http://informahealthcare.com/ddi ISSN: 0363-9045 (print), 1520-5762 (electronic) Drug Dev Ind Pharm, Early Online: 1–7 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2014.956111
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RESEARCH ARTICLE
Development of dry powder inhaler formulation loaded with alendronate solid lipid nanoparticles: solid-state characterization and aerosol dispersion performance Jafar Ezzati Nazhad Dolatabadi1,2, Hamed Hamishehkar3, and Hadi Valizadeh3,4 1
Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran, 2Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran, 3Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran, and 4 Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran Abstract
Keywords
Alendronate sodium is a bisphosphonate drug used for the treatment of osteoporosis and acts as a specific inhibitor of osteoclast-mediated bone resorption. Inhalable solid lipid nanoparticles (SLNs) of the alendronate were successfully designed and developed by spraydried and co-spray dried inhalable mannitol from aqueous solution. Emulsification technique using a simple homogenization method was used for preparation of SLNs. In vitro deposition of the aerosolized drug was studied using a Next Generation Impactor at 60 L/min following the methodology described in the European and United States Pharmacopeias. The Carr’s Index, Hausner ratio and angle of repose were calculated as suitable criteria for estimation of the flow behavior of solids. Scanning electron microscopy showed spherical particle morphology of the respirable particles. The proposed spray-dried nanoparticulate-on-microparticles dry powders displayed good aerosol dispersion performance as dry powder inhalers with high values in emitted dose, fine particle fraction and mass median aerodynamic diameter. These results indicate that this novel inhalable spray-dried nanoparticulate-on-microparticles aerosol platform has great potential in systemic delivery of the drug.
Alendronate, dry powder inhaler, fine particle fraction, solid lipid nanoparticle
Introduction Osteoporosis is the widespread metabolic disease affecting the bone, which leads to an enhanced bone fragility and risk of fracture1. Alendronate sodium is a bisphosphonate drug, which increase bone formation, osteoblast proliferation and inhibit osteoblast apoptosis2. It is used for the treatment of various bone diseases such as osteoporosis, Paget’s disease and metastatic bone disease3,4. However, gastrointestinal intolerance including gastric and esophageal lesions and ulcers is the main obstacle for oral delivery of alendronate5–7. On the other hand, its high hydrophilicity and complexation with divalent cations, like Ca2+ leads to poor oral absorption8. To overcome the poor bioavailability of alendronate sodium and the undesirable gastrointestinal effects, investigations on alternative route of administration have been made9. The pulmonary route is frequently being studied for the purpose of improving drug targeting and hence improving aerosol efficiency. In order to be more effective, pharmaceutical aerosols should be deposited in the targeted site at a therapeutic dose within the lung10. The aerodynamic diameter (mass median aerodynamic diameter [MMAD]) and physical characteristics of aerosol Address for correspondence: Hadi Valizadeh, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. Tel: +98(411) 3392649. Fax: +98(411) 334-4798. E-mail: [email protected]
History Received 28 April 2014 Revised 5 August 2014 Accepted 13 August 2014 Published online 15 September 2014
particles are important factors that affect the regional drug deposition of inhalable particles in the lungs and thus conditioning the therapeutic efficacy10,11. Sutton et al. reported a 25-fold increased bioavailability of alendronate following nasal administration in dogs and rats in comparison to peroral rout12. Inhalable nano-particulate systems are preferred approach because of their potential to achieve uniform distribution of dose among the alveoli, a sustained drug release that consequently reduces the dosing frequency, suitable for delivery of macromolecules and decreased incidence of side effects13. Sultana et al. demonstrated an antisolvent precipitation technique for the preparation of nonpolymeric alendronate nanoparticles. The MMAD and deposition pattern of prepared nanoparticles was well suited for lung delivery14. Application of carrier particles leads in easily emitting of drug particles from capsules and devices and increases the inhalation efficiency. Therefore, the carrier forms a significant component of the formulation and any change in the physicochemical properties of the carrier particles can alter the drug deposition profile. Thus, the proper carrier particle is essential for the development of dry powder inhalations15. Currently, various sugars as well as lactose, sorbitol and mannitol were used as excipient in dry powder inhalers (DPIs). Because of clinical considerations, lactose or other sugars cannot be used for drug delivery to diabetic patients. Moreover, lactose intolerance is a problem that necessitates the patient to use lactose-free formulations. But since mannitol does
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not have a reducing sugar function and is less hygroscopic, it can be potential alternatives to lactose for use in DPIs16,17. Cruz et al. prepared spray-dried powders for lung delivery of sodium alendronate by addition of leucine and ammonium bicarbonate, which leads to porous particles with rough surfaces. They carried out bronchoalveolar lavage study, which showed that the intratracheal administration of sodium alendronate dry powder does not induce significant increases of lung toxicity indicators. Besides, the intratracheal administration of sodium alendronate dry powder results in a 3.5-fold increase as compared to oral bioavailability. Finally, they suggested that sodium alendronate pulmonary delivery could be a promising administration route18. Recently, the solid lipid nanoparticles (SLNs) aerosol proposed a novel area of research and are suggested as carriers for pulmonary anticancer or peptide to improve their bioavailability. The biodistribution of inhaled radiolabeled SLNs showed a significant uptake into the lymphatics after inhalation11,19. A high rate of distribution in periaortic, axillar and inguinal lymph nodes was observed demonstrating that SLNs could be efficient colloidal carriers for lympho-scintigraphy or therapy on pulmonary delivery11,19,20. However, there is not any report related to spray-dried alendronate-loaded SLNs with and without mannitol for pulmonary delivery. Therefore, a systematic and comprehensive study was conducted for rational design, physicochemical characterization and optimization of novel respirable alendronate nanoparticles-on-microparticles for systemic delivery.
Materials and methods Materials Alendronate monosodium trihydrate was purchased from Modava Company (Tehran, Iran). Poloxamer 407, Tween 80, mannitol and o-phthalaldehyde (OPA) were obtained from Sigma Aldrich Co. (Poole, UK). Compritol 888 ATO and 2-Mercaptoethanol were acquired from Gattefosse (Nanterre, France) and Merck (Darmstadt, Germany), respectively. SLN preparation Hot homogenization method was used for preparation of alendronate-loaded SLNs. Briefly, 2 g of Compritol 888 ATOÕ (solid lipid matrix) with 100 ml of Tween 80 as a surfactant was melted at 80 C under continuous stirring (oil phase). In another container, 2 g of Poloxamer 407 was dissolved in 80 ml of deionized water and heated to 80 C (aqueous phase). Two milliliters of aqueous phase containing 200 mg of alendronate sodium was added into the oil phase under homogenization at 20 000rpm (Heidolph, Schwabach, Germany) to form the initial water-in-oil emulsion. The remaining hot aqueous phase was poured dropwise into the oil phase under the homogenization at 20 000 rpm, while maintaining the temperature at 80 C. The obtained nano-emulsion was cooled down in order to acquire nano-suspension. Ultrafiltration method through centrifugal devices (AmiconÕ Ultra-4 100 k, Millipore, Temecula, CA) was used to separate SLNs from unloaded drug solution21–26. The encapsulation efficiency (EE) of alendronate sodium in SLNs was determined after ultrafiltration through centrifugal devices (AmiconÕ Ultra-4 100 k, Millipore) with a 100 kDa molecular weight cut-off membrane. In brief, filters were filled with SLNs nano-suspensions and centrifuged at 5000 rpm for 10 min. The ultrafilterate solution was collected to measure unloaded drug concentration. Since alendronate has no UV-visible absorbing molecular functional group, samples were analyzed through derivatization reaction with OPA in 0.05 M NaOH followed by UV-vis spectrophotometry (UV-mini 1240; Shimadzu, Kyoto, Japan) at
Drug Dev Ind Pharm, Early Online: 1–7
333 nm. The samples of blank SLNs exhibited no UV interfering substances. The method was fully validated for linearity, accuracy and precision. EE was calculated using equation (1)3,27,28: EEð%Þ¼
ðCT CAP Þ 100 CT
ð1Þ
where CT ¼ total drug concentration in SLNs, CAP ¼ alendronate sodium concentration in aqueous phase. Preparation of the spray-dried DPI Prepared sodium alendronate-loaded SLNs nanosuspensions (50 ml) with or without 1 g mannitol were spray-dried using a Bu¨chi B-191 Mini Spray Dryer (Bu¨chi, Flawil, Switzerland) equipped with a 0.7 mm two-fluid nozzle, using the following operational conditions: inlet temperature of 110 C, outlet temperature of 65 C, aspiration at 70% and feed pump rate of 10 ml/min. The dried solid particles stored in sealed capped amber vials after collection from an electrostatic precipitator and were stored in a desiccator at room temperature13. Solid state flowability Flowability of the spray-dried powders was assessed by determining the angle of repose and the Carr’s compressibility index. The angle of repose was measured by a fixed funnel method29. The pile was carefully built up by dropping the material through a funnel till the formed pile touches the tip of the funnel (height, 2 cm). The angle of repose was calculated by inverting the ratio of height and radius of the formed pile. The Carr index is a measure of the propensity of a powder to consolidate. Changes occurring in packing arrangement during the taping procedure are expressed as the Carr’s compressibility index (CI)30,31. Tapped density was calculated using 2 g of the sample after 200 mechanical taps in a measuring cylinder and CI was computed from the tapped and bulk density of spray-dried powders by Equation (2)32–34. CI ¼ 1 B 100 ð2Þ T Hausner ratio was computed from tapped density and bulk density using equation (3). Hausner ratio ¼ T ð3Þ B where T is tapped bulk density of powder and B is freely bulk density of powder. Particle size examination Particle size of the SLNs and DPI formulations were measured by laser light scattering technique using particle size analyzer (SALD 2101, Shimadzu). DPI prepared with and without mannitol was dispersed in water and ethanol saturated with mannitol solution, respectively. Then, mean particle sizes based on volume diameter and standard deviations were recorded. All of measurements were performed at least three times. The span calculation is the most common format to express distribution width. The span calculation and its value were calculated through the following equation (4): Span ¼
Dv0:9 Dv0:1 Dv0:5
ð4Þ
The Dv0:5 , the median has been defined above as the diameter where half of the population lies below this value. Similarly,
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DOI: 10.3109/03639045.2014.956111
90% of the distribution lies below the Dv0:9 , and 10% of the population lies below the Dv0:1 . Scanning electron microscopy examination Scanning electron microscopy (SEM; Vega Tescan, Prague, Czech Republic) analysis of the preparations were performed to study the morphological behaviors like sphericity and aggregation. Gold coating of the particles was done under vacuum prior to imaging and the scanning was performed at an accelerating voltage of 5 kV.
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In vitro drug release study Phosphate-buffered saline (PBS; pH 7.4) was used as the release medium for in vitro alendronate release from the SLNs. An amount of 2 ml of alendronate-loaded SLNs was transferred into a dialysis bag (cutoff 10 kDa) and placed into a beaker containing 20 ml of PBS. The beaker was then placed in 25 and 37 C and stirred at 100 rpm. At predetermined time intervals, 2 ml samples were withdrawn from the incubation medium and analyzed for the drug content by UV spectrophotometry at max ¼ 333 nm for pH 7.413. In vitro aerosolization study
ED ¼
FPD 100 Initial particle mass in capsule
Initial particle mass in capsule Final mass remaining in capsule Initial particle mass in capsule
Solid-state characterization of spray dried powders The bulk and tapped density values were attained for the both spray-dried powders. The tapped density of each powder can be used to predict both its flow properties and respirable fraction. Lowering the tapped density of the dry powders considerably enhance the respirable fraction. The Carr’s Index, Hausner ratio and angle of repose commonly considered as proper criteria for estimation of the flow properties of solids32–34. Mannitol spraydried powder showed good flow properties and low density. Spray-dried SLNs showed intermediate flow properties. It was observed that DPI preparations composed of SLNs loaded on mannitol had relatively higher process yields because of the lower stickiness to the inner surface of the wall of spray drier cyclone. Improved flowability in the presence of mannitol could be ascribed to the higher crystalline content, lower surface energy and adhesion35–38. In addition, mannitol is a moderate mucolytic agent, which may decrease mucous viscosity. This effect may, in turn, improve the transport of therapeutic agents to the absorption membrane or receptors existing under the mucous lining the respiratory tract36. The physical properties of the two types of DPI formulations are summarized in Table 1. Characterization of particle size and morphology
The prepared powders were aerosolized by means of dry powder inhalation device (Easy haler). The in vitro deposition of the aerosolized drug was studied using Next Generation ImpactorÕ (NGI; Model 170, Apparatus E; British Pharmacopoeia, 2010, Norwich, NR). A total of 25 mg of alendronate-load SLNs and 30 mg of alendronate-load SLNs containing mannitol carrier spray-dried powders were loaded into hard gelatin capsules (size 3). In order to have a pressure of 4 k Pa, an air stream of 60 L/min was produced throughout the system by attaching the outlet of the using NGI to a vacuum pump for 4 s. The powder deposited in stages 1–8, the mouthpiece and the pre-separator device were collected by rinsing with concentrated dichloromethane solution and the drug was extracted by ultra-pure water. The drug content was then determined by aforementioned method. From drug deposition data the percentage of emitted dose (ED), fine particle fraction (FPF), mass MMAD and geometric standard deviation were calculated. CITDAS version 3.10, data processing software (Copley Scientific, Nottingham, UK) was used for data analysis13. The FPF and ED were calculated as follows (Equation 5 and 6): FPF ¼
3
ð5Þ
100
ð6Þ
Results and discussion Preparation and drug EE SLNs were prepared using Compritol 888 ATOÕ , a combination of Tween 80 as a surfactant and Poloxamer 407 as a co-surfactant. Several formulations were prepared with Compritol with EE from 73.7 to 85.6. The EE of prepared SLNs was improved by utilization of Tween 80. Probably due to the presence of this surfactant, which directly supports nanoparticle formation process, the minimum size and high drug loading was obtained. Consequently, the key for preparation of stable alendronateloaded SLNs was based on a selection of proper surfactant amount. The optimum SLN formulation was selected based on EE and particle size for the next examination24.
The particle size of prepared drug-loaded SLNs and spray-dried DPI preparations with and without mannitol was measured. SLN formulation exhibited a small particle size below 100 nm. The standard deviation was around 3.78 (polydispersity index below 0.25), indicating a narrow particle size distribution. Spray-dried DPI formulation containing SLNs with or without mannitol showed size distribution with a mean size of about 6 mm (Figure 1A–C). Furthermore, calculated span quantity confirmed the narrow distribution width, which were 2.08, 4.46 and 3.54 for drug-loaded SLNs, spray-dried DPI without and with mannitol, respectively. The SEM images indicated a regular and spheroidal shape with smooth surfaces for the spray-dried particles (Figure 2A–C). It is supposed that drying process from the surface part of the aqueous solution of mannitol results in spheroidal shape with smooth. The particles produced with and without mannitol were all spheroidal, which could be a valuable characteristic for the aerosol dispersion performance of DPI preparations35,39,40. Besides, a bit differences in the size attained from SEM and particle size analyzer origins from the fact that particle size analyzer calculate the mean of all particles presented in the solution, but SEM image is selective and maximum size in selective part contain very lower amount. In vitro drug release study To show the effect of temperature on the release of drug from SLNs, dialysis method has been used. Dialysis membrane retained lipid nanoparticles and allowed the transfer alendronate of in dissolution medium. Alendronate release percentage from SLNs was calculated and presented in Figure 3. As it is clear in Figure 3, drug was released from SLNs in a biphasical model with an initial moderate rate followed by a slower one. This moderate release could be due to the hydrophilic properties of alendronate. Furthermore, with increasing the temperature, the overall release of drug from SLNs slightly increased. The faster drug release in high temperature could be contributed to accelerating corrosion of SLNs. Since adverse gastrointestinal effects of alendronate and accumulation of it in non-calcified tissues have been reported, the low toxicity of sodium alendronate-loaded SLNs may be due to the moderate release properties of alendronate18,24,41.
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Table 1. The physical properties of inhaler spray-dried powders of alendronate-loaded SLNs with and without mannitol.
Powder type
Tapped density (g/cm3)
Angle of repose ( )
Carr’s compressibility index
Hausner ratio
0.082 ± 0.005 0.103 ± 0.007
0.102 ± 0.006 0.112 ± 0.006
40.74 ± 0.81 44.89 ± 0.32
19.60 ± 0.83 8.929 ± 0.54
1.24 ± 0.0 1.09 ± 0.0
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Spray-dried alendronate-loaded SLNs Spray-dried alendronate-loaded SLNs with mannitol
Bulk density (g/cm3)
Figure 1. The particle size distribution of prepared SLNs and related powders (A) alendronate-loaded SLNs, (B) spray-dried alendronate-loaded SLNs and (C) spray-dried alendronate-loaded SLNs with mannitol.
In vitro aerosol performance of spray-dried alendronate- loaded SLNs Aerosol performance of the spray-dried powders was studied in vitro using NGI equipped with EasyhalerÕ. The ED%, FPF%, MMAD and geometric standard deviation for DPI preparations
with and without mannitol are listed in Table 2. There was no considerable difference between the ED values of prepared formulations. This might be attributed to their improved flowability. Furthermore, one of the most important factors that influence deposition is particle size. It has effect both on the site of deposition and the mass of inhaled drug that deposits in the
Dry powder inhalation of alendronate-loaded SLNs
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DOI: 10.3109/03639045.2014.956111
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Figure 2. SEM images of SLN formulations (A) alendronate-loaded SLNs, (B) spray-dried alendronate-loaded SLNs and (C) spray-dried alendronate-loaded SLNs with mannitol. Figure 3. In vitro release profiles of alendronate from SLNs in 25 and 37 C.
Table 2. The emitted dose (ED) %, fine particle fraction (FPF) %, mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) quantities for powders prepared with and without mannitol. Powder type Spray-dried alendronate- loaded SLNs Spray-dried alendronate- loaded SLNs containing mannitol
ED %
FPF %
MMAD
GSD
96.34 ± 0.98 90.06 ± 0.48
11.87 ± 0.22 9.030 ± 0.13
6.385 ± 0.10 6.715 ± 0.12
2.03 ± 0.013 2.40 ± 0.019
respiratory tract. It is well distinguished that the fate of aerosol particles is mostly determined by the MMAD. The MMAD of the particles is diameter of a sphere of unit density (1 g/cm3) that has the same aerodynamic behavior as the particle under consideration. For therapeutic aerosols, particles 410 mm are already deposited in mouth/nose, pharynx and larynx by impaction and cannot enter the lungs. The larger the particles and the higher the air flow, the more efficient is the deposition by impaction, consequently the number of particles reaching lung periphery reduces. Particles between 0.1 and 1 mm in size are not well deposited in the lungs and a high fraction is usually exhaled11,19. The MMAD of the particles were 6.385 and 6.715 for spray-dried alendronate-loaded SLNs in the absence and presence of mannitol, respectively. Therefore, both prepared powder could be good candidates for DPI application. Figure 4 demonstrates the amount
of drug deposited on various stages of NGI. As it is evident, spray-dried DPI formulation of SLNs without mannitol had slightly higher FPF of 11.87% compared to the formulation containing mannitol (FPF ¼ 9.03%). The slight differences may be due to the larger size of the DPI, differences in hygroscopicity of the materials and surface properties and presence of agglomerated particles in the air stream during inspiration through the inhaler13,39,40. However, mannitol is less hygroscopic than other sugar-based carriers due to the fact that it does not have a reducing sugar function and offer lower adhesion and better release of the active ingredient. It provides a high sweet aftertaste, which can be used to verify to the patient that a dose has been successfully administered17,42,43. Besides, the safety and lack of toxicity of alendronate sodium loaded SLN to A549 cells was confirmed by various cytotoxicity methods and flow cytometry
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Figure 4. Drug deposition patterns of spray-dried alendronate-loaded SLNs with and without mannitol in the NGI.
analysis in our previous work24. Therefore, it is the most favorable candidate for DPI application than some of the other sugars42,44.
Conclusion This study demonstrates that spray drying with and without mannitol is a suitable technique for the preparation DPI formulation containing alendronate-loaded SLNs. The physicochemical characteristics, good flowability and acceptable aerosol dispersion performance of both powders indicate that they can be used as suitable vectors for alendronate delivery to the middle and deep regions of the lungs in a targeted fashion with the intention of improved systemic bioavailability. These results propose that the pulmonary delivery of alendronate could be a reliable and efficient administration method. Overall, this study might be useful for the development of novel alendronate DPIs with maximal fine particle dose.
Declaration of interest The authors report no conflicts of interest. Authors would like to thank Research Center for Pharmaceutical Nanotechnology (RCPN), Tabriz University of Medical Sciences for supporting this project (grant no: 5/87/260, which was a part of PhD thesis no: 90/014/104/2).
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RESEARCH ARTICLE
Development of dry powder inhaler formulation loaded with alendronate solid lipid nanoparticles: solid-state characterization and aerosol dispersion performance Jafar Ezzati Nazhad Dolatabadi1,2, Hamed Hamishehkar3, and Hadi Valizadeh3,4 1
Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran, 2Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran, 3Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran, and 4 Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran Abstract
Keywords
Alendronate sodium is a bisphosphonate drug used for the treatment of osteoporosis and acts as a specific inhibitor of osteoclast-mediated bone resorption. Inhalable solid lipid nanoparticles (SLNs) of the alendronate were successfully designed and developed by spraydried and co-spray dried inhalable mannitol from aqueous solution. Emulsification technique using a simple homogenization method was used for preparation of SLNs. In vitro deposition of the aerosolized drug was studied using a Next Generation Impactor at 60 L/min following the methodology described in the European and United States Pharmacopeias. The Carr’s Index, Hausner ratio and angle of repose were calculated as suitable criteria for estimation of the flow behavior of solids. Scanning electron microscopy showed spherical particle morphology of the respirable particles. The proposed spray-dried nanoparticulate-on-microparticles dry powders displayed good aerosol dispersion performance as dry powder inhalers with high values in emitted dose, fine particle fraction and mass median aerodynamic diameter. These results indicate that this novel inhalable spray-dried nanoparticulate-on-microparticles aerosol platform has great potential in systemic delivery of the drug.
Alendronate, dry powder inhaler, fine particle fraction, solid lipid nanoparticle
Introduction Osteoporosis is the widespread metabolic disease affecting the bone, which leads to an enhanced bone fragility and risk of fracture1. Alendronate sodium is a bisphosphonate drug, which increase bone formation, osteoblast proliferation and inhibit osteoblast apoptosis2. It is used for the treatment of various bone diseases such as osteoporosis, Paget’s disease and metastatic bone disease3,4. However, gastrointestinal intolerance including gastric and esophageal lesions and ulcers is the main obstacle for oral delivery of alendronate5–7. On the other hand, its high hydrophilicity and complexation with divalent cations, like Ca2+ leads to poor oral absorption8. To overcome the poor bioavailability of alendronate sodium and the undesirable gastrointestinal effects, investigations on alternative route of administration have been made9. The pulmonary route is frequently being studied for the purpose of improving drug targeting and hence improving aerosol efficiency. In order to be more effective, pharmaceutical aerosols should be deposited in the targeted site at a therapeutic dose within the lung10. The aerodynamic diameter (mass median aerodynamic diameter [MMAD]) and physical characteristics of aerosol Address for correspondence: Hadi Valizadeh, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. Tel: +98(411) 3392649. Fax: +98(411) 334-4798. E-mail: [email protected]
History Received 28 April 2014 Revised 5 August 2014 Accepted 13 August 2014 Published online 15 September 2014
particles are important factors that affect the regional drug deposition of inhalable particles in the lungs and thus conditioning the therapeutic efficacy10,11. Sutton et al. reported a 25-fold increased bioavailability of alendronate following nasal administration in dogs and rats in comparison to peroral rout12. Inhalable nano-particulate systems are preferred approach because of their potential to achieve uniform distribution of dose among the alveoli, a sustained drug release that consequently reduces the dosing frequency, suitable for delivery of macromolecules and decreased incidence of side effects13. Sultana et al. demonstrated an antisolvent precipitation technique for the preparation of nonpolymeric alendronate nanoparticles. The MMAD and deposition pattern of prepared nanoparticles was well suited for lung delivery14. Application of carrier particles leads in easily emitting of drug particles from capsules and devices and increases the inhalation efficiency. Therefore, the carrier forms a significant component of the formulation and any change in the physicochemical properties of the carrier particles can alter the drug deposition profile. Thus, the proper carrier particle is essential for the development of dry powder inhalations15. Currently, various sugars as well as lactose, sorbitol and mannitol were used as excipient in dry powder inhalers (DPIs). Because of clinical considerations, lactose or other sugars cannot be used for drug delivery to diabetic patients. Moreover, lactose intolerance is a problem that necessitates the patient to use lactose-free formulations. But since mannitol does
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not have a reducing sugar function and is less hygroscopic, it can be potential alternatives to lactose for use in DPIs16,17. Cruz et al. prepared spray-dried powders for lung delivery of sodium alendronate by addition of leucine and ammonium bicarbonate, which leads to porous particles with rough surfaces. They carried out bronchoalveolar lavage study, which showed that the intratracheal administration of sodium alendronate dry powder does not induce significant increases of lung toxicity indicators. Besides, the intratracheal administration of sodium alendronate dry powder results in a 3.5-fold increase as compared to oral bioavailability. Finally, they suggested that sodium alendronate pulmonary delivery could be a promising administration route18. Recently, the solid lipid nanoparticles (SLNs) aerosol proposed a novel area of research and are suggested as carriers for pulmonary anticancer or peptide to improve their bioavailability. The biodistribution of inhaled radiolabeled SLNs showed a significant uptake into the lymphatics after inhalation11,19. A high rate of distribution in periaortic, axillar and inguinal lymph nodes was observed demonstrating that SLNs could be efficient colloidal carriers for lympho-scintigraphy or therapy on pulmonary delivery11,19,20. However, there is not any report related to spray-dried alendronate-loaded SLNs with and without mannitol for pulmonary delivery. Therefore, a systematic and comprehensive study was conducted for rational design, physicochemical characterization and optimization of novel respirable alendronate nanoparticles-on-microparticles for systemic delivery.
Materials and methods Materials Alendronate monosodium trihydrate was purchased from Modava Company (Tehran, Iran). Poloxamer 407, Tween 80, mannitol and o-phthalaldehyde (OPA) were obtained from Sigma Aldrich Co. (Poole, UK). Compritol 888 ATO and 2-Mercaptoethanol were acquired from Gattefosse (Nanterre, France) and Merck (Darmstadt, Germany), respectively. SLN preparation Hot homogenization method was used for preparation of alendronate-loaded SLNs. Briefly, 2 g of Compritol 888 ATOÕ (solid lipid matrix) with 100 ml of Tween 80 as a surfactant was melted at 80 C under continuous stirring (oil phase). In another container, 2 g of Poloxamer 407 was dissolved in 80 ml of deionized water and heated to 80 C (aqueous phase). Two milliliters of aqueous phase containing 200 mg of alendronate sodium was added into the oil phase under homogenization at 20 000rpm (Heidolph, Schwabach, Germany) to form the initial water-in-oil emulsion. The remaining hot aqueous phase was poured dropwise into the oil phase under the homogenization at 20 000 rpm, while maintaining the temperature at 80 C. The obtained nano-emulsion was cooled down in order to acquire nano-suspension. Ultrafiltration method through centrifugal devices (AmiconÕ Ultra-4 100 k, Millipore, Temecula, CA) was used to separate SLNs from unloaded drug solution21–26. The encapsulation efficiency (EE) of alendronate sodium in SLNs was determined after ultrafiltration through centrifugal devices (AmiconÕ Ultra-4 100 k, Millipore) with a 100 kDa molecular weight cut-off membrane. In brief, filters were filled with SLNs nano-suspensions and centrifuged at 5000 rpm for 10 min. The ultrafilterate solution was collected to measure unloaded drug concentration. Since alendronate has no UV-visible absorbing molecular functional group, samples were analyzed through derivatization reaction with OPA in 0.05 M NaOH followed by UV-vis spectrophotometry (UV-mini 1240; Shimadzu, Kyoto, Japan) at
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333 nm. The samples of blank SLNs exhibited no UV interfering substances. The method was fully validated for linearity, accuracy and precision. EE was calculated using equation (1)3,27,28: EEð%Þ¼
ðCT CAP Þ 100 CT
ð1Þ
where CT ¼ total drug concentration in SLNs, CAP ¼ alendronate sodium concentration in aqueous phase. Preparation of the spray-dried DPI Prepared sodium alendronate-loaded SLNs nanosuspensions (50 ml) with or without 1 g mannitol were spray-dried using a Bu¨chi B-191 Mini Spray Dryer (Bu¨chi, Flawil, Switzerland) equipped with a 0.7 mm two-fluid nozzle, using the following operational conditions: inlet temperature of 110 C, outlet temperature of 65 C, aspiration at 70% and feed pump rate of 10 ml/min. The dried solid particles stored in sealed capped amber vials after collection from an electrostatic precipitator and were stored in a desiccator at room temperature13. Solid state flowability Flowability of the spray-dried powders was assessed by determining the angle of repose and the Carr’s compressibility index. The angle of repose was measured by a fixed funnel method29. The pile was carefully built up by dropping the material through a funnel till the formed pile touches the tip of the funnel (height, 2 cm). The angle of repose was calculated by inverting the ratio of height and radius of the formed pile. The Carr index is a measure of the propensity of a powder to consolidate. Changes occurring in packing arrangement during the taping procedure are expressed as the Carr’s compressibility index (CI)30,31. Tapped density was calculated using 2 g of the sample after 200 mechanical taps in a measuring cylinder and CI was computed from the tapped and bulk density of spray-dried powders by Equation (2)32–34. CI ¼ 1 B 100 ð2Þ T Hausner ratio was computed from tapped density and bulk density using equation (3). Hausner ratio ¼ T ð3Þ B where T is tapped bulk density of powder and B is freely bulk density of powder. Particle size examination Particle size of the SLNs and DPI formulations were measured by laser light scattering technique using particle size analyzer (SALD 2101, Shimadzu). DPI prepared with and without mannitol was dispersed in water and ethanol saturated with mannitol solution, respectively. Then, mean particle sizes based on volume diameter and standard deviations were recorded. All of measurements were performed at least three times. The span calculation is the most common format to express distribution width. The span calculation and its value were calculated through the following equation (4): Span ¼
Dv0:9 Dv0:1 Dv0:5
ð4Þ
The Dv0:5 , the median has been defined above as the diameter where half of the population lies below this value. Similarly,
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90% of the distribution lies below the Dv0:9 , and 10% of the population lies below the Dv0:1 . Scanning electron microscopy examination Scanning electron microscopy (SEM; Vega Tescan, Prague, Czech Republic) analysis of the preparations were performed to study the morphological behaviors like sphericity and aggregation. Gold coating of the particles was done under vacuum prior to imaging and the scanning was performed at an accelerating voltage of 5 kV.
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In vitro drug release study Phosphate-buffered saline (PBS; pH 7.4) was used as the release medium for in vitro alendronate release from the SLNs. An amount of 2 ml of alendronate-loaded SLNs was transferred into a dialysis bag (cutoff 10 kDa) and placed into a beaker containing 20 ml of PBS. The beaker was then placed in 25 and 37 C and stirred at 100 rpm. At predetermined time intervals, 2 ml samples were withdrawn from the incubation medium and analyzed for the drug content by UV spectrophotometry at max ¼ 333 nm for pH 7.413. In vitro aerosolization study
ED ¼
FPD 100 Initial particle mass in capsule
Initial particle mass in capsule Final mass remaining in capsule Initial particle mass in capsule
Solid-state characterization of spray dried powders The bulk and tapped density values were attained for the both spray-dried powders. The tapped density of each powder can be used to predict both its flow properties and respirable fraction. Lowering the tapped density of the dry powders considerably enhance the respirable fraction. The Carr’s Index, Hausner ratio and angle of repose commonly considered as proper criteria for estimation of the flow properties of solids32–34. Mannitol spraydried powder showed good flow properties and low density. Spray-dried SLNs showed intermediate flow properties. It was observed that DPI preparations composed of SLNs loaded on mannitol had relatively higher process yields because of the lower stickiness to the inner surface of the wall of spray drier cyclone. Improved flowability in the presence of mannitol could be ascribed to the higher crystalline content, lower surface energy and adhesion35–38. In addition, mannitol is a moderate mucolytic agent, which may decrease mucous viscosity. This effect may, in turn, improve the transport of therapeutic agents to the absorption membrane or receptors existing under the mucous lining the respiratory tract36. The physical properties of the two types of DPI formulations are summarized in Table 1. Characterization of particle size and morphology
The prepared powders were aerosolized by means of dry powder inhalation device (Easy haler). The in vitro deposition of the aerosolized drug was studied using Next Generation ImpactorÕ (NGI; Model 170, Apparatus E; British Pharmacopoeia, 2010, Norwich, NR). A total of 25 mg of alendronate-load SLNs and 30 mg of alendronate-load SLNs containing mannitol carrier spray-dried powders were loaded into hard gelatin capsules (size 3). In order to have a pressure of 4 k Pa, an air stream of 60 L/min was produced throughout the system by attaching the outlet of the using NGI to a vacuum pump for 4 s. The powder deposited in stages 1–8, the mouthpiece and the pre-separator device were collected by rinsing with concentrated dichloromethane solution and the drug was extracted by ultra-pure water. The drug content was then determined by aforementioned method. From drug deposition data the percentage of emitted dose (ED), fine particle fraction (FPF), mass MMAD and geometric standard deviation were calculated. CITDAS version 3.10, data processing software (Copley Scientific, Nottingham, UK) was used for data analysis13. The FPF and ED were calculated as follows (Equation 5 and 6): FPF ¼
3
ð5Þ
100
ð6Þ
Results and discussion Preparation and drug EE SLNs were prepared using Compritol 888 ATOÕ , a combination of Tween 80 as a surfactant and Poloxamer 407 as a co-surfactant. Several formulations were prepared with Compritol with EE from 73.7 to 85.6. The EE of prepared SLNs was improved by utilization of Tween 80. Probably due to the presence of this surfactant, which directly supports nanoparticle formation process, the minimum size and high drug loading was obtained. Consequently, the key for preparation of stable alendronateloaded SLNs was based on a selection of proper surfactant amount. The optimum SLN formulation was selected based on EE and particle size for the next examination24.
The particle size of prepared drug-loaded SLNs and spray-dried DPI preparations with and without mannitol was measured. SLN formulation exhibited a small particle size below 100 nm. The standard deviation was around 3.78 (polydispersity index below 0.25), indicating a narrow particle size distribution. Spray-dried DPI formulation containing SLNs with or without mannitol showed size distribution with a mean size of about 6 mm (Figure 1A–C). Furthermore, calculated span quantity confirmed the narrow distribution width, which were 2.08, 4.46 and 3.54 for drug-loaded SLNs, spray-dried DPI without and with mannitol, respectively. The SEM images indicated a regular and spheroidal shape with smooth surfaces for the spray-dried particles (Figure 2A–C). It is supposed that drying process from the surface part of the aqueous solution of mannitol results in spheroidal shape with smooth. The particles produced with and without mannitol were all spheroidal, which could be a valuable characteristic for the aerosol dispersion performance of DPI preparations35,39,40. Besides, a bit differences in the size attained from SEM and particle size analyzer origins from the fact that particle size analyzer calculate the mean of all particles presented in the solution, but SEM image is selective and maximum size in selective part contain very lower amount. In vitro drug release study To show the effect of temperature on the release of drug from SLNs, dialysis method has been used. Dialysis membrane retained lipid nanoparticles and allowed the transfer alendronate of in dissolution medium. Alendronate release percentage from SLNs was calculated and presented in Figure 3. As it is clear in Figure 3, drug was released from SLNs in a biphasical model with an initial moderate rate followed by a slower one. This moderate release could be due to the hydrophilic properties of alendronate. Furthermore, with increasing the temperature, the overall release of drug from SLNs slightly increased. The faster drug release in high temperature could be contributed to accelerating corrosion of SLNs. Since adverse gastrointestinal effects of alendronate and accumulation of it in non-calcified tissues have been reported, the low toxicity of sodium alendronate-loaded SLNs may be due to the moderate release properties of alendronate18,24,41.
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Table 1. The physical properties of inhaler spray-dried powders of alendronate-loaded SLNs with and without mannitol.
Powder type
Tapped density (g/cm3)
Angle of repose ( )
Carr’s compressibility index
Hausner ratio
0.082 ± 0.005 0.103 ± 0.007
0.102 ± 0.006 0.112 ± 0.006
40.74 ± 0.81 44.89 ± 0.32
19.60 ± 0.83 8.929 ± 0.54
1.24 ± 0.0 1.09 ± 0.0
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Spray-dried alendronate-loaded SLNs Spray-dried alendronate-loaded SLNs with mannitol
Bulk density (g/cm3)
Figure 1. The particle size distribution of prepared SLNs and related powders (A) alendronate-loaded SLNs, (B) spray-dried alendronate-loaded SLNs and (C) spray-dried alendronate-loaded SLNs with mannitol.
In vitro aerosol performance of spray-dried alendronate- loaded SLNs Aerosol performance of the spray-dried powders was studied in vitro using NGI equipped with EasyhalerÕ. The ED%, FPF%, MMAD and geometric standard deviation for DPI preparations
with and without mannitol are listed in Table 2. There was no considerable difference between the ED values of prepared formulations. This might be attributed to their improved flowability. Furthermore, one of the most important factors that influence deposition is particle size. It has effect both on the site of deposition and the mass of inhaled drug that deposits in the
Dry powder inhalation of alendronate-loaded SLNs
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DOI: 10.3109/03639045.2014.956111
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Figure 2. SEM images of SLN formulations (A) alendronate-loaded SLNs, (B) spray-dried alendronate-loaded SLNs and (C) spray-dried alendronate-loaded SLNs with mannitol. Figure 3. In vitro release profiles of alendronate from SLNs in 25 and 37 C.
Table 2. The emitted dose (ED) %, fine particle fraction (FPF) %, mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) quantities for powders prepared with and without mannitol. Powder type Spray-dried alendronate- loaded SLNs Spray-dried alendronate- loaded SLNs containing mannitol
ED %
FPF %
MMAD
GSD
96.34 ± 0.98 90.06 ± 0.48
11.87 ± 0.22 9.030 ± 0.13
6.385 ± 0.10 6.715 ± 0.12
2.03 ± 0.013 2.40 ± 0.019
respiratory tract. It is well distinguished that the fate of aerosol particles is mostly determined by the MMAD. The MMAD of the particles is diameter of a sphere of unit density (1 g/cm3) that has the same aerodynamic behavior as the particle under consideration. For therapeutic aerosols, particles 410 mm are already deposited in mouth/nose, pharynx and larynx by impaction and cannot enter the lungs. The larger the particles and the higher the air flow, the more efficient is the deposition by impaction, consequently the number of particles reaching lung periphery reduces. Particles between 0.1 and 1 mm in size are not well deposited in the lungs and a high fraction is usually exhaled11,19. The MMAD of the particles were 6.385 and 6.715 for spray-dried alendronate-loaded SLNs in the absence and presence of mannitol, respectively. Therefore, both prepared powder could be good candidates for DPI application. Figure 4 demonstrates the amount
of drug deposited on various stages of NGI. As it is evident, spray-dried DPI formulation of SLNs without mannitol had slightly higher FPF of 11.87% compared to the formulation containing mannitol (FPF ¼ 9.03%). The slight differences may be due to the larger size of the DPI, differences in hygroscopicity of the materials and surface properties and presence of agglomerated particles in the air stream during inspiration through the inhaler13,39,40. However, mannitol is less hygroscopic than other sugar-based carriers due to the fact that it does not have a reducing sugar function and offer lower adhesion and better release of the active ingredient. It provides a high sweet aftertaste, which can be used to verify to the patient that a dose has been successfully administered17,42,43. Besides, the safety and lack of toxicity of alendronate sodium loaded SLN to A549 cells was confirmed by various cytotoxicity methods and flow cytometry
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Figure 4. Drug deposition patterns of spray-dried alendronate-loaded SLNs with and without mannitol in the NGI.
analysis in our previous work24. Therefore, it is the most favorable candidate for DPI application than some of the other sugars42,44.
Conclusion This study demonstrates that spray drying with and without mannitol is a suitable technique for the preparation DPI formulation containing alendronate-loaded SLNs. The physicochemical characteristics, good flowability and acceptable aerosol dispersion performance of both powders indicate that they can be used as suitable vectors for alendronate delivery to the middle and deep regions of the lungs in a targeted fashion with the intention of improved systemic bioavailability. These results propose that the pulmonary delivery of alendronate could be a reliable and efficient administration method. Overall, this study might be useful for the development of novel alendronate DPIs with maximal fine particle dose.
Declaration of interest The authors report no conflicts of interest. Authors would like to thank Research Center for Pharmaceutical Nanotechnology (RCPN), Tabriz University of Medical Sciences for supporting this project (grant no: 5/87/260, which was a part of PhD thesis no: 90/014/104/2).
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