International Journal of Pharmaceutics 491 (2015) 292–298
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Dimethyl silicone dry nanoemulsion inhalations: Formulation study and anti-acute lung injury effect Lifei Zhua,b,1, Miao Lib,1, Junxing Dongb , Yiguang Jina,b,* a b
Anhui Medical University, Hefei 230001, China Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 April 2015 Received in revised form 15 June 2015 Accepted 23 June 2015 Available online 30 June 2015
Acute lung injury (ALI) is a severe disease, leading to death if not treated quickly. An emergency medicine is necessary for ALI therapy. Dimethyl silicone (DMS) is an effective agent to defoam the bubbles in the lung induced by ALI. However, DMS aerosols, a marketed formulation of DMS, affect environments and will be limited in the future. Here we firstly report a dry nanoemulsion inhalation for pulmonary delivery. Novel DMS dry nanoemulsion inhalations (DSNIs) were developed in this study. The optimal formulation of stable and homogenous DMS nanoemulsions (DSNs) was composed of Cremophor RH40/PEG 400/DMS (4:4:2, w/w/w) and water. The DSNs showed the tiny size of 19.8 nm, the zeta potential of 9.66 mV, and the low polydispersity index (PDI) of 0.37. The type of DSNs was identified as oil-in-water. The DSNs were added with mannitol followed by freeze-drying to obtain the DSNIs that were loose white powders, showed good fluidity, and were capable of rapid reconstitution to DSNs. The DSNs could adhere on the surfaces of lyophilized mannitol crystals. The aerodynamic diameter of DSNIs was 4.82 mm, suitable for pulmonary inhalation. The in vitro defoaming rate of DSNIs was 1.25 ml/s, much faster than those of the blank DSNIs, DMS, and DMS aerosols. The DSNIs showed significantly higher anti-ALI effect on the ALI rat models than the blank DSNIs and the DMS aerosols according to lung appearances, histological sections, and lung wet weight/dry weight ratios. The DSNIs are effective anti-ALI nanomedicines. The novel DMS formulation is a promising replacement of DMS aerosols. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Acute lung injury Defoaming Dimethyl silicone Dry powder inhalation Nanoemulsion
1. Introduction Acute lung injury (ALI) is a severe disease, which can be induced by many factors, such as acidic gas inhalation (Lim et al., 2007), and propellant poisoning (Cao et al., 2014). At the early stage of ALI, a large number of bubbles is produced and could block up the bronchus followed by hypoxia and dyspnea. Severe cases of bubble blocking can result in death if not treated quickly (Wheeler and Bernard, 2007). The mortality of ALI is reported to be 30–60% and more in the elders (Elder et al., 2012; Lv et al., 2014). An emergency medicine is necessary for ALI therapy. Dimethyl silicone (DMS) is an oily colorless transparent liquid with high flexibility of backbones (Gräbner et al., 2010). Besides widely industrial applications, the most important biological function of DMS is defoaming after entering the films of bubbles
* Corresponding author at: Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850, China. Fax: +86 10 68214653. E-mail address: [email protected] (Y. Jin). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijpharm.2015.06.041 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
and destroying the mechanical equilibrium of oriented films due to its weak surface tension, finally leading to the bubbles breaking (Watson, 2014). Therefore, DMS is mainly used as a defoaming agent to eliminate the bubbles in the various sites of the body, such as the gastro-enteric route and the lung. DMS tablets and powders are used as the defoamers to relieve flatulence (Jewell, 2007; Torrado et al., 1999), and make gastroscopic and enterscopic diagnoses easily in clinic (Cesaro et al., 2013; Leclercq et al., 2011; Spada et al., 2010). DMS aerosols are the effective defoamers for alleviating early pulmonary edema in the ALI. Dichlorodifluoromethane, the major component of DMS aerosols, is a propellant that reacts with ozone (O3) in the atmosphere. Ozone is known to absorb ultraviolet (UV) light in the sunlight. High UV light exposure increases prevalence of cataracts and skin cancer (Albert and Ostheimer, 2003; Gallagher and Lee, 2006; Yam and Kwok, 2014). More importantly, it probably influences the growth of many creatures followed by ecological imbalance (David, 2011). The European Union has decided to replace freon in all aerosol products before 2017. Many other countries have also claimed not to approve aerosol products containing freon and replace them with other effective preparations. Additionally, DMS aerosols were instable
L. Zhu et al. / International Journal of Pharmaceutics 491 (2015) 292–298
freeze-dried to form DMS dry powder inhalations (DSNIs) for pulmonary delivery. The in vitro defoaming ability of DSNIs was evaluated and compared to DMS aerosols. The anti-ALI effect of DSNIs was investigated on animal models.
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gly cer ol,
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Male Sprague–Dawley (SD) rats (180–200 g) from the Laboratory Animal Center of the Beijing Institute of Radiation Medicine (BIRM) were used. The handling and surgical procedures of animals were conducted strictly according to the Guiding Principles for the Use of Laboratory Animals. The animal experiments were approved by the Animal Care Committee of BIRM. The rats were sacrificed. The lung tissues were excised followed by hematoxylin and eosin (H&E) staining, or weighting to obtain the wet/dry weight ratio. All of the studies were conducted in accordance with the Declaration of Helsinki.
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Dimethyl silicone (DMS, 800 mm2/s of kinematic viscosity) and the marketed DMS aerosols were provided by Zigong Honghe Pharmaceutical Co., Ltd. (Sichuan, China). Mannitol was purchased from Nanning Chemical Pharmaceutical Co., Ltd. (Guangxi, China). Polyoxyethylene (40) hydrogenated castor oil (Cremophor RH40) was purchased from the BASF company (Germany). Propylene glycol, glycerol, poly(ethylene glycol) 400 (PEG 400), and the other reagents were of analytic grade. Methylene blue and methyl red were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China) and Beijing Great Wall Chemical Reagent Factory, respectively.
ter Wa
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and would explode when heated or bumped (Lexmond et al., 2014). A novel inhaled DMS formulation is necessary to replace DMS aerosols. However, the high viscous and oily liquid nature of DMS becomes the major hindrance for preparing DMS inhalations. Dry powder inhalations (DPIs) are directly pulmonary delivery preparations to transport drugs into the deep sites of the lung with patients’ active deep breath (Islam and Gladki, 2008). DPIs are portable and more effective than nebulizers which require routine maintenance (Thai et al., 2010). Organic solvents and other solubilizers are usually added in nebulizers and aerosols (Klyashchitsky and Owen, 1999), easily leading to local irritant potency (Onoue et al., 2012). Moreover, dry powders can improve drug stability during storage due to low chemical degradation in the solid state (Adi et al., 2008). Nanoemulsions are the thermodynamic stable colloidal systems with the particle sizes from 1 to 200 nm, which are generally made of surfactants, co-surfactants, oil, and water (Wu et al., 2013). Nanoemulsions can effectively improve the oral bioavailability of hydrophobic drugs because their high absorption is increased (Kotta et al., 2014). Some nanoemulsions were spray-dried or freeze-dried for oral administration or injection (Ahmed et al., 2008; Hatanaka et al., 2008; Wang et al., 2010). Some nebulized nanoemulsions for pulmonary delivery were also prepared (Amani et al., 2010; Laouini et al., 2014; Nasra et al., 2012; Nesamony et al., 2014). Herein, we firstly report the pulmonary delivery of a dry nanoemulsion inhalation in this paper. Silicone and its derivatives are highly viscous and hydrophobic liquids. A few emulsion formulations of them were reported (Nazir et al., 2014; Sharma et al., 2008; Somasundaran et al., 2006; Tan et al., 2013), though their biological applications were not concerned. In this study, DMS nanoemulsions (DSNs) were prepared after formulation optimization. The DSNs were further
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Fig. 1. Pseudo-ternary phase diagrams of the DMS/water/Cremophor RH40/co-surfactants mixtures. The types and ratios of co-surfactants are Cremophor RH40/propylene glycol (1:1) (A), Cremophor RH40/glycerol (1:1) (B), Cremophor RH40/PEG 400 (1:1) (C), Cremophor RH40/PEG 400 (1:2) (D), Cremophor RH40/PEG 400 (2:1) (E), and Cremophor RH40/PEG 400 (3:1) (F). The NE zones represent the area of nanoemulsions.
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2.3. Formulation screening of nanoemulsions
The optimal formulation of DMS nanoemulsions was composed of Cremophor RH40/PEG 400/DMS (4:4:2, w/w/w) and water. The surfactant and DMS mixture were stirred at 600 rpm for 2 min. Water was slowly added in drops until a nearly transparent solution was obtained. The primitive DMS emulsions were further processed with a high-pressure homogenizer (B.E.E. International, USA) for three cycles under 30,000 psi. A stable DMS nanoemulsion (DSN) was obtained. 2.5. Characterization of DMS nanoemulsions Methylene blue is a water-soluble dye and methyl red is an oil-soluble dye. They were used to identify the type of DSNs. The ethanol solutions of methylene blue (2 mg/ml) and methyl red (1 mg/ml) were prepared. One drop of the above solutions was added into the DSNs in the glass vial. After 1 h, the diffusion of dyes was observed. The structure of DSNs was also identified using a polarizing light microscope (PLM, Leica DM2500P, Germany). Optical observations were performed and images were acquired to judge whether a homogenous system was present (Rengstl et al., 2013). DSNs were observed on a Hitachi H-7650 80-kV transmission electron microscope (TEM). The samples were negatively stained using a sodium phosphotungstate solution (pH 7.2). Dynamic light scattering was performed on Zetasizer Nano ZS (Malvern, UK) at 25 C to measure the particle sizes, size distribution and zeta potentials of DSNs. 2.6. Preparation of DMS dry nanoemulsion inhalations Mannitol was dissolved in the DSNs with a 3:2 w/w ratio, where the amount of DSNs represented the non-water components
100
200
Viscosity
80
Conductivity 150
60 100 40 50
Conductivity (µs/cm)
2.4. Preparation of DMS nanoemulsions
Fig. 2. Effects of total surfactants/DMS ratios on the sizes and morphologies of DSNs. The inset pictures (A)–(D) are the TEM images of the DSNs with different ratios as the arrows indicate.
Viscosity (mpa/s)
The water titration method was used to make the pseudoternary phase diagrams of nanoemulsions (Chaiyana et al., 2010). A mixed surfactant was prepared with Cremophor RH40/one of co-surfactants (including propylene glycol, glycerol, and PEG 400) of 1:1 w/w ratio. DMS was added to the mixed surfactant with a series of DMS/mixed surfactant ratios (including 0:10, 1:9, 2:8, 3:7,4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and 10:0). The mixture was titrated with water under agitation at 600 rpm. Titration was stopped and the water volume was recorded when a nanoemulsion appeared as a visually clear liquid. The pseudo-ternary phase diagrams were drawn using the Origin 7.5 software (Origin Lab Co., Ltd., USA). The nanoemulsion area was calculated with the AUTOCAD2009 software (Autodesk Co., Ltd., USA). The formation of DSNs was confirmed with the dynamic measurement of electrical conductivities and viscosities of the mixtures of components in DSNs (Boonme et al., 2006; Chaiyana et al., 2010; Lawrence and Rees, 2012). A water titration method like the above processes was used. Dynamic electrical conductivities were measured on a conductivity meter (DDMS-307, Shanghai Leici Instrument Co., China) equipped with a DJS-1C electrode cell at 25 C. Dynamic viscosities were measured using a rotation viscometer (NDJ-9S, Shanghai Tianping Instruments Factory, China) equipped with a LV-1 spindle at 25 C and 60 rpm. The mixture (2 g) of Cremophor RH40/PEG 400/DMS (4:4:2, w/w/w) was put into a 20-ml flask inserted with a electrode cell or a spindle. Water was dropped with a syringe under agitation. The conductivities and viscosities were recorded when the water phase fraction reached to a certain value. At the same time, the states of the mixtures were observed by naked eyes.
20
0
0 40
50
60
70
75
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Water phase fraction (%) Fig. 3. Electrical conductivity and viscosity profiles of the mixtures of Cremophor RH40/PEG 400/DMS (4:4:2, w/w/w) with addition of water.
including Cremophor RH40/PEG 400/DMS. The final content of DMS in the DMS dry nanoemulsion inhalations (DSNIs) was 8% (w/w). The mixed liquid was spray-dried using a spray dryer (SD-1500, Shanghai Triowin Automation Machinery Co., Ltd., China) or freeze-dried using a lyophilizer (LGJ-30F, Beijing Songyuan Huaxing Technology Develop Co., Ltd., China). The optimal preparation process was chosen after comparing the particle sizes, repose angles, reconstituted nanoemulsion sizes, and the appearance of products. 2.7. Characterization of DMS dry nanoemulsion inhalations An aliquot (0.2 g) of DSNIs was put through a 180-mesh sieve. The obtained powders were dispersed in acetone (100 ml) under water ultrasound. The suspension was laid on a slide and the particle morphology was observed on a microscope. The volume diameters of the powders were measured using a BT2001 particle size analyzer (Bettersize Instruments Ltd., Dandong, China) based on the laser light diffraction method. The surface morphology of DSNIs was observed using a scanning electron microscope (SEM, S-4800, Hitachi, Japan). The
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of water, respectively. The aliquots (20 ml) of the suspensions were sprayed in the above foam-loaded flask. The marketed DMS aerosols (20 ml, containing 0.2 g of DMS) were also sprayed. The defoaming samples were stored without shaking. The second foam volume was 80 ml (V1) when the foam reached the record of 150 ml. At the same time, the time (s) was recorded and the defoaming rate (ml/s) was calculated as (V0 V1)/t. 2.9. In vivo anti-acute lung injury study
Fig. 4. Appearances of the DSNs. (A) The original DSNs, (B) the DSNs dropped with methylene blue, and (C) the DSNs dropped with methyl red.
DSNIs were mounted on metal stubs with an adhesive carbon tape, sputter-coated with gold and examined under the microscope at an acceleration voltage of 10 kV. A certain domain of the SEM sample was selected to perform the energy dispersive spectroscopic (EDS) measurement on the SEM to determine the abundance of specific elements, including C, O, and Si in this study. A lyophilized mannitol sample was obtained with the same method as the DSNIs, and then investigated with the SEM and EDS as above. The DSNIs (100 mg) were filled into one hard capsule (III Type). The emptying rate of DSNIs was determined by a glass twin stage impactor (TSI, made in our lab referred to China Pharmacopeia) at a flow rate of 60 l/min controlled by a vacuum pump. The angle ( ) of repose of DSNIs was calculated by the funnel method and the bulk density was calculated by the graduated flask method. The theoretical mass mean aerodynamic diameters (MMAD) of DSNIs are defined as d(r/r0X)1/2, where d is the geometric mean diameter (the D50 value obtained from the measurement of volume diameters), r0 is a reference density of 1 g/ml, X is the dynamic shape factor that is 1 for a sphere, and r is the tapped density (also the bulk density) (Ungaro et al., 2009). All of the measurements were performed in triplicates. 2.8. Investigation of defoaming ability A foaming liquid (1% sodium dodecyl sulphate aqueous solution, 50 ml) was added into a 500-ml graduated flask and shaken thoroughly with hand. The primitive foam volume was 450 ml (V0) when the foam reached the record of 500 ml. Blank DSNIs were prepared with the same method as DSNIs containing all of the excipients except DMS. DMS (0.4 g), blank DSNIs (2.5 g) and DSNIs (2.5 g, containing 0.2 g of DMS) were suspended in 20 ml
Rats were divided into six groups with 6 rats each group. A laryngoscope (MJ MediTech, Jiangsu, China) was used to visualize the opened tracheal route. A hydrochloric acid aqueous solution (pH 1.25) was sprayed into the rat lungs through the airway using an intratracheal MicroSprayer1 Aerosolizer (IA-1B, Penn-Century Inc., PA, USA) with the dose of 0.2 ml each rat to prepare the ALI rat models. The rats of Group A were the healthy rats that intratracheally administered with 0.9% NaCl solutions and the dose of 0.2 ml each rat. Group B contained the ALI rats without treatment. An aliquot (about 0.2 ml, containing 1.6 mg of DMS) of DMS aerosols was directly sprayed into the ALI rat lungs with the same method as mentioned above and the rats belonged to Group C. The defoaming agents including the blank DSNIs (20 mg each aliquot) and the DSNIs (20 mg each aliquot, containing 1.6 mg of DMS) were sprayed into the rat lungs using a Dry Powder InsufflatorTM (DP-4 M, Penn-Century Inc., PA, USA) through the tracheal without anesthesia. Groups D and E contained the rats treated with the blank DSNIs and the DSNIs, respectively. The rats were sacrificed after treatment for 4 h. The left lungs were excised, weighted as the wet weight (W), and then placed in an incubator at 80 C for 48 h to obtain the dry weight (D). The W/D ratio was calculated. The right lungs were harvested and fixed in 10% formaldehyde solutions, embedded in paraffin and cut into 5-mm thick sections. The slides were stained with hematoxylin and eosin. These samples were investigated on a light microscope. Student’s t-test was used for all of the statistical evaluations. Statistical significance was defined as a p < 0.05 (differences) or 0.01 (significant differences). 3. Results and discussion 3.1. Optimal formulation of DSNs The role of co-surfactants in nanoemulsions is to further reduce the interfacial tension on the basis of surfactants. Both of the types of co-surfactants and the ratios of surfactant/co-surfactant influence the formation of nanoemulsions. In this study, the nanoemulsion zones in the pseudo-ternary phase diagrams covered 18.1%, 18.4%, and 21.1% of the total area, respectively, when propylene glycol, glycerol, and PEG 400 of the 1:1 Cremophor RH40/co-surfactant ratio was used (Fig. 1). The ratios of PEG 400 were further screened in details. The nanoemulsion zone fractions were 19.5%, 17.9%, and 15.1% for the combinations of
Table 1 Comparative characteristics of DSNIs. Parameter
Appearance Reconstitution (s) Appearance after reconstitution Mean size of DSNIs (mm) Angle of repose ( ) Mean size of DSNs after reconstitution (nm) a
Preparation process Freeze drying
Spray drying
White, loose Very fast (1 h 455.7 51.2 324.3 21.0* 379.7 55.6 256.3 12.1#
– 0.71 0.08 0.99 0.06** 0.84 0.12 1.25 0.06#
a
The data are presented as means SDs (n = 3). p < 0.05, blank DSNIs vs. DMS. ** p < 0.01, blank DSNIs vs. DMS. # p < 0.01, DSNIs vs. the other defoamers and control. *
Cremophor RH40/PEG 400 of 1:2, 2:1, and 3:1 ratios, respectively. Therefore, the composition of Cremophor RH40/PEG 400 (1:1) was selected as the optimal surfactant/co-surfactant mixture. Another important impact factor influencing the preparation of DSNs was the fraction of DMS in DSNs. The sizes of DSNs were positively related to the fractions of DMS (Fig. 2). The mean sizes of DSNs were 13.5 2.5, 22.5 3.5, 44.4 1.8, and 75.5 2.7 nm (n = 3), respectively, as the ratios of total surfactants/DMS were 9:1, 8:2, 7:3, and 6:4. Theoretically, small size DSNs could go into the deep sites of the lung and well disperse. However, the smallest DSNs composed of 9:1 total surfactants/DMS had a lot of worm-like and spherical micelles besides the emulsion droplets
(Fig. 2A), indicating that the surfactants could be over-used far beyond the need of emulsification (Lawrence and Rees, 2012). In the cases of low ratios of total surfactants/DMS (7:3, 6:4), the sizes of DSNs were significantly large (Fig. 2C and D). The size of DSNs of 8:2 surfactants/DMS was appropriate and the DSNs kept stable for a long time. In summary, the 4:4:2 ratio of Cremophor RH40/PEG 400/DMS was optimal to prepare the DSN formulation. The electrical conductivities and viscosities of the DMS mixtures depended on the fractions of water phases (Fig. 3). The profiles could have the inflexion at about 65% fraction of water phases. Interestingly, before reaching the inflexion, i.e., lower than 65% of water phase, the samples were visually turbid, but they changed to clear (i.e., formation of nanoemulsions) after the inflexion. Therefore, when the fraction of water phases was lower than 65%, the O/W nanoemulsions could not be formed. At this time, the oil may be the continuous phase, leading to low conductivities and high viscosities. When the water fraction increased to a certain value, the O/W DSNs may be formed, leading to high conductivities and low viscosities. 3.2. Characteristics of DSNs The water-soluble methylene blue rapidly diffused in the DSNs but the oil-soluble methyl red remained on the top of the DSNs, indicating that the type of DSNs was oil-in-water (O/W) (Fig. 4).
Fig. 6. Appearances and histological section pictures (200) of rat lungs of different groups. (A) The normal lung of healthy rats, (B) the lung of ALI rats, (C) the ALI lung treated with DMS aerosols, (D) the ALI lung treated with blank DSNIs, and (E) the ALI lung treated with DSNIs.
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and no Si. However, the element weight percentages in the DSNIs were 50.55% C, 47.55% O, and 1.90% Si. Therefore, the presence of DMS in the DSNIs was further confirmed. The D50 of DSNIs was 10.52 mm according to the particle size measurement. The tapped density, aerodynamic diameter, repose angle, hygroscopicity, and emptying rate of DSNIs were 0.27 0.03 g/ml, 4.82 0.33 mm, 31.4 1.6 , 3.86 1.5%, and 98.0 1.2%, respectively. All of the data were presented as means SDs (n = 3). These characteristics indicated that the DSNIs were suitable as the DPIs to deliver DMS into the bronchioles and alveoli after pulmonary inhalation. The DSNIs were very stable after long-term storage. The reconstitution process of 6-month aged DSNIs was fast (
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Dimethyl silicone dry nanoemulsion inhalations: Formulation study and anti-acute lung injury effect Lifei Zhua,b,1, Miao Lib,1, Junxing Dongb , Yiguang Jina,b,* a b
Anhui Medical University, Hefei 230001, China Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 April 2015 Received in revised form 15 June 2015 Accepted 23 June 2015 Available online 30 June 2015
Acute lung injury (ALI) is a severe disease, leading to death if not treated quickly. An emergency medicine is necessary for ALI therapy. Dimethyl silicone (DMS) is an effective agent to defoam the bubbles in the lung induced by ALI. However, DMS aerosols, a marketed formulation of DMS, affect environments and will be limited in the future. Here we firstly report a dry nanoemulsion inhalation for pulmonary delivery. Novel DMS dry nanoemulsion inhalations (DSNIs) were developed in this study. The optimal formulation of stable and homogenous DMS nanoemulsions (DSNs) was composed of Cremophor RH40/PEG 400/DMS (4:4:2, w/w/w) and water. The DSNs showed the tiny size of 19.8 nm, the zeta potential of 9.66 mV, and the low polydispersity index (PDI) of 0.37. The type of DSNs was identified as oil-in-water. The DSNs were added with mannitol followed by freeze-drying to obtain the DSNIs that were loose white powders, showed good fluidity, and were capable of rapid reconstitution to DSNs. The DSNs could adhere on the surfaces of lyophilized mannitol crystals. The aerodynamic diameter of DSNIs was 4.82 mm, suitable for pulmonary inhalation. The in vitro defoaming rate of DSNIs was 1.25 ml/s, much faster than those of the blank DSNIs, DMS, and DMS aerosols. The DSNIs showed significantly higher anti-ALI effect on the ALI rat models than the blank DSNIs and the DMS aerosols according to lung appearances, histological sections, and lung wet weight/dry weight ratios. The DSNIs are effective anti-ALI nanomedicines. The novel DMS formulation is a promising replacement of DMS aerosols. ã 2015 Elsevier B.V. All rights reserved.
Keywords: Acute lung injury Defoaming Dimethyl silicone Dry powder inhalation Nanoemulsion
1. Introduction Acute lung injury (ALI) is a severe disease, which can be induced by many factors, such as acidic gas inhalation (Lim et al., 2007), and propellant poisoning (Cao et al., 2014). At the early stage of ALI, a large number of bubbles is produced and could block up the bronchus followed by hypoxia and dyspnea. Severe cases of bubble blocking can result in death if not treated quickly (Wheeler and Bernard, 2007). The mortality of ALI is reported to be 30–60% and more in the elders (Elder et al., 2012; Lv et al., 2014). An emergency medicine is necessary for ALI therapy. Dimethyl silicone (DMS) is an oily colorless transparent liquid with high flexibility of backbones (Gräbner et al., 2010). Besides widely industrial applications, the most important biological function of DMS is defoaming after entering the films of bubbles
* Corresponding author at: Department of Pharmaceutical Sciences, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850, China. Fax: +86 10 68214653. E-mail address: [email protected] (Y. Jin). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijpharm.2015.06.041 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
and destroying the mechanical equilibrium of oriented films due to its weak surface tension, finally leading to the bubbles breaking (Watson, 2014). Therefore, DMS is mainly used as a defoaming agent to eliminate the bubbles in the various sites of the body, such as the gastro-enteric route and the lung. DMS tablets and powders are used as the defoamers to relieve flatulence (Jewell, 2007; Torrado et al., 1999), and make gastroscopic and enterscopic diagnoses easily in clinic (Cesaro et al., 2013; Leclercq et al., 2011; Spada et al., 2010). DMS aerosols are the effective defoamers for alleviating early pulmonary edema in the ALI. Dichlorodifluoromethane, the major component of DMS aerosols, is a propellant that reacts with ozone (O3) in the atmosphere. Ozone is known to absorb ultraviolet (UV) light in the sunlight. High UV light exposure increases prevalence of cataracts and skin cancer (Albert and Ostheimer, 2003; Gallagher and Lee, 2006; Yam and Kwok, 2014). More importantly, it probably influences the growth of many creatures followed by ecological imbalance (David, 2011). The European Union has decided to replace freon in all aerosol products before 2017. Many other countries have also claimed not to approve aerosol products containing freon and replace them with other effective preparations. Additionally, DMS aerosols were instable
L. Zhu et al. / International Journal of Pharmaceutics 491 (2015) 292–298
freeze-dried to form DMS dry powder inhalations (DSNIs) for pulmonary delivery. The in vitro defoaming ability of DSNIs was evaluated and compared to DMS aerosols. The anti-ALI effect of DSNIs was investigated on animal models.
0.4
0.6
1.0
0.01.00 0.0
0.2
0.4 0.2
0.4
0.6
0.4
1.0
400 , 2: 1
400 , 1: 2
DMS
0.8
Cr em op ho rR H4 0/P EG
0.6
NE
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0.6
0.8
1.0
1:1 400 ,
1.00 0.0 0.0
0.00 1.0
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0.2
0.4
0.8
0.25
0.6
NE
0.4
0.2
0.2
0.4
0.6
DMS
0.8
1.0
0.6
0.8
1.0
0.0
DMS
(F)
0.75
0.01.00 0.0
0.4
0.50
0.00 1.0 0.8
0.25
0.6
NE
ter Wa
0.2
RH 40/ PE G
0.2
(E) 0.8
0.25
0.6
NE
0.75
ter Wa
Cr em op ho rR H4 0/P EG
0.00 1.0
0.75
1.00 0.0
0.8
0.25
0.50
0.4
0.00 1.0
DMS
ter Wa
0.50
0.6
NE
0.50
DMS
(D)
0.8
0.25
Cr em op ho r
gly cer ol,
0.8
(C)
400 , 3: 1
0.2
Male Sprague–Dawley (SD) rats (180–200 g) from the Laboratory Animal Center of the Beijing Institute of Radiation Medicine (BIRM) were used. The handling and surgical procedures of animals were conducted strictly according to the Guiding Principles for the Use of Laboratory Animals. The animal experiments were approved by the Animal Care Committee of BIRM. The rats were sacrificed. The lung tissues were excised followed by hematoxylin and eosin (H&E) staining, or weighting to obtain the wet/dry weight ratio. All of the studies were conducted in accordance with the Declaration of Helsinki.
0.75
0.2
1.00 0.0
2.2. Animals
Cr em op ho rR H4 0/P EG
Cr em op ho r
0.4
0.75
Dimethyl silicone (DMS, 800 mm2/s of kinematic viscosity) and the marketed DMS aerosols were provided by Zigong Honghe Pharmaceutical Co., Ltd. (Sichuan, China). Mannitol was purchased from Nanning Chemical Pharmaceutical Co., Ltd. (Guangxi, China). Polyoxyethylene (40) hydrogenated castor oil (Cremophor RH40) was purchased from the BASF company (Germany). Propylene glycol, glycerol, poly(ethylene glycol) 400 (PEG 400), and the other reagents were of analytic grade. Methylene blue and methyl red were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China) and Beijing Great Wall Chemical Reagent Factory, respectively.
ter Wa
NE
ter Wa
0.6
Cr em op ho rR H4 0/
0.8
0.25
0.50
(B)
2.1. Materials
0.00 1.0
1:1
0.00 1.0
2. Materials and methods
ter Wa
RH 40/ pro py len eg lyc ol, 1:1
and would explode when heated or bumped (Lexmond et al., 2014). A novel inhaled DMS formulation is necessary to replace DMS aerosols. However, the high viscous and oily liquid nature of DMS becomes the major hindrance for preparing DMS inhalations. Dry powder inhalations (DPIs) are directly pulmonary delivery preparations to transport drugs into the deep sites of the lung with patients’ active deep breath (Islam and Gladki, 2008). DPIs are portable and more effective than nebulizers which require routine maintenance (Thai et al., 2010). Organic solvents and other solubilizers are usually added in nebulizers and aerosols (Klyashchitsky and Owen, 1999), easily leading to local irritant potency (Onoue et al., 2012). Moreover, dry powders can improve drug stability during storage due to low chemical degradation in the solid state (Adi et al., 2008). Nanoemulsions are the thermodynamic stable colloidal systems with the particle sizes from 1 to 200 nm, which are generally made of surfactants, co-surfactants, oil, and water (Wu et al., 2013). Nanoemulsions can effectively improve the oral bioavailability of hydrophobic drugs because their high absorption is increased (Kotta et al., 2014). Some nanoemulsions were spray-dried or freeze-dried for oral administration or injection (Ahmed et al., 2008; Hatanaka et al., 2008; Wang et al., 2010). Some nebulized nanoemulsions for pulmonary delivery were also prepared (Amani et al., 2010; Laouini et al., 2014; Nasra et al., 2012; Nesamony et al., 2014). Herein, we firstly report the pulmonary delivery of a dry nanoemulsion inhalation in this paper. Silicone and its derivatives are highly viscous and hydrophobic liquids. A few emulsion formulations of them were reported (Nazir et al., 2014; Sharma et al., 2008; Somasundaran et al., 2006; Tan et al., 2013), though their biological applications were not concerned. In this study, DMS nanoemulsions (DSNs) were prepared after formulation optimization. The DSNs were further
(A)
293
0.4
0.75
0.01.00 0.0
0.2
0.2
0.4
0.6
0.8
1.0
0.0
DMS
Fig. 1. Pseudo-ternary phase diagrams of the DMS/water/Cremophor RH40/co-surfactants mixtures. The types and ratios of co-surfactants are Cremophor RH40/propylene glycol (1:1) (A), Cremophor RH40/glycerol (1:1) (B), Cremophor RH40/PEG 400 (1:1) (C), Cremophor RH40/PEG 400 (1:2) (D), Cremophor RH40/PEG 400 (2:1) (E), and Cremophor RH40/PEG 400 (3:1) (F). The NE zones represent the area of nanoemulsions.
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2.3. Formulation screening of nanoemulsions
The optimal formulation of DMS nanoemulsions was composed of Cremophor RH40/PEG 400/DMS (4:4:2, w/w/w) and water. The surfactant and DMS mixture were stirred at 600 rpm for 2 min. Water was slowly added in drops until a nearly transparent solution was obtained. The primitive DMS emulsions were further processed with a high-pressure homogenizer (B.E.E. International, USA) for three cycles under 30,000 psi. A stable DMS nanoemulsion (DSN) was obtained. 2.5. Characterization of DMS nanoemulsions Methylene blue is a water-soluble dye and methyl red is an oil-soluble dye. They were used to identify the type of DSNs. The ethanol solutions of methylene blue (2 mg/ml) and methyl red (1 mg/ml) were prepared. One drop of the above solutions was added into the DSNs in the glass vial. After 1 h, the diffusion of dyes was observed. The structure of DSNs was also identified using a polarizing light microscope (PLM, Leica DM2500P, Germany). Optical observations were performed and images were acquired to judge whether a homogenous system was present (Rengstl et al., 2013). DSNs were observed on a Hitachi H-7650 80-kV transmission electron microscope (TEM). The samples were negatively stained using a sodium phosphotungstate solution (pH 7.2). Dynamic light scattering was performed on Zetasizer Nano ZS (Malvern, UK) at 25 C to measure the particle sizes, size distribution and zeta potentials of DSNs. 2.6. Preparation of DMS dry nanoemulsion inhalations Mannitol was dissolved in the DSNs with a 3:2 w/w ratio, where the amount of DSNs represented the non-water components
100
200
Viscosity
80
Conductivity 150
60 100 40 50
Conductivity (µs/cm)
2.4. Preparation of DMS nanoemulsions
Fig. 2. Effects of total surfactants/DMS ratios on the sizes and morphologies of DSNs. The inset pictures (A)–(D) are the TEM images of the DSNs with different ratios as the arrows indicate.
Viscosity (mpa/s)
The water titration method was used to make the pseudoternary phase diagrams of nanoemulsions (Chaiyana et al., 2010). A mixed surfactant was prepared with Cremophor RH40/one of co-surfactants (including propylene glycol, glycerol, and PEG 400) of 1:1 w/w ratio. DMS was added to the mixed surfactant with a series of DMS/mixed surfactant ratios (including 0:10, 1:9, 2:8, 3:7,4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and 10:0). The mixture was titrated with water under agitation at 600 rpm. Titration was stopped and the water volume was recorded when a nanoemulsion appeared as a visually clear liquid. The pseudo-ternary phase diagrams were drawn using the Origin 7.5 software (Origin Lab Co., Ltd., USA). The nanoemulsion area was calculated with the AUTOCAD2009 software (Autodesk Co., Ltd., USA). The formation of DSNs was confirmed with the dynamic measurement of electrical conductivities and viscosities of the mixtures of components in DSNs (Boonme et al., 2006; Chaiyana et al., 2010; Lawrence and Rees, 2012). A water titration method like the above processes was used. Dynamic electrical conductivities were measured on a conductivity meter (DDMS-307, Shanghai Leici Instrument Co., China) equipped with a DJS-1C electrode cell at 25 C. Dynamic viscosities were measured using a rotation viscometer (NDJ-9S, Shanghai Tianping Instruments Factory, China) equipped with a LV-1 spindle at 25 C and 60 rpm. The mixture (2 g) of Cremophor RH40/PEG 400/DMS (4:4:2, w/w/w) was put into a 20-ml flask inserted with a electrode cell or a spindle. Water was dropped with a syringe under agitation. The conductivities and viscosities were recorded when the water phase fraction reached to a certain value. At the same time, the states of the mixtures were observed by naked eyes.
20
0
0 40
50
60
70
75
80
Water phase fraction (%) Fig. 3. Electrical conductivity and viscosity profiles of the mixtures of Cremophor RH40/PEG 400/DMS (4:4:2, w/w/w) with addition of water.
including Cremophor RH40/PEG 400/DMS. The final content of DMS in the DMS dry nanoemulsion inhalations (DSNIs) was 8% (w/w). The mixed liquid was spray-dried using a spray dryer (SD-1500, Shanghai Triowin Automation Machinery Co., Ltd., China) or freeze-dried using a lyophilizer (LGJ-30F, Beijing Songyuan Huaxing Technology Develop Co., Ltd., China). The optimal preparation process was chosen after comparing the particle sizes, repose angles, reconstituted nanoemulsion sizes, and the appearance of products. 2.7. Characterization of DMS dry nanoemulsion inhalations An aliquot (0.2 g) of DSNIs was put through a 180-mesh sieve. The obtained powders were dispersed in acetone (100 ml) under water ultrasound. The suspension was laid on a slide and the particle morphology was observed on a microscope. The volume diameters of the powders were measured using a BT2001 particle size analyzer (Bettersize Instruments Ltd., Dandong, China) based on the laser light diffraction method. The surface morphology of DSNIs was observed using a scanning electron microscope (SEM, S-4800, Hitachi, Japan). The
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of water, respectively. The aliquots (20 ml) of the suspensions were sprayed in the above foam-loaded flask. The marketed DMS aerosols (20 ml, containing 0.2 g of DMS) were also sprayed. The defoaming samples were stored without shaking. The second foam volume was 80 ml (V1) when the foam reached the record of 150 ml. At the same time, the time (s) was recorded and the defoaming rate (ml/s) was calculated as (V0 V1)/t. 2.9. In vivo anti-acute lung injury study
Fig. 4. Appearances of the DSNs. (A) The original DSNs, (B) the DSNs dropped with methylene blue, and (C) the DSNs dropped with methyl red.
DSNIs were mounted on metal stubs with an adhesive carbon tape, sputter-coated with gold and examined under the microscope at an acceleration voltage of 10 kV. A certain domain of the SEM sample was selected to perform the energy dispersive spectroscopic (EDS) measurement on the SEM to determine the abundance of specific elements, including C, O, and Si in this study. A lyophilized mannitol sample was obtained with the same method as the DSNIs, and then investigated with the SEM and EDS as above. The DSNIs (100 mg) were filled into one hard capsule (III Type). The emptying rate of DSNIs was determined by a glass twin stage impactor (TSI, made in our lab referred to China Pharmacopeia) at a flow rate of 60 l/min controlled by a vacuum pump. The angle ( ) of repose of DSNIs was calculated by the funnel method and the bulk density was calculated by the graduated flask method. The theoretical mass mean aerodynamic diameters (MMAD) of DSNIs are defined as d(r/r0X)1/2, where d is the geometric mean diameter (the D50 value obtained from the measurement of volume diameters), r0 is a reference density of 1 g/ml, X is the dynamic shape factor that is 1 for a sphere, and r is the tapped density (also the bulk density) (Ungaro et al., 2009). All of the measurements were performed in triplicates. 2.8. Investigation of defoaming ability A foaming liquid (1% sodium dodecyl sulphate aqueous solution, 50 ml) was added into a 500-ml graduated flask and shaken thoroughly with hand. The primitive foam volume was 450 ml (V0) when the foam reached the record of 500 ml. Blank DSNIs were prepared with the same method as DSNIs containing all of the excipients except DMS. DMS (0.4 g), blank DSNIs (2.5 g) and DSNIs (2.5 g, containing 0.2 g of DMS) were suspended in 20 ml
Rats were divided into six groups with 6 rats each group. A laryngoscope (MJ MediTech, Jiangsu, China) was used to visualize the opened tracheal route. A hydrochloric acid aqueous solution (pH 1.25) was sprayed into the rat lungs through the airway using an intratracheal MicroSprayer1 Aerosolizer (IA-1B, Penn-Century Inc., PA, USA) with the dose of 0.2 ml each rat to prepare the ALI rat models. The rats of Group A were the healthy rats that intratracheally administered with 0.9% NaCl solutions and the dose of 0.2 ml each rat. Group B contained the ALI rats without treatment. An aliquot (about 0.2 ml, containing 1.6 mg of DMS) of DMS aerosols was directly sprayed into the ALI rat lungs with the same method as mentioned above and the rats belonged to Group C. The defoaming agents including the blank DSNIs (20 mg each aliquot) and the DSNIs (20 mg each aliquot, containing 1.6 mg of DMS) were sprayed into the rat lungs using a Dry Powder InsufflatorTM (DP-4 M, Penn-Century Inc., PA, USA) through the tracheal without anesthesia. Groups D and E contained the rats treated with the blank DSNIs and the DSNIs, respectively. The rats were sacrificed after treatment for 4 h. The left lungs were excised, weighted as the wet weight (W), and then placed in an incubator at 80 C for 48 h to obtain the dry weight (D). The W/D ratio was calculated. The right lungs were harvested and fixed in 10% formaldehyde solutions, embedded in paraffin and cut into 5-mm thick sections. The slides were stained with hematoxylin and eosin. These samples were investigated on a light microscope. Student’s t-test was used for all of the statistical evaluations. Statistical significance was defined as a p < 0.05 (differences) or 0.01 (significant differences). 3. Results and discussion 3.1. Optimal formulation of DSNs The role of co-surfactants in nanoemulsions is to further reduce the interfacial tension on the basis of surfactants. Both of the types of co-surfactants and the ratios of surfactant/co-surfactant influence the formation of nanoemulsions. In this study, the nanoemulsion zones in the pseudo-ternary phase diagrams covered 18.1%, 18.4%, and 21.1% of the total area, respectively, when propylene glycol, glycerol, and PEG 400 of the 1:1 Cremophor RH40/co-surfactant ratio was used (Fig. 1). The ratios of PEG 400 were further screened in details. The nanoemulsion zone fractions were 19.5%, 17.9%, and 15.1% for the combinations of
Table 1 Comparative characteristics of DSNIs. Parameter
Appearance Reconstitution (s) Appearance after reconstitution Mean size of DSNIs (mm) Angle of repose ( ) Mean size of DSNs after reconstitution (nm) a
Preparation process Freeze drying
Spray drying
White, loose Very fast (1 h 455.7 51.2 324.3 21.0* 379.7 55.6 256.3 12.1#
– 0.71 0.08 0.99 0.06** 0.84 0.12 1.25 0.06#
a
The data are presented as means SDs (n = 3). p < 0.05, blank DSNIs vs. DMS. ** p < 0.01, blank DSNIs vs. DMS. # p < 0.01, DSNIs vs. the other defoamers and control. *
Cremophor RH40/PEG 400 of 1:2, 2:1, and 3:1 ratios, respectively. Therefore, the composition of Cremophor RH40/PEG 400 (1:1) was selected as the optimal surfactant/co-surfactant mixture. Another important impact factor influencing the preparation of DSNs was the fraction of DMS in DSNs. The sizes of DSNs were positively related to the fractions of DMS (Fig. 2). The mean sizes of DSNs were 13.5 2.5, 22.5 3.5, 44.4 1.8, and 75.5 2.7 nm (n = 3), respectively, as the ratios of total surfactants/DMS were 9:1, 8:2, 7:3, and 6:4. Theoretically, small size DSNs could go into the deep sites of the lung and well disperse. However, the smallest DSNs composed of 9:1 total surfactants/DMS had a lot of worm-like and spherical micelles besides the emulsion droplets
(Fig. 2A), indicating that the surfactants could be over-used far beyond the need of emulsification (Lawrence and Rees, 2012). In the cases of low ratios of total surfactants/DMS (7:3, 6:4), the sizes of DSNs were significantly large (Fig. 2C and D). The size of DSNs of 8:2 surfactants/DMS was appropriate and the DSNs kept stable for a long time. In summary, the 4:4:2 ratio of Cremophor RH40/PEG 400/DMS was optimal to prepare the DSN formulation. The electrical conductivities and viscosities of the DMS mixtures depended on the fractions of water phases (Fig. 3). The profiles could have the inflexion at about 65% fraction of water phases. Interestingly, before reaching the inflexion, i.e., lower than 65% of water phase, the samples were visually turbid, but they changed to clear (i.e., formation of nanoemulsions) after the inflexion. Therefore, when the fraction of water phases was lower than 65%, the O/W nanoemulsions could not be formed. At this time, the oil may be the continuous phase, leading to low conductivities and high viscosities. When the water fraction increased to a certain value, the O/W DSNs may be formed, leading to high conductivities and low viscosities. 3.2. Characteristics of DSNs The water-soluble methylene blue rapidly diffused in the DSNs but the oil-soluble methyl red remained on the top of the DSNs, indicating that the type of DSNs was oil-in-water (O/W) (Fig. 4).
Fig. 6. Appearances and histological section pictures (200) of rat lungs of different groups. (A) The normal lung of healthy rats, (B) the lung of ALI rats, (C) the ALI lung treated with DMS aerosols, (D) the ALI lung treated with blank DSNIs, and (E) the ALI lung treated with DSNIs.
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and no Si. However, the element weight percentages in the DSNIs were 50.55% C, 47.55% O, and 1.90% Si. Therefore, the presence of DMS in the DSNIs was further confirmed. The D50 of DSNIs was 10.52 mm according to the particle size measurement. The tapped density, aerodynamic diameter, repose angle, hygroscopicity, and emptying rate of DSNIs were 0.27 0.03 g/ml, 4.82 0.33 mm, 31.4 1.6 , 3.86 1.5%, and 98.0 1.2%, respectively. All of the data were presented as means SDs (n = 3). These characteristics indicated that the DSNIs were suitable as the DPIs to deliver DMS into the bronchioles and alveoli after pulmonary inhalation. The DSNIs were very stable after long-term storage. The reconstitution process of 6-month aged DSNIs was fast (
