- Email: [email protected]
Preparation of microsized spherical aggregates of ultrafine ciprofloxacin particles for dry powder inhalation (DPI)
Powder Technology 194 (2009) 81–86
Contents lists available at ScienceDirect
Powder Technology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p ow t e c
Preparation of microsized spherical aggregates of ultrafine ciprofloxacin particles for dry powder inhalation (DPI) Hong Zhao a, Yuan Le a, Haoying Liu a, Tingting Hu a, Zhigang Shen a,b, Jimmy Yun b, Jian-Feng Chen a,⁎ a b
Sin-China Nano Technology Center, Key Lab for Nanomaterials, Ministry of Education, Beijing University of Chemical. Technology, Beijing 100029, PR China Nanomaterials Technology Pte., Ltd., Singapore 139944, Singapore
a r t i c l e
i n f o
Article history: Received 17 November 2008 Received in revised form 20 February 2009 Accepted 17 March 2009 Available online 28 March 2009 Keywords: Ciprofloxacin Ultra-fine particles Reactive precipitation Fine particle fraction (FPF) Anti-solvent
a b s t r a c t In this paper, the ultrafine ciprofloxacin (CPF) particles were prepared by the reactive precipitation of ciprofloxacin hydrochloride (CPF·HCl) and NaOH aqueous solution with the presence of isopropyl alcohol (IPA) as the anti-solvent. Subsequently, microsized spherical CPF aggregates were generated using the prepared ultrafine particles as building blocks by spray drying method. The effect of the volume ratio of the CPF solution to anti-solvent (IPA) was investigated. The result shows that the ultra-fined particles of ciprofloxacin can be produced under the volume ratio of 1:15. After spray drying, 3 — 4 µm spherical aggregates with ultrafine primary CPF particles can be obtained and exhibited great improved aerosol performance with fine particle fraction (FPF) up to 60%. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Ciprofloxacin is a fluoroquinolone antibiotic that has demonstrated in vitro activity against Staphylococcus and Bacillus species and most gram-negative microorganisms including Pseudomonas species etc. It is often used in the treatment of inhalation anthrax and cystic fibrosis lung infection. Current delivery methods for ciprofloxacin include oral, injection and ocular deliveries. However, the bioavailability of ciprofloxacin administered orally is relatively low because half of the drug is metabolized in the gastrointestinal tract [1]. Moreover, a substantial proportion of patients suffer some severe symptoms such as nausea, inappetence or bellyache etc., which make oral and intranasal treatment unsatisfactory. Subcutaneous administration is an alternative, but it also has some untoward effects such as rubefaction, phlebitis and shock etc. [2]. The pulmonary delivery for ciprofloxacin can be a viable alternative for self-administration, whereby these limitations could be overcome. In recent years, the pulmonary delivery route has attracted much attention because of the following advantages over other routes: 1) the surface area of a lung is extremely large and the mucosal permeation of drug substances is comparatively easy, because the vascular system is well developed and the wall of the alveolus is extremely thin. 2) The pulmonary delivery route can avoid the first pass effect of hepar, which may highly enhance the bioavailability of administration [3]. The pulmonary delivery includes metered dose inhaler, dry powder inhaler and nebulizer. Dry powder inhalers (DPI) have received in⁎ Corresponding author. Tel.: +86 10 6444 6466; fax: +86 10 6443 4784. E-mail address: [email protected] (J.-F. Chen). 0032-5910/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.powtec.2009.03.031
creasing attention mainly because the devices do not require chlorofluorocarbon propellants to deliver the drug and can thus be regarded as safe delivery systems that do not affect the ozone layer. The particle size of the drug to be delivered is critical for its efficient deposition in the lower airways when delivering. It is feasible to deliver drug particles with the diameter of 1–5 µm to the specific site of the respiratory tract via DPIs [4]. These particles are accumulated in the deep lungs where they provide continuous absorption of drugs. However, particles measuring approximately smaller than 1 µm are exhaled during normal tidal breathings and those measuring approximately larger than 6 µm are deposited in the upper airways with a vigorously mucocilliary clearance [5,6]. Therefore, producing particles with demand size and specific structures is a significative work for DPI use. Conventional crystallization which can control the particle size includes jet-milling, pearl-ball mills, supercritical fluids and so on [7–11]. These techniques require significant energy input, which can induce disorder, defects or even amorphous regions in the drug particles, consequently causing physico–chemical instability of the product. Therefore, alternative production process is of great requirement for the pharmaceutical industry. Liquid precipitation technique, which includes reactive precipitation and liquid anti-solvent precipitation, has been developed in various pathways to effectively manipulate the size and morphology of pharmaceutical particles for different applications [12–20]. Moreover, the liquid precipitation technique has been demonstrated prospective in industrial fields because of its low cost, convenience in processing, as well as the ease of scale-up [21–23]. The aim of this investigation was to combine the liquid anti-solvent precipitation and the spray drying technique to generate spherical
82
H. Zhao et al. / Powder Technology 194 (2009) 81–86
Fig. 1. Reaction equation.
particles with desired size and structure for DPI use. Firstly, ultrafine CPF particles as the sub-units were prepared by the reactive precipitation with anti-solvent in presence. Subsequently, spray drying was employed to assemble the sub-units into uniform spherical aggregates. 2. Experimental 2.1. Materials The raw material of this project was market Ciprofloxacin hydrochloride product synthesized in China. All the chemicals and solvents used in following experiments are A.R. grade supplied by Beijing Chemical Agent Company. The water used during the whole experimental process is de-ionized water. 2.2. Particles preparation
was prepared by dropping one or two droplets of slurry on a small piece of slide directly and then dried under room conditions, which is in order to observe the shape and size of the particles in the slurry before drying to powder. The slide was fixed on aluminum stubs using double-sided adhesive tape. The dry powder sample was made by directly fixing it on aluminum stubs using double-sided adhesive tape. A small amount of ciprofloxacin particles were scattered on the stub and dispersed by tapping lightly on the edge of the stub with a spatula to break up any agglomerates. Both the slide and dry powder fixed on the aluminum stub were coated with Au at 50 mA for 30 s using a Pelco Model 3 sputter-coater under an Ar atmosphere. A Cambridge S250MK3 scanning electron microscope (SEM) (Cambridge Instruments Inc., U.K.) at an accelerating voltage of 10 kV with a secondary electron detector was used to obtain digital images of the samples. The particle size and its size distribution were determined by the Image-Pro Plus 5.0 via the obtained SEM photographs.
2.2.1. The combination of reaction precipitation and anti-solvent precipitation In this study, ultrafine ciprofloxacin particles were prepared by a method that is the reactive precipitation with the presence of the antisolvent (IPA). Ciprofloxacin (C17H18FN3O3, 1-cyclopropyl-6-fluoro-1, 4-dihydro4-oxo-7-(piperazin-1-yl) quinoline-3 -carboxylic acid, MW 331.4) was firstly dissolved in 0.1 M HCl solution (CPF·HCl) and then reacted with NaOH solution (Fig. 1) to obtain CPF precipitates. Isopropyl alcohol (IPA) defined as anti-solvent of ciprofloxacin was introduced into this reaction to generate a circumstance which will boost the supersaturation when the nucleation process takes place and inhibit the growth of the formed particles. The NaOH aqueous solution is at the concentration of 0.01 M. The volume ratio of NaOH solution to CPF·HCl solution is fixed at 10 to achieve the mol ratio of 1. The NaOH aqueous solution was firstly mixed with certain volume of anti-solvent (IPA) in a triflask, afterward, the 0.1 M CPF·HCl solution was added into via a Brand® pipette tip. A high shear mixer was combined with the whole solution adding process and the stir speed was set at 14 Krpm. The volume ratio of the CPF solution to IPA had been changed from 1:2 to 1:15. After the reaction was finished, the slurry was filtered and the filtrate was washed with IPA and then vacuum dried at 60 °C for 24 h. 2.2.2. Spray drying The spray drying was considered as the preferable method to produce the dry powder for inhalation. The ultrafine particles of CPF by vacuum oven was redispersed in water for spray drying. The spray drying was carried out under conditions of feed rate of 25 ml/min, atomization pressure of 0.6 MPa, and inlet and outlet temperatures of 190 and 90 °C. The spray dried powder was then transferred into a container and stored over silica gel until further use for characterization and testing. 2.3. Characterization of the particles 2.3.1. Particle morphology Particle size and morphology of the ciprofloxacin were determined by scanning electron microscopy (SEM). The wet sample for SEM study
Fig. 2. Illustration of the multi-stage liquid impingers (MSLI) equipment.
H. Zhao et al. / Powder Technology 194 (2009) 81–86
83
2.3.2. Particle crystallinity The XRD patterns for ciprofloxacin were measured by using a Lab X-ray diffractometer (SHIMADZU, Japan). The X-ray diffractometer was operated with an anode current of 30 mA and an accelerating voltage of 40 kV. Samples were slightly pressed on a aluminum sample tray using glass slide and exposed to Cu radiation at diffraction angles (2θ) from 3° to 50°(scan speed = 10.0000 (°/min); sampling pitch= 0.0200 (°)). 2.3.3. Fourier transform infrared spectroscopy (FT-IR) FT-IR spectra were recorded with a Bruker IFS66 spectrometer in the range 400–4000 cm− 1 using a resolution of 2 cm− 1 and 32 scans. Samples were diluted with KBr mixing powder at 1% and pressed to obtain self-supporting disks. 2.3.4. Differential scanning calorimetry (DSC) and thermal gravimetrical (TG) analysis Differential scanning calorimetry (DSC) was employed to determine the crystal form of ciprofloxacin. The calorimeter used was a STA 449C Thermal Analyzer (Netzsch, Germany) thermal analysis system. A sample (about 10 mg) was weighed into an aluminium pan, which was hermetically sealed and placed in the pre-equilibrated DSC furnace (ambient temperature). Each sample was allowed to equilibrate for 5 min at ambient temperatue before being heated to 300 °C at a introheating rate of 10 °C/min. Thermal gravimetric (TG) analysis was carried out using a STA 449C Thermal Analyzer (Netzsch, Germany) thermal analysis system. The sample (10 mg) was weighed into aluminum crucible and placed into a
Fig. 3. SEM photographs of ciprofloxacin particles: (a) in pure aqueous reaction medium; (b) in aqueous and IPA mixed reaction medium.
Fig. 4. Particle size distributions of ciprofloxacin: (a) volume ratio = 1:2 (b) volume ratio = 1:6 (c) volume ratio = 1:15.
pre-equilibrated furnace at ambient temperature. After equilibration at this temperature for at least 5 min, the sample was heated to 300 °C at a heating rate of 10 °C/min. The weight change with temperature was recorded. 2.4. Aerosol performance The dispersion behavior of the powder was assessed using an Aerolizer® (Novartis Pharmaceutical, Australia) coupled through a USP stainless steel throat to a multi-stage liquid impinger (MSLI, Copley, UK), operating at 60 L/min controlled by the flow meter (DMF 2000, Copley, UK) for 4 s. Multi-stage liquid impingers (MSLI)
Fig. 5. SEM images of CPF dried powder: (a) vacuum dried at 25 °C; (b) at 60 °C.
84
H. Zhao et al. / Powder Technology 194 (2009) 81–86
Fig. 8. The comparison of FT-IR spectrum.
drug particles smaller than 6.8 µm in the aerosol cloud is interpolated from the mass of drug collected from stages 3, 4 and filter while emitted dose is the drug mass collected from the throat, stages 1–4 and filter of the MSLI. 3. Results and discussion
were used to sizing aerosols (Fig. 2). About 18 mg powder was loaded to a capsule and dispersed using the DPI into the MSLI, operating at 60 L/min. The drug deposition in the throat, four stages and the filter was determined by UV-spectrophotometer at 277 nm (UV-2501PC, SHIMADZU). A calibration curve of Ciprofloxacin in HCl (approximately 0.01 m/L) was constructed linear in the concentration range of 0–35 µg/mL [ABS = 95.802 concentration (μg/mL) + 0.0043; R = 0.9999]. Fine particle fraction loaded (FPFloaded) is defined as the mass fraction of drug particles smaller than 6.8 µm in the aerosol cloud relative to the total mass recovered, and fine particle fraction emitted (FPFemitted) is the mass fraction of drug particles smaller than 6.8 µm in the aerosol cloud relative to the emitted dose. The mass of
It was illustrated that IPA as the anti-solvent of ciprofloxacin had a positive effect on controlling the particle growth when IPA was introduced into the reaction system. Comparing with pure CPF solution and NaOH reactant system, the particle size in slurry was rapidly decreased in all the three dimensions from 15–20 µm to 4–5 µm in length, 2–3 µm to 0.5–1 µm in width, and 2–3 µm to 0.1–0.5 µm in height, when the ratio of CPF solution to IPA was 1:15. (Fig. 3). Furthermore, the volume ratio of the aqueous solution to IPA was the most sensitive parameter to the particle size and its distribution in this process. With the volume ratio changed from 1:2 to 1:15, the particle size decreased significantly and the size distribution became much narrower (Fig. 4). The width was defined as the specific particle size when measuring. The median specific size of as-prepared ciprofloxacin from slurry was about 4 µm at volume ratio of CPF solution to the IPA = 1:2. The median specific size decreased to 2 µm and 1 µm when the ratio turned to 1:6 and 1:15, respectively. The particle size demonstrated a decreasing trend with the increase of the ratio increase, which can be attributed to the higher supersaturation and the spatial hinder effect to the growth of ciprofloxacin crystals introduced by the IPA. When the CPF·HCl solution was added into the mixture of NaOH and IPA solution, the previous aqueous soluble CPF·HCl will react with [OH−] to generate tiny CPF nuclei that insoluble in water or IPA. The IPA molecules created a surrounding that cannot react with CPF·HCl molecule but restrict the space
Fig. 7. SEM images of microsized CPF aggregates with ultrafine primary particles by spray drying.
Fig. 9. The comparison of XRD spectrum.
Fig. 6. TG-DSC curve of ciprofloxacin particles: (a) sample of vacuum dried at 25 °C; (b) sample of vacuum dried at 60 °C.
H. Zhao et al. / Powder Technology 194 (2009) 81–86
85
Fig. 10. The aerosol performance of different ciprofloxacin powders.
for nucleation and growth especially when the IPA volume is large. Therefore, the ultrafine CPF was obtained in the mixture. The filtrate was vacuum dried at 60 °C for 8 h. It is found that the previous needle-shape particles were broken down into short-rodshape particles (Fig. 5a). However, the ciprofloxacin particles can preserve the needle-shape if vacuum dried at lower temperature (such as 25 °C) (see Fig. 5b). Therefore, the breakdown phenomenon was assumed as the result of the loss of crystal water. When analyzed by TG and DSC, it explained the reason of this phenomenon well. When the ciprofloxacin particles, gained by vacuum oven at 25 °C (Fig. 5b), were heated, the powder lost 15% of its weight from 25 °C to 112 °C and an endothermic peak was shown at 112 °C (Fig. 6a), which demonstrated that the crystal water existed in ciprofloxacin molecules and assisted the molecules to form such needle-shape particles. However, the crystal water could be completely removed from the ciprofloxacin molecules when vacuum dried at 60 °C (P = − 0.09 MPa). Therefore, no weight loss and endothermic peak appeared by TG and DSC (Fig. 6b). Spray dry was considered as a proper drying technique to gain powder for inhalation. The vacuum dried particles had its size less than 1 µm, but the dispersity and fluidity was poor. If the vacuum dried powder was re-dispersed into water with solids contents of 10 mg/ml and then sprayed, uniform 3–4 µm spherical aggregates constituted by ultrafine primary particles could be obtained under given spray drying parameters (Fig. 7). During the spray drying process, the suspension was atomized into discrete droplets with ciprofloxacin particles interior. The liquid evaporated in the hot drying air, therefore, the suspension migrated from the droplet interior to the surface inducing the shrink of the droplet, and the distance between particles got smaller and smaller. Finally, the particles would come in contact with each other to form the spherical aggregates under the optimal spray drying parameters. The spherical ciprofloxacin particles could be testified as having the same chemical structure and crystal form as the standard sample by the IR spectrum and X-ray diffraction pattern as shown in Figs. 8 and 9, which proved that there was no change in the composition and molecular structure induced by the reaction, dispersion and drying process. An in vitro DPI dispersion test was conducted to evaluate the aerosol performance of the ciprofloxacin dry powder. The vacuum dried particles were milled and sieved before the DPI test, whereas the sprayed particles were not. The FPF value of the ciprofloxacin powder was significantly improved after spray drying. The DPI formulation of ciprofloxacin was a novel way to deliver ciprofloxacin. The aerosol performance of the ciprofloxacin powders was shown in Fig. 10. It indicated that, the emitted FPF of the dry powder by vacuum drying reached 23.36%, however when the dry powder was dispersed and dried by spray drying, the emitted FPF could reach 65.03%. Most of the dry powder (Fig. 4a) dried by vacuum drying stayed at stage 1, although the particle size is about 1 µm. The dispersity of ciprofloxacin powder obtained by vacuum drying was poor, and the ultrafine particles formed larger aggregates with irregular aggregating force. Approximately 60% of the powders exhibited the property of the
aerodynamic diameter Da N 13 µm. However, if this dry powder was re-dispersed and spray dried, the particles of the aggregates became uniform. Its dispersity became well established, and, most of it exhibited the property of the aerodynamic diameter Da b 5 µm. 4. Conclusions In this study, the ultra-fined ciprofloxacin particles for dry powder inhalation could be obtained by the combination of reactive precipitation and anti-solvent precipitation followed by spray drying at the optimal parameters. The anti-solvent had the positive effect on controlling the particle size. The result shows that the ultra-fined primary particles of ciprofloxacin (1–2 µm) can be prepared under the volume ratio about 1:15 of CPF·HCl solution to the IPA. Further, after controlled spray drying, CPF dry powder can form uniform spherical particles with diameter of 3–4 µm and exhibited great improved aerosol performance. Acknowledgments This work was financially supported by National Natural Science Foundation of China (Nos. 20806004 and 20821004) and the Talent Training Program of Beijing City (2007B022). References [1] Fengqian Li, Jinhong Hu, The new administration system of ciprofloxacin, World Pharm. 22 (2001) 33235. [2] Jiang Li, Lv. Ruihuang, The adverse reaction of ciprofloxacin, J. Clin. Pulm. Med. 10 (2005) 109. [3] J.L. Hoover, B.D. Rush, K.F. Wilkinson, J.S. Day, P.S. Burton, T.J. Vidmar, M.J. Ruwart, Peptides are better absorbed from the lung than the gut in the rat, Pharm. Res. 9 (1992) 1103–1106. [4] Fengqian Li, Jinhong Hu, Bi Lu n, Quanguang Zhu, Huajun Sun, Ciprofloxacin loaded bovine serum albumin microspheres: preparation and drug release characterization in vitro, J. Chin. Pharm. Sci. 10 (2001) 24–25. [5] R.U. Agu, M.I. Ugwoke, M. Armand, R. Kinget, N. verbeke, The lung as a route for systemic delivery of therapeutic proteins and peptides, Respir. Res. 2 (2001) 198–209. [6] J. Jones, Clearance of inhaled particles from the alceoli, in: S.W. Clarke (Ed.), Aerosol and the Lung: Clinical and Experimental Aspects, London, Butter-worth, 1984. [7] P. Chattopadhyay, R.B. Gupta, Production of griseofulvin nanoparticles using supercritical CO2 antisolvent with enhanced mass transfer, Int. J. Pharm. 228 (2001) 19. [8] C. Domingo, E. Berends, van G.M. Rosmalen, Precipitation of ultrafine organic crystals from the rapid expansion of supercritical solutions over a capillary and a frit nozzle, J. Supercrit. Fluids 10 (1997) 39. [9] D. Horn, J. Rieger, Organic nanoparticles in aqueous phasestheory, experiment, and use, Angew. Chem., Int. Ed. 40 (2001) 4330. [10] N. Rasenack, H. Steckel, B.W. Müller, Micronization of anti-Inflammatory drugs for pulmonary delivery by a controlled crystallization process, J. Pharm. Sci. 92 (1) (2003) 35. [11] H. Steckel, J. Thies, B.W. Müller, Micronizing of steroids for pulmonary delivery by supercritical carbon dioxide, Int. J. Pharm. 152 (1997) 99. [12] V. Uskokovic, E. Matijevic, Uniform particles of pure and silica-coated cholesterol, J. Coll. I. Sci., 315 (2007) 500–511. [13] G.A. Pozarnsky, E. Matijevic, Preparation of monodisperse colloids of biologically active compounds I, Naproxen. Coll. Surf. A 125 (1997) 47–52. [14] L. Joguet, E. Matijevic, Preparation of Finely Dispersed Drugs: III. Cyclosporine, J. Coll. I. Sci., 250 (2002) 503–506.
86
H. Zhao et al. / Powder Technology 194 (2009) 81–86
[15] S.D. Skapin, E. Matijevic, Preparation and coating of finely dispersed drugs: 4, Loratadine and danazol, J. Coll. I. Sci., 272 (2004) 90–98. [16] Z.Y. Yang, Y. Le, T.T. Hu, Z.G. Shen, J.F. Chen, J. Yun, Production of ultrafine sumatriptan succinate particles for pulmonary delivery, Pharm. Res. 25 (2008) 2012–2018. [17] J.F. Chen, C. Zheng, G.T. Chen, Interaction of macro-and micromixing on particle size distribution in reactive precipitation, Chem. Eng. Sci. 51 (1996) 1957–1966. [18] J.F. Chen, J.Y. Zhang, Z.G. Shen, J. Zhong, J. Yun, Preparation and characterization of amorphous cefuroxime axetil drug nanoparticles with novel technology: highgravity antisolvent precipitation, Ind. Eng. Chem. Res. 45 (2006) 8723–8727. [19] H. Zhao, J.F. Chen, Y. Zhao, L. Jiang, J.W. Sun, J. Yun, Hierarchical assembly of multilayered hollow microspheres from an amphiphilic pharmaceutical molecule of azithromycin, Adv. Mater. 20 (2008) 3682–3686.
[20] T.T. Hu, H. Zhao, L.C. Jiang, Y. Le, J.F. Chen, J. Yun, Engineering pharmaceutical fine particles of budesonide for dry powder inhalation (DPI), Ind. Eng. Chem. Res. 47 (2008) 9623–9627. [21] M.R. Violanto, H.W. Fischer 1989. Method for making uniformly sized particles from water-insoluble organic compounds, US Patent No. 4,826,689. [22] F. Ruch, E. Matijevíc, Preparation of micrometer size Budesonide particles by precipitation, J. Colloid Interface Sci. 229 (2000) 207–211. [23] B.L. Cushing, V.L. Kolesnichenko, C.J. O'Coonor, Recent advances in the liquidphase syntheses of inorganic nanoparticles, Chem. Rev. 104 (2004) 3893–3946.
Contents lists available at ScienceDirect
Powder Technology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p ow t e c
Preparation of microsized spherical aggregates of ultrafine ciprofloxacin particles for dry powder inhalation (DPI) Hong Zhao a, Yuan Le a, Haoying Liu a, Tingting Hu a, Zhigang Shen a,b, Jimmy Yun b, Jian-Feng Chen a,⁎ a b
Sin-China Nano Technology Center, Key Lab for Nanomaterials, Ministry of Education, Beijing University of Chemical. Technology, Beijing 100029, PR China Nanomaterials Technology Pte., Ltd., Singapore 139944, Singapore
a r t i c l e
i n f o
Article history: Received 17 November 2008 Received in revised form 20 February 2009 Accepted 17 March 2009 Available online 28 March 2009 Keywords: Ciprofloxacin Ultra-fine particles Reactive precipitation Fine particle fraction (FPF) Anti-solvent
a b s t r a c t In this paper, the ultrafine ciprofloxacin (CPF) particles were prepared by the reactive precipitation of ciprofloxacin hydrochloride (CPF·HCl) and NaOH aqueous solution with the presence of isopropyl alcohol (IPA) as the anti-solvent. Subsequently, microsized spherical CPF aggregates were generated using the prepared ultrafine particles as building blocks by spray drying method. The effect of the volume ratio of the CPF solution to anti-solvent (IPA) was investigated. The result shows that the ultra-fined particles of ciprofloxacin can be produced under the volume ratio of 1:15. After spray drying, 3 — 4 µm spherical aggregates with ultrafine primary CPF particles can be obtained and exhibited great improved aerosol performance with fine particle fraction (FPF) up to 60%. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Ciprofloxacin is a fluoroquinolone antibiotic that has demonstrated in vitro activity against Staphylococcus and Bacillus species and most gram-negative microorganisms including Pseudomonas species etc. It is often used in the treatment of inhalation anthrax and cystic fibrosis lung infection. Current delivery methods for ciprofloxacin include oral, injection and ocular deliveries. However, the bioavailability of ciprofloxacin administered orally is relatively low because half of the drug is metabolized in the gastrointestinal tract [1]. Moreover, a substantial proportion of patients suffer some severe symptoms such as nausea, inappetence or bellyache etc., which make oral and intranasal treatment unsatisfactory. Subcutaneous administration is an alternative, but it also has some untoward effects such as rubefaction, phlebitis and shock etc. [2]. The pulmonary delivery for ciprofloxacin can be a viable alternative for self-administration, whereby these limitations could be overcome. In recent years, the pulmonary delivery route has attracted much attention because of the following advantages over other routes: 1) the surface area of a lung is extremely large and the mucosal permeation of drug substances is comparatively easy, because the vascular system is well developed and the wall of the alveolus is extremely thin. 2) The pulmonary delivery route can avoid the first pass effect of hepar, which may highly enhance the bioavailability of administration [3]. The pulmonary delivery includes metered dose inhaler, dry powder inhaler and nebulizer. Dry powder inhalers (DPI) have received in⁎ Corresponding author. Tel.: +86 10 6444 6466; fax: +86 10 6443 4784. E-mail address: [email protected] (J.-F. Chen). 0032-5910/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.powtec.2009.03.031
creasing attention mainly because the devices do not require chlorofluorocarbon propellants to deliver the drug and can thus be regarded as safe delivery systems that do not affect the ozone layer. The particle size of the drug to be delivered is critical for its efficient deposition in the lower airways when delivering. It is feasible to deliver drug particles with the diameter of 1–5 µm to the specific site of the respiratory tract via DPIs [4]. These particles are accumulated in the deep lungs where they provide continuous absorption of drugs. However, particles measuring approximately smaller than 1 µm are exhaled during normal tidal breathings and those measuring approximately larger than 6 µm are deposited in the upper airways with a vigorously mucocilliary clearance [5,6]. Therefore, producing particles with demand size and specific structures is a significative work for DPI use. Conventional crystallization which can control the particle size includes jet-milling, pearl-ball mills, supercritical fluids and so on [7–11]. These techniques require significant energy input, which can induce disorder, defects or even amorphous regions in the drug particles, consequently causing physico–chemical instability of the product. Therefore, alternative production process is of great requirement for the pharmaceutical industry. Liquid precipitation technique, which includes reactive precipitation and liquid anti-solvent precipitation, has been developed in various pathways to effectively manipulate the size and morphology of pharmaceutical particles for different applications [12–20]. Moreover, the liquid precipitation technique has been demonstrated prospective in industrial fields because of its low cost, convenience in processing, as well as the ease of scale-up [21–23]. The aim of this investigation was to combine the liquid anti-solvent precipitation and the spray drying technique to generate spherical
82
H. Zhao et al. / Powder Technology 194 (2009) 81–86
Fig. 1. Reaction equation.
particles with desired size and structure for DPI use. Firstly, ultrafine CPF particles as the sub-units were prepared by the reactive precipitation with anti-solvent in presence. Subsequently, spray drying was employed to assemble the sub-units into uniform spherical aggregates. 2. Experimental 2.1. Materials The raw material of this project was market Ciprofloxacin hydrochloride product synthesized in China. All the chemicals and solvents used in following experiments are A.R. grade supplied by Beijing Chemical Agent Company. The water used during the whole experimental process is de-ionized water. 2.2. Particles preparation
was prepared by dropping one or two droplets of slurry on a small piece of slide directly and then dried under room conditions, which is in order to observe the shape and size of the particles in the slurry before drying to powder. The slide was fixed on aluminum stubs using double-sided adhesive tape. The dry powder sample was made by directly fixing it on aluminum stubs using double-sided adhesive tape. A small amount of ciprofloxacin particles were scattered on the stub and dispersed by tapping lightly on the edge of the stub with a spatula to break up any agglomerates. Both the slide and dry powder fixed on the aluminum stub were coated with Au at 50 mA for 30 s using a Pelco Model 3 sputter-coater under an Ar atmosphere. A Cambridge S250MK3 scanning electron microscope (SEM) (Cambridge Instruments Inc., U.K.) at an accelerating voltage of 10 kV with a secondary electron detector was used to obtain digital images of the samples. The particle size and its size distribution were determined by the Image-Pro Plus 5.0 via the obtained SEM photographs.
2.2.1. The combination of reaction precipitation and anti-solvent precipitation In this study, ultrafine ciprofloxacin particles were prepared by a method that is the reactive precipitation with the presence of the antisolvent (IPA). Ciprofloxacin (C17H18FN3O3, 1-cyclopropyl-6-fluoro-1, 4-dihydro4-oxo-7-(piperazin-1-yl) quinoline-3 -carboxylic acid, MW 331.4) was firstly dissolved in 0.1 M HCl solution (CPF·HCl) and then reacted with NaOH solution (Fig. 1) to obtain CPF precipitates. Isopropyl alcohol (IPA) defined as anti-solvent of ciprofloxacin was introduced into this reaction to generate a circumstance which will boost the supersaturation when the nucleation process takes place and inhibit the growth of the formed particles. The NaOH aqueous solution is at the concentration of 0.01 M. The volume ratio of NaOH solution to CPF·HCl solution is fixed at 10 to achieve the mol ratio of 1. The NaOH aqueous solution was firstly mixed with certain volume of anti-solvent (IPA) in a triflask, afterward, the 0.1 M CPF·HCl solution was added into via a Brand® pipette tip. A high shear mixer was combined with the whole solution adding process and the stir speed was set at 14 Krpm. The volume ratio of the CPF solution to IPA had been changed from 1:2 to 1:15. After the reaction was finished, the slurry was filtered and the filtrate was washed with IPA and then vacuum dried at 60 °C for 24 h. 2.2.2. Spray drying The spray drying was considered as the preferable method to produce the dry powder for inhalation. The ultrafine particles of CPF by vacuum oven was redispersed in water for spray drying. The spray drying was carried out under conditions of feed rate of 25 ml/min, atomization pressure of 0.6 MPa, and inlet and outlet temperatures of 190 and 90 °C. The spray dried powder was then transferred into a container and stored over silica gel until further use for characterization and testing. 2.3. Characterization of the particles 2.3.1. Particle morphology Particle size and morphology of the ciprofloxacin were determined by scanning electron microscopy (SEM). The wet sample for SEM study
Fig. 2. Illustration of the multi-stage liquid impingers (MSLI) equipment.
H. Zhao et al. / Powder Technology 194 (2009) 81–86
83
2.3.2. Particle crystallinity The XRD patterns for ciprofloxacin were measured by using a Lab X-ray diffractometer (SHIMADZU, Japan). The X-ray diffractometer was operated with an anode current of 30 mA and an accelerating voltage of 40 kV. Samples were slightly pressed on a aluminum sample tray using glass slide and exposed to Cu radiation at diffraction angles (2θ) from 3° to 50°(scan speed = 10.0000 (°/min); sampling pitch= 0.0200 (°)). 2.3.3. Fourier transform infrared spectroscopy (FT-IR) FT-IR spectra were recorded with a Bruker IFS66 spectrometer in the range 400–4000 cm− 1 using a resolution of 2 cm− 1 and 32 scans. Samples were diluted with KBr mixing powder at 1% and pressed to obtain self-supporting disks. 2.3.4. Differential scanning calorimetry (DSC) and thermal gravimetrical (TG) analysis Differential scanning calorimetry (DSC) was employed to determine the crystal form of ciprofloxacin. The calorimeter used was a STA 449C Thermal Analyzer (Netzsch, Germany) thermal analysis system. A sample (about 10 mg) was weighed into an aluminium pan, which was hermetically sealed and placed in the pre-equilibrated DSC furnace (ambient temperature). Each sample was allowed to equilibrate for 5 min at ambient temperatue before being heated to 300 °C at a introheating rate of 10 °C/min. Thermal gravimetric (TG) analysis was carried out using a STA 449C Thermal Analyzer (Netzsch, Germany) thermal analysis system. The sample (10 mg) was weighed into aluminum crucible and placed into a
Fig. 3. SEM photographs of ciprofloxacin particles: (a) in pure aqueous reaction medium; (b) in aqueous and IPA mixed reaction medium.
Fig. 4. Particle size distributions of ciprofloxacin: (a) volume ratio = 1:2 (b) volume ratio = 1:6 (c) volume ratio = 1:15.
pre-equilibrated furnace at ambient temperature. After equilibration at this temperature for at least 5 min, the sample was heated to 300 °C at a heating rate of 10 °C/min. The weight change with temperature was recorded. 2.4. Aerosol performance The dispersion behavior of the powder was assessed using an Aerolizer® (Novartis Pharmaceutical, Australia) coupled through a USP stainless steel throat to a multi-stage liquid impinger (MSLI, Copley, UK), operating at 60 L/min controlled by the flow meter (DMF 2000, Copley, UK) for 4 s. Multi-stage liquid impingers (MSLI)
Fig. 5. SEM images of CPF dried powder: (a) vacuum dried at 25 °C; (b) at 60 °C.
84
H. Zhao et al. / Powder Technology 194 (2009) 81–86
Fig. 8. The comparison of FT-IR spectrum.
drug particles smaller than 6.8 µm in the aerosol cloud is interpolated from the mass of drug collected from stages 3, 4 and filter while emitted dose is the drug mass collected from the throat, stages 1–4 and filter of the MSLI. 3. Results and discussion
were used to sizing aerosols (Fig. 2). About 18 mg powder was loaded to a capsule and dispersed using the DPI into the MSLI, operating at 60 L/min. The drug deposition in the throat, four stages and the filter was determined by UV-spectrophotometer at 277 nm (UV-2501PC, SHIMADZU). A calibration curve of Ciprofloxacin in HCl (approximately 0.01 m/L) was constructed linear in the concentration range of 0–35 µg/mL [ABS = 95.802 concentration (μg/mL) + 0.0043; R = 0.9999]. Fine particle fraction loaded (FPFloaded) is defined as the mass fraction of drug particles smaller than 6.8 µm in the aerosol cloud relative to the total mass recovered, and fine particle fraction emitted (FPFemitted) is the mass fraction of drug particles smaller than 6.8 µm in the aerosol cloud relative to the emitted dose. The mass of
It was illustrated that IPA as the anti-solvent of ciprofloxacin had a positive effect on controlling the particle growth when IPA was introduced into the reaction system. Comparing with pure CPF solution and NaOH reactant system, the particle size in slurry was rapidly decreased in all the three dimensions from 15–20 µm to 4–5 µm in length, 2–3 µm to 0.5–1 µm in width, and 2–3 µm to 0.1–0.5 µm in height, when the ratio of CPF solution to IPA was 1:15. (Fig. 3). Furthermore, the volume ratio of the aqueous solution to IPA was the most sensitive parameter to the particle size and its distribution in this process. With the volume ratio changed from 1:2 to 1:15, the particle size decreased significantly and the size distribution became much narrower (Fig. 4). The width was defined as the specific particle size when measuring. The median specific size of as-prepared ciprofloxacin from slurry was about 4 µm at volume ratio of CPF solution to the IPA = 1:2. The median specific size decreased to 2 µm and 1 µm when the ratio turned to 1:6 and 1:15, respectively. The particle size demonstrated a decreasing trend with the increase of the ratio increase, which can be attributed to the higher supersaturation and the spatial hinder effect to the growth of ciprofloxacin crystals introduced by the IPA. When the CPF·HCl solution was added into the mixture of NaOH and IPA solution, the previous aqueous soluble CPF·HCl will react with [OH−] to generate tiny CPF nuclei that insoluble in water or IPA. The IPA molecules created a surrounding that cannot react with CPF·HCl molecule but restrict the space
Fig. 7. SEM images of microsized CPF aggregates with ultrafine primary particles by spray drying.
Fig. 9. The comparison of XRD spectrum.
Fig. 6. TG-DSC curve of ciprofloxacin particles: (a) sample of vacuum dried at 25 °C; (b) sample of vacuum dried at 60 °C.
H. Zhao et al. / Powder Technology 194 (2009) 81–86
85
Fig. 10. The aerosol performance of different ciprofloxacin powders.
for nucleation and growth especially when the IPA volume is large. Therefore, the ultrafine CPF was obtained in the mixture. The filtrate was vacuum dried at 60 °C for 8 h. It is found that the previous needle-shape particles were broken down into short-rodshape particles (Fig. 5a). However, the ciprofloxacin particles can preserve the needle-shape if vacuum dried at lower temperature (such as 25 °C) (see Fig. 5b). Therefore, the breakdown phenomenon was assumed as the result of the loss of crystal water. When analyzed by TG and DSC, it explained the reason of this phenomenon well. When the ciprofloxacin particles, gained by vacuum oven at 25 °C (Fig. 5b), were heated, the powder lost 15% of its weight from 25 °C to 112 °C and an endothermic peak was shown at 112 °C (Fig. 6a), which demonstrated that the crystal water existed in ciprofloxacin molecules and assisted the molecules to form such needle-shape particles. However, the crystal water could be completely removed from the ciprofloxacin molecules when vacuum dried at 60 °C (P = − 0.09 MPa). Therefore, no weight loss and endothermic peak appeared by TG and DSC (Fig. 6b). Spray dry was considered as a proper drying technique to gain powder for inhalation. The vacuum dried particles had its size less than 1 µm, but the dispersity and fluidity was poor. If the vacuum dried powder was re-dispersed into water with solids contents of 10 mg/ml and then sprayed, uniform 3–4 µm spherical aggregates constituted by ultrafine primary particles could be obtained under given spray drying parameters (Fig. 7). During the spray drying process, the suspension was atomized into discrete droplets with ciprofloxacin particles interior. The liquid evaporated in the hot drying air, therefore, the suspension migrated from the droplet interior to the surface inducing the shrink of the droplet, and the distance between particles got smaller and smaller. Finally, the particles would come in contact with each other to form the spherical aggregates under the optimal spray drying parameters. The spherical ciprofloxacin particles could be testified as having the same chemical structure and crystal form as the standard sample by the IR spectrum and X-ray diffraction pattern as shown in Figs. 8 and 9, which proved that there was no change in the composition and molecular structure induced by the reaction, dispersion and drying process. An in vitro DPI dispersion test was conducted to evaluate the aerosol performance of the ciprofloxacin dry powder. The vacuum dried particles were milled and sieved before the DPI test, whereas the sprayed particles were not. The FPF value of the ciprofloxacin powder was significantly improved after spray drying. The DPI formulation of ciprofloxacin was a novel way to deliver ciprofloxacin. The aerosol performance of the ciprofloxacin powders was shown in Fig. 10. It indicated that, the emitted FPF of the dry powder by vacuum drying reached 23.36%, however when the dry powder was dispersed and dried by spray drying, the emitted FPF could reach 65.03%. Most of the dry powder (Fig. 4a) dried by vacuum drying stayed at stage 1, although the particle size is about 1 µm. The dispersity of ciprofloxacin powder obtained by vacuum drying was poor, and the ultrafine particles formed larger aggregates with irregular aggregating force. Approximately 60% of the powders exhibited the property of the
aerodynamic diameter Da N 13 µm. However, if this dry powder was re-dispersed and spray dried, the particles of the aggregates became uniform. Its dispersity became well established, and, most of it exhibited the property of the aerodynamic diameter Da b 5 µm. 4. Conclusions In this study, the ultra-fined ciprofloxacin particles for dry powder inhalation could be obtained by the combination of reactive precipitation and anti-solvent precipitation followed by spray drying at the optimal parameters. The anti-solvent had the positive effect on controlling the particle size. The result shows that the ultra-fined primary particles of ciprofloxacin (1–2 µm) can be prepared under the volume ratio about 1:15 of CPF·HCl solution to the IPA. Further, after controlled spray drying, CPF dry powder can form uniform spherical particles with diameter of 3–4 µm and exhibited great improved aerosol performance. Acknowledgments This work was financially supported by National Natural Science Foundation of China (Nos. 20806004 and 20821004) and the Talent Training Program of Beijing City (2007B022). References [1] Fengqian Li, Jinhong Hu, The new administration system of ciprofloxacin, World Pharm. 22 (2001) 33235. [2] Jiang Li, Lv. Ruihuang, The adverse reaction of ciprofloxacin, J. Clin. Pulm. Med. 10 (2005) 109. [3] J.L. Hoover, B.D. Rush, K.F. Wilkinson, J.S. Day, P.S. Burton, T.J. Vidmar, M.J. Ruwart, Peptides are better absorbed from the lung than the gut in the rat, Pharm. Res. 9 (1992) 1103–1106. [4] Fengqian Li, Jinhong Hu, Bi Lu n, Quanguang Zhu, Huajun Sun, Ciprofloxacin loaded bovine serum albumin microspheres: preparation and drug release characterization in vitro, J. Chin. Pharm. Sci. 10 (2001) 24–25. [5] R.U. Agu, M.I. Ugwoke, M. Armand, R. Kinget, N. verbeke, The lung as a route for systemic delivery of therapeutic proteins and peptides, Respir. Res. 2 (2001) 198–209. [6] J. Jones, Clearance of inhaled particles from the alceoli, in: S.W. Clarke (Ed.), Aerosol and the Lung: Clinical and Experimental Aspects, London, Butter-worth, 1984. [7] P. Chattopadhyay, R.B. Gupta, Production of griseofulvin nanoparticles using supercritical CO2 antisolvent with enhanced mass transfer, Int. J. Pharm. 228 (2001) 19. [8] C. Domingo, E. Berends, van G.M. Rosmalen, Precipitation of ultrafine organic crystals from the rapid expansion of supercritical solutions over a capillary and a frit nozzle, J. Supercrit. Fluids 10 (1997) 39. [9] D. Horn, J. Rieger, Organic nanoparticles in aqueous phasestheory, experiment, and use, Angew. Chem., Int. Ed. 40 (2001) 4330. [10] N. Rasenack, H. Steckel, B.W. Müller, Micronization of anti-Inflammatory drugs for pulmonary delivery by a controlled crystallization process, J. Pharm. Sci. 92 (1) (2003) 35. [11] H. Steckel, J. Thies, B.W. Müller, Micronizing of steroids for pulmonary delivery by supercritical carbon dioxide, Int. J. Pharm. 152 (1997) 99. [12] V. Uskokovic, E. Matijevic, Uniform particles of pure and silica-coated cholesterol, J. Coll. I. Sci., 315 (2007) 500–511. [13] G.A. Pozarnsky, E. Matijevic, Preparation of monodisperse colloids of biologically active compounds I, Naproxen. Coll. Surf. A 125 (1997) 47–52. [14] L. Joguet, E. Matijevic, Preparation of Finely Dispersed Drugs: III. Cyclosporine, J. Coll. I. Sci., 250 (2002) 503–506.
86
H. Zhao et al. / Powder Technology 194 (2009) 81–86
[15] S.D. Skapin, E. Matijevic, Preparation and coating of finely dispersed drugs: 4, Loratadine and danazol, J. Coll. I. Sci., 272 (2004) 90–98. [16] Z.Y. Yang, Y. Le, T.T. Hu, Z.G. Shen, J.F. Chen, J. Yun, Production of ultrafine sumatriptan succinate particles for pulmonary delivery, Pharm. Res. 25 (2008) 2012–2018. [17] J.F. Chen, C. Zheng, G.T. Chen, Interaction of macro-and micromixing on particle size distribution in reactive precipitation, Chem. Eng. Sci. 51 (1996) 1957–1966. [18] J.F. Chen, J.Y. Zhang, Z.G. Shen, J. Zhong, J. Yun, Preparation and characterization of amorphous cefuroxime axetil drug nanoparticles with novel technology: highgravity antisolvent precipitation, Ind. Eng. Chem. Res. 45 (2006) 8723–8727. [19] H. Zhao, J.F. Chen, Y. Zhao, L. Jiang, J.W. Sun, J. Yun, Hierarchical assembly of multilayered hollow microspheres from an amphiphilic pharmaceutical molecule of azithromycin, Adv. Mater. 20 (2008) 3682–3686.
[20] T.T. Hu, H. Zhao, L.C. Jiang, Y. Le, J.F. Chen, J. Yun, Engineering pharmaceutical fine particles of budesonide for dry powder inhalation (DPI), Ind. Eng. Chem. Res. 47 (2008) 9623–9627. [21] M.R. Violanto, H.W. Fischer 1989. Method for making uniformly sized particles from water-insoluble organic compounds, US Patent No. 4,826,689. [22] F. Ruch, E. Matijevíc, Preparation of micrometer size Budesonide particles by precipitation, J. Colloid Interface Sci. 229 (2000) 207–211. [23] B.L. Cushing, V.L. Kolesnichenko, C.J. O'Coonor, Recent advances in the liquidphase syntheses of inorganic nanoparticles, Chem. Rev. 104 (2004) 3893–3946.