- Email: [email protected]
Preparation and characterization of cefquinome sulfate microparticles for transdermal delivery by negative-pressure cavitation antisolvent precipitation
Powder Technology 294 (2016) 429–436
Contents lists available at ScienceDirect
Powder Technology journal homepage: www.elsevier.com/locate/powtec
Preparation and characterization of cefquinome sulfate microparticles for transdermal delivery by negative-pressure cavitation antisolvent precipitation Xinxuan Du 1, Suchong Zu 1, Fengli Chen, Zaizhi Liu, Xinran Li, Lei Yang ⁎, Yuangang Zu, Xiuhua Zhao ⁎, Lin Zhang Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, China
a r t i c l e
i n f o
Article history: Received 15 October 2015 Received in revised form 22 February 2016 Accepted 5 March 2016 Available online 8 March 2016 Keywords: Cefquinome sulfate Microparticle Negative-pressure cavitation antisolvent precipitation Preparation Transdermal delivery
a b s t r a c t The objective of this study was to prepare micronized cefquinome sulfate by negative-pressure cavitation antisolvent precipitation to improve its permeability and transdermal absorbability. Dimethylsulfoxide and ethanol were used as the solvent and anti-solvent, respectively. The effects of operating parameters, including cefquinome sulfate concentration, volume ratio of antisolvent to solvent, antisolvent precipitation time, and applied negative pressure on the characteristics of the precipitate crystals, were evaluated using an orthogonal array design. Under the optimized conditions, micronized cefquinome sulfate with a mean particle size of 415.8 nm was obtained at a yield of 78%. The micronized cefquinome sulfate was characterized by scanning electron microscopy, X-ray diffraction, and differential scanning calorimetry. No obvious change in its chemical structure was observed, but crystallinity was reduced. The physicochemical properties and transdermal absorbability of crystalline cefquinome sulfate improved as a result of the particle size reduction by physical modification. This negative-pressure cavitation antisolvent precipitation process offers a promising technique for improving the physicochemical properties and anti-inflammatory activity of cefquinome sulfate. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Cow mastitis, the most commonly occurring inflammation in dairy cows, is difficult to cure, and is therefore a major topic of research by international scholars [1]. Cow mastitis symptoms include inflammations of udder parenchyma and interstitial tissue, which are usually caused by mechanical stimulation, immersion of pathogenic microorganisms, and chemicophysical damage [2]. The main symptoms manifest as redness, swelling, and heat or pain of the udder, which directly result from reduced or completely halted lactation [3]. Cows are reared widely in many countries, and they contribute to a major portion of milk production in countries like China. In China, the livestock industry suffers huge economic losses because of low milk production caused by bacterial diseases, including mastitis [4]. Oral anti-inflammatory drugs are often mixed in with the animal feedstocks, but, because the cow has a complex four-chambered stomach, this leads to excessive drug loss in the metabolic process [5,6]. Injection of drugs is difficult because of the thick hides of the animals and requires professional skill. Use of transdermal preparations for treating mastitis can reduce the drug in the body's metabolism, reduce injury to the liver, has the advantage of convenient use, and does not ⁎ Corresponding authors. E-mail addresses: [email protected] (L. Yang), [email protected] (X. Zhao). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.powtec.2016.03.008 0032-5910/© 2016 Elsevier B.V. All rights reserved.
easily produce drug resistance [7]. The poor solubility and low dissolution rate of active pharmaceutical ingredients in water is one of the most difficult and as-yet unsolved problems in pharmaceutical technology [8]. This drawback limits medical developments, such as new dosage forms for transdermal absorption. Cefquinome sulfate (Fig. 1) is a synthetic compound, the poor solubility of which leads to low oral absorption [9]. Cefquinome sulfate, a fourth-generation cephalosporin, has been solely researched for veterinary use owing to its antimicrobial activity against a broad spectrum of gram-positive and gram-negative bacterial species [10,11]. It has been also applied for treatment of acute mastitis and foot rot in cattle, and respiratory tract diseases in pigs, cows, and horses [12]. Cefquinome sulfate has been shown to have wide antibacterial activity and can achieve the effect of sterilization by inhibiting cell synthesis. In vitro antibacterial use of cefquinome sulfate indicates that it can inhibit both positive and negative common bacteria: for example, Escherichia coli, Bacillus, Klebsiella bacteria, Pasteurella, Salmonella species, Serratia marcescens, Streptococcus, the Bacteroides fragilis group, Clostridium prazmowski, Actinobacillus, and Erysipelothrix rhusiopathiae [11]. Cefquinome sulfate is an effective agent against many in vitro and in vivo bacteria. It is mainly used for treating cow mastitis, which is caused by sensitivity to bacteria, respiratory infections in pigs, and mastitis–metritis–agalactia syndrome in sows [10]. Microparticles have been popularly applied in enhancing the dissolution rate of drugs that have poor solubility in water. Owing to the
430
X. Du et al. / Powder Technology 294 (2016) 429–436
China). N-Methyl-2-pyrrolidone, acetone, methanol, dichloromethane, trichloromethane, and dimethyl sulfoxide (DMSO) (analytically pure) were purchased from Bodi Chemical Reagent Company (Tianjin, China). 2.2. Preparation of skin The skin permeation study was performed and approved by the Institutional Animal Ethics Committee. The thoracic skin of a dairy cow was obtained from Mengniu Pasture, Zhaodong, China. The skin was washed with normal saline and the hair on the skin was removed with an electric shaver before use. Fig. 1. Molecular structures of cefquinome sulfate.
reduced particle size of microparticles as compared with that of the raw drug particles, the interfacial surface area is increased, leading to improved solubility in water [13]. Bioavailability and oral efficacy of microparticles can be consequently improved by reduction in particle size, thereby allowing for direct usage of the poorly water-soluble drugs at smaller dosages or with more rapid response times [14,15]. Traditionally, many techniques have been applied for the preparation of microparticles, including ball milling [16], air jet pulverization [17,18], supercritical antisolvent technology [13–15], high pressure homogenization [19,20], reactive crystallization [21], and spray drying [22,23]. The process of mechanical comminution [16–18], a method of micronizing drugs, is now widely used in pharmaceutical manufacturing. Supercritical antisolvent technology has the disadvantage of large capital equipment investment, high pressure homogenization is limited by expensive equipment and solvent requirements, while reactive crystallization can only be applied to the preparation of acidic or alkaline drugs. In contrast, liquid antisolvent precipitation has the advantages of lower reagent consumption, higher process efficiency, and easy operation [24,25]. Cavitation is the phenomenon of sequential formation, growth and collapse of millions of tiny vapor bubbles (voids) in a liquid. It can occur whenever a liquid is used in a machine that induces pressure and velocity fluctuations in the fluid (e.g. pumps, turbines and propellers) [26]. Negative-pressure cavitation extraction has been used for extraction of biological active compounds from plant materials centuries. Negative-pressure cavitation antisolvent precipitation is based on a technique that uses negative pressure as its driving force. In this technique, a solvent and an anti-solvent are fully stirred by pumping air through the bottom of a reactor in which they are contained. The characteristic bubbles that form under conditions of negative pressure cavitation are used to keep the particle size of the powder uniform and tiny for a relatively long time. The aim of this study was to prepare cefquinome sulfate microparticles using a negative-pressure cavitation antisolvent precipitation process and to evaluate their physicochemical properties and transdermal delivery in vitro. To optimize the negative-pressure cavitation antisolvent precipitation process, the effects of cefquinome sulfate concentration, volume ratio of antisolvent to solvent, antisolvent precipitation time, and applied negative pressure on the yield and mean particle size (MPS) of the cefquinome sulfate microparticles were studied using an orthogonal array design. The microparticles were further characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and X-ray diffraction (XRD). The transdermal delivery performance in vitro of cefquinome sulfate microparticles prepared was also evaluated. 2. Materials and methods 2.1. Drugs and chemicals Cefquinome sulfate powder (purity of 98.5%) was provided by Zhejiang Hisun Pharmaceutical Co. Ltd. of China. Ethanol (analytically pure) was purchased from Tianli Chemical Reagent Company (Tianjin,
2.3. Negative-pressure cavitation antisolvent precipitation apparatus Micronized cefquinome sulfate was prepared by antisolvent precipitation. A schematic diagram of the apparatus is shown in Fig. 2. This consisted of an injection system (10–14), a negative-pressure cavitation reaction tank (8), and a vacuum system (1, 2, 6). The negative pressure valve (6) was first opened to maintain the pressure of the reaction tank (8) at − 0.04 to − 0.08 MPa, which measured by a vacuum gauge (YZ-40, Qingdao Dongfang Instrument Co., Ltd., China). The inlet valve (7) was then opened to allow the ethanol antisolvent to fill the negative-pressure cavitation reaction tank (8). The inlet valve (7) was then closed and the piston pump (2J-X 52/4.5, Zhijiang Science and Technology Instrument Factory, China) (19) was opened to control the air inflow speed in the range from 1 to 10 m3/h. The intake valve (16) was then opened, which allowed air to flow through the intake branch pipeline (14), an annular conduit (10), and a nozzle (FD-1-1/ 8-Brass, Dalian Baoyuan Purification Technology Co., Ltd., China) (11). The liquid flow meter (LZZ-32, Changzhou Chengfeng Flowmeter Co., Ltd., China) (18) and piston pump (19) were then simultaneously opened to guide the mixed liquids (containing cefquinome sulfate and DMSO) into the negative-pressure cavitation reaction tank (8) by means of a branched pipeline (13), an annular conduit (12), and a nozzle (11). The mixed liquids then began to cavitate and become suspended. The intake valve (16) was used to control the air inflow and the piston pump (19) was employed to maintain the inlet fluid velocity. The mixed liquids were suddenly blended with ethanol antisolvent contained in reaction tank (8) when they were ejected through a microporous nozzle. Cefquinome sulfate submicroscopic particulates were finally separated out, suspended in the DMSO–ethanol solution under the effects of cavitation and suspension. During these procedures, secondary aggregation could not occur and so tiny, uniformly dispersed cefquinome sulfate particles were obtained. After importing the mixed liquids, the liquid flow meter (18) was closed and the piston pump (19) was switched off, having achieved its technical requirements with respect to the impacts of continuous cavitation and suspension. The intake valve (16), negative pressure valve (6), and relief valve (5) were then closed to balance the pressure in the reaction tank (8). The material discharge valve (15) was opened to export the residue solutions contained in the reaction tank. The suspension was centrifuged at 5000 rpm for 5 min, and the particles were then freeze-dried at − 50 °C for 24 h. Each experiment was performed in triplicate. 2.4. Optimization of the antisolvent precipitation process An L16(4)5 orthogonal experiment, performed with four factors and four levels, was selected for optimization of the operational parameters for cefquinome sulfate micronization using this process. Based on the results of preliminary experiments, we optimized the operating conditions and analyzed the statistical experimental results using an orthogonal array design with four factors applied using Design-Expert 7.0 software (Stat-Ease, Minneapolis, USA). The boundaries of the four factors were 100 to 400 mg/mL cefquinome sulfate concentration, 3 to 9 mL/mL volume ratio of antisolvent to solvent, −0.02 to − 0.08 MPa
X. Du et al. / Powder Technology 294 (2016) 429–436
431
Fig. 2. Schematic drawing of negative-pressure cavitation antisolvent precipitation apparatus to prepare the micronized cefquinome sulfate. 1. Vacuum gauge; 2. Negative pressure pipeline; 3. Relief pipeline; 4. Inlet pipeline; 5. Relief valve; 6. Negative pressure valve; 7. Inlet valve; 8. Negative-pressure cavitation reaction tank; 9. Viewing port; 10. Annular conduit; 11. Nozzle; 12. Annular conduit; 13. Branch pipeline; 14. Intake branch pipeline; 15. Material discharge valve; 16. Intake valve; 17. Inlet valve; 18. Liquid flow meter; 19. Piston pump; 20. Gas flow meter; 21. Liquid pipeline; 22. Intake pipeline; 23. Material discharge pipeline.
negative pressure, and 5 to 20 min antisolvent precipitation time. The levels of the factors for the orthogonal array design are indicated in Table 1. Micronized cefquinome sulfate was obtained from the above 16 tests, the yield and MPS of which were variable-dependent. Generally speaking, MPS and yield are selected as the two indexes used to evaluate the advantages and disadvantages of the antisolvent precipitation technique. In the orthogonal design, a scoring system was applied to comprehensively investigate the proposed process. MPS and yield were considered as equally important, with each assigned a value of 0.5. The detailed appraisals and the relative marking criteria with respect to MPS and yield are presented in Table 2.
2.5. Powder characterization 2.5.1. Dynamic light scattering (DLC) The diameter distributions of the cefquinome sulfate powders were measured using a dynamic light scattering analyzer (ZetaPALS, Brookharen Instrument Corporation, USA). Samples were suspended in deionized water and treated by sonication for 3 min at 100 W (KQ250DB, Kunshan Ultrasonic Instruments Co., China) to eliminate dust and to prevent the aggregation of particles. The suspension was then placed in a cuvette and measured at room temperature. Each experiment was performed in triplicate.
Table 1 Factors and levels for orthogonal test. Factors
(A) Cefquinome sulfate concentration (mg/mL) (B) Volume ratio of antisolvent to solvent (mL/mL) (C) Negative pressure (MPa) (D) Antisolvent precipitation time (min)
2.5.2. Scanning electron microscopy The surface morphologies of the unprocessed and processed cefquinome sulfate were investigated by SEM (Quanta 200, FEI, Hillsboro, USA). Before examination, the samples were sputter-coated with a thin layer of gold–palladium (5 to 10 nm; 10 mA; 30 s) at room temperature using a sputter coater. 2.5.3. X-ray diffraction Powder XRD patterns were collected in transmission mode using an X-ray diffractometer with a rotating anode (Philips, Xpert-Pro, Holland) with Cu Kα1 radiation generated at 30 mA and 40 kV. Powders of the unprocessed and processed cefquinome sulfates were filled to the same depth inside the sample holder by leveling with a spatula. The range of 2θ diffraction angles from 5 to 80° was examined, with steps of 0.02° and a measuring time of 0.2 s per step. 2.5.4. Differential scanning calorimetry The unprocessed and processed cefquinome sulfate powders were analyzed by DSC (TA instruments, Q100, New Castle, USA). About 5 mg of sample was placed in an open aluminum pan and heated from ambient temperature to 250 °C at a rate of 5 °C/min. 2.5.5. Transdermal delivery tests As shown in Fig. 3, a drug transdermal diffusion instrument (TT-6, Zhengtong Technology Co., Ltd., Tianjin, China) and an improved Franz diffusion pool method [27,28] were applied in the present study. The Table 2 Relationship between process score and particle size and yield.
Levels 1
2
3
4
Particle size (nm)
Score
Yield (%)
Score
100
200
300
400
3
5
7
9
−0.02 5
−0.04 10
−0.06 15
−0.08 20
˂200 200–400 400–600 600–800 800–1000 ˃1000
10 8 6 4 2 1
˃ 80 60–80 40–60 20–40 1–20
10 8 6 4 1
432
X. Du et al. / Powder Technology 294 (2016) 429–436
volume of the reception tank was 5 mL with 0.785 cm2 of effective surface. The skin of the udder was cut, the subcutaneous fat layer and connective tissue were carefully peeled off, and the area rinsed thoroughly with normal saline. Two slices of skin with the same area were prepared using a hole puncher. The skin was placed with the inner side downwards in the reaction vessel and fixed with this side facing into the diffusion chamber and the outer skin facing towards the supply side of the apparatus. Then, 1.0 mL each of 10 mg/mL unprocessed and processed cefquinome sulfate suspensions were separately added into the supply chamber. Five mL ethanol was also added as a receptor fluid to ensure good contact with the skin. A constant interlayer temperature in the diffusion chamber of 37 ± 0.5 °C was maintained. Several rotors were added to the receiving chamber and stirred at a constant speed of 900 rpm. Timing was started 15 min after adding the samples: 5 mL aliquots of fluids in the receptor were taken separately at predetermined times and simultaneously replaced by an equal volume of ethanol. The aliquots were centrifuged, following which an appropriate volume of liquid supernatant was sampled to determine the cefquinome sulfate content and to calculate its cumulative penetration into the skin.
2.5.6. Determination of cefquinome sulfate content by high-performance liquid chromatography (HPLC) Both the unprocessed and processed cefquinome sulfate present in the permeate liquors generated in the transdermal delivery test were analyzed by HPLC using a Waters 1525–2489 series system (Waters, USA), equipped with a HiQ sil-C18 reversed-phase column (4.6 mm × 250 mm, 5 μm, KYA TECH). The mobile phase consisted of 14% volume fraction acetonitrile. The injection volume was 20 μL and was delivered at 0.8 mL/min. The response signal was monitored at a wavelength of 220 nm [29]. 2.5.7. Residual solvent determination The residual DMSO in the micronized cefquinome sulfate was analyzed using gas chromatography (GC) (Agilent 7890 A, Palo Alto, CA, USA) equipped with a G1540N-210 flame-ionization detector and an HP-5 capillary column (film thickness 0.25 μm, 30 m × 320 μm). The micronized cefquinome sulfate samples (10 mg) were suspended in 1 mL methanol and centrifuged at 10 000 rpm for 5 min. Ten microliters of the supernatant was then injected. DMSO analysis was carried out using the following GC conditions: detector temperature, 280 °C; injector temperature, 200 °C; initial oven temperature, 40 °C for 5 min, following which the temperature was increased to 240 °C at 40 °C/min and maintained for a further 5 min; hydrogen flow rate, 30 mL/min; airflow velocity, 400 mL/min; carrier gas, nitrogen; carrier gas velocity, 2.2 mL/min; and split ratio, 20:1. 3. Results and discussion 3.1. Screening of solvent and antisolvent The first step in developing the antisolvent precipitation method was to select the solvent and antisolvent, making sure that the solute has high solubility in the solvent and that the antisolvent has little solubility in solvent but is miscible with it. Seven common solvents were evaluated, including N-methyl-2-pyrrolidone, ethanol, acetone, methanol, dichloromethane, trichloromethane, and DMSO, with respect to solubility of cefquinome sulfate, so as to choose the most suitable combination of solvent and antisolvent. Cefquinome sulfate has a solubility at 25 °C that differs greatly in the different solvents, as shown in Table 3: it has a rather low solubility (below 0.5 mg/mL) in ethanol, acetone, methanol, dichloromethane, and trichloromethane; however, it has a low solubility (40 mg/mL) in N-methyl-2-pyrrolidone and relatively higher solubility in DMSO (140 mg/mL). Based on these results, ethanol was chosen as the antisolvent and DMSO as the solvent. 3.2. Orthogonal test design The various operational parameters exert different influences on the preparation process, each of which should be systematically optimized. For the antisolvent precipitation process, the cefquinome sulfate concentration, the volume ratio of antisolvent to solvent, the antisolvent precipitation time, and the negative pressure applied are generally considered to be the most important parameters. An orthogonal L16(45) test design was applied to optimize the preparation conditions for Table 3 Solubility of cefquinome sulfate in the different solvents at 25 °C.
Fig. 3. Transdermal procedures in vitro. a: Skin of the udder; b: micronized cefquinome sulfate suspension; c: transdermal diffusion of unprocessed and processed cefquinome sulfate suspensions in Franz diffusion pool.
Solvents
Solubility (mg/mL)
N-Methyl-2-pyrrolidone Ethanol Acetone Methanol Dichloromethane Chloroform Dimethyl sulfoxide (DMSO)
40 10 b0.5 6 b0.5 b0.5 140
X. Du et al. / Powder Technology 294 (2016) 429–436
micronized cefquinome sulfate. Sixteen experiments were carried out in triplicate and run in randomized order to avoid any personal or subjective bias. Based on the pre-experimental results, the values of the level settings of the four factors (A, B, C, and D) used in the orthogonal arrayed design are listed in Table 1. The process relationship scores with respect to particle size and yield are listed in Table 2. The results of the 16 experimental trials carried out under these different conditions are shown in Table 4. Under the orthogonal conditions, the particle sizes of the drug ranged from 340 nm to 1316 nm, while the yields ranged from 0.5% to 75%. Experiment 11 achieved the highest composite score, with a particle size of 555.9 nm and yield of 75%. As Table 4 also shows, the scores range from A N D N B N C, indicating that the degree of influence on the process follows a decreasing order from cefquinome sulfate concentration, antisolvent precipitation time, volume ratio of antisolvent to solvent, to negative pressure applied. Considering the experimental data and the above analysis, the optimum conditions are represented by the combination A2B2C3D3, for which the cefquinome sulfate concentration is 200 mg/mL, the volume ratio of antisolvent to solvent is 5 mL/mL, the antisolvent precipitation time is 15 min, and the pressure is − 0.06 MPa. A confirmatory test performed under these conditions gave a MPS of 415.8 nm and yield of 78% for the obtained powder. 3.3. Scanning electron microscopy Electron micrographs of the cefquinome sulfate surfaces are shown in Fig. 4. Fig. 4a shows an overall view of unprocessed cefquinome sulfate, with a corresponding enlarged view in Fig. 4c. The unprocessed cefquinome sulfate morphology comprises large, irregular crystalline flakes. Fig. 4b and d show analogous views of micronized cefquinome sulfate, indicating the presence of porous spheres with a relatively regular size of 10 to 15 μm. Inspection at greater magnification showed that these consisted of tiny sheet structures with a particle size of 300 to 450 nm. These results confirm that the particle size of cefquinome sulfate is modified by the antisolvent precipitation process. The main reason for the multi-aperture spherical appearance of the micronized power is probably that the antisolvent volatilizes during the drying
433
process and the fines reunite to form the powder observed at the end of the reaction. 3.4. X-ray diffraction analysis To further investigate the occurrence of possible crystal structural changes, XRD analyses were undertaken. The results for unprocessed and processed cefquinome sulfates are shown in Fig. 5. Several strong peaks for the unprocessed cefquinome sulfate sample, occurring at 2θ diffraction angles from 8.1° to 27.7°, reveal that the drug is present in a crystalline form. In contrast, the processed cefquinome sulfate shows significantly reduced intensity of peaks. The reduction of diffraction intensity of the peaks for the processed sample is attributed to reduced particle crystallinity or decreased particle size. This result indicates that cefquinome sulfate particles with a smaller MPS and lower crystallinity were obtained following antisolvent precipitation processing. This micronization can increase the solubility of cefquinome sulfate drugs and thereby enhance the therapeutic effect of percutaneous absorption. 3.5. Differential scanning calorimetry As seen in Fig. 6, the DSC curves for unprocessed and processed cefquinome sulfate show that the unprocessed drug produced an endothermic melting peak at 177.3 °C but that of processed cefquinome sulfate occurred at 178.5 °C. These results indicate that both unprocessed and processed cefquinome sulfate have essentially the same melting point; however, the unprocessed sample shows slightly higher exothermicity. This confirms that the processed cefquinome sulfate has lower crystallinity, consistent with the XRD results. 3.6. Residual solvent analysis DMSO acts as a Class III solvent with low toxicity and is limited to a maximum of 0.5% in a drug, according to the International Conference on Harmonization (ICH) [30,31]. As shown in Fig. 7, the residual DMSO content in the cefquinome sulfate (prepared using DMSO as the solvent), observed at a retention time of 8.77 min by GC analysis, has
Table 4 Arrangement, results and analysis of variance (ANOVA) of L16(45) orthogonal test (n = 3). Design ID number
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 K1jc K2j K3j K4j Rd Oe a b c d e
Responsea
Factors
Score
Ab
B
C
D
Particle size (nm)
Yield (%)
Yield score
Particle size score
Composite score
100 100 100 100 200 200 200 200 300 300 300 300 400 400 400 400 6.250 7.250 4.250 2.500 4.750 A2
5 7 3 9 3 5 9 7 7 3 9 5 3 5 9 7 4.500 6.000 4.000 5.750 2.000 B2
−0.04 −0.06 −0.02 −0.08 −0.04 −0.02 −0.06 −0.08 −0.02 −0.06 −0.04 −0.08 −0.08 −0.06 −0.02 −0.04 4.750 4.500 6.000 5.000 1.500 C3
10 15 5 20 15 20 10 5 10 20 5 15 10 5 15 20 6.250 3.750 6.500 3.750 2.750 D3
340.0 1168.2 792.0 895.0 530.0 1014.7 1316.2 1211.4 769.4 525.7 555.9 1013.9 550.0 677.5 772.2 942.1
10.0 3.0 1.0 1.4 20.0 25.0 16.0 32.8 3.3 2.7 75.0 60.0 0.5 2.5 5.1 4.0
1 1 4 1 4 4 2 4 1 1 8 8 1 1 1 1
8 1 4 2 6 1 1 1 4 6 6 1 6 4 4 2
9 2 8 3 10 5 3 5 5 7 14 9 7 5 5 3
Response values were comprised of mean of three independent experiments. A: Cefquinome sulfate concentration (mg/mL); B: Volume ratio of antisolvent to solvent (mL/mL); C: Negative pressure (MPa); D: Antisolvent precipitation time (min). Logesh Kij = (1/4) Σ mean composite score at factor j (j = A, B, C, D). Logesh R = max (Kij) − min (Kij), j and i mean factor and setting level here, respectively. O means the optimum condition. The optimum combination of conditions is A2B2C3D3.
434
X. Du et al. / Powder Technology 294 (2016) 429–436
Fig. 4. Scanning electron micrographs of (a, c) unprocessed and (b, d) processed cefquinome sulfate (processed cefquinome sulfate is the micronized cefquinome sulfate by negativepressure cavitation antisolvent precipitation under optimum condition).
a standard curve represented by y = 341.37x + 1.84 (R2 = 0.9996), where y is the peak area and x is the DMSO concentration. According to this regression equation, the residual DMSO concentration in the micronized cefquinome sulfate was 0.39%. The micronized cefquinome sulfate therefore conforms to the ICH standard and is suitable for industrial veterinary drug utilization. 3.7. Results of transdermal delivery tests The cumulative absorptions of the unprocessed and processed cefquinome sulfate suspensions by the cowhide pieces in the transdermal tests are shown in Fig. 8. The processed cefquinome sulfate suspension was able to pass through the skin from exposure times greater than 0.5 h and the unit area cumulative penetration amount varied almost
Fig. 5. XRD patterns of unprocessed and processed cefquinome sulfate (processed cefquinome sulfate is the micronized cefquinome sulfate by negative-pressure cavitation antisolvent precipitation under optimum condition).
linearly with respect to the exposed surface area. After 12 h, the unit area cumulative penetration of this suspension was 53.69 μg/cm2. When using the unprocessed cefquinome sulfate suspension, however, only a small amount passed through the skin within 1 h of exposure, but diffusion stopped thereafter. The reason is that the unprocessed cefquinome sulfate had a higher MPS of 1166 nm, and was therefore unable to pass through the skin. These results reveal that processed cefquinome sulfate exhibits considerably higher transdermal delivery ability than unprocessed cefquinome sulfate, and therefore possesses potential as a new transdermal dosage form that can be further developed as a drug for curing inflammation of cows' udders.
Fig. 6. DSC curves of unprocessed and processed cefquinome sulfate (processed cefquinome sulfate is the micronized cefquinome sulfate by negative-pressure cavitation antisolvent precipitation under optimum condition).
X. Du et al. / Powder Technology 294 (2016) 429–436
435
Fig. 7. Gas chromatograms of cefquinome sulfate microparticles, indicating the presence of residual DMSO (cefquinome sulfate microparticles is the micronized cefquinome sulfate by negative-pressure cavitation antisolvent precipitation under optimum condition).
4. Conclusions In this work, micronized cefquinome sulfate was successfully prepared using a negative-pressure cavitation antisolvent precipitation process. A sample of micronized cefquinome sulfate with a MPS of 415.8 nm was obtained when the cefquinome sulfate concentration was 200 mg/mL, the volume ratio of antisolvent to solvent was 5 mL/mL, a pressure − 0.06 MPa was applied, and using an antisolvent precipitation time of 15 min. The morphologies of the unprocessed and processed cefquinome sulfate particles were investigated using SEM, XRD, and DSC. All of these analyses indicated that the micronized cefquinome sulfate existed as a lower crystallinity form and that no degradation occurred following the negative-pressure cavitation antisolvent precipitation process. When compared with the unprocessed higher crystallinity form, the lower crystallinity cefquinome sulfate microparticles showed better performance in transdermal delivery. In addition, the residual solvent in the cefquinome sulfate microparticle powder met the specification for pharmaceutical use. These results suggest that the negativepressure cavitation antisolvent precipitation process may be a promising approach for preparation of cefquinome sulfate microparticles having low crystallinity, low MPS, and thereby improved transdermal delivery. Acknowledgments The authors thank the Special Fund for Forestry Scientific Research in the Public Interest (201404616) for financial support.
Fig. 8. Comparison of unit area cumulative volume of processed and unprocessed cefquinome sulfate microparticles absorbed in transdermal tests (processed cefquinome sulfate is the micronized cefquinome sulfate by negative-pressure cavitation antisolvent precipitation under optimum condition).
References [1] R. Pilla, C. Bonura, M. Malvisi, G.G. Snel, R. Piccinini, Methicillin-resistant Staphylococcus pseudintermedius as causative agent of dairy cow mastitis, Vet. Rec. 173 (2013) 19. [2] Y. Lu, Y.L. Hu, X.F. Kong, D.Y. Wang, Selection of component drug in activating blood flow and removing blood stasis of Chinese herbal medicinal formula for dairy cow mastitis by hemorheological method, J. Ethnopharmacol. 116 (2008) 313–317. [3] C.W. Rougoor, W.J. Hanekamp, A.A. Dijkhuizen, M. Nielen, J.B. Wilmink, Relationships between dairy cow mastitis and fertility management and farm performance, Prev. Vet. Med. 39 (1999) 247–264. [4] X. Zhou, C. Yang, Y. Li, X. Liu, Y. Wang, Potential of berberine to enhance antimicrobial activity of commonly used antibiotics for dairy cow mastitis caused by multiple drug-resistant Staphylococcus epidermidis infection, Genet. Mol. Res. 14 (2015) 9683–9692. [5] K.L. Ingvartsen, K.M. Moyes, Factors contributing to immunosuppression in the dairy cow during the periparturient period, Jpn. J. Vet. Res. 63 (2015) S15–S24. [6] A. Zonca, M. Gallo, C. Locatelli, S. Carli, P. Moroni, R. Villa, P. Cagnardi, Cefquinome sulfate behavior after intramammary administration in healthy and infected cows, J. Dairy Sci. 94 (2011) 3455–3461. [7] Y.T. Zhang, M.Q. Han, L.N. Shen, J.H. Zhao, N.P. Feng, Solid lipid nanoparticles formulated for transdermal aconitine administration and evaluated in vitro and in vivo, J. Biomed. Nanotechnol. 11 (2015) 351–361. [8] S. Zu, L. Yang, J. Huang, C. Ma, W. Wang, C. Zhao, Y. Zu, Micronization of taxifolin by supercritical antisolvent process and evaluation of radical scavenging activity, Int. J. Mol. Sci. 13 (2012) 8869–8881. [9] Q. Fu, H.L. Fu, L. Huan, W. Zhang, G. Shu, M.J. Liu, F.Y. Deng, J. Hu, Preparation of cefquinome sulfate proliposome and its pharmacokinetics in rabbit, Iran. J. Pharm. Res. 12 (2013) 611–621. [10] M. Limbert, D. Isert, N. Klesel, A. Markus, K. Seeger, G. Seibert, E. Schrinner, Antibacterial activities in vitro and in vivo and pharmacokinetics of cefquinome (HR 111V), a new broad-spectrum cephalosporin, Antimicrob. Agents Chemother. 35 (1991) 14–19. [11] N.X. Chin, J.W. Gu, W. Fang, H.C. Neu, In vitro activity of cefquinome, a new cephalosporin, compared with other cephalosporin antibiotics, Diagn. Microbiol. Infect. Dis. 15 (1992) 331–337. [12] N.Y. Shpigel, D. Levin, M. Winkler, A. Saran, G. Ziv, A. Böttner, Efficacy of cefquinome for treatment of cows with mastitis experimentally induced using Escherichia coli, J. Dairy Sci. 80 (1997) 318–323. [13] Y. Zu, Q. Zhang, X. Zhao, D. Wang, W. Li, X. Sui, Y. Zhang, S. Jiang, Q. Wang, C. Gu, Preparation and characterization of vitexin powder micronized by a supercritical antisolvent (SAS) process, Powder Technol. 228 (2012) 47–55. [14] X. Sui, W. Wei, L. Yang, Y. Zu, C. Zhao, L. Zhang, F. Yang, Z. Zhang, Preparation, characterization and in vivo assessment of the bioavailability of glycyrrhizic acidmicroparticles by supercritical anti-solvent process, Int. J. Pharm. 423 (2012) 471–479. [15] L. Yang, Z. Sun, Y. Zu, C. Zhao, X. Sun, Z. Zhang, L. Zhang, Physicochemical properties and oral bioavailability of ursolic acid nanoparticles using supercritical anti-solvent (SAS) process, Food Chem. 132 (2012) 319–325. [16] D. Chen, D.Y. Li, Y.Z. Zhang, Z.T. Kang, Preparation of magnesium ferrite nanoparticles by ultrasonic wave-assisted aqueous solution ball milling, Ultrason. Sonochem. 20 (2013) 1337–1340. [17] M. Aoki, H. Nishimura, A. Mimura, S. Kita, T. Yasuzawa, K. Terada, Identification of the degradation products of the steroid sulfatase inhibitor KW-2581 in jet millmicronized powder, J. Pharm. Sci. 102 (2013) 1760–1772. [18] L.W. Chan, C.C. Lee, P.W. Heng, Ultrafine grinding using a fluidized bed opposed jet mill: effects of feed load and rotational speed of classifier wheel on particle shape, Drug Dev. Ind. Pharm. 28 (2002) 939–947.
436
X. Du et al. / Powder Technology 294 (2016) 429–436
[19] Y. Li, X. Zhao, Y. Zu, Y. Zhang, Preparation and characterization of paclitaxel nanosuspension using novel emulsification method by combining high speed homogenizer and high pressure homogenization, Int. J. Pharm. 490 (2015) 324–333. [20] A. Homayouni, F. Sadeghi, J. Varshosaz, G.H. Afrasiabi, A. Nokhodchi, Promising dissolution enhancement effect of soluplus on crystallized celecoxib obtained through antisolvent precipitation and high pressure homogenization techniques, Colloids Surf. B 122 (2014) 591–600. [21] Y. Gao, J. Wang, Y. Wang, Q. Yin, B. Glennon, J. Zhong, J. Ouyang, X. Huang, H. Hao, Crystallization methods for preparation of nanocrystals for drug delivery system, Curr. Pharm. Des. 21 (2015) 3131–3139. [22] K. Anand, S. Varghese, T. Kurian, Preparation of ultra-fine dispersions of zinc oxide by simple ball-milling: optimization of process parameters, Powder Technol. 271 (2015) 187–192. [23] L.B. de Paiva, A.M. de Oliveira, R.R. Gavioli, Preparation and properties of rubber powder from modified-SBR latex by spray drying process, Powder Technol. 264 (2014) 507–513. [24] A. Fodor-Kardos, J. Toth, J. Gyenis, Preparation of protein loaded chitosan microparticles by combined precipitation and spherical agglomeration, Powder Technol. 244 (2013) 16–25. [25] C.A. Ober, L. Kalombo, H. Swai, R.B. Gupta, Preparation of rifampicin/lactose microparticle composites by a supercritical antisolvent-drug excipient mixing technique for inhalation delivery, Powder Technol. 236 (2013) 132–138.
[26] W. Liu, Y. Fu, Y. Zu, Y. Kong, L. Zhang, B. Zu, T. Efferth, Negative-pressure cavitation extraction for the determination of flavonoids in pigeon pea leaves by liquid chromatography–tandem mass spectrometry, J. Chromatogr. A 1216 (2009) 3841–3850. [27] J.P. Venter, D.G. Müller, J. du Plessis, C. Goosen, A comparative study of an in situ adapted diffusion cell and an in vitro Franz diffusion cell method for transdermal absorption of doxylamine, Eur. J. Pharm. Sci. 13 (2001) 169–177. [28] O. Guillard, B. Fauconneau, F. Favreau, A. Marrauld, A. Pineau, An analytical procedure for the determination of aluminum used in antiperspirants on human skin in Franz™ diffusion cell, Toxicol. Mech. Methods 22 (2012) 205–210. [29] A. Dołhań, A. Jelilńska, M. Manuszewska, Stability-indicating HPLC method for the determination of cefquinome sulfate, Acta Pol. Pharm. 71 (2014) 249–254. [30] X. Zhao, Y. Zu, Q. Li, M. Wang, B. Zu, X. Zhang, R. Jiang, C. Zu, Preparation and characterization of camptothecin powder micronized by a supercritical antisolvent (SAS) process, J. Supercrit. Fluids 51 (2010) 412–419. [31] Y. Zu, W. Sun, X. Zhao, W. Wang, Y. Li, Y. Ge, Y. Liu, K. Wang, Preparation and characterization of amorphous amphotericin B nanoparticles for oral administration through liquid antisolvent precipitation, Eur. J. Pharm. Sci. 53 (2014) 109–117.
Contents lists available at ScienceDirect
Powder Technology journal homepage: www.elsevier.com/locate/powtec
Preparation and characterization of cefquinome sulfate microparticles for transdermal delivery by negative-pressure cavitation antisolvent precipitation Xinxuan Du 1, Suchong Zu 1, Fengli Chen, Zaizhi Liu, Xinran Li, Lei Yang ⁎, Yuangang Zu, Xiuhua Zhao ⁎, Lin Zhang Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, China
a r t i c l e
i n f o
Article history: Received 15 October 2015 Received in revised form 22 February 2016 Accepted 5 March 2016 Available online 8 March 2016 Keywords: Cefquinome sulfate Microparticle Negative-pressure cavitation antisolvent precipitation Preparation Transdermal delivery
a b s t r a c t The objective of this study was to prepare micronized cefquinome sulfate by negative-pressure cavitation antisolvent precipitation to improve its permeability and transdermal absorbability. Dimethylsulfoxide and ethanol were used as the solvent and anti-solvent, respectively. The effects of operating parameters, including cefquinome sulfate concentration, volume ratio of antisolvent to solvent, antisolvent precipitation time, and applied negative pressure on the characteristics of the precipitate crystals, were evaluated using an orthogonal array design. Under the optimized conditions, micronized cefquinome sulfate with a mean particle size of 415.8 nm was obtained at a yield of 78%. The micronized cefquinome sulfate was characterized by scanning electron microscopy, X-ray diffraction, and differential scanning calorimetry. No obvious change in its chemical structure was observed, but crystallinity was reduced. The physicochemical properties and transdermal absorbability of crystalline cefquinome sulfate improved as a result of the particle size reduction by physical modification. This negative-pressure cavitation antisolvent precipitation process offers a promising technique for improving the physicochemical properties and anti-inflammatory activity of cefquinome sulfate. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Cow mastitis, the most commonly occurring inflammation in dairy cows, is difficult to cure, and is therefore a major topic of research by international scholars [1]. Cow mastitis symptoms include inflammations of udder parenchyma and interstitial tissue, which are usually caused by mechanical stimulation, immersion of pathogenic microorganisms, and chemicophysical damage [2]. The main symptoms manifest as redness, swelling, and heat or pain of the udder, which directly result from reduced or completely halted lactation [3]. Cows are reared widely in many countries, and they contribute to a major portion of milk production in countries like China. In China, the livestock industry suffers huge economic losses because of low milk production caused by bacterial diseases, including mastitis [4]. Oral anti-inflammatory drugs are often mixed in with the animal feedstocks, but, because the cow has a complex four-chambered stomach, this leads to excessive drug loss in the metabolic process [5,6]. Injection of drugs is difficult because of the thick hides of the animals and requires professional skill. Use of transdermal preparations for treating mastitis can reduce the drug in the body's metabolism, reduce injury to the liver, has the advantage of convenient use, and does not ⁎ Corresponding authors. E-mail addresses: [email protected] (L. Yang), [email protected] (X. Zhao). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.powtec.2016.03.008 0032-5910/© 2016 Elsevier B.V. All rights reserved.
easily produce drug resistance [7]. The poor solubility and low dissolution rate of active pharmaceutical ingredients in water is one of the most difficult and as-yet unsolved problems in pharmaceutical technology [8]. This drawback limits medical developments, such as new dosage forms for transdermal absorption. Cefquinome sulfate (Fig. 1) is a synthetic compound, the poor solubility of which leads to low oral absorption [9]. Cefquinome sulfate, a fourth-generation cephalosporin, has been solely researched for veterinary use owing to its antimicrobial activity against a broad spectrum of gram-positive and gram-negative bacterial species [10,11]. It has been also applied for treatment of acute mastitis and foot rot in cattle, and respiratory tract diseases in pigs, cows, and horses [12]. Cefquinome sulfate has been shown to have wide antibacterial activity and can achieve the effect of sterilization by inhibiting cell synthesis. In vitro antibacterial use of cefquinome sulfate indicates that it can inhibit both positive and negative common bacteria: for example, Escherichia coli, Bacillus, Klebsiella bacteria, Pasteurella, Salmonella species, Serratia marcescens, Streptococcus, the Bacteroides fragilis group, Clostridium prazmowski, Actinobacillus, and Erysipelothrix rhusiopathiae [11]. Cefquinome sulfate is an effective agent against many in vitro and in vivo bacteria. It is mainly used for treating cow mastitis, which is caused by sensitivity to bacteria, respiratory infections in pigs, and mastitis–metritis–agalactia syndrome in sows [10]. Microparticles have been popularly applied in enhancing the dissolution rate of drugs that have poor solubility in water. Owing to the
430
X. Du et al. / Powder Technology 294 (2016) 429–436
China). N-Methyl-2-pyrrolidone, acetone, methanol, dichloromethane, trichloromethane, and dimethyl sulfoxide (DMSO) (analytically pure) were purchased from Bodi Chemical Reagent Company (Tianjin, China). 2.2. Preparation of skin The skin permeation study was performed and approved by the Institutional Animal Ethics Committee. The thoracic skin of a dairy cow was obtained from Mengniu Pasture, Zhaodong, China. The skin was washed with normal saline and the hair on the skin was removed with an electric shaver before use. Fig. 1. Molecular structures of cefquinome sulfate.
reduced particle size of microparticles as compared with that of the raw drug particles, the interfacial surface area is increased, leading to improved solubility in water [13]. Bioavailability and oral efficacy of microparticles can be consequently improved by reduction in particle size, thereby allowing for direct usage of the poorly water-soluble drugs at smaller dosages or with more rapid response times [14,15]. Traditionally, many techniques have been applied for the preparation of microparticles, including ball milling [16], air jet pulverization [17,18], supercritical antisolvent technology [13–15], high pressure homogenization [19,20], reactive crystallization [21], and spray drying [22,23]. The process of mechanical comminution [16–18], a method of micronizing drugs, is now widely used in pharmaceutical manufacturing. Supercritical antisolvent technology has the disadvantage of large capital equipment investment, high pressure homogenization is limited by expensive equipment and solvent requirements, while reactive crystallization can only be applied to the preparation of acidic or alkaline drugs. In contrast, liquid antisolvent precipitation has the advantages of lower reagent consumption, higher process efficiency, and easy operation [24,25]. Cavitation is the phenomenon of sequential formation, growth and collapse of millions of tiny vapor bubbles (voids) in a liquid. It can occur whenever a liquid is used in a machine that induces pressure and velocity fluctuations in the fluid (e.g. pumps, turbines and propellers) [26]. Negative-pressure cavitation extraction has been used for extraction of biological active compounds from plant materials centuries. Negative-pressure cavitation antisolvent precipitation is based on a technique that uses negative pressure as its driving force. In this technique, a solvent and an anti-solvent are fully stirred by pumping air through the bottom of a reactor in which they are contained. The characteristic bubbles that form under conditions of negative pressure cavitation are used to keep the particle size of the powder uniform and tiny for a relatively long time. The aim of this study was to prepare cefquinome sulfate microparticles using a negative-pressure cavitation antisolvent precipitation process and to evaluate their physicochemical properties and transdermal delivery in vitro. To optimize the negative-pressure cavitation antisolvent precipitation process, the effects of cefquinome sulfate concentration, volume ratio of antisolvent to solvent, antisolvent precipitation time, and applied negative pressure on the yield and mean particle size (MPS) of the cefquinome sulfate microparticles were studied using an orthogonal array design. The microparticles were further characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), and X-ray diffraction (XRD). The transdermal delivery performance in vitro of cefquinome sulfate microparticles prepared was also evaluated. 2. Materials and methods 2.1. Drugs and chemicals Cefquinome sulfate powder (purity of 98.5%) was provided by Zhejiang Hisun Pharmaceutical Co. Ltd. of China. Ethanol (analytically pure) was purchased from Tianli Chemical Reagent Company (Tianjin,
2.3. Negative-pressure cavitation antisolvent precipitation apparatus Micronized cefquinome sulfate was prepared by antisolvent precipitation. A schematic diagram of the apparatus is shown in Fig. 2. This consisted of an injection system (10–14), a negative-pressure cavitation reaction tank (8), and a vacuum system (1, 2, 6). The negative pressure valve (6) was first opened to maintain the pressure of the reaction tank (8) at − 0.04 to − 0.08 MPa, which measured by a vacuum gauge (YZ-40, Qingdao Dongfang Instrument Co., Ltd., China). The inlet valve (7) was then opened to allow the ethanol antisolvent to fill the negative-pressure cavitation reaction tank (8). The inlet valve (7) was then closed and the piston pump (2J-X 52/4.5, Zhijiang Science and Technology Instrument Factory, China) (19) was opened to control the air inflow speed in the range from 1 to 10 m3/h. The intake valve (16) was then opened, which allowed air to flow through the intake branch pipeline (14), an annular conduit (10), and a nozzle (FD-1-1/ 8-Brass, Dalian Baoyuan Purification Technology Co., Ltd., China) (11). The liquid flow meter (LZZ-32, Changzhou Chengfeng Flowmeter Co., Ltd., China) (18) and piston pump (19) were then simultaneously opened to guide the mixed liquids (containing cefquinome sulfate and DMSO) into the negative-pressure cavitation reaction tank (8) by means of a branched pipeline (13), an annular conduit (12), and a nozzle (11). The mixed liquids then began to cavitate and become suspended. The intake valve (16) was used to control the air inflow and the piston pump (19) was employed to maintain the inlet fluid velocity. The mixed liquids were suddenly blended with ethanol antisolvent contained in reaction tank (8) when they were ejected through a microporous nozzle. Cefquinome sulfate submicroscopic particulates were finally separated out, suspended in the DMSO–ethanol solution under the effects of cavitation and suspension. During these procedures, secondary aggregation could not occur and so tiny, uniformly dispersed cefquinome sulfate particles were obtained. After importing the mixed liquids, the liquid flow meter (18) was closed and the piston pump (19) was switched off, having achieved its technical requirements with respect to the impacts of continuous cavitation and suspension. The intake valve (16), negative pressure valve (6), and relief valve (5) were then closed to balance the pressure in the reaction tank (8). The material discharge valve (15) was opened to export the residue solutions contained in the reaction tank. The suspension was centrifuged at 5000 rpm for 5 min, and the particles were then freeze-dried at − 50 °C for 24 h. Each experiment was performed in triplicate. 2.4. Optimization of the antisolvent precipitation process An L16(4)5 orthogonal experiment, performed with four factors and four levels, was selected for optimization of the operational parameters for cefquinome sulfate micronization using this process. Based on the results of preliminary experiments, we optimized the operating conditions and analyzed the statistical experimental results using an orthogonal array design with four factors applied using Design-Expert 7.0 software (Stat-Ease, Minneapolis, USA). The boundaries of the four factors were 100 to 400 mg/mL cefquinome sulfate concentration, 3 to 9 mL/mL volume ratio of antisolvent to solvent, −0.02 to − 0.08 MPa
X. Du et al. / Powder Technology 294 (2016) 429–436
431
Fig. 2. Schematic drawing of negative-pressure cavitation antisolvent precipitation apparatus to prepare the micronized cefquinome sulfate. 1. Vacuum gauge; 2. Negative pressure pipeline; 3. Relief pipeline; 4. Inlet pipeline; 5. Relief valve; 6. Negative pressure valve; 7. Inlet valve; 8. Negative-pressure cavitation reaction tank; 9. Viewing port; 10. Annular conduit; 11. Nozzle; 12. Annular conduit; 13. Branch pipeline; 14. Intake branch pipeline; 15. Material discharge valve; 16. Intake valve; 17. Inlet valve; 18. Liquid flow meter; 19. Piston pump; 20. Gas flow meter; 21. Liquid pipeline; 22. Intake pipeline; 23. Material discharge pipeline.
negative pressure, and 5 to 20 min antisolvent precipitation time. The levels of the factors for the orthogonal array design are indicated in Table 1. Micronized cefquinome sulfate was obtained from the above 16 tests, the yield and MPS of which were variable-dependent. Generally speaking, MPS and yield are selected as the two indexes used to evaluate the advantages and disadvantages of the antisolvent precipitation technique. In the orthogonal design, a scoring system was applied to comprehensively investigate the proposed process. MPS and yield were considered as equally important, with each assigned a value of 0.5. The detailed appraisals and the relative marking criteria with respect to MPS and yield are presented in Table 2.
2.5. Powder characterization 2.5.1. Dynamic light scattering (DLC) The diameter distributions of the cefquinome sulfate powders were measured using a dynamic light scattering analyzer (ZetaPALS, Brookharen Instrument Corporation, USA). Samples were suspended in deionized water and treated by sonication for 3 min at 100 W (KQ250DB, Kunshan Ultrasonic Instruments Co., China) to eliminate dust and to prevent the aggregation of particles. The suspension was then placed in a cuvette and measured at room temperature. Each experiment was performed in triplicate.
Table 1 Factors and levels for orthogonal test. Factors
(A) Cefquinome sulfate concentration (mg/mL) (B) Volume ratio of antisolvent to solvent (mL/mL) (C) Negative pressure (MPa) (D) Antisolvent precipitation time (min)
2.5.2. Scanning electron microscopy The surface morphologies of the unprocessed and processed cefquinome sulfate were investigated by SEM (Quanta 200, FEI, Hillsboro, USA). Before examination, the samples were sputter-coated with a thin layer of gold–palladium (5 to 10 nm; 10 mA; 30 s) at room temperature using a sputter coater. 2.5.3. X-ray diffraction Powder XRD patterns were collected in transmission mode using an X-ray diffractometer with a rotating anode (Philips, Xpert-Pro, Holland) with Cu Kα1 radiation generated at 30 mA and 40 kV. Powders of the unprocessed and processed cefquinome sulfates were filled to the same depth inside the sample holder by leveling with a spatula. The range of 2θ diffraction angles from 5 to 80° was examined, with steps of 0.02° and a measuring time of 0.2 s per step. 2.5.4. Differential scanning calorimetry The unprocessed and processed cefquinome sulfate powders were analyzed by DSC (TA instruments, Q100, New Castle, USA). About 5 mg of sample was placed in an open aluminum pan and heated from ambient temperature to 250 °C at a rate of 5 °C/min. 2.5.5. Transdermal delivery tests As shown in Fig. 3, a drug transdermal diffusion instrument (TT-6, Zhengtong Technology Co., Ltd., Tianjin, China) and an improved Franz diffusion pool method [27,28] were applied in the present study. The Table 2 Relationship between process score and particle size and yield.
Levels 1
2
3
4
Particle size (nm)
Score
Yield (%)
Score
100
200
300
400
3
5
7
9
−0.02 5
−0.04 10
−0.06 15
−0.08 20
˂200 200–400 400–600 600–800 800–1000 ˃1000
10 8 6 4 2 1
˃ 80 60–80 40–60 20–40 1–20
10 8 6 4 1
432
X. Du et al. / Powder Technology 294 (2016) 429–436
volume of the reception tank was 5 mL with 0.785 cm2 of effective surface. The skin of the udder was cut, the subcutaneous fat layer and connective tissue were carefully peeled off, and the area rinsed thoroughly with normal saline. Two slices of skin with the same area were prepared using a hole puncher. The skin was placed with the inner side downwards in the reaction vessel and fixed with this side facing into the diffusion chamber and the outer skin facing towards the supply side of the apparatus. Then, 1.0 mL each of 10 mg/mL unprocessed and processed cefquinome sulfate suspensions were separately added into the supply chamber. Five mL ethanol was also added as a receptor fluid to ensure good contact with the skin. A constant interlayer temperature in the diffusion chamber of 37 ± 0.5 °C was maintained. Several rotors were added to the receiving chamber and stirred at a constant speed of 900 rpm. Timing was started 15 min after adding the samples: 5 mL aliquots of fluids in the receptor were taken separately at predetermined times and simultaneously replaced by an equal volume of ethanol. The aliquots were centrifuged, following which an appropriate volume of liquid supernatant was sampled to determine the cefquinome sulfate content and to calculate its cumulative penetration into the skin.
2.5.6. Determination of cefquinome sulfate content by high-performance liquid chromatography (HPLC) Both the unprocessed and processed cefquinome sulfate present in the permeate liquors generated in the transdermal delivery test were analyzed by HPLC using a Waters 1525–2489 series system (Waters, USA), equipped with a HiQ sil-C18 reversed-phase column (4.6 mm × 250 mm, 5 μm, KYA TECH). The mobile phase consisted of 14% volume fraction acetonitrile. The injection volume was 20 μL and was delivered at 0.8 mL/min. The response signal was monitored at a wavelength of 220 nm [29]. 2.5.7. Residual solvent determination The residual DMSO in the micronized cefquinome sulfate was analyzed using gas chromatography (GC) (Agilent 7890 A, Palo Alto, CA, USA) equipped with a G1540N-210 flame-ionization detector and an HP-5 capillary column (film thickness 0.25 μm, 30 m × 320 μm). The micronized cefquinome sulfate samples (10 mg) were suspended in 1 mL methanol and centrifuged at 10 000 rpm for 5 min. Ten microliters of the supernatant was then injected. DMSO analysis was carried out using the following GC conditions: detector temperature, 280 °C; injector temperature, 200 °C; initial oven temperature, 40 °C for 5 min, following which the temperature was increased to 240 °C at 40 °C/min and maintained for a further 5 min; hydrogen flow rate, 30 mL/min; airflow velocity, 400 mL/min; carrier gas, nitrogen; carrier gas velocity, 2.2 mL/min; and split ratio, 20:1. 3. Results and discussion 3.1. Screening of solvent and antisolvent The first step in developing the antisolvent precipitation method was to select the solvent and antisolvent, making sure that the solute has high solubility in the solvent and that the antisolvent has little solubility in solvent but is miscible with it. Seven common solvents were evaluated, including N-methyl-2-pyrrolidone, ethanol, acetone, methanol, dichloromethane, trichloromethane, and DMSO, with respect to solubility of cefquinome sulfate, so as to choose the most suitable combination of solvent and antisolvent. Cefquinome sulfate has a solubility at 25 °C that differs greatly in the different solvents, as shown in Table 3: it has a rather low solubility (below 0.5 mg/mL) in ethanol, acetone, methanol, dichloromethane, and trichloromethane; however, it has a low solubility (40 mg/mL) in N-methyl-2-pyrrolidone and relatively higher solubility in DMSO (140 mg/mL). Based on these results, ethanol was chosen as the antisolvent and DMSO as the solvent. 3.2. Orthogonal test design The various operational parameters exert different influences on the preparation process, each of which should be systematically optimized. For the antisolvent precipitation process, the cefquinome sulfate concentration, the volume ratio of antisolvent to solvent, the antisolvent precipitation time, and the negative pressure applied are generally considered to be the most important parameters. An orthogonal L16(45) test design was applied to optimize the preparation conditions for Table 3 Solubility of cefquinome sulfate in the different solvents at 25 °C.
Fig. 3. Transdermal procedures in vitro. a: Skin of the udder; b: micronized cefquinome sulfate suspension; c: transdermal diffusion of unprocessed and processed cefquinome sulfate suspensions in Franz diffusion pool.
Solvents
Solubility (mg/mL)
N-Methyl-2-pyrrolidone Ethanol Acetone Methanol Dichloromethane Chloroform Dimethyl sulfoxide (DMSO)
40 10 b0.5 6 b0.5 b0.5 140
X. Du et al. / Powder Technology 294 (2016) 429–436
micronized cefquinome sulfate. Sixteen experiments were carried out in triplicate and run in randomized order to avoid any personal or subjective bias. Based on the pre-experimental results, the values of the level settings of the four factors (A, B, C, and D) used in the orthogonal arrayed design are listed in Table 1. The process relationship scores with respect to particle size and yield are listed in Table 2. The results of the 16 experimental trials carried out under these different conditions are shown in Table 4. Under the orthogonal conditions, the particle sizes of the drug ranged from 340 nm to 1316 nm, while the yields ranged from 0.5% to 75%. Experiment 11 achieved the highest composite score, with a particle size of 555.9 nm and yield of 75%. As Table 4 also shows, the scores range from A N D N B N C, indicating that the degree of influence on the process follows a decreasing order from cefquinome sulfate concentration, antisolvent precipitation time, volume ratio of antisolvent to solvent, to negative pressure applied. Considering the experimental data and the above analysis, the optimum conditions are represented by the combination A2B2C3D3, for which the cefquinome sulfate concentration is 200 mg/mL, the volume ratio of antisolvent to solvent is 5 mL/mL, the antisolvent precipitation time is 15 min, and the pressure is − 0.06 MPa. A confirmatory test performed under these conditions gave a MPS of 415.8 nm and yield of 78% for the obtained powder. 3.3. Scanning electron microscopy Electron micrographs of the cefquinome sulfate surfaces are shown in Fig. 4. Fig. 4a shows an overall view of unprocessed cefquinome sulfate, with a corresponding enlarged view in Fig. 4c. The unprocessed cefquinome sulfate morphology comprises large, irregular crystalline flakes. Fig. 4b and d show analogous views of micronized cefquinome sulfate, indicating the presence of porous spheres with a relatively regular size of 10 to 15 μm. Inspection at greater magnification showed that these consisted of tiny sheet structures with a particle size of 300 to 450 nm. These results confirm that the particle size of cefquinome sulfate is modified by the antisolvent precipitation process. The main reason for the multi-aperture spherical appearance of the micronized power is probably that the antisolvent volatilizes during the drying
433
process and the fines reunite to form the powder observed at the end of the reaction. 3.4. X-ray diffraction analysis To further investigate the occurrence of possible crystal structural changes, XRD analyses were undertaken. The results for unprocessed and processed cefquinome sulfates are shown in Fig. 5. Several strong peaks for the unprocessed cefquinome sulfate sample, occurring at 2θ diffraction angles from 8.1° to 27.7°, reveal that the drug is present in a crystalline form. In contrast, the processed cefquinome sulfate shows significantly reduced intensity of peaks. The reduction of diffraction intensity of the peaks for the processed sample is attributed to reduced particle crystallinity or decreased particle size. This result indicates that cefquinome sulfate particles with a smaller MPS and lower crystallinity were obtained following antisolvent precipitation processing. This micronization can increase the solubility of cefquinome sulfate drugs and thereby enhance the therapeutic effect of percutaneous absorption. 3.5. Differential scanning calorimetry As seen in Fig. 6, the DSC curves for unprocessed and processed cefquinome sulfate show that the unprocessed drug produced an endothermic melting peak at 177.3 °C but that of processed cefquinome sulfate occurred at 178.5 °C. These results indicate that both unprocessed and processed cefquinome sulfate have essentially the same melting point; however, the unprocessed sample shows slightly higher exothermicity. This confirms that the processed cefquinome sulfate has lower crystallinity, consistent with the XRD results. 3.6. Residual solvent analysis DMSO acts as a Class III solvent with low toxicity and is limited to a maximum of 0.5% in a drug, according to the International Conference on Harmonization (ICH) [30,31]. As shown in Fig. 7, the residual DMSO content in the cefquinome sulfate (prepared using DMSO as the solvent), observed at a retention time of 8.77 min by GC analysis, has
Table 4 Arrangement, results and analysis of variance (ANOVA) of L16(45) orthogonal test (n = 3). Design ID number
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 K1jc K2j K3j K4j Rd Oe a b c d e
Responsea
Factors
Score
Ab
B
C
D
Particle size (nm)
Yield (%)
Yield score
Particle size score
Composite score
100 100 100 100 200 200 200 200 300 300 300 300 400 400 400 400 6.250 7.250 4.250 2.500 4.750 A2
5 7 3 9 3 5 9 7 7 3 9 5 3 5 9 7 4.500 6.000 4.000 5.750 2.000 B2
−0.04 −0.06 −0.02 −0.08 −0.04 −0.02 −0.06 −0.08 −0.02 −0.06 −0.04 −0.08 −0.08 −0.06 −0.02 −0.04 4.750 4.500 6.000 5.000 1.500 C3
10 15 5 20 15 20 10 5 10 20 5 15 10 5 15 20 6.250 3.750 6.500 3.750 2.750 D3
340.0 1168.2 792.0 895.0 530.0 1014.7 1316.2 1211.4 769.4 525.7 555.9 1013.9 550.0 677.5 772.2 942.1
10.0 3.0 1.0 1.4 20.0 25.0 16.0 32.8 3.3 2.7 75.0 60.0 0.5 2.5 5.1 4.0
1 1 4 1 4 4 2 4 1 1 8 8 1 1 1 1
8 1 4 2 6 1 1 1 4 6 6 1 6 4 4 2
9 2 8 3 10 5 3 5 5 7 14 9 7 5 5 3
Response values were comprised of mean of three independent experiments. A: Cefquinome sulfate concentration (mg/mL); B: Volume ratio of antisolvent to solvent (mL/mL); C: Negative pressure (MPa); D: Antisolvent precipitation time (min). Logesh Kij = (1/4) Σ mean composite score at factor j (j = A, B, C, D). Logesh R = max (Kij) − min (Kij), j and i mean factor and setting level here, respectively. O means the optimum condition. The optimum combination of conditions is A2B2C3D3.
434
X. Du et al. / Powder Technology 294 (2016) 429–436
Fig. 4. Scanning electron micrographs of (a, c) unprocessed and (b, d) processed cefquinome sulfate (processed cefquinome sulfate is the micronized cefquinome sulfate by negativepressure cavitation antisolvent precipitation under optimum condition).
a standard curve represented by y = 341.37x + 1.84 (R2 = 0.9996), where y is the peak area and x is the DMSO concentration. According to this regression equation, the residual DMSO concentration in the micronized cefquinome sulfate was 0.39%. The micronized cefquinome sulfate therefore conforms to the ICH standard and is suitable for industrial veterinary drug utilization. 3.7. Results of transdermal delivery tests The cumulative absorptions of the unprocessed and processed cefquinome sulfate suspensions by the cowhide pieces in the transdermal tests are shown in Fig. 8. The processed cefquinome sulfate suspension was able to pass through the skin from exposure times greater than 0.5 h and the unit area cumulative penetration amount varied almost
Fig. 5. XRD patterns of unprocessed and processed cefquinome sulfate (processed cefquinome sulfate is the micronized cefquinome sulfate by negative-pressure cavitation antisolvent precipitation under optimum condition).
linearly with respect to the exposed surface area. After 12 h, the unit area cumulative penetration of this suspension was 53.69 μg/cm2. When using the unprocessed cefquinome sulfate suspension, however, only a small amount passed through the skin within 1 h of exposure, but diffusion stopped thereafter. The reason is that the unprocessed cefquinome sulfate had a higher MPS of 1166 nm, and was therefore unable to pass through the skin. These results reveal that processed cefquinome sulfate exhibits considerably higher transdermal delivery ability than unprocessed cefquinome sulfate, and therefore possesses potential as a new transdermal dosage form that can be further developed as a drug for curing inflammation of cows' udders.
Fig. 6. DSC curves of unprocessed and processed cefquinome sulfate (processed cefquinome sulfate is the micronized cefquinome sulfate by negative-pressure cavitation antisolvent precipitation under optimum condition).
X. Du et al. / Powder Technology 294 (2016) 429–436
435
Fig. 7. Gas chromatograms of cefquinome sulfate microparticles, indicating the presence of residual DMSO (cefquinome sulfate microparticles is the micronized cefquinome sulfate by negative-pressure cavitation antisolvent precipitation under optimum condition).
4. Conclusions In this work, micronized cefquinome sulfate was successfully prepared using a negative-pressure cavitation antisolvent precipitation process. A sample of micronized cefquinome sulfate with a MPS of 415.8 nm was obtained when the cefquinome sulfate concentration was 200 mg/mL, the volume ratio of antisolvent to solvent was 5 mL/mL, a pressure − 0.06 MPa was applied, and using an antisolvent precipitation time of 15 min. The morphologies of the unprocessed and processed cefquinome sulfate particles were investigated using SEM, XRD, and DSC. All of these analyses indicated that the micronized cefquinome sulfate existed as a lower crystallinity form and that no degradation occurred following the negative-pressure cavitation antisolvent precipitation process. When compared with the unprocessed higher crystallinity form, the lower crystallinity cefquinome sulfate microparticles showed better performance in transdermal delivery. In addition, the residual solvent in the cefquinome sulfate microparticle powder met the specification for pharmaceutical use. These results suggest that the negativepressure cavitation antisolvent precipitation process may be a promising approach for preparation of cefquinome sulfate microparticles having low crystallinity, low MPS, and thereby improved transdermal delivery. Acknowledgments The authors thank the Special Fund for Forestry Scientific Research in the Public Interest (201404616) for financial support.
Fig. 8. Comparison of unit area cumulative volume of processed and unprocessed cefquinome sulfate microparticles absorbed in transdermal tests (processed cefquinome sulfate is the micronized cefquinome sulfate by negative-pressure cavitation antisolvent precipitation under optimum condition).
References [1] R. Pilla, C. Bonura, M. Malvisi, G.G. Snel, R. Piccinini, Methicillin-resistant Staphylococcus pseudintermedius as causative agent of dairy cow mastitis, Vet. Rec. 173 (2013) 19. [2] Y. Lu, Y.L. Hu, X.F. Kong, D.Y. Wang, Selection of component drug in activating blood flow and removing blood stasis of Chinese herbal medicinal formula for dairy cow mastitis by hemorheological method, J. Ethnopharmacol. 116 (2008) 313–317. [3] C.W. Rougoor, W.J. Hanekamp, A.A. Dijkhuizen, M. Nielen, J.B. Wilmink, Relationships between dairy cow mastitis and fertility management and farm performance, Prev. Vet. Med. 39 (1999) 247–264. [4] X. Zhou, C. Yang, Y. Li, X. Liu, Y. Wang, Potential of berberine to enhance antimicrobial activity of commonly used antibiotics for dairy cow mastitis caused by multiple drug-resistant Staphylococcus epidermidis infection, Genet. Mol. Res. 14 (2015) 9683–9692. [5] K.L. Ingvartsen, K.M. Moyes, Factors contributing to immunosuppression in the dairy cow during the periparturient period, Jpn. J. Vet. Res. 63 (2015) S15–S24. [6] A. Zonca, M. Gallo, C. Locatelli, S. Carli, P. Moroni, R. Villa, P. Cagnardi, Cefquinome sulfate behavior after intramammary administration in healthy and infected cows, J. Dairy Sci. 94 (2011) 3455–3461. [7] Y.T. Zhang, M.Q. Han, L.N. Shen, J.H. Zhao, N.P. Feng, Solid lipid nanoparticles formulated for transdermal aconitine administration and evaluated in vitro and in vivo, J. Biomed. Nanotechnol. 11 (2015) 351–361. [8] S. Zu, L. Yang, J. Huang, C. Ma, W. Wang, C. Zhao, Y. Zu, Micronization of taxifolin by supercritical antisolvent process and evaluation of radical scavenging activity, Int. J. Mol. Sci. 13 (2012) 8869–8881. [9] Q. Fu, H.L. Fu, L. Huan, W. Zhang, G. Shu, M.J. Liu, F.Y. Deng, J. Hu, Preparation of cefquinome sulfate proliposome and its pharmacokinetics in rabbit, Iran. J. Pharm. Res. 12 (2013) 611–621. [10] M. Limbert, D. Isert, N. Klesel, A. Markus, K. Seeger, G. Seibert, E. Schrinner, Antibacterial activities in vitro and in vivo and pharmacokinetics of cefquinome (HR 111V), a new broad-spectrum cephalosporin, Antimicrob. Agents Chemother. 35 (1991) 14–19. [11] N.X. Chin, J.W. Gu, W. Fang, H.C. Neu, In vitro activity of cefquinome, a new cephalosporin, compared with other cephalosporin antibiotics, Diagn. Microbiol. Infect. Dis. 15 (1992) 331–337. [12] N.Y. Shpigel, D. Levin, M. Winkler, A. Saran, G. Ziv, A. Böttner, Efficacy of cefquinome for treatment of cows with mastitis experimentally induced using Escherichia coli, J. Dairy Sci. 80 (1997) 318–323. [13] Y. Zu, Q. Zhang, X. Zhao, D. Wang, W. Li, X. Sui, Y. Zhang, S. Jiang, Q. Wang, C. Gu, Preparation and characterization of vitexin powder micronized by a supercritical antisolvent (SAS) process, Powder Technol. 228 (2012) 47–55. [14] X. Sui, W. Wei, L. Yang, Y. Zu, C. Zhao, L. Zhang, F. Yang, Z. Zhang, Preparation, characterization and in vivo assessment of the bioavailability of glycyrrhizic acidmicroparticles by supercritical anti-solvent process, Int. J. Pharm. 423 (2012) 471–479. [15] L. Yang, Z. Sun, Y. Zu, C. Zhao, X. Sun, Z. Zhang, L. Zhang, Physicochemical properties and oral bioavailability of ursolic acid nanoparticles using supercritical anti-solvent (SAS) process, Food Chem. 132 (2012) 319–325. [16] D. Chen, D.Y. Li, Y.Z. Zhang, Z.T. Kang, Preparation of magnesium ferrite nanoparticles by ultrasonic wave-assisted aqueous solution ball milling, Ultrason. Sonochem. 20 (2013) 1337–1340. [17] M. Aoki, H. Nishimura, A. Mimura, S. Kita, T. Yasuzawa, K. Terada, Identification of the degradation products of the steroid sulfatase inhibitor KW-2581 in jet millmicronized powder, J. Pharm. Sci. 102 (2013) 1760–1772. [18] L.W. Chan, C.C. Lee, P.W. Heng, Ultrafine grinding using a fluidized bed opposed jet mill: effects of feed load and rotational speed of classifier wheel on particle shape, Drug Dev. Ind. Pharm. 28 (2002) 939–947.
436
X. Du et al. / Powder Technology 294 (2016) 429–436
[19] Y. Li, X. Zhao, Y. Zu, Y. Zhang, Preparation and characterization of paclitaxel nanosuspension using novel emulsification method by combining high speed homogenizer and high pressure homogenization, Int. J. Pharm. 490 (2015) 324–333. [20] A. Homayouni, F. Sadeghi, J. Varshosaz, G.H. Afrasiabi, A. Nokhodchi, Promising dissolution enhancement effect of soluplus on crystallized celecoxib obtained through antisolvent precipitation and high pressure homogenization techniques, Colloids Surf. B 122 (2014) 591–600. [21] Y. Gao, J. Wang, Y. Wang, Q. Yin, B. Glennon, J. Zhong, J. Ouyang, X. Huang, H. Hao, Crystallization methods for preparation of nanocrystals for drug delivery system, Curr. Pharm. Des. 21 (2015) 3131–3139. [22] K. Anand, S. Varghese, T. Kurian, Preparation of ultra-fine dispersions of zinc oxide by simple ball-milling: optimization of process parameters, Powder Technol. 271 (2015) 187–192. [23] L.B. de Paiva, A.M. de Oliveira, R.R. Gavioli, Preparation and properties of rubber powder from modified-SBR latex by spray drying process, Powder Technol. 264 (2014) 507–513. [24] A. Fodor-Kardos, J. Toth, J. Gyenis, Preparation of protein loaded chitosan microparticles by combined precipitation and spherical agglomeration, Powder Technol. 244 (2013) 16–25. [25] C.A. Ober, L. Kalombo, H. Swai, R.B. Gupta, Preparation of rifampicin/lactose microparticle composites by a supercritical antisolvent-drug excipient mixing technique for inhalation delivery, Powder Technol. 236 (2013) 132–138.
[26] W. Liu, Y. Fu, Y. Zu, Y. Kong, L. Zhang, B. Zu, T. Efferth, Negative-pressure cavitation extraction for the determination of flavonoids in pigeon pea leaves by liquid chromatography–tandem mass spectrometry, J. Chromatogr. A 1216 (2009) 3841–3850. [27] J.P. Venter, D.G. Müller, J. du Plessis, C. Goosen, A comparative study of an in situ adapted diffusion cell and an in vitro Franz diffusion cell method for transdermal absorption of doxylamine, Eur. J. Pharm. Sci. 13 (2001) 169–177. [28] O. Guillard, B. Fauconneau, F. Favreau, A. Marrauld, A. Pineau, An analytical procedure for the determination of aluminum used in antiperspirants on human skin in Franz™ diffusion cell, Toxicol. Mech. Methods 22 (2012) 205–210. [29] A. Dołhań, A. Jelilńska, M. Manuszewska, Stability-indicating HPLC method for the determination of cefquinome sulfate, Acta Pol. Pharm. 71 (2014) 249–254. [30] X. Zhao, Y. Zu, Q. Li, M. Wang, B. Zu, X. Zhang, R. Jiang, C. Zu, Preparation and characterization of camptothecin powder micronized by a supercritical antisolvent (SAS) process, J. Supercrit. Fluids 51 (2010) 412–419. [31] Y. Zu, W. Sun, X. Zhao, W. Wang, Y. Li, Y. Ge, Y. Liu, K. Wang, Preparation and characterization of amorphous amphotericin B nanoparticles for oral administration through liquid antisolvent precipitation, Eur. J. Pharm. Sci. 53 (2014) 109–117.