employed (Table 2). When the two vibrating-mesh devices were compared, VMD of the droplets was highly similar but the Aeroneb Pro (active mesh) nebulizer tended to generate aerosols with narrower size distribution (i.e. small span values) (Table 2), indicating that active mesh vibrations of the Aeroneb Pro may provide more control on the polydispersity of the aerosols compared with the passive vibrations of the Omron nebulizer. This contrasts with previous reports using liposomes (Elhissi et al., 2006) and niosomes (Elhissi et al., 2013) where the Omron MicroAir generated droplets with lower polydispersity, suggesting that this device behaves differently when different delivery systems are used. Nebulizers should generate small droplets with high output to ensure that a high proportion of the drug can potentially reach the peripheral airways. Thus, FPF of the aerosol was calculated by multiplying the aerosol output by the aerosol fraction ≤5.4 m. This figure was selected because it is the closest value to 5 m as presented by the Spraytec software. For each nebulizer, dendrimer generation did not affect the FPF (Fig. 7) which was consistent
with the findings of VMD and span (Table 2). However, the type of nebulizer significantly influenced FPF of the aerosols (p < 0.05) and the values were in the following order: Pari (air-jet) > Aeroneb Pro (active mesh) > Omron MicroAir (passive mesh) (Fig. 7). Despite the fact that passive and active vibrating mesh nebulizers both involve the presence of vibrating plates with multiple apertures, it is evident that since they operate according to different technologies they have significant differences in aerosol output (Fig. 6) and FPF (Fig. 7). 4. Conclusions BDP was successfully complexed with PAMAM dendrimers, and complexation was found to be dependent on generation and concentration of dendrimers and the pH of the dispersion medium. This work confirms the potential use of full generation PAMAM dendrimers as carriers for poorly soluble drugs such as BDP. The amine terminated dendrimers (G3, G4 and G4(12)) formed more stable complexes with BDP when compared with the ester terminated (half-generation; G4.5) dendrimers. The generation of dendrimer was also of great importance at enhancing the solubility of BDP. The highest solubility of BDP was found with G4(12) dendrimers, emphasizing that solubility of this hydrophobic drug directly correlates with the hydrophobicity of the dendrimer core. The sustained release profile and the high aerosol output and FPF suggests that dendrimers are a promising nanocarrier system for pulmonary delivery of BDP using air-jet or vibrating-mesh nebulizers. Pari LC Sprint (air-jet) and Aeroneb Pro (active mesh) nebulizers were more suitable for delivery of PAMAM dendrimers, producing high output and superior FPF. Overall, nebulization studies revealed that the aerosol properties of the dendrimer-BDP complexes were dependent on nebulizer design rather than dendrimer generation.
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