Preparation and characterization of amorphous amphotericin B nanoparticles for oral administration through liquid antisolvent precipitation

European Journal of Pharmaceutical Sciences 53 (2014) 109–117

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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps

Preparation and characterization of amorphous amphotericin B nanoparticles for oral administration through liquid antisolvent precipitation Yuangang Zu, Wei Sun, Xiuhua Zhao ⇑, Weiguo Wang, Yong Li, Yunlong Ge, Ying Liu, Kunlun Wang Key Laboratory of Forest Plant Ecology, Northeast Forestry University, Ministry of Education, Harbin 150040, Heilongjiang, China

a r t i c l e

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Article history: Received 17 July 2013 Received in revised form 7 November 2013 Accepted 8 December 2013 Available online 15 December 2013 Keywords: Amphotericin B Nanoparticles LAP Oral Preparation Characterization

a b s t r a c t We prepared amphotericin B (AmB) nanoparticles through liquid antisolvent precipitation (LAP) and by freeze-drying to improve the solubility of AmB for oral administration. The LAP was optimized through a single-factor experiment. We determined the effects of surfactants and their concentration, the stirring time, the precipitation temperature, the stirring intensity, the drug concentration and the volume ratio of antisolvent to solvent on the mean particle size (MPS) of the AmB nanoparticles. Increased stirring intensity and precipitation time favored AmB nanoparticles with smaller MPS, but precipitation times exceeding 30 min did not further reduce the MPS. Increased Tween-80 concentration and the drug concentration decreased the MPS of the AmB nanoparticles. Increased precipitation temperature and antisolvent to solvent volume ratio initially decreased the MPS of the AmB nanoparticles, which increased thereafter. Optimum conditions produced AmB nanoparticles with an MPS of 135.1 nm. The AmB nanoparticles were characterized through scanning electron microscopy (SEM), mass spectrometry (MS), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), differential scanning calorimetry (DSC), thermal gravimetric analysis (TG), solvent residue, drug purity test, and dissolution testing. The analyses indicated that the chemical structure of AmB remained unchanged in the nanoparticles, but the structure was changed from crystalline to amorphous. The residual DMSO in the nanoparticles was 0.24% less than the standard set by the International Conference on Harmonization limit for class III solvents. The AmB nanoparticles exhibited 2.1 times faster dissolution rates and 13 times equilibrium solubility compared with the raw drug. The detection results indicate that the AmB nanoparticles potentially improved the oral absorption of AmB. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction Amphotericin B (AmB) was isolated from Streptomyces nodosus in 1955 (Kleinberg, 2006). AmB (Fig. 1) is the classic broad-spectrum agent for systemic fungal infections and it remains the gold standard therapy for fungal infections (Antoniadou and Dupont, 2005). The mechanism of action of this highly effective antifungal is binding to the ergosterol in the cell membrane of fungi and some protozoan parasites, which disrupts cell membrane integrity (Lin et al., 2010; Sachs-Barrable et al., 2008). Membrane pore formation damages membrane permeability, which causes the leakage of important materials, leading to fungal cell death (Pereira et al., 2013). However, the poor water solubility of AmB limits its application. Since 1958, it was dissolve and formulated in deoxycholate (FungizoneÒ), which required intravenous administration and associated with infusion and various acute side effects, which ⇑ Corresponding author. Tel.: +86 451 82191517; fax: +86 451 82102082. E-mail address: [email protected] (X. Zhao). 0928-0987/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejps.2013.12.005

restricts the clinical benefits of AmB therapy (Falamarzian and Lavasanifar, 2010; Italia et al., 2009). To reduce the toxicity and side effects, three lipid AmB formulations for intravenous therapy was made commercially available: liposomal amphotericin B (AmbisomeÒ), amphotericin B colloidal dispersion (AmphocilÒ), and amphotericin B lipid complex (AbelcetÒ) (Antoniadou and Dupont, 2005). However, the high cost is the main barrier to the widespread use of these formulations. AmB is unavailable to the poor consumers in developing countries. With the development of drug technology, lipid nanosomal AmB and nanosphere AmB (LNS-AmB) have been investigated for intravenous administration (Fukui et al., 2003; Sheikh et al., 2010). However, the hospitalization required for injection therapy still limits the widespread use of these formulations. These formulations are only secondary choices for clinical use. Consequently, an oral delivery system is highly desirable. Oral administration improves patient compliance and the pharmacokinetics of drugs. Different groups have investigated oral formulations for AmB delivery, including nanosuspensions and emulsions (Italia et al., 2009; Kayser et al., 2003; Lemke et al., 2010).

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Fig. 1. Chemical structure of amphotericin B (AmB).

The AmB nanosuspension reported by Lemke and Kayser were produced through high-pressure homogenization (Kayser et al., 2003; Lemke et al., 2010) The nanosuspension improved the solubility of AmB; however, high-pressure homogenization produces heat and AmB is heat sensitive. In addition, the high-pressure homogenization entails high equipment costs. Oral emulsion formulations to improve the oral bioavailability of AmB have attracted attention, such as AmB in Peceol/distearoylphosphatidylethanolamine– poly(ethylene glycol) (Peceol/DSPE-PEG) and self-emulsifying drug delivery systems (SEDDS) viz., Peceol–AmB (Ibrahim et al., 2012; Italia et al., 2009; Jaffe et al., 2010; Wasan et al., 2009; SachsBarrable et al., 2008; Van de Ven et al., 2012). These formulations improved the oral absorption of AmB; however, emulsions are unstable and require excipients. Furthermore, the SEDDS and Peceol/DSPE-PEG formulations are lipid-based, which are expensive. Therefore, the development of an inexpensive, convenient and effective preparation for oral administration is paramount for treating fungal diseases, especially in developing countries. Amorphous preparations and reducing the particle size reportedly improve solubility and bioavailability of crystals (Kim et al., 2008). Nanoparticles enhance dissolution and improve oral bioavailability (Das and Suresh, 2011). Many methods have been attempted to produce nanoparticles, including antisolvent precipitation technique, supercritical fluid technique, and mechanical attrition such as media milling, jet-milling, and high-pressure homogenization (Antoniadou and Dupont, 2005; Chiou et al., 2007; Zhang et al., 2009). Mechanical attrition methods need high energy inputs and usually result in broad particle size distributions, pharmaceutical contamination, and electrostatic effects (Zhang et al., 2009). In addition, the supercritical fluid technique has a low yield and high equipment cost (Oz et al., 2013; Wang et al., 2007). Liquid antisolvent precipitation (LAP) is a simple and effective method for producing ultrafine nanosized particles. LAP has the potential to be applied in the pharmaceutical industry, which allows rapidity, convenience, low cost, and high yield preparation (Zhang et al., 2009). The technique is based on the change from supersaturation caused by the mixing of miscible solvent and antisolvent. LAP has successfully been applied to spironolactone, megestrol acetate microcrystal, fenofibrate, and so on (Cho et al., 2010; Dong et al., 2010; Hu et al., 2011). Moreover, AmB nanoparticles have not been prepared using LAP. This study aims to employ LAP to produce amorphous AmB nanoparticles for oral treatment to increase the water solubility of AmB. This method could potentially improve the oral bioavailability of AmB.

AmB nanoparticles were prepared through LAP. We investigated the effects of various surfactants and their concentration, the stirring time, the precipitation temperature, the stirring intensity, the drug concentration, and the antisolvent to solvent volume ratio on particle size and morphology. The AmB nanoparticles were characterized through scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), mass spectrometry (MS), powder X-ray diffraction (XRD), differential scanning calorimetry (DSC), thermal gravimetric analysis (TG), drug purity test, solvent residual determination, and dissolution testing.

2. Materials and methods 2.1. Materials AmB (purity = 97.0%) was kindly provided by Zhejiang Hisun Pharmaceutical Co., Ltd. Mannitol, Tween-80, poloxamer, ethanol, and dimethyl sulfoxide (DMSO) were obtained from Sigma. Methanol, acetonitrile, and acetic acid were all of HPLC grade.

Fig. 2. Diagram of the experimental processes to prepare the AmB nanoparticles.

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2.2. Preparation of amorphous AmB nanoparticles Amorphous AmB nanoparticles were produced through LAP and were freeze-dried. The preparation of the AmB nanoparticles is shown in Fig. 2. DMSO was used as the solvent and ethanol was used as the antisolvent. A certain concentration of AmB was completely dissolved in DMSO. At room temperature, the DMSO solution was completely poured into the ethanol solution (containing a certain concentration of surfactant) with vigorous magnetic stirring. AmB nanoparticles immediately precipitated from the solution upon mixing. The stirring intensity and temperature were controlled by a temperature-controlled magnetic stirrer. After stirring for a predetermined time, the nanoparticle suspension was centrifuged at 10,000 r/min for 15 min and washed three times with ethanol. To study the influence of LAP, the condition of freeze-drying was stationary. The obtained nanoparticles were redispersed in deionized water containing 50% mannitol, prefrozen at 40 °C for 2 hours, and subsequently lyophilized at 40 °C for 48 hours to obtain the AmB nanoparticles powder. AmB nanoparticles without mannitol were lyophilized under the same freezedrying conditions. Every experiment was repeated three times.

2.3. Optimization of the LAP process Single-factor experiments were performed to confirm the optimum conditions for the preparation of the amorphous AmB nanoparticles. Six factors were chosen for analysis in a preliminary experiment: surfactants and their concentration, stirring time, precipitation temperature, stirring intensity, drug concentration, and antisolvent to solvent volume ratio. One factor was changed at a time while the other factors were kept constant. During testing of the surfactants, the stirring time was 60 min, the precipitation temperature was 22 °C, the stirring intensity was 1000 r/min, the drug concentration was 20 mg/mL, and the antisolvent to solvent volume ratio was 13/1. The same method as above was applied to study the other factors. Surfactants were Tween80 and poloxamer188. The surfactant concentration was studied at 0.15%, 0.25%, 0.35%, and 0.45%. The stirring time was studied at 1, 5, 15, 30, 60, 90, and 120 min. The precipitation temperature was investigated at 2, 12, 22, and 32 °C. The stirring intensity was studied at 500, 1000, 1500, and 2000 r/min. The drug concentration was studied at 20, 40, 60, and 80 mg/mL. The antisolvent to solvent volume ratio was varied at 1/1, 7/1, 13/1and 19/1. Finally, the optimum condition for every factor was determined based on the smallest particle size.

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2.4.3. Fourier transform infrared spectroscopy (FTIR) The molecular structures of the samples were recorded through FTIR spectroscopy (Shimadzu Corporation, Japan) at wave numbers ranging from 400 cm 1 to 4000 cm 1 at a resolution of 2 cm 1. The AmB samples were properly diluted with KBr mixing powder at 1%. The IR spectra were obtained in a KBr disc. 2.4.4. Mass spectrum (MS) The quantitative analyses of AmB and AmB nanoparticles were monitored using a triple quadrupole mass spectrometer (API300, MDS Sciex, USA) equipped with an APCI interface. The samples were dissolved in methanol. The mass spectrometer was operated in electrospray ionization (ESI) positive ion mode with the ion spray voltage 5500 V. The other parameters were as follows: collision gas was at 6 L/min, the curtain gas was at 10 L/min, and the nebulizing gas was at 12 L/min. The m/z ranged from 100 amu to 1000 amu. 2.4.5. X-ray diffraction studies (XRD) The crystal forms of the samples were detected using an X-ray diffractometer (Philips, Xper t-Pro; The Netherlands). The current and voltage using Cu Kal radiation were 30 mA and 40 kV, respectively. The angular range was scanned from 5° to 70° of 2h, with a step size of 0.02° at a rate of 5°/min. 2.4.6. Differential scanning calorimetry (DSC) The DSC thermal profiles of samples were determined using DSC (TA instruments, model, DSC 204). The samples were operated at temperatures ranging from 20 °C to 220 °C at a scanning rate of 10 °C/min under a N2 atmosphere. 2.4.7. Thermal gravimetric analysis (TG) Thermal gravimetric analyses of samples were performed using a Thermogravimetric analyzer (Diamond TG/DTA from Perkin–Elmer, Waltham, MA, USA) at a heating rate of 10 °C/min using a nitrogen purge. The heating temperature of samples weighing 3 mg to 5 mg ranged from 25 °C to 250 °C.

2.4.1. Scanning electron microscopy (SEM) The external morphology of AmB and the AmB nanoparticles was examined through SEM (Quanta 200, FEI; The Netherlands). Particles of the representative samples were fixed on a SEM stub using double-sided adhesive tape. Before observation, the samples were coated with a thin layer of gold.

2.4.8. Drug purity test The drug purity experiments of raw AmB and AmB nanoparticles without mannitol were carried out through HPLC. The AmB samples (5 mg) were dissolved in 2.5 mL of DMSO and then diluted with methanol in a 10 mL volumetric flask. Finally, the samples were diluted 100 times with methanol, and then centrifuged at 8000 rpm for 5 min. The samples (10 lL) were directly injected into the HPLC system. The drug concentration was determined using a Waters HPLC (Waters Corporation, Milford, MA, USA) consisting of a pump (Waters 1525 binary), an autosampler (Waters 717 plus), and a UV detector (Waters 2478 Tunable Absorbance Detector), equipped with a DIKMA Diamonsil C18 column (5 lm, 4.6 mm  250 mm). The integrator system was Empower Pro. The mobile phase, consisting of 37% acetonitrile, 58.7% deionized water, and 4.3% acetic acid (v/v), was delivered at 1.0 mL/min. The samples were detected at 407 nm and the retention time of the drug was 7.3 min. The experiment was conducted in triplicate.

2.4.2. Particle size analysis The mean particle size (MPS) of the freshly precipitation and freeze-dried AmB nanoparticles were measured through dynamic laser light scattering (ZetaPALS, Brookhaven, USA). Before measurement, the freshly precipitated AmB nanoparticles were diluted with the corresponding proportion of solvent and antisolvent for each measurement, and the freeze-dried AmB nanoparticles were diluted with deionizer water. The measurements were performed in triplicate.

2.4.9. Residual solvent determination The residual DMSO in the AmB nanoparticles was analyzed using a gas chromatograph (GS). The AmB nanoparticles (10 mg) were dissolved in 1 mL of methanol and centrifuged at 8000 rpm for 5 min. Then, 10 lL of the supernatant was injected into the GS (Agilent 7890A, Palo Alto, CA, USA) equipped with a G1540N-210 FID. The column was a HP-5 capillary column (5% phenyl methyl siloxane, 30.0 m  320 lm  0.25 lm). The detection conditions for the GC analysis of DMSO were as follows: initial oven temperature of 40 °C for 5 min, which was increased to 240 °C at 40 °C/min and

2.4. Characterization of AmB nanoparticles

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maintained for 5 min. The injector temperature was 200 and the detector temperature was 280 °C. The H2 gas flow rate was 30 mL/ min and the airflow rate was 400 mL/min. N2 was used as the carrier gas at a flow rate of 2.2 mL/min. The samples (1 lL) were directly injected in split mode at a split ratio 20:1. 2.4.10. Equilibrium solubility test The dissolution of raw AmB, AmB nanoparticles, and physical mixture of raw AmB with mannitol were tested using a USP apparatus (II) paddle method. Simulated intestinal fluid with pancreatic enzymes (SIFe) (pH = 7.5) was used as the dissolution medium. SIFe, which is based on the US Pharmacopeia (USP28) method consisted of 0.2 M NaOH, 10 g/L pancreatin, and 6.8 g/L monobasic potassium phosphate, adjusted to pH 7.5 with NaOH. The stirring speed was set to 100 rpm and the bath temperature was set to 37.0 ± 0.5 °C. Raw AmB (2 mg), physical mixture of raw AmB with mannitol (containing 2 mg AmB), AmB nanoparticles (containing 2 mg of AmB) were added to 200 mL of SIFe dissolution medium and allowed to dissolve for 48 h. After 48 h, samples (1 mL) were withdrawn and filtered using a 0.22 lm filter. The filtered samples were directly injected into the HPLC system and assayed for AmB concentration. The analysis conditions were the same as in Section 2.4.8. 2.4.11. Dissolution rate study The dissolution rate experiment of raw AmB and AmB nanoparticles were carried out using the USP apparatus (II) paddle dissolution method. The stirring speed was 100 rpm and the bath temperature was 37.0 ± 0.5 °C. SIFe (pH = 7.5) was used as the dissolution medium. Raw AmB (2 mg) and AmB nanoparticles (containing 2 mg AmB) were added to 200 mL of SIFe dissolution medium with 0.3% Tween-80. Then, 1 mL of the release medium were collected after 10, 20 30, 45, 60, 90 and 120 min, centrifuged at 8000 r/min for 5 min, and immediately supplemented with the same volume of fresh SIFe. All of the raw AmB and AmB nanoparticles samples were injected into the HPLC system for drug concentration analysis. The analysis conditions were the same as in Section 2.4.8. 3. Results and discussion 3.1. Optimization study 3.1.1. The effect of the surfactants The effect of Tween80 and poloxamer188 and their concentrations on the MPS are shown in Fig. 3a. The MPS of AmB

nanoparticles obviously increased from 164.5 nm to 280.5 nm because the concentration of Tween80 changed from 0.15% to 0.45%. The MPS of poloxamer188 decreased from 265 nm to 211 nm; however, the decrease was inconspicuous. The critical micelle concentration (CMC) of Tween80 is 1.4 * 10 2 mol/L (0.6%) and that of poloxamer188 is 1.74 * 10 3 mol/L to 1.85 * 10 3 mol/ L (1.3–1.7%). The concentrations of Tween80 and poloxamer188 were changed from 0.15% to 0.45% within the limits of their CMC. The surfactants exhibited different effects during AmB recrystallization. The optimum surfactant was 0.15% Tween80. Tween80 reduces the MPS because the surface of the drug particle has multiple hydrophobic domains (Cho et al., 2010). It prevents crystal from aggregation. However, increasing the concentration also increased the viscosity. Increasing the Tween80 concentration slowed the diffusion between the solvent and the antisolvent, which lead to a slower nucleation rate. Hence, higher Tween80 concentrations increase the MPS. Therefore, 0.15% Tween80 may provide the most suitable steric hindrance for inhibiting particle growth and aggregation. 3.1.2. Effect of stirring time The relationship between stirring time and MPS is shown in Fig. 3b. The MPS at a stirring time of 5 min is about 185.4 nm. The particle size decreased gradually with increasing stirring time. Particles size did not significantly change after 30 min of stirring. Stirring times less than 30 min caused agglomeration and subsequent precipitation. Extending the stirring time breaks up the bigger crystals into smaller particles (Zhang et al., 2009). Therefore, the optimum stirring time for obtain the smallest AmB nanoparticles is 30 min. 3.1.3. Effect of precipitation temperature The relationship of MPS with precipitation temperature is shown in Fig. 3c. The MPS of the AmB nanoparticles remained almost unchanged when the precipitation temperature was increased from 2 °C to 22 °C. However, the MPS obviously increased from 164.5 nm to 311.4 nm when the precipitation temperature increased from 22 °C to 32 °C. The change in solubility and supersaturation with temperature is shown in Fig. S1 (Supplementary material). Increasing the temperature from 2 °C to 22 °C increased the solubility of AmB. Further increasing the temperature from 22 °C to 32 °C did not significantly affect the solubility. However, the change of supersaturation with temperature exhibited the opposite trend: low temperature resulted in a high degree of supersaturation. The relationship of viscosity to temperature is

Fig. 3. The effect of each parameter on the MPS of AmB nanoparticles. (a) a1 is Tween 80, a2 is poloxamer188; A1 = 0.15%, A2 = 0.25%, A3 = 0.35%, A4 = 0.45%; (b) B1 = 1 min, B2 = 5 min, B3 = 15 min, B4 = 30 min, B5 = 60 min, B6 = 90 min, B7 = 120 min; (c) C1 = 2 °C, C2 = 12 °C, C3 = 22 °C, C4 = 32 °C; (d) D1 = 500 r/min, D2 = 1000 r/min, D3 = 1500 r/min, D4 = 2000 r/min; (e) E1 = 20 mg/ml, E2 = 40 mg/ml, E3 = 60 mg/ml, E4 = 80 mg/ml; (f) F1 = 1/1, F2 = 7/1, F3 = 13/1, F4 = 19/1 (v/v).

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given in Fig. S2 (Supplementary material). The viscosity of the mixture did not significantly change with increasing temperature. Therefore, viscosity has little impact on precipitation. Precipitation has two main steps: nucleation and crystal growth. The nucleation is mainly dependent on supersaturation, and the nucleation rate greatly affects the final particle size. Low temperatures increase the supersaturation level and accelerate nucleation, resulting in smaller particles. Simultaneously, larger numbers of nuclei slows down the diffusion from solvent to antisolvent, causing particle aggregation. (Wang et al., 2007; Yeo and Lee, 2004; Zhang et al., 2009) According to the experimental results, the crystals grew at precipitation temperatures below 4 °C. Therefore, the optimum precipitation temperature is 12 °C. 3.1.4. Effect of stirring intensity The MPS decreased from 174.6 nm to 135.1 nm when the increasing stirring intensity was increased from 500 r/min to 2000 r/min. The crystals from recrystallization under highintensity stirring were smaller than those under low-intensity stirring. Increasing the stirring intensity enhances the micromixing between the multiphases, which results in highly homogeneous supersaturation within a short time. Rapid nucleation rates help prepare drugs with smaller particles within a narrower size distribution (Wang et al., 2007). Therefore, the optimum stirring intensity is 2000 r/min. 3.1.5. Effect of drug concentration The effect of drug concentration on the MPS is shown in Fig. 3e. Increasing the AmB concentration from 20 mg/mL to 40 mg/mL increased the MPS from 135.1 nm to 224.3 nm. However, the MPS did not significantly change at concentrations higher than 40 mg/ mL. The relationship of viscosity to drug concentration is shown in Fig. S3 (Supplementary material). Viscosity increased with increasing drug concentration. Increasing the viscosity slowed the diffusion between solvent and antisolvent, which lowered the nucleation rate. Under the appropriate concentration, higher supersaturation produces smaller particles. However, increasing drug concentrations also increase the viscosity of drug solution. Higher concentrations hinder the diffusion between solvent and antisolvent, which increases the particles size. (Cho et al., 2010; Zhu et al., 2010). Hence, the AmB particles began to grow and aggregate. The smallest particles were obtained at 20 mg/mL. 3.1.6. Effect of antisolvent to solvent volume ratio As is shown in Fig. 3f, the MPS of AmB nanoparticles significantly decreased from 317.8 nm to 135.1 nm as the antisolvent to solvent volume ratio increased from 1/1 to 13/1. However, the particle size increased to 186.4 nm when the volume ratio was increased to 19/1. Hence, the antisolvent to solvent volume ratio has an important role in the process. Increasing the volume ratio in-

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creases the supersaturation, which reduces the MPS. However, the nucleation rate reaches its maximum value with further increases in supersaturation (Wang et al., 2007). Therefore, the optimum volume ratio is 13/1. The single-factor experiments indicated that the optimal conditions for AmB preparation are as follows: 0.15% Tween-80, stirring time of 30 min, precipitation temperature of 12 °C, stirring intensity of 2000 r/min, drug concentration of 20 mg/mL, and antisolvent to solvent volume ratio of 13/1. The optimum conditions obtained the smallest nanoparticles, which were called AmB nanoparticles. The subsequent characteristics of the optimum sample were obtained under this condition. 3.2. Characterization of AmB nanoparticles 3.2.1. Morphology and particle size The raw AmB formed irregular crystals with an MPS of 13.5 lm (Fig. 4a). The AmB nanoparticles produced through were significantly smaller and more uniform than the raw AmB, which indicates better solubility (Kocbek et al., 2006). The AmB nanoparticles were almost spherical (Fig 4b). The normal distribution curve of the freshly precipitated and the freeze-dried AmB nanoparticles under optimum condition are shown in (Fig. 4c). The freshly precipitated and freeze-dried AmB nanoparticles were 135.1 nm and 215.6 nm, respectively. The increase in MPS could have been caused by the aggregation of particles during the freeze-drying. 3.2.2. Chemical structure characterization The molecular structures of the raw AmB, AmB nanoparticles, and mannitol were examined the rough Fourier transform infrared (FTIR) spectroscopy from 400 cm 1 to 4000 cm 1. The FTIR spectra of the raw AmB and the AmB nanoparticles without mannitol exhibited no changes in the AmB molecular structure before and

Fig. 5. FTIR spectra of mannitol, raw AmB and AmB nanoparticles. (a) Mannitol; (b) raw AmB; (c) AmB nanoparticles without mannitol; and (d) AmB nanoparticles.

Fig. 4. SEM images and normal distribution curve of samples. (a) SEM image of raw AmB; (b) SEM image of AmB nanoparticles under the optimum conditions: 12 °C, 2000 r/min, 20 mg/ml, 13:1; (c) normal distribution curve of samples: (c1) the freshly precipitated AmB nanoparticles under optimum condition, and (c2) the freeze-dried AmB nanoparticles under optimum condition.

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Fig. 6. MS fragments of (a) raw AmB and (b) AmB nanoparticles.

Fig. 7. XRD patterns of mannitol, raw AmB and AmB nanoparticles. (a) Mannitol; (b) raw AmB; (c) AmB nanoparticles without mannitol; and (d) AmB nanoparticles.

Fig. 8. DSC curves of mannitol, raw AmB and AmB nanoparticles. (a) Mannitol; (b) raw AmB; (c) AmB nanoparticles without mannitol; and (d) AmB nanoparticles.

after the precipitation (Fig. 5b and d). The FTIR spectra of the AmB nanoparticles presented remarkable absorption peaks at 1083 cm 1 and 1007 cm 1 because of mannitol (Fig. 5d).

Fig. 9. TG results of (a) raw AmB and (b) AmB nanoparticles.

Fig. 10. HPLC chromatogram of (a) AmB nanoparticles without mannitol and (b) raw AmB.

The mass spectra of the samples are shown in Fig. 6. The molecular weight of the raw AmB and AmB nanoparticles are 924.6. The results are consistent with those in the literature (Deshpande et al.,

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2010). According to FTIR and MS, the molecular structure of AmB nanoparticles remained unchanged.

3.2.3. Physical structure characterization X-ray diffraction was performed to determine the crystalline structure of particles. The corresponding results for mannitol, raw AmB, AmB nanoparticles, and AmB nanoparticles without mannitol are shown in Fig. 7. The mannitol and raw AmB were highly crystallized and showed intense crystalline peaks (Fig. 7a

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and b). However, the AmB nanoparticles and the AmB nanoparticles without mannitol did not present an obvious peak in Fig. 7c and d, which indicated that the AmB nanoparticles might be present in the desired amorphous form. DSC analysis was used to confirm the results of the XRD. The results are shown in Fig. 8. The peak at 171 °C in Fig. 8a is the melting point of mannitol crystals, which is consistent with the literature (Li et al., 2012). The curve of raw AmB in Fig. 8b showed two endothermic peaks, a peak at 108 °C and a peak at 169 °C. The first peak could be attributed to water loss and the second peak was closer to the melting point of AmB crystal, which is consistent with the literature (Salerno et al., 2013). The crystallinity of mannitol is stronger than the crystallinity of AmB. The DSC curve of the AmB nanoparticles and AmB nanoparticles without mannitol did not exhibit obvious melting processes, which implies their amorphous forms (Figs. 8c and 7d). This finding further proves that the noncrystalline state of AmB nanoparticles was not caused by mannitol, which is consistent with our XRD analysis. Many studies reported that the amorphous form enhances dissolution and bioavailability (Kim et al., 2008). The TG curves of the raw AmB and AmB nanoparticles are shown in Fig. 9. The raw AmB showed obvious thermal weight losses at 168 °C, whereas the AmB nanoparticles began to lose weight from 127 °C. This may be due to the higher specific surface area of the smaller AmB nanoparticles compared with the raw AmB, which leads to an easier vaporization and quickly thermal decomposition. Generally, amorphous powders have poor heat stability and are difficult to preserve. However, the AmB nanoparticles are almost consistent with the raw AmB. Thus, the mannitol may have protected the AmB nanoparticles. 3.2.4. Drug purity result Fig. 10 shows the photographs of the HPLC of raw AmB and AmB nanoparticles. The purity of the raw AmB was 97.0%, which increased to 99.6% in the AmB nanoparticles. Therefore, the LAP increased the purity of the drugs. Recrystallizing the AmB through LAP dissolved the AmB and the impurities remained dissolved in the solvent. The impurities were removed after centrifugation. Therefore, the purity of AmB nanoparticles was increased.

Fig. 11. Gas chromatograms of (a) 0.0791 mg/mL DMSO standard solution and (b) AmB nanoparticles.

3.2.5. Solvent residue analysis Dimethyl sulfoxide (DMSO) is a class III solvent with low toxicity and limited to 0.5% in International Conference on

Fig. 12. Results of (a) equilibrium solubility and (b) dissolution profiles of samples. (a1) raw AmB; (a2) the corresponding physical mixture of raw AmB with mannitol; and (a3) AmB nanoparticles.

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Harmonization (ICH). The AmB nanoparticles were prepared through LAP using DMSO as the solvent. Fig. 11 shows the residual DMSO using gas chromatograph (GC), with a retention time of 8.8 min and a standard curve represented by y = 338.62x + 2.0641 (R2 = 0.9998), where x is the DMSO concentration and y is the peak area. The DMSO residue in the AmB nanoparticles is 0.24% according to the regression equation. Therefore, the AmB nanoparticles meet the ICH standard and are suitable for pharmaceutical use.

3.2.6. Equilibrium solubility The equilibrium solubility of raw AmB, which corresponds to AmB with mannitol and AmB nanoparticles, is shown in Fig. 12a. The terminal solubility of the raw AmB was 0.07 lg/mL, that of the raw AmB with mannitol was 0.071 lg/mL, and that of the AmB nanoparticles was 0.932 lg/mL. The equilibrium solubility of the raw AmB was almost the same as that of the raw AmB with mannitol. However, the AmB nanoparticles contained 13 times more AmB than the amount raw drug. Therefore, the increase of solubility of AmB nanoparticles is due to the decreased particle size rather than the mannitol, which acted as a cryoprotectant.

3.2.7. Dissolution rate The dissolution profiles of the raw AmB and AmB nanoparticles are shown in Fig. 12b. At 10 min, 0.048 lg/mL of AmB was dissolved from the raw drug, whereas 0.075 lg/mL was dissolved from the AmB nanoparticles. After 30 min, only a small amount of the drug was dissolved from raw AmB, whereas the AmB nanoparticles continued to dissolve. The AmB nanoparticles exhibited a 2.1 times faster dissolution rate within 2 h compared with the raw AmB. According to the Noyes–Whitney equation, the drug dissolution rate is directly proportional to the surface area exposed to the dissolution medium (Dong et al., 2010). The accelerated dissolution rate of the AmB nanoparticles could be mainly attributed to their greater surface area from the greatly reduced particles size (Kocbek et al., 2006). The amorphous form of the AmB nanoparticles also contributed to the increased dissolution rate (Italia et al., 2009). Therefore, amorphous AmB nanoparticles have a higher dissolution rate and solubility than the raw AmB. In addition, the AmB nanoparticle powder can be made into oral tablets, which would improve the oral bioavailability of AmB.

4. Conclusions Amorphous AmB nanoparticles were successfully prepared using LAP, followed by freeze-drying. DMSO was used as the solvent and ethanol was used as the antisolvent. Tween80 was used as the surfactant to inhibit aggregation and mannitol was used as the cryoprotectant to inhibit particle growth. The optimal conditions are as follows: 0.15% Tween-80, stirring time of 30 min, precipitation temperature of 12 °C, stirring intensity of 2000 r/min, drug concentration of 20 mg/mL, antisolvent to solvent volume ratio of 13/1. Under these optimal conditions, AmB nanoparticles with an MPS of 135.1 nm were obtained. In addition, FTIR and MS analyses demonstrated that the molecular structure of the AmB nanoparticles was unchanged after LAP. The XRD and DSC analyses indicated that the prepared AmB nanoparticles were amorphous. The residual DMSO in the nanoparticles was 0.24% lower than the ICH limit for class III solvents. The AmB nanoparticles exhibited 2.1 times faster dissolution rate and 13 times the equilibrium solubility of the raw drug, indicating potentially enhanced AmB bioavailability. LAP is a simple and inexpensive method for preparing AmB nanoparticles for oral administration.

Acknowledgments The authors would like to acknowledge the financial support from the Fundamental Research Funds for the Central Universities (DL13CB08), the National Natural Science Foundation of China (No. 21203018) and the Agricultural Science and Technology Achievements Transformation Fund Programs of the Ministry of Science and Technology (2011GB23600016). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ejps.2013.12.005. References Antoniadou, A., Dupont, B., 2005. Lipid formulations of amphotericin B: Where are we today? J. Mycol. Med. 15, 230–238. Chiou, H., Li, L., Hu, T., Chan, H., Chen, J., Yun, J., 2007. Production of salbutamol sulfate for inhalation by high-gravity controlled antisolvent precipitation. Int. J. Pharm. 331, 93–98. Cho, E., Cho, W., Cha, K.-H., Park, J., Kim, M.-S., Kim, J.-S., Park, H.J., Hwang, S.-J., 2010. 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