Hydrous icaritin nanorods with excellent stability improves the in vitro and in vivo activity against breast cancer

Abstract Due to their various biological activities that are beneficial to human health and antitumor effect, flavonoid compounds have attracted much attention in recent years. Hydrous icaritin (HICT) was such a flavonoid that can inhibit the growth of breast cancer and cancer stem cells. In order to overcome the insolubility problem, HICT was fabricated into nanorods (NRs) through anti-solvent precipitation in this paper using D-α tocopherol acid polyethylene glycol succinate and sodium oleate as a co-stabilizer meanwhile using the mixture of ethanol and acetone (1:2, v/v) as the organic solvent. The obtained HICT NRs showed an average particle size 222.0 nm with a small polydispersity index value of 0.124 and a high zeta potential of – 49.5 mV. HICT NRs could maintain similar particle size in various physiological medium and could be directly lyophilized without the addition of any cytoprotectants and then reconstituted into a colloidal system of similar size. The resultant HICT NRs had a high drug loading content of 55.6% and released HICT in a steady and constant pattern. MTT assay indicated NRs enhanced HICT’s antitumor activity to ninefold against MCF-7 breast carcinoma cells. In vivo studies demonstrated oral administration free HICT had almost no tumor inhibitory effect while HICT NRs showed a tumor inhibition rate of 47.8%. When intravenously injected, HICT NRs displayed similar therapeutic efficacy to paclitaxel injections (70.4% vs. 74.5%, TIR). This may be partly due to the high accumulation of the injected HICT NRs in tumor ranking only second to that in the liver but much higher than in other organs. These results demonstrated that HICT NRs could be a promising antitumor agent for the treatment of breast cancer in clinic.


Introduction
The burden of cancer is increasing worldwide due to the aging of population and the increasing of unhealthy lifestyles (Torre et al., 2015). Breast cancer has been the highest incidence of cancer among women (Torre et al., 2015;Chen et al., 2018). However, the current therapeutic methods, including hormone therapy, radiotherapy, surgery, and chemotherapy, are not satisfied due to the accompanied side effect and multiple drug resistance (Peng et al., 2009;Akram et al., 2017;Chatterjee & Bivona, 2019), which will in return aggravate the patient's depression (Rao et al., 2019), the most widely occurred symptom among the breast cancer patients (Zainal et al., 2013;Shah et al., 2014;Stanton & Bower, 2015).
Natural products prove to be one of the sources of antitumor drugs, such as paclitaxel and hydroxylcamptothecin. Flavonoids are a major class of polyphenols naturally abundant in various fruits, vegetables, tea, and plants. Due to their various biological activities beneficial to human health and antitumor effect, flavonoid compounds are attracting much attention in recent years (Perez-Vizcaino & Fraga, 2018;Rauf et al., 2018).
Hydrous icaritin (HICT) is just such a flavonoid compound that can not only effectively inhibit the growth of breast cancer cells (Pan et al., 2007;Pan et al., 2010;Guo et al., 2011;Lai et al., 2013;Wang et al., 2017;Zhang et al., 2018) and cancer stem cells (Guo et al., 2011), but also relieve depression (Pan et al., 2007;Pan et al., 2010). However, like most flavonoids, HICT is water-insoluble, thus difficult to be effectively delivered in vivo, and meanwhile results into low bioavailability and limited effectiveness (Lewin et al., 2013;Guo et al., 2017). Encapsulation of hydrophobic drugs into nanoparticles, micelles or liposomes can well resolve the insolubility problem of this kind of drugs and achieve passive targeting through the enhanced permeability and retention (EPR) effect when intravenously administrated (Hashizume et al., 2000;Torchilin, 2011;Maeda et al., 2013;Prabhakar et al., 2013), and is thus suitable for hydrophobic antitumor agents. In this paper, HICT was successfully fabricated into nanorods (NRs) with good stability and high drug loading content (DLC). As far as we know, it is the first report on HICT NRs and the first report on NRs for flavonoid compounds.
More and more investigations have proved that the shape has a fundamental effect on the in vivo fate of nanoparticles through their interaction with the plasma components, immune system and the various physiological barriers and so on. NRs show longer blood circulation time, higher cellular uptake and higher tumor accumulation than disc and spherical nanoparticles (Toy et al., 2014;Banerjee et al., 2016;Yang et al., 2016). Probably due to these reasons, the obtained HICT NRs showed significantly increased the in vitro antitumor effect in comparison with free HICT, much significantly enhanced tumor inhibition rate in contrast with free HICT when oral administrated and a similar in vivo antitumor therapeutic efficacy to that of paclitaxel (PTX) injections. This demonstrated that HICT NRs are promising to be an effective antitumor drug in the future.

Animals and cell lines
The MCF-7 (breast carcinoma) cell line was supplied by China infrastructure of cell line resource. The cells were cultured in DMEM medium with 10% fetal calf serum (Thermo Fisher Scientific), streptomycin (100 U/mL), and penicillin (100 U/mL) at 37 C with 5% CO 2 in cell incubator (Sanyo, Osaka, Japan). Female NU/NU nude mice (20 ± 2 g, 6-8 weeks old) were obtained from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The animals were acclimated under 12 h light-dark cycle environment with relative humidity of 70 ± 5% at 25 C for one week. All animal experiments are conducted according to guidelines for Ethical and Regulatory

Preparation of HICT NRs
Screening for suitable stabilizer HICT NRs were fabricated by the antisolvent precipitation method. Preliminary test showed HICT had much higher solubility in dimethyl sulfoxide (DMSO) (>50 mg/mL) than in other solvents such as ethanol, acetone, and methanol, so DMSO was firstly selected as the organic solvent. Briefly, 5 mg of HICT powder and 5 mg of stabilizer were co-dissolved in 0.3 mL of DMSO according to a fixed HICT/stabilizer feeding ratio (1:1, weight ratio), then slowly instilled into 5 mL of deionized water under ultrasonic conditions (250 W, 25 C), followed by centrifugation at 13000 r/min for 10 min. The sediment was resuspended in 5 mL of deionized water, sonicated for 10 min, and then homogenized (25 C, 1500 bar) for 10 cycles to obtain HICT NRs. When mPEG -PCL 2000, mPEG 2000-PLA 2000 , Oleic acid, TPGS and SPC were used as a stabilizer, respectively, they were dissolved together with HICT in DMSO; while BSA was dissolved in the deionized water when used as a stabilizer.
Improvement of HICT nanorods stability by alteration of organic solvent 5 mg of TPGS and 5 mg of HICT were dissolved in 0.5 mL of acetone and ethanol (2:1, v/v), then instilled into 5 mL of deionized water under ultrasonic conditions (250 W, 25 C), followed by rotary evaporation at 40 C to remove the organic solvents, then the mixture was homogenized (25 C, 1500 bar) for 10 cycles. Then the particle size of the resultant HICT NRs was monitored during their incubation in various physiological medium at 37 C.

The optimization of HICT NR preparation
Different HICT/TPGS feeding ratio (2:1, 3:1, 4:1) was tried according to the method described in the method 'Improvement of HICT nanorod's stability by alteration of organic solvent' and their particle size and polydispersity index (PDI) values were measured to select the optimal feeding ratio.
In order to reduce the intestinal metabolism of HICT when orally administrated, SO was added in the formulation of HICT nanosuspension. Specifically, 9 mg of HICT and 3 mg of TPGS were co-dissolved in 0.9 mL of acetone and ethanol (2:1, v/v), then instilled into 9 mL of deionized water containing 3 mg of SO, followed by rotary evaporation at 40 C to remove the organic solvents, then the mixture was homogenized (25 C, 1500 bar) for 10 cycles to obtain HICT NRs.

Physicochemical characterization of HICT NRs
Dynamic light scattering measurement The mean particle diameter, PDI, and zeta potential of HICT NRs were measured by a dynamic light scattering method (Zetasizer Nano ZS, Malvern Instruments, UK) at 25 C. Each sample was measured three times and all data were expressed in the form of mean ± standard deviation (SD).

Morphology of HICT NRs
The morphological characterization of HICT NRs was performed by a JEM-1400 transmission electron microscope (JEOL, Tokyo, Japan). A drop of particle dispersion was dropped onto the surface of the 300-mesh copper mesh, dyed with 2% (w/v) uranyl acetate, and then the morphology of HICT NRs was observed under the electron microscope.
The stability of HICT NRs in various physiological medium HICT NRs were, respectively, mixed with 1.8% NaCl, 10% glucose, 2 Â PBS (1:1, v/v), or mixed with plasma, simulated gastric fluid (1% pepsin in 1 mol/L diluted HCl), and simulated intestinal fluid (1% pancreatin in pH 6.8 PBS, 0.01 M) (1:4, v/ v) and incubated at 37 C. Particle size and PDI value of the sample were measured at specific time intervals. The above experiments were conducted in triplicates.

Lyophilization of HICT NRs
20 mL of HICT NRs solution was taken in a beaker and covered with lens paper frozen for 6 h under -80 C freezer (MELING DW-HW50, China) and lyophilized for 12 h in a vacuum freeze dryer (FD, Christ-Alpha 2-4 LD plus, Germany) under 0.12 mbar, -40 C. The freeze-dried powder was dispersed in 20 mL deionized water and the particle size, PDI, and zeta potential were measured as described in the method of 'dynamic light scattering measurement.'

Chromatographic condition of HICT
The concentration of HICT was measured using HPLC system (DIONEX Ultimate 3000, USA) (HPLC, DIONEX Ultimate 3000, Germering, Germany). A column C 18 (4.6 mm Â 250 mm, 5 lm; Dr. Maisch GmbH, Germany) was used at 25 C for chromatographic separation. The mobile phase was constituted of acetonitrile and 0.1% acetate water (68:32, v/v) with a flow rate of 0.6 mL/min. The detection wavelength UV was 270 nm.

Drug loading content of HICT NRs
To determine DLC of HICT NRs, the lyophilized HICT NRs were weighed and dissolved in methanol. The concentration of HICT was determined using HPLC system. The DLC of HICT NRs is expressed as follows: where V is the volume of methanol, C is the concentration of HICT, and W is the weight of lyophilized powder of HICT NRs.

In vitro drug release behavior
The release behavior of HICT NRs (300 lg/mL, 10 mL) was performed in triplicate in Float-A-Lyzer dialysis cassettes (MWCO: 20KD, Spectrum Labs, Rancho Dominguez, CA, USA). Then the dialysis cassettes were suspended in 2.0 L PBS (PH 7.4 ± 0.1, 2.0 L) containing 0.5% (w/v) tween 80 to assure the sink conditions at 37 C with constant stirring (100 rpm). 200 lL of internal dialysate were withdrawn at fixed time intervals, the same volume of fresh release medium was replenished and the external release medium was replaced every 24 h. HICT concentration in the dialysis cassettes was determined by HPLC. The cumulative release of drug was calculated by reduction of HICT NRs inside the dialysis cassettes.

Differential scanning calorimetry (DSC) characterization
DSC thermal profiles of the powder samples were tested through a differential scanning calorimeter (Q200, TA Instruments, New Castle, DE). Samples of approximately 10 mg were placed in standard aluminum pans, sealed with a lid and measured from 0 to 300 C at a scanning rate of 10 C/min under nitrogen atmosphere.

X-ray diffraction (XRD) measurements
An X-ray diffractometer (DX-2700, China) was applied to conduct X-ray powder diffraction with a generator set at 40 kV and 100 mA. Samples were scanned over an angular range of 3-80 of 2h, with a step size of 0.02 and a count time of three per step. Samples were rotated at 30 rpm during the analyses.

The pharmacological evaluation
In vitro cytotoxicity assay The cytotoxicity of HICT and HICT NRs were determined by MTT assay. First, the MCF-7 cells in the logarithmic growth phase were cultured in 96-well plates (8000 cell/well), and cultured in 37 C, 5% CO 2 environment for 24 h. Then HICT NR and HICT DMSO solution (dissolved in DMSO, final concentration of DMSO 0.1%) was diluted into different concentrations by medium and added to each well. After 48 h of continuous culture, 20 lL MTT solution (5 mg/mL in PBS) was added to each well and incubated for 4 h. Then the supernatant solution was decanted, 150 lL of DMSO was added dropwise to each well to dissolve the bottom precipitate, and the optical density (OD) value was measured at a maximum absorption wavelength of 570 nm by ELISA plate reader (Biotech, Winooski, VT, USA). The cell inhibition rate of HICT NRs and HICT DMSO solution is calculated by the following formula: where OD e is the mean OD of experimental group and OD c is the mean OD of control group. The half-maximal inhibitory concentration (IC 50 ) of HICT NRs was calculated from GraphPad Prism software, version 5.0 (GraphPad Software, La Jolla, CA, USA), by the sigmoidal dose-response variable curve-fitting method.

In vivo antitumor efficacy and biodistribution
In vivo antitumor efficacy of HICT NRs was investigated using MCF-7 NU/NU tumor-bearing nude mice. Female NU/NU nude mice were inoculated with MCF-7 cells (4.0 Â 10 7 /mL, 0.2 mL/ mouse) at the right forelimb. When the tumor volume reached 100 mm 3 , the nude mice were randomly divided into five groups (six mice in each group). Three groups of mice were, respectively, administered normal saline (negative control, 0.2 mL/mouse), PTX injection (positive control, 8 mg/kg) and HICT-NRs (35 mg/kg) via tail vein every other day for 14 days. The other two groups of mice were, respectively, given daily gavage of HICT-NRs (35 mg/kg) and free HICT (HICT, TPGS and SO were composed of suspension dispersed in water at a mass ratio of 3:1:1) for 14 days. The weight of mice and length (a) and width (b) of the mice tumor were measured every other day. The tumor volume was calculated by the formula: V ¼ (aÁb 2 )/2. At the end of the experiment (12 h after the last dose), the mice were sacrificed by spine. The tumor, liver and spleen were dissected and weighed, respectively. The tumor inhibition rate and liver and spleen index are calculated according to the following formulas..
The tumor inhibition rate (TIR): where W e is the mean tumor weight of experimental group and W n is the mean tumor weight of negative control group. Liver index rate (LIR): where W Li is the liver weight of experimental group and W mi is the nude mice weight of group. Splenic index rate (SIR): where W Si is the spleen weight of experimental group and W mi is the nude mice weight of group. In order to know the biodistribution and tumor-targetability of HICT NR, Dir-labeled HICT NRs were prepared according to the finally determined method just by incorporating a bit of Dir (1:40, Dir: HICT, weight ratio) into the organic phase, and then the resultant Dir-labeled HICT NRs were intravenously injected through the tail vein of mice for the last dose. At the end of the experiment, the major organs such as heart, liver, spleen, lung, kidney and brain together with tumor were put to measure the fluorescence using the IVIS Living Image software, version 4.4 (Caliper Life Sciences, Hopkinton, MA, USA).

Data analysis
Statistical analysis of the experimental data was performed using Statistical Package for the Social Sciences software, and IC 50 values were calculated by GraphPad Prism software, version 6.01 (GraphPad Software, La Jolla, CA, USA). In vitro and in vivo results were analyzed by t-test and one-way analysis of variance. The value of p < .05 was considered statistically significant.

Preparation and optimization of HICT NRs
Screening for suitable stabilizer Preliminary test showed HICT had much higher solubility in DMSO (> 50 mg/mL) than in ethanol, acetone, and methanol, so DMSO was firstly selected as the organic solvent to prepare HICT NRs. As shown in Table 1, each of the listed stabilizers could alone help to fabricate HICT into NRs with small particle size (<230 nm) and narrow particle distribution (PDI < 0.2). Although the obtained HICT NRs basically maintained their particle size in normal saline (Table 1), they were all unstable in PBS, 5% glucose, simulated gastric fluid and simulated intestinal fluid with significantly increased particles size and aggregation (Supplementary Tables S1-S6), among which TPGS showed the best performance (Supplementary Table S4). Only when the NRs are stably present in the body fluids, the advantages of NRs can be achieved. So, it is necessary to tune the preparation of HICT NRs.

Improvement of HICT NR stability in physiological medium
There are many factors influencing the self-assembly of hydrophobic molecules and stabilizer during the antisolvent precipitation process, such as the type of stabilizer, the type Table 1. The particle size, PDI, and zeta potential of HICT nanorods before and after 10 h of incubation in normal saline.

Initial
In normal saline for 10 h of organic solvent, the feeding ratio of drug and stabilizer, the removal rate of the organic solvent and so on. Among all the tested mixing systems composed of ethanol, acetone, and methanol, the mixture of ethanol and acetone (1:2, v/v) showed the best solubility for HICT (about 8.5 mg/mL), and thus was tried as the good solvent for the preparation of HICT NRs through antisolvent precipitation. Fortunately, as shown in Table 2, HICT NRs obtained in this way proved to be able to stably exist in various physiological medium with similar particle size and acceptable PDI values after 10 h incubation, and no aggregation or abnormal phenomena was observed. Thus, the mixture of ethanol and acetone (1:2, v/v) was chosen as the good solvent for the preparation of HICT NRs in the subsequent study.
It was speculated that the instability of the HICT NRs prepared using DMSO as the good solvent was partly ascribed to the specific self-assemble mode of HICT and stabilizer when the DMSO solution was diluted with deionized water, and partly ascribed to the much less of reservation of the feeding stabilizer, most of which remained in the supernatant during the centrifugation. In order to explore the reason behind, three different HICT NRs were prepared using TPGS as a stabilizer with a HICT/TPGS feeding ratio of 1:1, all through antisolvent precipitation method. The first one used the mixture of ethanol and acetone (1:2, v/v) as the good solvent to prepare HICT NRs (named as M1) and then removed the solvent by vacuum rotary evaporation; the second one used the mixture of ethanol and acetone (1:2, v/ v) as the good solvent (named as M2) and then removed the solvent by centrifugation; the third one used DMSO as the good solvent (named as M3) and then removed DMSO by centrifugation. The particle size of the three HICT NRs after incubation in the physiological medium at 37 C for 10 h was examined. As shown in Figure 1, M2 NRs showed significantly increased particle size in PBS, simulated gastric and simulated intestinal fluids in contrast to M1, indicating that removal of partial stabilizer that retained in the supernatant reduced the stability of the M2 NRs. Removal of the organic solvent by vacuum rotatory evaporation is a relatively slow process, thus probably allow M1 NRs to 'repair' their structural flaws during the solvent evaporation. However, during the centrifugation, the self-assembly NRs fail to 'repair' their structural flaws due to an abrupt loss of the organic solvent and the co-existed stabilizer that was abundant in the supernatant. The all above factors may result in the less stability of M2 and M3 HICT NRs in comparison with M1 NRs. In addition, the self-assembly of HICT and TPGS in the ethanol-acetone-water system may also differ from that in the DMSO-water system.

Formulation optimization and lyophilization
Small particle size, good stability in the physiological medium and high DLC are all that we want for HICT NRs. As seen in Figure 2, it was evident the HICT/TPGS feeding ratio of 3:1 well balanced the small particle size and presentable DLC and meanwhile achieved good stability in various physiological medium. So, the HICT/TPGS feeding ratio of 3:1 was chosen to prepare HICT NRs in the subsequent study.
It has been reported that most flavonoids are subject to phase II metabolism and the widely occurred phase II metabolism becomes one of the main reasons for the low oral bioavailability for flavonoids . Thus, SO, a strong phase II metabolism inhibitor (He et al., 2018), was added in the formulation of HICT NRs with the aim of improving the oral bioavailability of HICT. As seen in Table 3, the incorporation of SO not only has little effect on the particle size and PDI values of HICT NRs, but also increased their zeta potential, which is beneficial to the stability of NRs. Unexpectedly, through the incorporation of SO, the resultant HICT NRs could be lyophilized without any cryoprotectant and then be reconstituted into a clear colloidal system with the similar particle size just by re-suspending into water followed by gentle shaking (Table 3).
So far, the final formulation of HICT/TPGS/SO (3:1:1) was set with the specific preparation method as follows: HICT and TPGS were co-dissolved in acetone and ethanol (2:1, v/ v), then the organic solution was instilled into 10 volume of deionized water containing SO, followed by rotary evaporation at 40 C to remove the organic solvents, and then homogenization (25 C, 1500 bar) for 10 cycles to obtain HICT NRs.

Characterization of HICT NRs and stability of physiological medium
The self-assembly of HICT, TPGS and SO into NRs was illustrated in Figure 3(A). The resultant HICT NRs had an average particle size of 222.0 ± 7.4 nm with a small PDI of 0.124 ± 0.035, and a high zeta potential of (-44.5 ± 3.37) mV (Figure 3(B)). TEM photography displayed that HICT NRs were NRs in shape (Figure 3(C)), which was very seldom seen for flavonoid nanoparticles. It was determined by HPLC assay that the actual DLC of HICT NRs was 55.6%, a litter lower than the theoretic value of 60%.
In the DSC diagram, bulk HICT powders showed sharp and strong diffraction peaks at 245.28 C (Figure 3(D)-e1) corresponding to the melting point of HICT, indicating its crystal characteristics. However, this peak was shifted in the physical mixture of HICT, TPGS and SO (Figure 3(D)-b1), and disappeared in HICT NRs (Figure 3(D)-a1), indicating the alteration of the original crystalline form of HICT and the fact that HICT Table 3. The particle size, PDI, and zeta potential of HICT NRs before and after lyophilization and reconstitution.

Formulation
Size (  are easy to interact with TOGS and SO during the heating process. And the total disappearance of this peak in the DSC diagram of HICT NRs (Figure 3(D)-a1) suggested HICT may be in the amorphous form in HICT NRs.
In the XRD spectrum of the physical mixture (Figure 3(E)-b2), there is a lot of sharp diffraction peaks corresponding to those of HICT bulk powder (Figure 3(E)-e2). However, in the XRD spectrum of HICT NRs (Figure 3(E)-a2), no significant diffraction peaks of HICT could be observed, indicating the transform of HICT crystalline into amorphous form during the preparation of HICT NRs, which was in accordance with the result of DSC. High energy state of HICT (amorphous form) in HICT NRs meant the probably relatively fast drug release in vitro and in vivo.

The stability of HICT NRs in various physiological medium and drug release in vitro
In order to achieve effective in vivo drug delivery, HICT NRs must be stable in various physiological medium. As seen in Figure 4(A, B), the obtained HICT NRs well maintained their original particle size with similar PDI values in normal saline, PBS, 5% glucose, plasma, simulated gastric and intestinal fluids with no turbidity or aggregation at all. The only difference was that HICT NRs in plasma was smaller than that in deionized water. This was because that blank plasma itself contained some tiny nanoparticles (probably composed of serum proteins) and nanovesicles (e.g. exosomes), and their presence in the mixture led to the relatively smaller particle size and relatively higher PDI value as a system when HICT NRs mixed with plasma. Similar phenomena have been observed and reported (Allen et al., 1999;Yang & Alexandridis, 2000).
The in vitro release of HICT NRs was investigated in PBS solution (pH ¼ 7.4) containing 0.5% Tween 80. As shown in Figure 4(C), HICT NRs displayed a steady and constant drug release pattern and the cumulative release reached about 90% in 140 h. The sustained release could help avoid drug leakage in circulation and was beneficial for drug to be effectively delivered into tumor through EPR effect.  In vitro cytotoxicity experiment MTT assay was used to examine the in vitro inhibitory effect of HICT NRs against breast cancer MCF-7 cell using free HICT as a control. As shown in Figure 4(D), in the concentration range of 0.5-100 ug/mL, both HICT NRs and free HICT displayed a good dose-dependent inhibition against the growth of MCF-7 cells; however, HICT NRs showed stronger inhibitory effect than free HICT at each concentration. It was calculated the IC 50 of HICT NRs was 1.1 mg/mL, much lower than that of free HICT (10.1 mg/mL, p < .01). This demonstrated that after fabrication in NRs, the in vitro antitumor activity of HICT was enhanced to more than ninefold, which may be due to that the unspecific interaction between NRs and cells promoted cellular uptake of HICT nanoparticles and the shape of NR further facilitated this process.

The in vivo antitumor treatment and biodistribution
Antitumor treatment was performed on MCF-7 tumor-bearing nude mice model using PTX injection as a positive control (Fujioka et al., 2017;Zheng et al., 2017). As seen in Figure 5(A, B), daily oral administration of free HICT (35 mg/ kg) showed no antitumor effect with a similar tumor growth profile to that of normal saline group, probably due to its poor solubility and oral bioavailability of HICT suspensions even in the presence of TPGS and SO. After fabrication into NRs, HICT displayed a significant tumor inhibitory effect with much slower tumor growth and a much higher tumor inhibition rate (TIR, 47.8%, p < .001). When intravenously injected ( Figure 5(A)-(D)), HICT NRs dosed every 2 days further demonstrated significantly slower tumor growth and significantly lower TIR (70.4%) than that of daily oral administration (p < .05) even dosed every 2 days, close to that for 8 mg/kg of PTX injections (74.5%, TIR), probably due to the high bioavailability and EPR effect.
Dir, a widely used near-red fluorescent dye, was incorporated into HICT NRs and intravenously injected to mice for the last dose to examine the biodistribution of HICT NRs. As shown in Figure 5(F), it was clear that the injected HICT NRs mainly accumulated in liver and tumor, followed by in spleen, with much higher fluorescence than in other organs, demonstrating good tumor targetability.
Although the high biodistribution of HICT NRs in the liver and spleen, there was no difference in the liver index and spleen index between saline group and HICT NRs (i.v.) group ( Figure 5(G, H)), suggesting good biosafety of HICT NRs. The similar body weight growth profiles between normal saline group and HICT NRs groups (i.g. and i.v.) ( Figure 5(E)) also indicated HICT NRs did not bring mice significant toxicity whatever orally or intravenously administrated.
The good therapeutic efficacy for breast cancer, accumulation in tumor tissue, preliminary good safety, together with potential anti-depression activity, demonstrated that HICT NRs could be a promising antitumor agent for the treatment of breast cancer in clinic.

Conclusions
Flavonoid compound with presentable antitumor activity is attracting more and more attention, as their various concomitant biological activities beneficial to human health, which may significantly improve the living quality of tumor patients and help them restore their physical power. In our preliminary screening, HICT displayed good growth inhibition against a number of tumor cell lines and immune-enhancing effects (Pan et al., 2007(Pan et al., , 2010Lai et al., 2013;Zhang et al., 2018), but the poor solubility restricted its in vivo drug delivery. In this work, we encapsulated HICT into nanoparticles with high DLC and optimize the formulation to achieve good stability in storage and in various physiological medium, thus suitable to oral or intravenous administration. TEM observation that the obtained HICT nanoparticles were NRs in shape, which was never seen before. TEM observation verified that the obtained HICT nanoparticles were NRs in shape, and this is the first NRs report for the nanoparticles of flavonoid compounds. Through the incorporation of small amount of SO, the obtained HICT NRs could be directly lyophilized and then go back to the NR colloidal system of similar size. NR significantly enhanced the cytotoxicity of HICT against MCF-7 breast cancer cells, enhanced the in vivo inhibition rate from nearly 0 to 47.8% when orally dosed. Intravenously injection of HICT NRs mainly accumulated liver and tumor, and even achieved similar antitumor therapeutic efficacy to that of PTX injections. It seemed that HICT NRs are promising to be a novel antitumor agent along with much health-helpful effect that may benefit tumor patients in clinic. However, further study and more work are needed to provide more evidence.

Disclosure statement
No potential conflict of interest was reported by the authors.