The pure anlotinib powder (a reference substance with an HPLC purity >98.7%) was chemically synthesized by Shuang Cao in the Wuhan Institute of Technology (Wuhan City, Hubei, China). Poloxamer 188 was purchased from the BASF Corporation (Ludwigshafen, Germany). Tween 80 was purchased from Serva (Heidelberg, Germany). The egg yolk lecithin for injections was purchased from Lipoid (Ludwigshafen, Germany). The cell line used in the study, MHCC97-H, was a gift from Dr. Yan Chen at the Center of Therapeutic Research for Liver Cancer (Beijing the 302 Hospital, Beijing, China) and has been described in previous publications (37, 38). The BalB/c nude mice used in the experiments (BalB/c strain lacking a thymus [absence of mature T cells] immunodeficiency) were purchased from Beijing-Si-bei-fu Company (Beijing China). The serum and culture medium for cell culture experiments were purchased from Thermo (USA; Invitrogen brand), and the laboratory consumables such as cell culture flasks and cell culture dishes were purchased from Corning (NY, USA). The high-performance liquid chromatograph (HPLC), transmission electron microscope (transmission electron microscope, TEM) and other equipment are products of Hitachi (Japan); the liquid chromatography-mass spectrometer (ESI/TOF/API4000) was purchased from Applied Biosystems/MDS Analytical Technologies USA Products; the laser Zeta potential particle size analyzer (Zetasizer) is a product of Malvern (United Kingdom); the wet mill is a product of the Swiss WAB-Group (Basel Region, Switzerland); and, the T25 type dispersing and emulsifying homogenizer is a product of the German IKA company (Janke & Kunkel OHG, Germany).

We prepared the anlotinib nanocrystal injection using a ball milling method (39, 40), the specific steps were the following: We used prescribed amounts of charge and space stabilizer (poloxamer-188 and egg yolk lecithin); we added part of the prescription amount of distilled water and magnetically stirred the mixture to make it evenly dispersed; next, we added the prescribed amount of anlotinib. The concentration of anlotinib in the prepared anlotinib nanocrystal was approximately 10 mg/mL; we used the T25 high shear emulsification homogenizer at 12000 rpm for 15 minutes to disperse the crystals. Next, we poured the dispersed suspension into the grinding chamber and added the remaining water, using 0.3 mm zirconia beads as the grinding spheres. The speed of the grinding chamber was gradually increased from 1500 to 4000 rpm taking samples at 30, 60, 90, and 120 minutes and measuring the average particle sizes at each time point. When the mean particle size of anlotinib was less than 300 nm, we stopped the grinding and collected the formulation.

We prepared a solution of anlotinib to use as a control. The anlotinib solution was prepared following published methods (41–44). Briefly, the anlotinib powder was dissolved in organic solvents, PEG400, TWEEN80, and DMSO. Next, the anlotinib organic solvents solution was dissolved in PBS to a final anlotinib solution concentration of ~2 mg/ml (almost 2mg/ml).

We poured 0.5 mL of the above anlotinib nanocrystal (10 mg/mL) suspension into a dialysis bag (45). We placed the dialysis bag in 200 mL of release medium (0.5% sodium lauryl sulfate, 0.9% sodium chloride, 20 nM phosphate buffer, pH 7.0) and kept it at 37°C on a shaker platform rotating at 50 rpm. We took 1 mL samples of release medium at different time points and replaced the volume with blank medium. Each medium sample taken was passed through a 0.22-μm microporous membrane, and the anlotinib content was determined by HPLC. The HPLC liquid phase conditions included: an Agilent ZOBAX C18 column (250 × 4.6 mm, 5 µm), the mobile phase was an acetonitrile-ammonium acetate buffer (40:60, pH 8.50 ± 0.05 adjusted with ammonia), and the flow rate was 1.0 mL•min^-1. The detection wavelength was 247 nm, the column temperature was set at 40°C, and the injection volume was 20 μL.

For the disposition of Anlotinib-nanocrystal injection, twelve healthy male Vistar rats were taken, weighed, and randomly divided into two groups, each with 6 rats. The Anlotinib-nanocrystal injection was administered intravenously at a dose of 7 mg/kg. One group was decapitated 3h and 9h after administration for another group, blood was drained quickly, heart, liver, spleen, lung, kidney and brain tissues were taken, washed with normal saline, blotted dry with filter paper, and stored at -80°C for later use. For sample determination, take 0.5g of homogenized sample, centrifuge at 16112×g for 3min, taken 200μL of the supernatant for HPLC.

We cultured MHCC97-H cells and injected them into subcutaneous tissue of nude mice to form tumors (46–49). The amount of cells injected in each animal is exactly the same (5×10⁶ cells for every mice). Next, we grouped the mice randomly. Mice in each model group received a different treatment: oral administration of crude anlotinib suspension (concentrations: 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, or 0.2 mg/kg); a tail-vein injection of anlotinib nanocrystals (concentrations: 0.5 mg/kg, 0.2 mg/kg, 0.1 mg/kg, 0.05 mg/kg, or 0.02 mg/kg); and a tail-vein injection of anlotinib solution (concentrations: 0.5 mg/kg, 0.2 mg/kg, 0.1 mg/kg, 0.05 mg/kg, or 0.02 mg/kg). After 21 days of treatment (approximately 10 doses of treatment), we collected the subcutaneous tumor tissues and measured their volumes and weights according to published methods (50, 51). Each group consists of 6-10 animals, each animal has one subcutaneous tumor tissue. As a control, another three groups of nude mice were respectively inoculated with MHCC97-H cells, and were treated with stabilizer (as the control of Anlotinib-nanocrystal) or solvent control (as the control of Anlotinib solution).

For the liver orthotopic tumor model, MHCC97-H cells were first cultured and inoculated subcutaneously in nude mice to form tumor tissues. After the tumor tissue is collected, the part with good surface properties of the tumor tissue is stripped off to prepare a tissue micro-block (52–54). Trim the tissue micro-blocks to make the weight basically the same Supplementary Table 1 ). After that, the nude mice were inhaled anesthetized with isoflurane, the aforementioned tumor tissue micro-blocks were inoculated into the nude mice’s liver by open surgery, and finally the nude mice’s wounds were sutured. The drug treatment strategy is basically the same as the above, using microPET to perform live imaging of animals (55) and quantitative analysis of mouse liver images (47, 49): determining the intensity of the microPET image based on the area and pixel density (i.e., brightness) of the image in the liver region in the microPET images; determine the size of the lesion based on the ratio of the area of the lesions to the area of the entire liver organ (47, 49, 54).

The statistical analyses were performed using SAS, version 9.4 (SAS Institute, Inc., Cary, NC, USA) and performed a one-way analysis of variance to compare the means and standard deviations of each treatment method and calculate the p values.

We successfully prepared the anlotinib nanocrystals ( Figures 1 , 2 ). As shown in Figure 1 , the longer the grinding time, the smaller the mean particle size of the anlotinib nanocrystals; and after 90 minutes’ grinding, the smallest nanocrystals reached approximately 200 nm, an appropriate size for intravenous administration. We determined that the optimal formulation process for preparing anlotinib nanocrystals includes the stabilizers Poloxamer 188 and egg yolk lecithin E80 in a mixture ground continuously for 90 to 120 minutes to obtain an anlotinib-nanocrystalline injection preparation with a uniform particle size distribution and an average particle size of approximately 200 nm.

The TEM anlotinib nanocrystal images on Figure 2A show the uniform particle size distribution of the anlotinib nanocrystals, with an average particle size of approximately 200 nm ( Figure 2B ). The TEM microscopic morphology analysis results showed well-formed nanocrystals that were regular in shape and uniform in size, without large drug particles ( Figure 2A ). The Zeta potential of the nanocrystals was approximately -30 mV ( Figure 2C ), indicating that the surface of the nanocrystal was negatively charged and that the potential value was moderate, which is beneficial for maintaining the physical stability of the nanocrystal. Moreover, results from an X-ray powder diffraction analysis showed that the crystal structure of anlotinib nanocrystals did not change during the preparation process ( Figure 2D ). The powder diffraction pattern of the bulk drug showed multiple characteristic peaks, and those same characteristic peaks were still present in the nanocrystal preparation, as shown in Figure 2D . These results indicate that our efforts to obtain anlotinib nanocrystals were successful.

Figure 3 shows the results of the anlotinib nanocrystals’ in vitro release. Anlotinib was completely released from the anlotinib nanocrystals within 2 h, and the release rate reached more than 90% within 1 h. These suggest that the prepared anlotinib nanocrystals can effectively release the anlotinib. Moreover, as shown in Supplementary Figure 1 , Anlotinib-nanocrystals have the characteristic of being selectively enriched in the liver and the selective concentration of Anlotinib after Anlotinib-nanocrystal injected in the liver is beneficial to the treatment of HCC. The accumulation of Anlotinib in the kidney may be due to the excretion and clearance of Anlotinib.

We examined the subcutaneous growth of HCC cells to further confirm the antitumor effects of anlotinib nanocrystals on HCC. As shown in Figure 4 , both the crude oral anlotinib suspension administration (3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, or 0.2 mg/kg) and the intravenous anlotinib nanocrystal administration (0.5 mg/kg, 0.2 mg/kg, 0.1mg/kg, 0.05mg/kg, or 0.02 mg/kg) inhibited the subcutaneous growth of the HCC cell line, MHCC97-H, in a dose-dependent manner. The intravenous anlotinib nanocrystal injection at a dose of 0.05 mg/kg can inhibit the subcutaneous growth of MHCC97-H cells in nude mice; whereas the dose of orally administered crude anlotinib suspension required to obtain equivalent inhibitory results was 1 mg/kg. Thus, the anlotinib nanocrystal preparation achieved an anti-tumor activity against HCC at very small doses.

Next, we compared the antitumor effects of the anlotinib nanocrystals injection with the anlotinib solution injection to further confirm the effects of the anlotinib nanocrystal injection preparation. As shown in Figure 5 , both the intravenous injection of anlotinib nanocrystals or of anlotinib solution inhibited the subcutaneous growth of MHCC97-H cells. However, the antitumor effect of the nanocrystal preparation was better than that if the anlotinib solution.

To further examine the antitumor activation of Anlotinib-nanocrystal, the intrahepatic tumor model was examined. As shown in Supplementary Figure 2 , the MHCC97-H cells could form the intrahepatic lesions/nodules in nude mice’s liver organs. These lesions could be examined by the microPET imaging ( Supplementary Figure 2 ). The anti-tumor activity of the drug was reflected by the intensity of the microPET image and the size of the tumor lesion ( Supplementary Figure 2 ). The stabilizer did not have anti-tumor activity ( Supplementary Figure 2 ). Tail vein injection of Anlotinib-nanocrystal at a dose of 0.2 mg/kg and Anlotinib solution at a dose of 0.2 mg/kg have the same anti-tumor activity as oral Anlotinib crude suspension at a dose of 3 mg/kg ( Supplementary Figure 2 ). These results confirm the advantages of the anlotinib nanocrystal preparation.

To examine whether the Anlotinib nanocrystal can also relieve adverse-effects, we examined the animal body weights, main organ weights, and blood routine indicators of the treated animals. As shown in Table 1 , oral administration of crude anlotinib suspension significantly induced a decrease in hematological markers (leukocyte and red blood cell numbers, hemoglobin content, and platelet counts) and a decrease in body or major organs’ weights (heart weight, liver weight, lung weight, kidney weight or spleen weight) of the nude mice mentioned in Figure 4 . The intravenous injection of anlotinib nanocrystals produced adverse effects after injection in the mice, but those were milder than the effects observed after oral administration of crude anlotinib suspension ( Table 1 ). Moreover, the adverse effects of anlotinib nanocrystals were less severe than those after the intravenous injection of anlotinib solution ( Table 1 ). These results further confirm the advantages of the anlotinib nanocrystal preparation.

The above results were all using Anlotinib. In order to further confirm the specificity of the system, we also tested the stabilizer and solvent controls. The results are shown in Supplementary Figure 3 . The stabilizer and solvent controls do not have any anti-tumor activity by themselves. The stabilizer has no obvious toxicity to nude mice, while the solvent control can significantly damage the mice, which is reflected in: the solvent control but not stabilizer induced a decrease in body or major organs’ weights (heart weight, liver weight, lung weight, kidney weight or spleen weight) of the nude mice mentioned in Supplementary Figure 3 ( Supplementary Table 2 ). These results further confirmed the activation of Anlotinib-nanocrystal.