Controlling Nucleation and Fabricating Nanoparticulate Formulation of

Res. , 2018, 57 (6), pp 1903–1911. DOI: 10.1021/acs.iecr.7b04103. Publication Date (Web): January 30, 2018. Copyright © 2018 American Chemical Soci...
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Controlling nucleation and fabricating nanoparticulate formulation of sorafenib using high gravity rotating packed bed Kai Wu, Haoran Wu, Tianchen Dai, Xingzheng Liu, Jian-Feng Chen, and Yuan Le Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.7b04103 • Publication Date (Web): 30 Jan 2018 Downloaded from http://pubs.acs.org on February 4, 2018

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Controlling nucleation and fabricating nanoparticulate formulation of sorafenib using high gravity rotating packed bed

Kai Wu a, Haoran Wu a, Tianchen Dai a, Xingzheng Liu a, Jian-Feng Chen a, b, Yuan Le a∗∗ a

State Key Laboratory of Organic-Inorganic Composites, Beijing University of Chemical Technology, Beijing 100029, PR China;

b

Research Center of the Ministry of Education for High Gravity Engineering and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China;



Corresponding authors. Yuan Le: Tel: +86-10-64447274. Fax: +86-10-64423474. E-mail address: [email protected](Y. Le)

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Abstract Nucleation is the initial step of the crystallization process and is the significant step to prepare nanometer-sized crystalline materials. In this work, we systematically investigated the nucleation kinetics of poorly water-soluble drug sorafenib when precipitated by liquid antisolvent precipitation in high-gravity rotating packed bed. We found that high-gravity field tremendously promoted the nucleation rate and it was increased by 2~3 orders of magnitude over that in the stir tank reactor. Moreover, polymer excipients have a significant impact on nucleation, especially, PVP could increase nucleation rate by 3 orders of magnitude over that without excipient. Finally, stable amorphous sorafenib nanoparticulate formulation with particle size of 80 nm was obtained by controlling nucleation in RPB. Compared to the coarse drug, the nanoparticulate formulation performed faster drug release and much better cytotoxicity. In vivo pharmacokinetics of the nanoparticulate formulation displayed the increase in AUC0-∞ and Cmax, which demonstrated nanoparticulate formulation could enhance the bioavailability and exhibit extensive potential in pharmaceutical industry.

Keywords:

Sorafenib,

high-gravity

rotating

packed

nanoparticlulate formulation

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bed,

nucleation

rate,

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1. Introduction In recent years, pharmaceutical industry has shown an increasing desire to formulate poorly-water soluble drugs as nanometer-sized particles (nanoparticulate formulation) with the goal of improving dissolution rate, enhancing bioavailability, eliminating food effects, and improving efficacy and safety.1-5 To address the need of nanoparticulate formulation development, top-down processes which involve breaking down larger particles by wet ball milling and high pressure homogenization have been exploited as relatively simple and efficient methods to produce nanoparticles.6-8 The top-down processes have been extensively applied in the preparation of some commercial drugs such as Rapamune® (sirolimus), Emend® (aprepitant), Tricor® (fenofibrate), Megace® ES (megesterol acetate) and Invega Sustenna® (paliperidone palmitate).9 However, a high energy input and a relatively long process time restrict these techniques widespread use, which cause the deformation of crystals and degradation of chemicals. More seriously, the particle size of nanoparticles prepared by top-down processes is about 150-300 nm.10 Therefore, top-down processes exhibit the limitation in terms of particle size reduction, which intimately links with the resulting dissolution performance according to Noyes– Whitney equation.11-13 Bottom-up method, especially liquid precipitation process, has lately attracted considerable attention for producing drug nanoparticles due to its low-cost, convenience in the processing, and easy scaling up.10,14 Importantly, liquid precipitation break down the barrier of the final particle size and the size distribution by tuning the nucleation and growth kinetics. Furthermore, a higher nucleation rate

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resulting in a low and negligible growth rate generally facilitates the fabrication of nanoparticles with narrow size distribution, which demands process with very short characteristic mixing time to achieve the homogenous supersaturation level.15,16 Clearly, a tight control of the mixing process is very vital for homogeneous precipitation of drugs calling for uniformity of spatial distribution of active pharmaceutical ingredient (API) in the molecular scale. High-gravity technology, implemented by rotating packed bed (RPB), performs excellent mixing efficiency.17 The fluids going through the packing of RPB are spread or split into very fine droplets, threads, and thin films by the strong shear force, resulting in a significant intensification of micromixing and mass transfer between the fluid elements and hence benefits the form of uniform concentration distribution.18 RPB has been proved to be a promising device for the preparation of nanoparticles and successfully applied in the pharmaceutical industry. Our group have successfully prepared nanosized drugs by high-gravity technology, such as cefuroxime axetil, itraconazole, glibenclamide, and cefixime.19-22 However, theses researches are basically to regulate the particle size by operating the conditions of RPB without deep study of the performance of RPB on the nucleation, which contributed to poor control of particle size of final product. Herein, the objective of this work was to investigate the impact of RPB on the nucleation process during liquid antisolvent precipitation and fabricate drug nanoparticulate formulation using RPB. Sorafenib (SFN), an oral multiple kinase inhibitor for the treatment of patients with advanced renal cell carcinoma and

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advanced hepatocellular carcinoma, classified as the biopharmaceutics classification system (BCS) class II (low solubility, high permeability), is taken as a model drug.23-25 The effects of high-gravity levels (β), polymer excipients, and antisolvent phase/solvent phase flow rate ratio (ASP/SP FRR) on the nucleation rates were investigated when drug precipitated in RPB. Moreover, solid-state properties and dissolution performance of SFN nanoparticulate formulation (SFN NF) were characterized. In vitro antitumor activity and in vivo pharmacokinetic study were taken to evaluate the oral bioavailability of SFN NF. 2. Theory 2.1 Nucleation kinetics The nucleation rate (J), that is, the number of nuclei formed per unit time per unit volume, is defined as eq 126,27 J = N 0υ exp(

−∆G ∗ ) kT

(1)

where N0 is the number of molecules in a unit volume, υ is the frequency of molecular transport at the nucleus-liquid interface, k is the Boltzmann’s constant, and ∆G ∗ is the Gibbs free energy change for the formation of critical clusters. Derived

from the classical theory of homogeneous nucleation, ∆G ∗ can been given by eq 228

∆G ∗ =

16π v3γ 3 3(kT ln( S ))2

(2)

where v is the molecular volume of the crystallizing solute and γ is the interfacial energy per unit area between the cluster and the surrounding solvent. The supersaturation ratio (S) is defined as

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S=

C C*

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(3)

where C is the actual concentration in the solution and C* is the equilibrium solubility. And the nucleation rate J becomes

J = N 0υ exp(

−16π v 2γ 3 ) 3(kT )3 (ln( S )2

(4)

3. Experiment section 3.1. Materials and equipment Sorafenib tosylate (≥ 99%) was purchased from Beijing Zhongshuo Medicine Science and Technology Development Co., Ltd (Beijing, China). Hydroxypropyl methyl cellulose (HPMC K86, MW = 86000) and polyvinylpyrrolidone (PVP K30, MW = 58000) were provided from Tianjin Heowns Biochemical Technology Co., Ltd (Tianjin, China). Cell counting kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies, INC (Japan). Methanol (MeOH, AR grade) and all buffer salts used for dissolution medium were purchased from Beijing Chemical Works (Beijing, China). All the other chemicals and solvents were of chromatographic and analytical grade. Deionized water was purified by Hitech-K flow water purification system (Hitech Instrument Co., Ltd. Shanghai, China). 0.22 µm membrane filter was sourced from Tianjin Jinteng Experiment Equipment Co., Ltd (Tianjin, China). The experimental setup for high-gravity antisolvent precipitation process is schematically displayed in Figure 1. The key equipment parameters of the RPB are listed in Table 1.

3.2. Preparation of SFN nanoparticulate formulation The basic principle of preparation of SFN NF by high-gravity technology is to

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carry out the precipitation process under a high-gravity environment via the action of centrifugal force in RPB, which is reflected by the value of β and β is determined by eq 529 r2

β=

(2π n) 2 ∫ 2π r 2 dr r1

r2

g ∫ 2π rdr

=

8π 2 n 2 ( r12 + r1r2 + r22 ) 3 ( r1 + r2 ) g

(5)

r1

where n is the rotating speed of RPB (r/s), g is the acceleration of gravity (m/s2), r1 is the inner radius of packing (m), and r2 is outer radius of packing (m). Briefly, the solvent phase (SP) was prepared by adding SFN (750 mg) into MeOH (50 mL), followed by filtering. The drug excipient (1875 mg) was dissolved in water to form the antisolvent phase (ASP). SP and ASP were added into the storage container 2 and 3, followed by pumped into RPB using two pumps (5, 7) at 45 mL/min and at a range of 225 mL/min to 900 mL/min, respectively. These two phases were mixed in the packed rotator through the liquid distributors at the temperature of 20 °C and SFN suspension was obtained immediately. The slurry was collected through the outlet followed by lyophilization utilizing a lyophilizer (LGJ-18S, Beijing, China). Finally, the dried nanoparticulate formulation was stored in a vacuum desiccator at room temperature until further use for characterization and testing. As a control experiment, the SFN precipitation process was taken in the stirred tank reactor (STR),which was composed of a 1000 mL beaker and a stirrer, under other same operating conditions.

3.3. Estimation of supersaturation In order to estimate the S generated after mixing, it is necessary to determine the solubility of SFN in the organic aqueous solution in presence or absence of excipient

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according to eq 3. In a typical experiment, an excess amount of drug was dissolved in 5 mL of MeOH, to which were added different volume of aqueous solution (25 mL, 35 mL, 50 mL, and 100 mL) in presence or absence of 187.5 mg of PVP or HPMC. The glass containers were sealed and mechanically stirred at 20 °C for 24 h, followed by filtered and diluted. The drug concentration of the filtrate was determined by a UV spectrophotometer (UV-2501, Shimadzu, Japan) at 265 nm. The solubility was in turn determined from a calibration model (regression coefficient R2 > 0.99) relating absorbance intensity to SFN concentration.

3.4. Nucleation rate determination Initially, the particle size distribution was represented by the population density (ni) through eq 630

ni =

∆ ( wt % ) M T kv ρc L3i ∆Li

(6)

where Li (nm) is the average size of particle, kv is the volumetric shape factor for a sphere (π/6), and ρc is the density of the particle (1454 kg m-3). ∆Li (nm) and ∆(wt%) (dimensionless) is the size width and mass fraction of the size range i (nm), respectively. MT is the suspension density for the continuous flow system determined by eq 730  Qsolvent MT =   Qsolvent + Qantisolvent

  Csolute 

(7)

where Csolute (kg m-3) is the mass concentration of SFN in the SP. Qsolvent (L min-1) and Qantisolvent (L min-1) is the volumetric flow rate of the SP and ASP, respectively. Note eq 6 is based on the assumption that the weight fraction of crystals of size Li is same

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as the volume fraction found from the laser diffraction measurements. Finally, J was calculated by from the particle size distribution using eq 7

16,31

, since

Mixed-Suspension-Mixed-Product Removal crystallizer (MSMPR model) based on the size-independent crystal growth, where straight-line population density plots are usually encountered, did not fit well to the population densities calculated (as can be seen from Figure 2, Figure 3 and Figure 4).16, 32 J=



Li +1

Li

ni ( Li ) dLi tsamp

(8)

where tsamp (s) is the sampling time.

3.5. Characterization The morphology of SFN sample was observed by scanning electronic microscope (SEM, Hitachi S4800) operated at an accelerating voltage of 5 kV. The Mastersizer (MS2000) was carried out to measure the particle size of micron-sized SFN particles, and the Zetasizer (Nano-ZS) was used to measure particle size in the submicron range. The crystal type of SFN was performed using an XRD-6000 diffractometer (Shimadzu Inc., Japan). The sample was scanned from 5° to 50° with a scan speed of 5 degree/min. The thermal behavior was analyzed by differential scanning calorimetry (Pyris 1, PerkinElmer, USA) at a heating rate of 10 °C/min between 40 °C and 250 °C in an aluminum pan under a nitrogen atmosphere, which was maintained by purging nitrogen gas at flow rate of 50 mL/min. The calibration of instrument with respect to temperature and enthalpy was achieved using high purity standard of indium.

3.6. Dissolution evaluation

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In vitro dissolution behaviors of SFN raw drug, SFN and PVP physical mixture (SFN PM, WSFN : WPVP = 1 : 2.5) and SFN NF, investigated by a model D-800LS dissolution apparatus (Tianjin, China), was in accordance with the USP Apparatus II (paddle) method. The dissolution flask with 900 mL of phosphate buffer (pH 6.8) dissolution medium was immersed in a water bath at 37 ± 0.5 °C, and the medium was continuously stirred at 100 rpm. Then, the samples equivalent to 274 mg SFN were weighed and put into the vessel. The sample (5 mL) was withdrawn at specific time intervals and immediately filtered through a membrane filter. In the meantime, fresh medium (5 mL) at 37 °C was added to keep constant volume. Quantification of the samples was determined with a UV method at the wavelength of 265 nm. The dissolution test of each sample was performed in triplicate.

3.7. Cell viability assay The in vitro cytotoxicity of SFN NF and SFN PM were determined in Hep G2 cells. In a typical experiment, cells were seeded into 96-well plate at a density of 10000 cells per well and cultured in Dulbecco’s modified eagle’s medium (DMEM) (Gibco, USA) culture media overnight at 37 °C and a 5% CO2 atmosphere. Then, the SFN NF and SFN PM were respectively added to each well at predeterminded concentrations and incubated for 24 h and 48 h. The SFN concentration of SFN PM was equivalent to SFN NF. Subsequently, 10 µL of CCK-8 solution was added into each well to evaluate cell viability and incubated with cells for 2 h before measuring absorbance at 450 nm with a microplate reader (Multiskan Spectrum, Thermo Electron Co., Vantaa, Finland).

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3.8. Pharmacokinetics study The pharmacokinetic performances of the SFN NF and SFN PM were evaluated in male Sprague-Dawley rats (weighing 180-220g). Six rats were fasted overnight with free access to water and were randomly divided into two groups prior to the experiment. The two groups were orally administered with SFN NF and SFN PM by gavage at a single dose 20 mg/kg expressed as SFN equivalents, respectively. Blood samples were collected via orbit vein at 5 min, and 0.25, 0.5, 1, 2, 4, 6, and 24 h after dosing. Blood samples were centrifuged at 3000 rpm for 10 min and the resultant plasma was transferred into capped tubes and stored at -20 °C until HPLC LC/MS analysis (Applied Biosystems, Foster City, USA). The rat pharmacokinetic study was performed in accordance with the standards recommended by the Guide for Care and Use for Laboratory Animals (Institute of Animal Laboratory Resources, 1995).

3.9. Pharmacokinetic and statistical analysis The pharmacokinetic parameters of the samples based on a non-compartmental method implemented in Phoenix® WinNonlin® version 7.0 (Certara, St Louis, MO, USA) including the area under the plasma concentration time curve (AUC), the elimination half-life (t1/2), and mean residence time (MRT), the maximum plasma concentration (Cmax) and time to reach the Cmax (tmax) obtained directly from the SFN plasma concentration versus time profiles. Student’s t-test or ANOVA were used for statistical analysis. Values of p SFN NF at 24 h and 48 h. Thereby, the antiproliferation activity was significantly improved by nanoparticulate formulation.

4.7. Pharmacokinetic study To investigate the effect of nanoparticulate formulation on the bioavailability of SFN, an in vivo test was carried out in rats and pharmacokinetic parameters of SFN were compared. The plasma concentration-time profiles are presented in Figure 9 and the corresponding pharmacokinetic parameters are summarized in Table 4. The Cmax value of SFN NF (16900±1955 µg/mL) was higher than that of SFN PM (12900±943 µg/mL), a 1.31 fold increase was observed. SFN NF exhibited considerable

enhancement in the oral bioavailability with an increase in AUC0-∞ (465936±41554 vs 208122±12436 µg ⋅ h/mL). Similarly, nanoparticle had a significant increase in mean residence time (MRT) of SFN (27.3±6.3 h) in comparison to SFN PM (14.3±0.7 h).

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The relative bioavailability of the SFN nanoparticle was approximately 224% in comparison to SFN PM. These results indicated that SFN nanoparticle was easier to be absorbed in vivo.

5. Conclusion In this study, nucleation rate of the poorly water-soluble model drug (SFN) precipitated in the RPB was calculated by population densities determined from particle size distribution data. Furthermore, the effects of high-gravity level, polymer excipients, and ASP/SP flow rate ratio on the nucleation rate and subsequently on particle size have been discussed. We found that nucleation rate obviously enhanced with the enhancement of high-gravity level. The addition of PVP significantly promoted the nucleation process of SFN more sharply than that by addition of HPMC. In addition, the nucleation rate first increased first and then decreased with the increase of ASP/SP FRR from 5 to 20. Eventually, the SFN nanoparticulate formulation with particle size of 80 nm was successfully fabricated through controlling nucleation in the RPB. Physicochemical characterization of SFN NF indicated the change of SFN from crystallinity to amorphous state and SFN NF showed good stability. Besides, the dissolution rate of SFN was significantly improved by nanoparticulate formulation. The SFN NF had promising in-vitro cytotoxicity and pharmacokinetic experiment showed that SFN NF improved the oral bioavailability based on its AUC0-∞, Cmax, and MRT results. These results present a promise in application of controlling nucleation for crystallizing nanosized drug molecules.

ASSOCIATED CONTENT

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Supporting Information Experimental particle size distribution; the calculated suspension density and population density gathered in this work. The Supporting Information is available free of charge on via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author Tel:

+86-10-64447274.

Fax:

+86-10-64423474.

E-mail

address:

[email protected].

ACKNOWLEDGEMENTS This work was supported by National Key Basic Research Program of China (2015CB932100) and National Natural Science Foundation of China (21622601).

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nanomatrix for oral administration to rat. Int. J. Pharmaceut. 2011,419, 339-346. (26) Rodriguez-Hornedo, N.; Murphy, D. Significance of controlling crystallization mechanisms and kinetics in pharmaceutical systems. J. Pharm. Sci.1999, 88 ,656-660. (27) Brouwers, J.; Brewster, M. E.; Augustijns, P. Supersaturating drug delivery systems: the answer to solubility-limited oral bioavailability? J. Pharm. Sci.2009, 98, 2549-2572. (28) Zhang, R. Y.; Khalizov, A.; Wang, L.; Hu, M.; Xu, Nucleation and growth of nanoparticles in the atmosphere. Chem. Rev. 2012, 112, 1957-2011 (29) Wu, K.; Xie, M. L.; Chen, J. F.; Le, Y. A novel routine for fabrication of Y-type oxotitanium phthalocyanine nanocrystals in high-gravity rotating packed beds. Ind. Eng. Chem. Res. 2016, 55,6753-6759.

(30) Jarmer, D. J.; Lengsfeld, C. S.; Randolph, T. W. Nucleation and growth rates of poly(L-lactic acid) microparticles during precipitation with a compressed-fluid antisolvent. Lagmuir. 2004, 20, 7254-7264. (31) Zarladas, D. M.; Sirkar, K. K. Antisolvent crystallization in porous hollow fiber devices. Chem. Eng. Sci. 2006, 61, 5030-5048. (32) Falope, G. O.; Jones, A. G.; Zauner, R. On modelling continuous agglomerative crystal precipitation via Monte Carlo simulation. Chem. Eng. Sci. 2001, 56, 2567-2574. (33) Guo, K.; Guo, F.; Feng, Y. D.; Chen, J. F.; Zhang, C.; Gardner, N. C. Synchronous visual and RTD study on liquid flow in rotating packed-bed contactor. Chem. Eng. Sci.

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(34) Zhang, X. H.; Chu, G. W.; Kong, D. J.; Luo, Y.; Zhang, J. P.; Zou, H. K.; Zhang, L. L.; Chen, J. F. Mass transfer intensification in a rotating packed bed with surface-modified nickel foam packing. Chem. Eng. J. 2016, 285, 236-242. (35) Li, C. X.; Wang, H. B.; Oppong, D.; Wang, J. X.; Chen, J. F.; Le, Y. Excipient-assisted vinpocetine nanoparticles: Experiments and molecular dynamic simulations. Mol. Pharmaceut. 2014, 11, 4023-4035. (36) Truong, D. H.; Tran, T. H.; Ramasamy, T.; Choi, J. Y.; Choi, H. G.; Yong, C. S,; Kim, J. O. Preparation and characterization of solid dispersion using a novel amphiphilic copolymer to enhance dissolution and oral bioavailability of sorafenib. Powder. Technol. 2015, 283, 260-265.

(37) Muller, R. H.; Jacobs, C.; Kayser, O. Nanosuspensions as particulate drug formulations in therapy: Rationale for development and what we can expect for future. Adv. Drug. Deliv. Rev. 2001, 47, 3-19.

(38) Dolenc, A.; Kristl, J.; Baumgartner, S.; Planinsek, O. Advantages of celecoxib nanosuspension formulation and transformation into tables. Int. J. Pharm. 2009, 376, 204-212.

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Figure 1. (A) Schematic of the high-gravity antisolvent precipitation setup (1, RPB reactor; 2 and 3, SP and ASP storage container; 4, water tank; 5, 6 and 7, pump; 8 and 9, flow meters; 10, product storage tank). (B) Structure of RPB (11, casing; 12, liquid distributors; 13, outlet; 14, packed rotator; 15, seal ring; 16, motor).

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Figure 2. Population density plots for SFN precipitated without excipient under ASP/SP ratio of 7 in STR or RPB at different high-gravity levels

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Figure 3. Population density plots for SFN precipitated under ASP/SP flow rate ratio of 7 with different drug excipients in RPB at high-gravity of 171

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Figure 4. (A) Population density plots for SFN precipitated with PVP in RPB at high-gravity of 171 under different ASP/SP flow rate ratios, and (B) Effect of ASP/SP flow rate ratio on the terminal solubility of SFN and the terminal value of S.

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Figure 5. SEM image of (A) raw drug, (B) fresh SFN NF, (C) redispersed SFN NF after freeze-drying nanoparticles, and (D) SFN NF stored for 160 days.

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Figure 6. (A) X-ray power diffraction of raw SFN, SFN PM, fresh SFN NF and SFN NF stored for 160 days. (B) DSC curves of raw SFN and SFN NF.

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Figure 7. Dissolution rate test for raw SFN, SFN PM, and SFN NF.

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Figure 8. Concentration-dependent cytotoxicity of SFN PM and SFN NF in Hep G2 cell at (A) 24 h and (B) 48 h

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Figure 9. Plasma drug concentration-time curve of SFN in rats after oral administration of SFN PM and SFN NF equivalent to 20 mg/kg of SFN.

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Table 1. The equipment parameters of RPB used in this study. rotor

equipment

RPB

packing

inner diameter

out diameter

axial height

(mm)

(mm)

(mm)

40

84

15

material wire mesh

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operation parameter porosity

rotating speed

(%)

(r/min)

95

0-2800

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Table 2. Summary of SFN nucleation rate and particle size under various processing conditions. Particle size

D10/D50/D90

Nucleation rate J (1/m3s)

40.95 µm

3.1/37.2/74.3 µm

9.20×10

96

26.27 µm

9.4/21.4/49.1 µm

1.18×10

171

23.35 µm

6.2/16. 3/49.1 µm

1.66×10

HPMC

440 nm

275.1/427.4/663.8 nm

1.78×10

PVP

110 nm

75.7/104.6/149.1 nm

8.68×10

5/1

170 nm

134.2/164.4/199.9 nm

2.75×10

10/1

80 nm

55.6/75.6/103.5 nm

1.26×10

20/1

210 nm

132.1/205.2/318.7 nm

5.91×10

Precipitation in STR

β in RPB

Excipient

AS/S FRR

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9

12

12

14

15

15

16

14

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Table 3. IC50 comparsion of SFN PM and SFN NF IC50 (µmol/L) 24 h SFN PM SFN NF

> 45 22.15 ± 0.26* * respresent significantly different compared with SFN PM (P < 0.05)

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48 h 26.82 ± 0.34 14.30 ± 0.48*

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Table 4. Pharmacokinetic parameters of SFN PM and SFN NF after single oral administration of 20 mg/kg body weight in rats. Parameters Cmax(µg/mL) Tmax (h) AUC0-∞ (µg ⋅ h/mL) t1/2 (h)

SFN PM

SFN NF

12900 ± 943 4±0 208122 ± 12436 9.7 ± 0.5

16900 ± 1955* 4±0 465936 ± 41554* 18.9 ± 4.5*

14.3 ± 0.7 * respresent significantly different compared with SFN PM (P < 0.05)

MRT (h)

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27.3 ± 6.3*

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