Materials
CA was obtained from professor Song Fuxing (Beijing Technology and Business University, purity >99%). Disodium salt of glycyrrhizic acid (Na2GA) was purchased from Shanxi Pioneer Biotech Co. Ltd. (Xian, China, purity >98%). Acetonitrile was obtained from Tedia Company, Inc. (Fairfield, OH, USA, HPLC grade). Formic acid with purity >88% was purchased from Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China). Roswell Park Memorial Institute 1640 (RPMI-1640) cell culture medium, fetal bovine serum (FBS) and penicillin/ streptomycin were all purchased from Gibco BRL (Gaithersburg, MD, USA).
Cells and animals
The mouse melanoma cell line B16-F10 was purchased from the China Center for Type Culture Collection (Wuhan, China) and cultured in RPMI-1640 containing 10% FBS and 1% antibiotics (penicillin/ streptomycin).
Female ICR mice (5-6 weeks of age, 18-20 g body weight) and female C57BL/6 mice (5 to 6 weeks of age, 16-18g) used in the experiments were provided by the Zhejiang Academy of medical Science, conducting with the approval of the animal experiment center of Zhejiang University of Technology. All the animals were performed in strict compliance with the PR China legislation for the use and care of laboratory animals.
Fabrication of nanoparticles by mechanochemical treatment
The roll mill ML-007 (Wiggens, German) was used to prepare samples. Briefly, 0.15 g CA and 14.85 g Na2GA (weight ratio 1/99) were added to 300 mL vial with 660.0 g zirconium balls (diameter 22 mm) with milling time of 3 h, rotation speed 30 rpm and samples were picked out at 0.5, 1, 1.5, 2, 2.5 h, and 3 h, respectively. In addition, a mixture of Na2GA and CA (weight ratio was same as above) by ordinary physical treating without ball milling, were prepared for comparing with the ball milling products. At last, the ball-milling products with different milling time were described as BM-0.5 h, BM-1.0 h, BM-1.5 h, BM-2.0 h, BM-2.5 h, BM-3 h, and the physical milling product was described as Na2GA/CA-PM.
Analysis of chrysomycin A by HPLC
The appropriate amounts of samples were dissolved completely in a mixture solution (deionized water to acetonitrile, 1:1, v/v) respectively, and filtered through a 0.22 μm filter paper. Then, the filterate was determined by a high performance liquid chromatography (HPLC, Aglient 1260 infinity Ⅱ) equipped with column Inertisil O DS-3 C18 (250 mm×4.6 mm, 5 μm, GL Science Inc., Japan) at 25 ℃, and a UV detector set at a wavelength of 254 nm. Acetonitrile-0.1%formate water (40:60) was used as eluent (pH=2.6-2.8) with the flow rate of 1.0 ml/min.
Solubility determination
To determine the solubility, an overdose of samples and CA, were put into 500 μL of deionized water respectively and stirred for 12 h at 25 ℃. Finally, these solutions were filtered and analyzed by HPLC.
Powder X-ray diffraction (XRD)
X-ray diffraction test of samples was implemented with a Bruker D2 Phase diffractometer (Buker, Germany) by using CuKα radiation. Step range: 3°-40°. Counter speed: 3.7°/min. All the data were analyzed through GraphPad Prism 7.
Polarized light microscopy (PLM)
To distinguish the refraction phenomenon of samples, a small amount of solid powder was placed on microscope slide and observed by an Olympus CX41 polarized microscope (Japan) with a CCD camera (HTC1600, China). All the pictures were obtained at 10× resolution.
Scanning electron microscopy (SEM)
After samples were coated with platinum by a Leica EM ACE200 Vacuum Coater (Germany), SEM (ZEISS Gemini500, Germany) was performed to acquire electronic images. The Coating parameter: amperage 30 mA, spraying time 100 s.
Particle characterization and zeta potential
The physicochemical properties of samples containing hydrodynamic diameter, polydispersity index (PDI), and zeta potential, were detected using dynamic light scattering (DLS) instrument (Zetasizer NanoZS, Malvern Instruments, Malvern, UK) at 25 ℃. Before being measured, all samples were dissolved in deionized water at the concentration of 1 mg/mL, then filtered by a 0.22 μm filter.
Transmission electron microscopy (TEM)
To observe the morphology of micelle, samples were configured into 1mg/ml solution. One drop of sample was dripped on a carbon Formvar-coated cooper grid for a minute, and then were dried below the infrared light. Finally, TEM (Hitachi HT700 EXALENS, Japan) was at a working voltage of 100 kV to form the morphology of samples.
Pharmacokinetic evaluation
Ten female ICR mice were randomly divided into two groups (CA and the ball-milling produc to evaluate the pharmacokinetic of samples. The samples were dispersed in deionized water and were intragastriclly administered to the mice at the equivalent dose of 50mg/kg CA. Next, 0.2 mL of blood was collected into prepared heparinized tubes at different time points (0.25, 0.5, 1, 2, 4, 8, 12, and 24 h) after administration, and then centrifuged at 5,000 rpm, 4 ℃ for 5 minutes to obtain plasma supernatant. After taking plasma to a cleaning tube, a certain volume of acetonitrile was added to the supernatant (the volume ratio was 3:1). When protein precipitates generated, the mixture was vortexed for 2 minutes, and centrifugated at 10,000 rpm, 4 ℃ for 10 minutes. Then, supernatant from the mixture was extracted and stored at -80 ℃ for 2 h for furth use. After being thawed, samples were centrifugated (10,000 rpm, 4 ℃) for 10 minutes and take out. At last, the sample was filtered by a 0.22 μm filter for HPLC analysis.
In vivo tissue biodistribution study
To investigate the tissue biodistribution of CA and the ball-milling product, ten female ICR mice were stochastically divided into two groups. The ball-milling sample and CA were formulated as suspensions at a concentration of 5 mg/mL. The dose for each intragastric administration was 50 mg/kg equivalent to the concentration of CA. At the set time points (2 h, 6 h, 12 h), major organs containing heart, liver, spleen, lung, kidney, brain, skeletal muscle were resected and wash with 10mM phosphate buffered saline (PBS). After being dried and weighted, the organs were divided into small pieces and homogenized with deionized water at ratio of 1:2 (g /mL). To extract CA from tissues, the homogenate was added with acetonitrile (the ratio was 1:3). Then the mixture was vortexed for 1 minutes and centrifuged at 10,000 rpm, 4 ℃ for 10 minutes. Ultimately, supernatant was removed from the mixture to a clean tube and stored at -80 ℃ for HPLC analysis.
In vivo antitumor efficacy
The tumor-bearing model was established by subcutaneously injecting 1×106 B16-F10 cells in 100μL of PBS into female C57BL/6 mice at the right flank. When the tumor volume reached to about 35-60 mm3, the mice were casually divided into 3 groups (n= 6 /group). Each mouse was intragastrically administered with an equivalent dose of 50mg/kg CA every 2-3 days, whereas the control group was given PBS.
The tumor growth and body weight change were monitored every 2-3 days. The tumor volume was measured with a caliper and was calculated as follows: tumor volume = 0.5×length×width2. On the 12th day, the mice were sacrificed, then the tumor and major organs (hearts, lungs, livers, kidneys and spleens) were washed with PBS and weighed. Moreover, tumor paraffin sections of three groups were stained with H&E staining to observe pathological changes.
Statistical analysis
Data were reported as mean±standard error of the mean, using the unpaired Student’s t-test. Values of *p < 0.05 and ***p < 0.001 calculated by GraphPad Prism 7 were considered significant and extremely significant, respectively.