Large-Scale Preparation of Amorphous Cefixime Nanoparticles by

Article pubs.acs.org/IECR

Large-Scale Preparation of Amorphous Cefixime Nanoparticles by Antisolvent Precipitation in a High-Gravity Rotating Packed Bed Yun-Yun Kuang,†,§ Zhi-Bing Zhang,§ Miao-Ling Xie,§ Jie-Xin Wang,*,†,‡ Yuan Le,†,‡ and Jian-Feng Chen*,†,‡ †

State Key Laboratory of Organic−Inorganic Composites, Beijing University of Chemical Technology, Beijing 100029, PR China Research Center of the Ministry of Education for High Gravity Engineering and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China § Beijing Engineering Research Center of nano-micro structured drugs, Beijing Winsunny Pharmaceutical Co., Ltd, Beijing 100022, PR China ‡

ABSTRACT: To enhance the solubility and dissolution rate, and thus potentially improve the oral bioavailability of cefixime (CFX), amorphous CFX nanoparticles were prepared via high-gravity antisolvent precipitation (HGAP) without the aid of any pharmaceutical additives in a rotating packed bed (RPB). The effects of operating variables on particle size and distribution were investigated. Compared to raw CFX, the mean size of prepared nanoparticles decreased greatly from about 2.1 μm to 57 nm, and the saturation solubility increased tremendously from 0.289 to 0.951 mg/mL. CFX nanoparticles showed good stability and were capable of generating a maximum supersaturation level, reaching up to ∼22.8 times of raw CFX’s saturation solubility. Further, CFX nanoparticles achieved 100% drug dissolution within 2 min, while the raw drug did not dissolve completely after 45 min, suggesting that the solubility and dissolution properties of CFX nanoparticles were significantly improved. Since the drug recovery ratio achieved 99.9%, and the production capacity of lab-scale RPB with a continuous operation reached 1.8 kg/h, the HGAP method might offer a general and facile platform for mass production of CFX nanoparticles.

1. INTRODUCTION Amorphous drug nanoparticles represent an effective oral bioavailability enhancement strategy for water insoluble drugs.1 Nanoparticles show improved dissolution rate, while an amorphous form allows for high supersaturation of the metastable state (up to 1600 times) relative to the crystalline equilibrium solubility.2−4 An increase in supersaturation in the gastrointestinal tract would lead to greater flux through biomembranes and higher bioavailability.5−7 More importantly, the increased dissolution rate can minimize the time window for solution-mediated crystallization of the remaining solid phase,8 which unfavorably tends to occur in microscale amorphous solid dispersions due to their slower dissolution rate. Amorphous drug nanoparticles have been prepared by various techniques, including antisolvent precipitation,1,7,9,10 reactive precipitation,11 evaporative precipitation,12 pH-shift precipitation,13 and sonoprecipitation.14 Among these techniques, the antisolvent precipitation technique has promising properties, especially from an industrial viewpoint, such as its low-cost, time-saving, convenience in processing, as well as the ease for scale-up. For antisolvent precipitation of insoluble drug, instant and homogeneous mixing of the solvent and the antisolvent is crucial to ensure the particles in nanorange and with narrow particle size distribution. In this respect, the traditional stirred tank is apparently not suitable for a continuous precipitation of drug nanoparticles at a large scale due to the intermittent batch production and ununiform distribution of spatial concentration caused by poor mixing effect.15 © XXXX American Chemical Society

Various methods and equipment of process intensification are being developed to gain excellent mixing efficiency to precipitate nanoparticles.16−20 High-gravity antisolvent precipitation (HGAP) technology, implemented by rotating packed bed (RPB), is prestigious for its continuous massive production and easy scale-up for industrialization.21,22 RPB can generate a high-gravity environment to strongly intensify the mixing and mass transfer processes. The magnitude of mixing efficiency and mass transfer rate in a RPB are much larger than those in a stirred tank, which are very helpful for generating higher supersaturation, faster nucleation rate, and more uniform spatial concentration.23 Therefore, it can provide good control of particle size and size distribution. HGAP technology has been successfully applied to prepare nanosized armorphous drugs such as cefuroxime axetil,24 itraconazole,25 and glibenclamide.26 However, the supersaturation generation and saturation solubility properties of these nanoparticles were not mentioned. Cefixime (CFX), an oral third generation cephalosporin antibiotic, issued to treat infections caused by bacteria such as pneumonia, bronchitis, and gonorrhea and ear, lung, throat, and urinary tract infections.27 CFX exhibits poor aqueous solubility and low dissolution rate, which limits its effective absorption and bioavailability.28 Literature survey reveals that after the administration of CFX oral tablets, only ∼40% of the drug is bioavailable.29 To improve drug solubility and dissolution performance, CFX solid dispersions and cyclodextrin comReceived: April 28, 2015 Revised: June 30, 2015 Accepted: August 3, 2015

A

DOI: 10.1021/acs.iecr.5b01584 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research

Figure 1. (a) Schematic representation of HGAP process (1, casing; 2, packed rotator; 3, motor; 4, liquid distributors; 5, flow meters; 6, seal ring; 7, outlet; 8, pump; 9 and 10, liquid storage containers). (b) Structure of RPB. (c) Illustration of the fluid pattern of HGAP process within RPB.

Table 1. Parameters of RPB Used in This Study rotor

packing

operation parameters

inner diameter (mm)

out diameter (mm)

axial height (mm)

material

porosity (%)

volumetric flow rate (L/min)

rotating speed (r/min)

production capacity (kg/h)

35

80

18

wire-mesh

95%

0.2−4

0−2800

0.15−3

plexes have been produced.29−31 In addition, the supercritical fluid technology has also been fundamentally investigated.28 However, no attempts have been done to prepare CFX amorphous nanoparticles. In this study, HGAP technology was proposed to produce amorphous CFX nanoparticles with enhanced solubility and dissolution rate, which could generate a high supersaturation environment when dissolved in water. The particles were characterized by SEM, XRD, BET, TG, and FT-IR. The supersaturation generation, saturation solubility, redispersibility, solid-state stability, and dissolution performance of CFX nanoparticles were also evaluated.

2.3. Particle Size and Morphology. The size and morphology of CFX samples were observed by scanning electron microscopy (SEM), JSM-6701 (JEOL Inc., Japan). The dry powder or a glass slide with sample was fixed on an aluminum stub using double-sided adhesive tape and coated with gold. The column chart of the particle size distribution (PSD) was generated using the Image-Pro 5.1 (Media Cybernetics, Inc.) according to the obtained SEM images. The diameter or width of the particle was prescribed as the specific particle size. Gauss fitting curves were also constructed to give an apparent illustration for PSD comparison. 2.4. Specific Surface Area. The specific surface area was measured using a N2 adsorption method. In this method, calculation was implemented by a Surface Area Analyzer (Quadrasorb SI, Quantachrome, USA) based on the BET equation. Before measuring, sample powder was degassed for at least 4 h. 2.5. Chemical Composition and Physical Characteristics. FT-IR analysis was carried out to evaluate the molecular states of raw CFX and CFX nanoparticles. FT-IR spectra were recorded with a Nicolet model 8700 spectrometer (Nicolet instrument corporation) in the range of 400−4000 cm−1 using a resolution of 2 cm−1 and 32 scans. Samples were diluted with 1% of KBr mixing powder and pressed to obtain self-supporting disks. X-ray diffraction (XRD) was employed to detect any changes in physical characteristics and crystallinity of CFX particles using a XRD-6000 diffractometer (Shimadzu Inc., Japan). The sample powder was placed in an aluminum sample holder. Cu Kα1 radiation was generated at 30 mA and 40 kV. The scanning speed was 5°/min from 5° to 50° with a step size of 0.05°. Thermal gravimetric (TG) analysis was implemented using a thermogravimetrical analyzer (TGS-2, PerkinElmer). The experiment was operated under a heating rate of 10 °C/min using nitrogen flow (50 mL/min) and samples were weighed (approximately 5 mg) in open aluminum pans. The percentage weight loss of the samples was monitored from 30 to 300 °C.

2. EXPERIMENTAL SECTION 2.1. Materials and Equipment. Raw CFX was supplied by Wuhan Hezhong Bio-Chemical Manufacture Co., Ltd. (Hubei, China). Tetrahydrofuran (THF) and isopropyl ether (IPE) (AR grade) were purchased from Chemical Reagent Company (Beijing, China). The experimental setup for HGAP process is schematically displayed in Figure 1. The key part of the RPB is a packed rotator (Table 1), which could be referenced to our previous work for more details.15,22 2.2. Preparation of CFX Nanoparticles. In a typical process, THF and IPE were used as solvent and antisolvent, respectively. Raw CFX was dissolved in THF at the concentration of 100 mg/mL, and the solution was filtrated through 0.45 μm pore-size membranes to remove the impure particulates. Then, the drug solution and IPE were added into the storage containers 9 and 10, respectively. After that, the two liquids were pumped through the liquid distributors 4 into the RPB, separately. Both liquids were sprayed onto the inside edge of the rotator and mixed in the packed-bed zone to yield nanoparticles immediately. The slurry finally left the equipment through the liquid exit for collection and then was filtered. Finally, the filter cake was dried in a vacuum oven at 50 °C for 12 h to yield the drug powder. The dried nanoparticles were stored in a vacuum desiccator at room temperature until further use for characterization and testing. B


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Figure 2. SEM images of CFX nanoparticles prepared at different AS/S ratios: (A) AS/S = 5; (B) AS/S = 7; (C) AS/S = 10; and (D) AS/S = 15. (E) Particle size distributions of CFX particles prepared at different ratios. (F) Effect of AS/S volume ratio on the terminal solubility of CFX in the THF−IPE mixture and the terminal value of S.

2.6. Dissolution Test. Dissolution tests were carried out using a dissolution apparatus (D-800LS, Tianjin, China) following the USP Apparatus II (paddle) method. The paddle speed and bath temperature were set at 100 rpm and 37.0 ± 0.5 °C, respectively. Phosphate buffer saline (PBS) solution (pH 7.2) was employed as the dissolution medium. The dissolution rate tests were performed at sink conditions. Raw CFX or nanosized CFX powder (∼100 mg) was respectively added into different vessels containing 900 mL dissolution medium. Then a 5 mL aliquot was taken each time at specific time intervals and immediately filtered through a 0.10 μm syringe filter. In the meantime, fresh medium (5 mL) was added to keep constant volume. Quantification of the samples was determined with a

UV spectrophotometer at 288 nm. The dissolution test of each sample was performed in triplicate. 2.7. Supersaturation Generation. To evaluate the supersaturation generation capability of CFX nanoparticles, saturation solubility of raw CFX (Crs) was first determined by incubating raw CFX in excess in 20 mL deionized water at 37 °C for 24 h. Afterward, the dispersion was filtered and diluted. CFX concentration in the filtrate was then quantified by a UV spectrophotometer (UV-2501, Shimadzu, Japan) at 288 nm, from which Crs was obtained. The supersaturated CFX solution was generated by adding an excess amount of CFX nanoparticles into 50 mL of deionized water in a capped vial, which was then placed in a water bath at 37 °C with magnetic stirring. Afterward, 1.0 mL aliquot was withdrawn at fixed time intervals C


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Figure 3. SEM images of CFX nanoparticles prepared under (A) 700, (B) 1400, and (C) 2800 rpm. (D) Particle size distributions of CFX particles prepared under different rotating speeds.

over a 6 h period. The aliquot was filtered immediately through a 0.10 μm syringe filter and diluted 100 times to prevent drug precipitation from the supersaturated solution. Drug concentration in the aliquot was assayed by UV−vis spectrophotometer to determine the supersaturated concentration of CFX nanoparticles (Cns) from which the supersaturation level, defined as the ratio of Cns to Crs, was determined. The measurements were repeated three times.

where σ is the interfacial tension, Vs is the volume of a solute molecule, k is the Boltzmann constant, T is the temperature, S is the supersaturation defined as the ratio of the drug solvent concentration over the saturation solubility in mixed solvents. The nucleation rate depends more strongly on the degree of supersaturation and greatly affects the final particle size. When the AS/S ratio increased, the supersaturation level increased due to the decrease of saturation concentration. As shown in Figure 2F, the solubility decreased greatly before the AS/S ratio of 5 and then slowly decreased. Accordingly, a higher level of supersaturation led to a faster nucleation rate and a smaller critical nucleus size. As a result, the number of formed particles increased with the increase of the AS/S ratio. On the other hand, the particle growth rate can be expressed as33

3. RESULTS AND DISCUSSION 3.1. Effect of AS/S Ratio. Since the driving force of antisolvent precipitation is the supersaturation of a solution induced by mixing of an antisolvent (AS) and drug solution (S), AS/S ratio can be listed as a key factor that affects the ultimate products. To explore the effect of AS/S ratio, the flow rate of antisolvent was fixed at 3 L/min, while the flow rate of drug solution was regulated to achieve various AS/S ratios. Figure 2 displays SEM images of CFX nanoparticles prepared at different AS/S ratios and the corresponding particle size distributions (PSDs), as well as the effect of AS/S volume ratio on the terminal solubility of CFX in THF−IPE mixture and the terminal value of S. As shown in Figure 2 (panels A−E), it could be clearly observed that CFX nanoparticle size decreased from 152 to 74 nm and had a narrower PSD with the AS/S ratio increased from 5 to 10. However, when AS/S volume ratio was further increased, only slight change in the mean size and the PSD was observed. This can be explained as follows. Precipitation mainly includes two steps: nucleation and particle growth. The nucleation rate is given by eq 1:32 ⎛ 16πσ 3V 2 ⎞ dN ∝ exp⎜ − 3 3 S 2 ⎟ dt ⎝ 3k T (ln S) ⎠

dl = K g (Ci − C*)b dt

(2)

where Kg is the particle growth rate constant and Ci and C* are the solute concentration on the particle surface and saturation concentration, respectively. The value of the parameter b is usually between 1 and 3 and decreases with reduction of temperature. The increased AS/S ratio virtually decreased the solute concentration on the formed CFX particle surface. Therefore, the decreased value of Ci − C* resulted in a slower particle growth rate, thereby leading to a smaller ultimate mean particle size. However, when AS/S ratio was higher than 10, the value of Ci − C* achieved a relatively constant level, and hence no obvious further size decrease was observed. Once the AS/S ratio was fixed at 10, the theoretical yield of the ultimate nanoparticles could reach up to 99.94% and the production capacity of lab-scale RPB achieved 1.8 kg/h.

(1) D


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Figure 4. SEM images of CFX nanoparticles prepared at (A) 20 °C and (B) 40 °C.

Figure 5. SEM images of (A) raw CFX particles, (B) vacuum-dried particles, (C) redispersed particles, (D) spray-dried particles, and (E) the corresponding particle size distributions of CFX nanoparticles.

E


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Industrial & Engineering Chemistry Research 3.2. Effect of RPB Rotating Speed. In order to obtain nanoparticles with a narrow PSD during the precipitation process, uniform spatial distribution of the concentration and supersaturation must be created and kept before nucleation.19 Hence, intensification of micromixing to reach the region of tm < τ should be taken, where tm is the characteristic time of micromixing for species reaching a maximum mixed state at the molecular level, and τ is the induction time of nucleation (normally on the order of 1 ms or less). In the case of typical stirred tank, tm ≫ τ, while tm is estimated to be on the order of 5−50 ms.19 However, in HGAP process, the fluids going through RPB were split into thin films, threads, and very fine droplets. The mass transfer rate in a RPB was 1−3 orders of magnitude larger than that in a conventional stirred tank. Therefore, the theoretical requirement of tm < τ could be fulfilled in the RPB. It is accepted that the rotating speed of RPB has a significant influence on the magnitude of micromixing. Figure 3 shows SEM images of CFX nanoparticles prepared under different RPB rotating speeds and the corresponding PSDs. It was clear that the particle size decreased from 429 to 60 nm, while PSD became narrower with the increase of the rotating speed from 700 to 2800 rpm. Owing to the enhanced rotating speed, the mass transfer was intensified and higher supersaturation level was thus induced. As a result, the nucleation rate was accelerated and particle growth was suppressed, thereby generating nanoparticles with smaller size and narrower PSD. 3.3. Effect of Temperature. Figure 4 exhibits SEM images and the corresponding PSDs of CFX nanoparticles precipitated at different temperatures. Apparently, with the temperature increased from 20 to 40 °C, the mean particle size increased from 57 to 72 nm and there was an increased portion of particles conglutinated together to form irregular bigger ones. The following reasons may be responsible for this phenomenon. First, lower temperature results in higher supersaturation level, which leads to smaller critical nuclei size. Second, the liquid precipitation is a diffusion-limited process, lower temperature would decrease the diffusion rate, and accordingly the particle growth rate. Third, the probability of particle collision and subsequently conglutination in liquid phase can be minimized at a lower temperature. As a result, room temperature (∼20 °C) could fulfill the requirement of preparation of uniform CFX nanoparticles. 3.4. Effect of Drying Method. Freeze-drying and spraydrying are commonly utilized to convert drug nanosuspension into dry powder with sufficient stability, and the redispersibility of dried nanoparticles is an important factor in the preparation process for practical applications. Figure 5 presents SEM images of raw CFX particles, vacuum-dried particles, redispersed particles, spray-dried particles, and the corresponding particle size distributions of CFX nanoparticles. For normally used freeze-dryer, it is impossible to maintain the temperature below −60 °C. Also, the freezing points of IPE and THF are −86 °C and −108 °C, respectively. In this case, freeze-drying is unsuitable for this system. However, when CFX nanoparticles were vacuum-dried, the obtained powder was incompact, and nearly no changes in particle size and morphology were observed (Figure 5B). In contrast, raw CFX had an irregular morphology and a mean particle size of ∼2.1 μm with a wide PSD from 0.2 to 10 μm (Figure 5A). The dried nanoparticles could be well-redispersed in deionized water with nearly no change in morphology and slight increase in particle size (from

57 to 66 nm, Figure 5, panels C and E), and this might be due to the particle agglomeration or recrystallization. When CFX nanosuspension was spray-dried by a lab-scale spray dryer (B290, Buchi, Switzerland) with an inlet temperature of 100 °C and a flow rate of 8 mL/min, the size of dried particles was increased to 141 nm due to the increase in collision rate under such a relatively high temperature (Figure 5, panels D and E). Furthermore, the recovery ratio of the spray drying process was very low (

Large-Scale Preparation of Amorphous Cefixime Nanoparticles by

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