Preparation, characterization, and evaluation of the antitumor effect of kaempferol nanosuspensions

Kaempferol (KAE) is a naturally occurring flavonoid compound with antitumor activity. However, the low aqueous solubility, poor chemical stability, and suboptimal bioavailability greatly restrict its clinical application in cancer therapy. To address the aforementioned limitations and augment the antitumor efficacy of KAE, we developed a kaempferol nanosuspensions (KAE-NSps) utilizing D-α-tocopherol polyethylene glycol 1000 succinate (TPGS) as a stabilizing agent, screened the optimal preparation process, and conducted a comprehensive investigation of their fundamental properties as well as the antitumor effects in the study. The findings indicated that the particle size was 186.6 ± 2.6 nm of the TPGS-KAE-NSps optimized, the shape of which was fusiform under the transmission electron microscope. The 2% (w/v) glucose was used as the cryoprotectant for TPGS-KAE-NSps, whose drug loading content was 70.31 ± 2.11%, and the solubility was prominently improved compared to KAE. The stability and biocompatibility of TPGS-KAE-NSps were favorable and had a certain sustained release effect. Moreover, TPGS-KAE-NSps clearly seen to be taken in the cytoplasm exhibited a stronger cytotoxicity and suppression of cell migration, along with increased intracellular ROS production and higher apoptosis rates compared to KAE in vitro cell experiments. In addition, TPGS-KAE-NSps had a longer duration of action in mice, significantly improved bioavailability, and showed a stronger inhibition of tumor growth (the tumor inhibition rate of high dose intravenous injection group was 68.9 ± 1.46%) than KAE with no obvious toxicity in 4T1 tumor-bearing mice. Overall, TPGS-KAE-NSps prepared notably improved the defect and the antitumor effects of KAE, making it a promising nanodrug delivery system for KAE with potential applications as a clinical antitumor drug.


Introduction
Cancer, the incidence and mortality of which have been rising in recent years, is the second leading cause of death worldwide as well as a critical threat to human health.The current strategies employed for the treatment of cancer encompass chemotherapy, radiotherapy, and surgical resection [1,2]; nevertheless, these interventions frequently exhibit suboptimal efficacy and may result in adverse reactions.Compared to conventional cancer therapies, natural products not only have similarly significant antitumor efficacy, but also have the advantages of abundant candidate resources, low-cost, less side effects, and favorable safety [3].Active compounds extracted from natural products, notably flavonoids and polyphenols, have been employed in the treatment of cancers by hindering the activity of numerous enzymes implicated in cell proliferation and the signaling pathways of tumor cells [4].The gradual introduction of natural products into the field of cancer treatment has played a crucial role in the development of various antitumor agents, which have been receiving heightened attention nowadays [5,6].
Nanotechnology has garnered significant attention in recent times, owing to its potential in addressing challenges associated with drug delivery [22][23][24].Nanodrug delivery systems such as liposomes, nanoemulsions, nanosuspensions, nanogels, micelles, and polymer-based nanoparticles have emerged as promising solutions for enhancing the solubility of hydrophobic drugs and improving drug delivery efficacy in vivo.Consequently, these nanodrug delivery systems have gained prominence in contemporary pharmaceutical research [25,26].Nanosuspensions are colloidal dispersion system composed of drug nanoparticles and trace stabilizers, offering comparable benefits to other nanodrug delivery systems including the improved pharmacokinetics in vivo, enhanced oral availability, and the better therapeutic effectiveness.Additionally, it exhibits characteristics of simple production process, good dispersibility, high drug loading capacity, and suitability for the production of various compounds [27,28].There are some hydrophobic drugs such as curcumin [29], ticagrelor [30], and quercetin [31] that have already been formulated as nanosuspensions to improve their effects, and kaempferol nano-formulations have been developing.For instance, Tao et al. have synthesized the ovalbumin and sodium alginate composite carriers to protect and encapsulate the tannic acid (TA) and kaempferol, which improved the bioavailability of TA/KAE [32].Gupta et al. have encapsulated kaempferol into albumin nanoparticles to prepare a nanofibrous mat, which was conducive to the drug release [33].Kazmi et al. have formulated kaempferol-loaded nanoparticles using the method of quasi-emulsifying solvent diffusion technology with high drug loading and good drug release properties [34].Other kaempferol nano-formulations like nanostructured lipid carrier [35] and nanoliposome [36] also have been synthesized.
It appears evident that the implementation of nanodrug delivery systems has the potential to circumvent the aforementioned limitations of kaempferol, thereby enhancing its antitumor efficacy.While previous research has reported the preparation process, physicochemical characterization, and oral bioavailability of kaempferol nanosuspensions [37], the particle size of formulation prepared was too large, and the study of essential physicochemical characterizations and other basic properties was not sufficiently comprehensive.In addition, to the best of our knowledge, there was no antitumor studies of kaempferol nanosuspensions currently.Herein, we prepared a new kaempferol nanosuspension and optimized the preparation process and investigated their physicochemical characterization, stability, solubility, lyophilization process, hemolysis, drug release, and pharmacokinetic and antitumor activity to establish an effective kaempferol nanodrug delivery system against tumor, which offered a feasible strategy to enhance the efficacy of KAE or similar insoluble natural drugs on tumors.

Preparation and process optimization of nanosuspensions
Anti-solvent precipitation and high-pressure homogenization reported were the common methods of preparing nanosuspensions [38].In this study, kaempferol nanosuspensions (KAE-NSps) were prepared by anti-solvent precipitation combined with high-pressure homogenization.Briefly, a certain amount of KAEs and stabilizers was dissolved in organic solvent together; the mixture was added to deionized water dropwise under ultrasonication at 45 ℃ with ultrasonic instrument (Shanghai Kedao Co., Ltd).Then the organic solvent was evaporated under vacuum at 50 ℃ by rotary evaporator (Shanghai Yarong Co., Ltd).Subsequently, the resulting solutions were subjected to homogenization (ATS Engineering Inc., Shanghai, China) to produce KAE-NSps.Finally, the lyophilization process of KAE-NSps was conducted by LD-A80-21L freeze dryer (Shanghai Binglin Electronic Technology Co., Ltd.) for further characterization.There were many factors including different stabilizer, organic solvent, ultrasonic power, drug-load ratio, homogenization pressure, and homogenization times that affected the particle size of KAE-NSps during the preparation process.Here, we took the particle size of KAE-NSps prepared as the final index as well as the method of single-factor analysis which was applied to optimize the preparation process.

Physicochemical characterization of nanosuspensions
The method of dynamic light scattering (DLS) was applied to determine the particle size, polydispersity index (PDI), and zeta potential (ZP) of TPGS-KAE-NSps prepared using Zetasizer Nano ZS90 (Malvern Instruments Ltd, Malvern, England).Diluting the specimens (60-fold) by deionized water before measurement to obtain the particle densities required for DLS, and all the tests were repeated in triplicate under room temperature.JEM-2100F transmission electron microscope (TEM) (JEOL, Japan) was used to observe the morphology of TPGS-KAE-NSps and TPGS, the sample dilution liquid of which was placed on a 200-mesh copper grid.The X-ray diffraction (XRD) of samples was detected using Ultima IV X-ray diffractometer (Rigaku, Japan) and scanned at a diffraction angle range of 5 ~ 60° using a Cu-Ka radiation generator set at 200 mA and 40 kV, with step length of 0.02°and speed of 5°/min.Fourier transform infrared spectroscopy (FTIR) spectra of the samples were detected using AVATAR370 spectrometer (Nicolet, USA) with the scanning wavelength set 4000-500 cm −1 .Thermogravimetric analysis (TG), derivative thermogravimetric analysis (DTG), and differential scanning calorimetry (DSC) of samples were detected by STA449F3 simultaneous thermal analyzer (NETZSCH, Germany).Approximately 10 mg of each sample placed and sealed in standard aluminum pan was detected at 10 °C/min from 30 to 600 °C under nitrogen environment [39].

Stability of nanosuspensions
The stability of nanosuspensions mainly includes storage and various physiological media stabilities.The particle size and PDI of TPGS-KAE-NSps stored in a vial and placed at room temperature were tested on the 1st, 3rd, 5th, 7th, 10th, and 14th days to evaluate the storage stability.TPGS-KAE-NSps prepared was mixed with 2 × phosphate buffer solution (PBS, pH 7.4), 1.8% NaCl solution, and 10% glucose solution, respectively (1:1, v/v), to get an isotonic solution, while the nanosuspensions were also mixed with plasma (4:1, v/v), artificial intestinal fluid (in PBS (pH 6.8) with 1% trypsin), and artificial gastric fluid (in 1 mol/L diluted HCl with 1% pepsin).All the samples were placed in a water bath at 37 ℃, and the particle size was determined at 0, 2, 4, 8, and 12 h to evaluate the physiological media stability.Then the protein adsorption test was conducted to investigate whether the increased particle size of TPGS-KAE-NSps prepared in plasma was related to the adsorption of proteins.

Optimization of lyophilization for nanosuspensions
Three parts of TPGS-KAE-NSps freshly prepared were accurately absorbed into 10 mL vials, as well as 2% w/v glucose, sucrose, and PVP which were added into for lyophilized protectors to obtain lyophilized powder by freeze-drying at −90 ℃ for 24 h using the LD-A80-21L freeze dryer (Shanghai Binglin Electronic Technology Co., Ltd.).0.5%, 1%, 2%, and 5% w/v glucose were added into TPGS-KAE-NSps freshly prepared, lyophilized as before, respectively.All the lyophilized powder dissolved in 2 mL of deionized water was determined the particle size by DLS.

Hemolysis evaluation of nanosuspensions
The hemolysis experiment of TPGS-KAE-NSps was performed to evaluate the biocompatibility and whether it was applied to intravenous injection [40].The whole blood was derived from Balb/c mice orbit.Whereafter, the supernatant plasma was removed by centrifugation at 5000 rpm to obtain 4% red blood cell suspension (v/v), which was mixed with 0.5 mL 1 × PBS, 0.5 mL deionized water, and 0.5 mL TPGS-KAE-NSps of specific concentrations, respectively.All of samples were incubated in a water bath at 37 °C for 3 h and centrifuged at 4000 rpm for 10 min, and then the supernatants of each were determined at 540 nm using microplate reader (Thermo, USA).The hemolysis ratio was obtained by the formula as follows: where A, A PBS , and A H2O are the absorbances of the sample at 540 nm with TPGS-KAE-NSps, PBS, and deionized water treatment, respectively.

High-performance liquid chromatography (HPLC) analysis
Kaempferol absorption wavelength was determined on ultraviolet spectrophotometer (U-3010, Hitachi, Japan).The quantification of kaempferol was performed using HPLC (Shimadzu, 20 AT, Japan), the chromatographic column of which was the C18 column (250 × 4.6 mm, 5 μm).The mobile phase consists of methanol and 0.4% phosphoric with a ratio of 70:30 (v/ v).The sample was under isokinetic elution conditions, the flow rate was 1 mL/min, the detection wavelength was 366 nm, the column temperature was 30 °C, and the injection volume was 20 μL.The mass concentration (X) of kaempferol reference solution with the peak area (Y) was used to obtain the standard calibration curve.

Drug loading capacity (DLC)
TPGS-KAE-NSps lyophilized powder was completely destroyed in methanol and centrifuged at 12,000 rpm for 10 min.Then the supernatants of each were passed through a 0.22-μm filter membrane to obtain the filtrate, whose concentration was measured by HPLC.The DLC was obtained via the following formula: where W 1 and W 2 are the weight of drug in TPGS-KAE-NSps and the weight of TPGS-KAE-NSps, respectively.

Solubilization capacity of nanosuspensions
Respectively, 1 × PBS (pH 7.4), PBS containing 0.5, and 1% SDS and PBS containing 0.5 and 1% T-80 were added with excessive TPGS-KAE-NSps and KAE in a vial.All the mixed samples, which were oscillated at 150 rpm for 72 h at 37 °C, centrifuged at 12,000 rpm for 10 min, and then the supernatants were determined by HPLC to obtain the concentration of KAE.

In vitro drug release behavior of nanosuspensions
Two mL of TPGS-KAE-NSps was put into a dialysis membrane (cellulose ester, MWCO of 14 kDa) and then immersed it in 250 mL of PBS (pH 7.4) containing 1% T-80 to dialysis under stirring at 37 ℃ (200 rpm).The dialysate was withdrawn regularly, while an equal volume of fresh PBS solution was refilled, and change the release medium every 24 h.Finally, the dialysate was quantified using HPLC for the concentration of KAE.Each time point was performed in triplicate.

Cell culture and cytotoxicity assay
Tumor cells were ordered from the Chinese Academy of Sciences (Shanghai, China), including human hepatoma carcinoma cell line (HepG2 cells), glioma cell line (U251 cells), gastric cancer cell line (SGC-7901 cells), and mouse breast cancer cell line (4T1 cells).Fetal bovine serum (FBS), Roswell Park Memorial Institute-1640 (RPMI 1640) cell culture medium, Dulbecco's modified Eagle's medium (DMEM) cell culture medium, and penicillin-streptomycin were supplied by Dingguo Biological Technology Co., Ltd.(Shanghai, China).Tumor cells were cultured in DMEM or RPMI 1640 cell culture medium containing 1% penicillin-streptomycin and 10% FBS at 37 °C in a 5% CO 2 atmosphere.
The cytotoxicity study of nanosuspensions was conducted using a CCK-8 Kit (DOJINDO Laboratories, Japan) in accordance with the manufacturer's instructions.Briefly, 4T1, U251, SGC-7901, and HepG2 cells seeded evenly in 96-well plates (5 × 10 3 cells per well) were incubated for 24 h and treated with TPGS-KAE-NSps and KAE at the set concentrations for another 24 h, and then, CCK-8 Kit was added to each well, the cell viability of which was determined using microplate reader (Thermo, USA) at 450 nm.Finally, the cell viability (CV, %) was obtained by the formula as follows: OD S , OD B , and OD N are the optical density (OD) values of the samples, blank control, and negative control, respectively.

Cell scratch
The migration potential of cancer cells was evaluated by the scratch method [41].4T1 and U251 cells were seeded evenly in 6-well plates (2.5 × 10 5 cells per well).After 24 h of incubation, scrape each well with a 200 μL pipette tip while washing the wound with D-Hanks to remove loose and floating cells.Cells were then treated with specific concentrations of TPGS-KAE-NSps and KAE, as well as cells untreated served as controls.The conditional of wound closure was observed by an inverted microscope (XDS-1B, USA) and photographed using a camera 24 h later.Images were analyzed using ImageJ software, and wound healing was calculated via the following formula: where L 0 and L 1 are wound width at 0 h and 24 h.

Cellular uptake
A lipid-soluble infrared fluorescence probe DIR was added into kaempferol (1:40, w/w), and then the nanosuspensions was prepared by the same method to obtain TPGS-KAE-NSps labeled with DIR.4T1 and U251 cells seeded evenly in confocal plates (2.5 × 10 5 cells per well) were incubated for 24 h, and then the TPGS-KAE-NSps labeled with DIR were added and incubated for 6 h.Subsequently, cells were subject to fixing with paraformaldehyde solution and staining with DAPI.Finally, the fluorescence intensity was observed by FV1000 confocal laser scanning microscopy (CLSM, Japan).

The detection of intracellular reactive oxygen species (ROS)
The intracellular ROS levels were quantified using the method of DCFH-DA.Briefly, 4T1 and U251 cells seeded evenly in confocal plates (2.5 × 10 5 cells per well) were maintained for 24 h, and then the cells were treated with TPGS-KAE-NSps and KAE, incubated for 24 h, while cells untreated served as the control.Subsequently, the ROS assay kit (Beyotime Laboratories, China) was used in accordance with the instructions.4T1 and U251 cells were dyed with DCFH-DA for 15 min and imaged by CLSM using the FITC channel [42].

Cell apoptosis
The test of cell apoptosis was conducted by annexin V-FITC/ PI Apoptosis Detection Kit (BD, USA).4T1 and U251 cells seeded evenly in 6-well plates (2.5 × 10 5 cells per well) were maintained for 24 h, and it was cultured with TPGS-KAE-NSps and KAE for another 24 h, and then the cells were treated in accordance with the instructions and analyzed using Beckman Coulter flow cytometer (San Jose, CA).Each experiment was performed three times.

In vivo antitumor efficacy study
Female Balb/c mice were ordered from Shanghai Jiesijie Experimental Animal Co., Ltd.The establishment of the 4T1 tumor-bearing mice model was to inquire the in vivo antitumor efficacy.All mice were inoculated with 0.1 mL of 4T1 cell suspension (1.0 × 10 6 cells) subcutaneously to prepare tumor-bearing mice, which were divided into six groups randomly when the tumor volume grew to nearly 200 mm 3  and (f) TPGS-KAE-NSps, 15 mg•kg −1 , iv.The intravenous group was treated every other day, as well as the gavage group was daily for 2 weeks.The body weight of mice and tumor size were measured every second day.After treatment, the mice were sacrificed, while the subcutaneous tumor and major organ including the heart, liver, spleen, lung, kidney, and brain were dissected completely as well as blood was harvested.Each major organ was weighed, and the blood was centrifuged for subsequent study.Tumor volume and the tumor inhibition rate (TIR) were obtained via the following formula: where L and W are the tumor length and tumor width, while V 0 and V are the tumor volume of control group and experimental group, respectively.

In vivo biodistribution study
The establishment of 4T1 tumor-bearing mice was the same as the antitumor efficacy study.TPGS-KAE-NSps labeled with DIR was injected intravenously when the tumor grew to a certain extent, and then, the mice were sacrificed at a specific time point set (0.5, 1, 2, 4, 8, and 24 h); the tumor and main organs including the heart, liver, spleen, lung, kidney, and brain were harvested for ex vivo imaging.The biodistribution of TPGS-KAE-NSps was monitored using UVP iBox ® Explorer2TM in vivo imaging system (Analytik Jena, Germany).

In vivo pharmacokinetic study
A total of 120 Balb/c mice were randomly assigned into three groups, including the oral pure kaempferol group, the kaempferol nanosuspension group, and the intravenous kaempferol nanosuspension group, with each group consisting of 40 mice.Prior to the experiment, mice were fasted for 24 h, and the dosage of kaempferol administered was 15 mg•kg −1 , which was calculated based on the drug loading.Orbital blood was collected at predetermined time points of 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h post-administration and transferred to centrifuge tubes containing heparin sodium.The collected blood samples were centrifuged at 4000 r/min for 15 min to separate the supernatant and obtain plasma.Subsequently, 80 μL of plasma samples was fully mixed with 400 μL of methanol and centrifuged at 8000 r/min for 5 min.The supernatant was filtered through a 0.45-μm aqueous phase filter membrane and subsequently analyzed using HPLC, and the pharmacokinetic parameters were calculated by DAS 2.0 software.

In vivo safety evaluation study
In the antitumor efficacy study, primary tissues were gathered in sacrificed mice, and all the blood samples were centrifuged at 3000 rpm for 20 min to obtain serum, whose alkaline phosphatase (ALP), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and alanine aminotransferase (ALT) levels were measured to assess liver function, as well as serum creatinine (CRE) and urea nitrogen (UREA) levels were determined to assess nephrotoxicity.
All biochemical indicators of serum were measured by commercial kits (Biosino Biotechnolgy and Science Inc., China) using Microlab 300 (VITALAB, Netherlands) along with all experiments of histological which were conducted by standard inspection procedure.Specimens were obtained by embedding primary organs fixed with 4% (v/v) paraformaldehyde in paraffin blocks and then sectioned at 5 μm thickness and mounted on glass slides.Sections were stained with hematoxylin and eosin (H&E) and examined under an upright microscope (Nikon Corporation, Japan).

Statistical analysis
All results were presented as mean ± standard deviation.t test and one-way ANOVA were applied to establish the statistical differences between groups.The difference was considered significant at P < 0.05, and all statistical analyses were performed by the GraphPad Prism 8.0.

Preparation and process optimization of KAE-NSps
As briefly noted earlier, KAE-NSps, the preparation process of which was shown in Fig. S1B, were prepared by anti-solvent precipitation combined with high-pressure homogenization, and the optimal preparation process was confirmed using the method of single-factor analysis by changing the reaction conditions (Fig. S2A).TPGS and KAE were dissolved in methanol at a drug loading ratio of 3:1, and the deionized water was injected into it under 350 W ultrasonic power at 45 ℃.Then the methanol was removed by rotary evaporator, and the resulting solutions were homogenized 30 times under 1500 bar pressure to obtain TPGS-KAE-NSps, the particle size of which was the smallest under these conditions.The TPGS-KAE-NSps prepared was pale yellow liquid; the particle size was 186.6 ± 2.6 nm (Fig. 1A), which was signally reduced compared to the kaempferol nanosuspensions prepared in previous research [37]; the PDI and zeta potential was 0.158 ± 0.017 and −19.5 ± 0.9 mV, respectively.

Stability analysis
The particle size of nanocarriers, which affected stability, biodistribution, and cellular uptake, was very critical [43].From Fig. 1B, we could see that the particle size of nanosuspensions remained unchanged on the whole within 2 weeks.It could be concluded that the TPGS-KAE-NSps prepared had good stability under room temperature, which was convenient for storage and use.The particle size variation of nanosuspensions after being incubated with various physiological media was shown in Fig. 1C, which showed that the particle size of all mediums except plasma increased at the beginning and then changed not significantly.This demonstrated that TPGS-KAE-NSps prepared could exist in physiological media stably.However, plasma contained various serum albumins and enzymes that might be adsorbed on the surface of the nanoparticle, which led to the increase in particle size.The method of Coomassie brilliant blue was carried out to investigate whether the protein adsorption occurred, whose experimental result was shown in Fig. 1D.We could see that the absorbance of the nanosuspensions group increased compared with the control group, manifesting that some proteins were adsorbed onto TPGS-KAE-NSps.Therefore, it could be confirmed that the particle size of nanosuspensions increased after incubation in plasma was caused by the adsorption of protein.In general, TPGS-KAE-NSps prepared exhibited good stability whether in storage or in various physiological media, which was not only conducive to the development of subsequent study, but also provided a fundamental guarantee for the transportation, storage, and clinical use of drugs.

Lyophilization process of TPGS-KAE-NSps
The method of freeze-drying was often applied to achieve the nanosuspensions in solid form, which was conducive to storage, transportation, and accelerating later commercial production.However, the agglomeration of nanoparticles occurred during the lyophilization, which made it difficult for them to return to the initial state after re-dispersing in water.It was necessary that cryoprotectants should be added to prevent the clustering of drug nanoparticles and keep the original structure of the nanosuspensions [44].In this study, the TPGS-KAE-NSps lyophilized powders were prepared with different concentration cryoprotectants, the particle size of which was measured to screen out the optimal lyophilized prescription, and the results were presented in Fig. 1E and F. From the results, we could see that the lyophilized powders with glucose as cryoprotectant exhibited an excellent redispersibility, and the effect was impacted by the concentration.Hence, the 2% (w/v) glucose was selected for the cryoprotectant of TPGS-KAE-NSps.

Hemolysis test of TPGS-KAE-NSps
Hemolysis test was of great importance to the safety evaluation of drugs before they entered the market.In this experiment, the nanosuspensions were directly contacted with red blood cells to explore the safety of TPGS-KAE-NSps.The results of hemolysis test (Figs.1G and S2B) revealed that the hemolysis rates of TPGS-KAE-NSps with different concentrations (50, 100, 200 μg/mL) were all lower than 5%, which was permissible for biomaterials [45].This indicated that the TPGS-KAE-NSps had good biocompatibility, which was suitable for intravenous administration in vivo.

Solubility and drug release analysis
Here, we established a standard curve for the determination of kaempferol content by HPLC firstly.The full wavelength scan of kaempferol was shown in Fig. S3A that kaempferol had a maximum absorption peak at 366 nm.From Fig. S3B and C, we could see that the retention time of the kaempferol reference solution was 5.455 min as well as the blank solvent did not interfere with the determination of kaempferol by HPLC, which indicated the specificity met the requirements.The standard curve (Fig. S3D) obtained was y = 93818x −36287, R 2 = 0.9999, the linearity of which was great.The DLC calculated by the standard curve was 70.31 ± 2.11%, less than 75% of the theoretical drug loading slightly, which might be caused by the loss in the process of rotary evaporation and homogenization.The solubility of KAE and TPGS-KAE-NSps in different solvents was shown in Fig. 1H that the solubility of kaempferol was improved significantly after preparation of nanosuspensions.The reason why we selected the PBS containing 1% T-80 as the medium for drug release in vitro was that KAE had a maximum solubility in it.The drug release curve was shown in Fig. 2A that the release rate of TPGS-KAE-NSps was always lower than KAE, and its cumulative release rate of 19.94 ± 0.41% at 120 h was still much lower than KAE (44.44 ± 0.7%), which illustrated that TPGS-KAE-NSps had a certain sustained release effect and could be more effective for long-term controlled drug release.

Physicochemical characterization
The morphology evaluation of TPGS and TPGS-KAE-NSps was carried out by TEM.As shown in Figs.2B-D and S4, it could be seen from the TEM image that the TPGS was irregular circular shape as well as the TPGS-KAE-NSps was fusiform shape before and after the high press homogeneous.The average particle size of TPGS-KAE-NSps in the aqueous phase was 209.59 nm, which was nearly consistent with a previous result of DLS.FTIR spectroscopic analysis was conducted to estimate the latent interactions between KAE and TPGS.As can be seen from Fig. 2E, the FTIR peaks of TPGS-KAE-NSps did not change significantly compared with KAE, indicating that there was no interaction between KAE and TPGS, and also suggested that the chemical structure of KAE did not alter when preparing nanosuspensions, which was of great significance for ensuring the biological activity of KAE.XRD analysis was performed to assess the physical states of KAE, TPGS, physical mixture, and lyophilized TPGS-KAE-NSps.As shown in Fig. 2F, on the one hand, the XRD pattern of lyophilized TPGS-KAE-NSps largely retained the characteristic peak of KAE, suggesting that the crystalline nature of KAE remained unchanged.On the other hand, the relative intensity of the diffraction peaks of nanosuspensions decreased or disappeared compared with KAE, indicating that the crystallinity was reduced after the preparation of nanosuspensions, which had more amorphous structure than KAE.Moreover, this phenomenon might be related to the process of high-pressure homogenization, leading to the transformation of the crystal structure, which might increase the solubility and bioavailability of KAE subsequently [44].The DSC result of KAE, TPGS, physical mixture, and freeze-dried TPGS-KAE-NSps were displayed in Fig. 2G, respectively.As can be seen from the DSC curve, KAE has two endothermic peaks at 135.1 ℃ and 286 ℃ and an exothermic peak at 372.9 ℃, while compared with TPGS-KAE-NSps, the exothermic peak of which disappeared, the endothermic peak of 135.1 ℃ shifted to 110 ℃, and the endothermic peak of 286 ℃ shifted to 276.4 ℃ as well as the peak intensity was obviously weakened.This phenomenon might be caused by the tight encapsulation of the drug particles by the stabilizer.Therefore, it could be concluded that some crystal structures of the drug should be transformed in nanosuspensions, which was consistent with the XRD results.Thermogravimetric analysis experiment was conducted to study the decomposition temperature and mass changes of substances.TG and DTG curves were shown in Fig. 2H and I; TPGS-KAE-NSps had a mass loss of 2.53% between 97.8 and 114.6 °C, which might be due to the loss of bound water during the continuous heating process.The mass rapidly lost 23.44% between 334.4 and 380.9 ℃, suggesting that decomposition has occurred at this time, and its remaining mass remained at 39.04% until 600 °C.Compared with the physical mixture, the residual mass of which remained at 12.22% after being heated to 600 °C, showing that the thermal stability of the nanosuspensions was better than the physical mixture significantly.In short, the method of four characterizations (FTIR, XRD, DSC, and TG) showed that the characteristic peaks of KAE and TPGS-KAE-NSps had no significant differences, which strongly suggested that KAE was still in a crystalline state even within the nanosuspensions.

In vitro cytotoxicity, cell scratch, and cellular uptake
CCK-8 assay was conducted to assess the cytotoxicity of TPGS-KAE-NSps and KAE.The viability of tumor cells (4T1, U251, HepG2, SGC-7901) treated with the TPGS-KAE-NSps for 24 h was generally lower than those treated with the KAE (Fig. 3A), which had a significant difference in the 4T1 and U251group at all concentration.The results showed that the toxicity of TPGS-KAE-NSps on tumor cells was higher than that of KAE at the same concentrations with dose-dependent.There are two reasons to explain this phenomenon.On one hand, TPGS is a well-known inhibitor of P-glycoprotein (P-gp), which is an efflux pump that can expel drugs out of cells and decrease their effectiveness.TPGS can improve the intracellular accumulation of drugs by inhibiting overexpressed P-gp [46,47].Therefore, TPGS-KAE-NSps may have better intracellular drug delivery and higher cytotoxicity.On the other hand, nanoparticle delivery systems like TPGS-KAE-NSps can enhance the cellular uptake of drugs through endocytosis, a process where cells engulf extracellular material into vesicles [48].This improved cellular uptake can lead to higher intracellular drug concentrations and stronger cytotoxicity.The scratch test was carried out for tumor cells treated with TPGS-KAE-NSps and KAE to determine whether nanosuspensions could retard the migration of 4T1 and U251 cells.As showed in Fig. 3B, the wound healing rate of 4T1 and U251 cells in the control group was 66.35 ± 7.56% and 57.04 ± 3.98% at 24 h after scratching, respectively.The wound healing rates of 4T1 and U251 treated with KAE and TPGS-KAE-NSps were 38.85 ± 4.69% and 6.04 ± 1.22% and 33.65 ± 3.18% and 19.08 ± 1.86% at 24 h, respectively.It was concluded that tumor cells possessed high metastatic potential motility, and TPGS-KAE-NSps group displayed a better metastatic inhibition effect than KAE.TPGS-KAE-NSps labeled with DIR was prepared to investigate the cellular uptake of nanosuspensions by utilizing the fluorescence properties of DIR, and CLSM was exploited to observe the cellular uptake of 4T1 and HepG2.From Fig. 4A and B, we could see that the red fluorescence of TPGS-KAE-NSps group was located around the cell nuclei dyed blue by DAPI, revealing that TPGS-KAE-NSps could be taken in by 4T1 and HepG2 cells and distributed in the cytoplasm, thus realizing transmembrane transfer.

Cell apoptosis and ROS detection
We preliminarily explored the antitumor mechanism of kaempferol nanosuspensions.Apoptosis was a major mechanism of cancer cell death [49].Flow cytometry and annexin V-FITC/PI staining were applied to appraise the effect of KAE and TPGS-KAE-NSps on 4T1 and U251 cells apoptosis.As shown in Fig. 5A, the apoptosis rates of 4T1 and U251 cells with TPGS-KAE-NSps were 22.47 ± 1.08% and 19.22 ± 0.98%, which was notably higher than the control group (1.82 ± 0.2%, 2.12 ± 0.04%) and KAE group (2.69 ± 0.11%, 5.39 ± 0.4%), indicating that TPGS-KAE-NSps prominently promoted the apoptosis of 4T1 and U251 cells.Studies [50,51] have demonstrated that drugs have the potential to induce apoptosis in tumor cells by augmenting the levels of intracellular ROS, which in turn modulate downstream cellular signaling pathways.Consequently, the fluorescence probe DCFH-DA was employed to evaluate the generation of ROS within cells.Whether 4T1 or U251 cells, as shown in Fig. 5B, that green fluorescence barely detectable in the control group was signally enhanced after treating with KAE and TPGS-KAE-NSps, while the results of fluorescence semi-quantitative further revealed the generation of intracellular ROS, where the ROS in TPGS-KAE-NSps group was significantly higher than those in the KAE group.The results above indicated that TPGS-KAE-NSps led to an elevation in intracellular ROS, which subsequently facilitated tumor cell apoptosis and inhibited tumor growth (Fig. S5).

Antitumor efficacy in vivo
The flow chart of in vivo antitumor efficacy study was shown in Fig. 6A, which consisted of the establishment of 4T1 tumor-bearing mouse model, treatment, and anatomy of mouse.KAE was insoluble in water, which was easy to cause a vascular blockage if it was injected through the tail vein.Thus, gavage was suitable, while KAE nanosuspensions  can be administered by gavage or intravenous injection.The tumor-bearing mice were treated with different administration methods and doses to assess the antitumor efficacy in vivo.The growth of tumor volume in each group over time was shown in Fig. 6B.The results indicated that the growth of tumor volume from fast to slow was KAE (15 mg•kg −1 , ig), TPGS-KAE-NSps (15 mg•kg −1 , ig), TPGS-KAE-NSps (5 mg•kg −1 , iv), TPGS-KAE-NSps (10 mg•kg −1 , iv), and TPGS-KAE-NSps (15 mg•kg −1 , iv) in order, and the differences were significant compared with the control group, which suggested that KAE and its nanosuspensions were effective in treating the breast cancer.In order to evaluate the therapeutic effect of TPGS-KAE-NSps intuitionistic, mice were put to death after the last treatment while the tumor tissues were achieved.The TPGS-KAE-NSps (15 mg•kg −1 , iv) group had the smallest tumor volume in all treatment groups, which demonstrated the best therapeutic efficiency (Fig. 6C), while could also be concluded by the weight of the tumors in the mice (Fig. 6D).The TIR calculated was based on the final volume of tumor collected (Fig. 6E), where KAE (15 mg•kg −1 , ig) and TPGS-KAE-NSps (5 mg•kg −1 , iv) showed an inhibition rate of 31.02 ± 2.96% and 46.2 ± 5.14%, respectively.This indicated that KAE administered orally was much less bioavailable than intravenous injection.Moreover, the TIR of TPGS-KAE-NSps (15 mg•kg −1 , ig) and TPGS-KAE-NSps (15 mg•kg −1 , iv) was 38.1 ± 5.54% and 68.9 ± 1.46%, which revealed that the bioavailability of KAE for oral administration was limited even if it was prepared as a nanosuspensions.The main reason was that there was a strong intestinal metabolism after oral administration of drugs was metabolized a large proportion by the intestinal enzyme system before absorption, which further reflected the advantage of nanosuspension that can be injected intravenously.In addition, the TIR of TPGS-KAE-NSps (15 mg•kg −1 , ig) compared with KAE (15 mg•kg −1 , ig) at the same dose and through the same administration route, however, was improved slightly, but the difference was not significant.We hypothesized that it might be related to the concentration of the drug or the limitations of low bioavailability of oral administration.In short, KAE was prepared into nanosuspensions and administered by intravenous injection, improved the bioavailability, and enhanced the antitumor efficacy in vivo, which might be a feasible preparation form for clinical application and had a good development prospect in the treatment of breast cancer.

Biodistribution in vivo
In order to assess biodistribution in vivo, mice bearing tumors were injected with TPGS-KAE-NSps labeled with DIR intravenously.The fluorescence image was shown in Fig. 6F that TPGS-KAE-NSps were primarily distributed in the liver and tumor after administration.The average intensity of fluorescence signal was shown in Fig. 6G that the fluorescence signal of the tumor tissue was always in a strong state from 0.5 to 8 h, which indicated that TPGS-KAE-NSps could rapidly reach to the tumor tissue through blood circulation and maintain for a period of time.Even after 24 h, the intensity of TPGS-KAE-NSps was still significantly different from that of control group.These results demonstrated that TPGS-KAE-NSps prepared had a certain passive targeting effect and could continuously reach the tumor site through EPR effect [52].

Pharmacokinetic study
The plasma concentration-time profiles were depicted in Fig. 6H, which revealed that TPGS-KAE-NSps reached peak concentration in vivo faster after intravenous injection, and achieved a much higher maximum blood concentration in vivo (803.32 ± 65.60 ng/mL) compared to oral administration.The critical pharmacokinetic parameters (Table 1) demonstrated that the AUC 0-t of TPGS-KAE-NSps (15 mg•kg −1 , ig) group and TPGS-KAE-NSps (15 mg•kg −1 , iv) group were 4.83-fold and 8.81-fold higher than that of KAE (15 mg•kg −1 , ig) group, respectively.These results indicate that the bioavailability of KAE was significantly enhanced upon formulation into nanosuspension, irrespective of the route of administration.Notably, the relative bioavailability of TPGS-KAE-NSps via intravenous injection was 182.52% as compared to oral TPGS-KAE-NSps, underscoring the superior efficacy of the intravenous route of administration.Furthermore, the half-life (T 1/2 ) and average retention time (MRT 0-t ) of

Safety evaluation in vivo
In terms of safety evaluation in vivo, the body weight change and organ coefficients of mice in each group were shown in Figs.6I and S6.There was no significant difference in body weight between each group and the control group, the same as the organ coefficients.It could be concluded that mice treated with KAE and TPGS-KAE-NSps had no negative side effects.Additionally, to further evaluate the safety, blood biochemistry experiment and histological analysis were carried out.As shown in Fig. 7A, the blood biochemical analysis results in each index were within the normal range and had no significant difference among the groups, revealing that there was no obvious damage to the functions of liver and kidney.The histopathological slices images of the main organs including the heart, liver, spleen, lungs, kidney, and brain were shown in Fig. 7B, which further confirmed the low toxicity.No obvious signs of necrosis in main organs were observed, implying that TPGS-KAE-NSps had negligible systemic toxicity and good biocompatibility in vivo [53].

Conclusion
In this research, we developed a kaempferol nanosuspensions via the method of anti-solvent precipitation combined with high-pressure homogenization successfully and adopted single-factor analysis method to obtain the optimal preparation process.The particle size of TPGS-KAE-NSps optimized, which was 186.6 ± 2.6 nm, was greatly reduced compared to the previous study.Furthermore, TPGS-KAE-NSps prepared had a high drug loading capacity and solubility, good stability, and biocompatibility.Two percent (w/v) glucose was chosen as the cryoprotectant for TPGS-KAE-NSps, which had a certain sustained release effect.Most important of all, TPGS-KAE-NSps prepared markedly improved the antitumor effect of KAE in vitro and in vivo, alongside a superior pharmacokinetic behavior in vivo compared to KAE.In all, our work not only provided some new ideas for the research and development of kaempferol nanoparticles, but also provided a certain theoretical foundation for the potential application of KAE in clinical antitumor.

Fig. 2 A
Fig. 2 A In vitro drug release of KAE and TPGS-KAE-NSps (mean ± SD, n = 3).B TEM images of TPGS.C TEM images of TPGS-KAE-NSps after the high press homogeneous.The scale bar was 500 nm.D Frequency diameter distribution (%) of TPGS-KAE-

Fig. 6 A
Fig. 6 A The flow chart of in vivo antitumor efficacy study.B The growth of tumor volume (mean ± SD, n = 5, *** P < 0.001).C The images of mice tumor tissues after the last treatment.D The tumor weight of mice (mean ± SD, n = 5, * P < 0.05).E The TIR of mice was based on the final volume of tumor (mean ± SD, n = 5, ** P < 0.01, *** P < 0.001).F Fluorescence distribution of major organs at 0.5, 1, 2,

Fig. 7 A
Fig. 7 A The blood biochemical analysis of ALT, AST, ALP, UREA, CRE, and LDH (mean ± SD, n = 3).B The images of histopathological slices of the main organs including the heart, liver, spleen, lungs, kidney, and brain after staining with H&E.The scale bar was 200 μm

Table 1
TPGS-KAE-NSps were longer, while the clearance rate (CL) was lower than KAE, whether orally or intravenously, indicating that the nanosuspension formulation could prolong the drug action time by reducing the clearance rate of KAE in mice.