Highly Biocompatible Apigenin-Loaded Silk Fibroin Nanospheres: Preparation, Characterization, and Anti-Breast-Cancer Activity

Breast cancer is among the most common fatal diseases among women. Low-toxicity apigenin (AGN) is of interest due to its good antitumor activity, but its clinical application is severely limited due to its poor water solubility and low bioavailability. An effective strategy to enhance the anti-breast-cancer activity of AGN is to develop it as a nanodelivery system. Silk fibroin (SF) is an ideal drug carrier with good biocompatibility, biodegradability, and a simple extraction process. This paper develops a novel and efficient apigenin-loaded silk fibroin nanodelivery system (SF-AGN) by nanoprecipitation with SF as a carrier. The system was characterized in terms of morphology, zeta potential, particle size, ultraviolet (UV), infrared (IR), and synchronous thermal analyses (TG-DSC), and the in vitro cytotoxicity and in vivo pharmacokinetics were examined. Finally, the chronic toxicity of SF-AGN in mice was studied. The SF-AGN nanodelivery system has good dispersibility, a hydrated particle size of 163.35 nm, a zeta potential of −18.5 mV, an average drug loading of 6.20%, and good thermal stability. MTT studies showed that SF-AGN significantly enhanced the inhibitory effect of AGN on 4T1 and MDA-MB-231 cells. Pharmacokinetic studies have demonstrated that SF-AGN can dramatically improve the bioavailability of AGN. The results of toxicity experiments showed that SF-AGN is biocompatible and does not alter normal tissues or organs. In sum, the SF-AGN nanodelivery system is a promising drug-delivery system for the clinical treatment of breast cancer.


Introduction
Globally, cancer is a major public health problem; it is a disease in which certain cells of the body grow uncontrollably and spread to other parts of the body, and it has high morbidity and mortality [1]. Breast cancer (BC) is the leading cause of cancer-related disability-adjusted life years and death in women worldwide, with 2.1 million new cases diagnosed globally each year. It is estimated that 627,000 deaths occurred in 2018, accounting for 15% of the annual cancer deaths among women [2]. Breast cancer is clinically characterized by high incidence, high invasiveness, and poor prognosis. The most effective treatment for early-stage breast cancer patients is surgery combined with other adjuvant treatments. As for breast cancer patients who are clinically diagnosed with cancer spread or recurrence, especially those in an advanced stage, chemotherapy is usually used to significantly prolong the survival time of patients [3]. Unfortunately, chemotherapy is usually associated with adverse effects such as side effects and drug resistance as well as long treatment cycles and high treatment costs [4]. Compared with other medications, plant-derived  Figure 1 shows the process of SF extraction from cocoons [18,19]. A solution of Na 2 CO 3 at a concentration of 0.02 M was prepared accurately, and the cocoons were boiled in Na 2 CO 3 for 30 min. This process was repeated three times, replacing the solution with fresh Na 2 CO 3 . Next, the solution was centrifuged at 3000 rpm for 10 min, the supernatant was discarded, the degummed silk fibers were rinsed with distilled water, and the excess water was squeezed. The silk fibers were then placed in an oven and dried at 50 • C to obtain the silk fibers. The extracted filament fibers were dissolved in Ajisawa reagent (CaCl 2 : ethanol: water, 1:2:8 molar ratio) at 60 • C for 4 h. The insoluble impurities were removed by filtration; the fibers were transferred to a dialysis bag with a cut-off molecular weight of 14,000 Da for 48 h using deionized water as the dialysis medium. The retention solution was transferred to a beaker and dried in an oven to obtain SF. The retained solution was transferred to a beaker and dried in an oven (50 • C) to obtain a lumpy solid, which was gently ground to obtain SF flowable powder.

Extraction of Silk Fibroin
Polymers 2023, 14, x FOR PEER REVIEW 3 of 12 Experiment Center of Jinzhou Medical University, China (Jinzhou, China). The animal study protocol was approved by the Animal Ethics Committee of Jinzhou Medical University of China (20220311001). All the water used was double-distilled water. Figure 1 shows the process of SF extraction from cocoons [18,19]. A solution of Na2CO3 at a concentration of 0.02 M was prepared accurately, and the cocoons were boiled in Na2CO3 for 30 min. This process was repeated three times, replacing the solution with fresh Na2CO3. Next, the solution was centrifuged at 3000 rpm for 10 min, the supernatant was discarded, the degummed silk fibers were rinsed with distilled water, and the excess water was squeezed. The silk fibers were then placed in an oven and dried at 50 °C to obtain the silk fibers. The extracted filament fibers were dissolved in Ajisawa reagent (CaCl2: ethanol: water, 1:2:8 molar ratio) at 60 °C for 4 h. The insoluble impurities were removed by filtration; the fibers were transferred to a dialysis bag with a cut-off molecular weight of 14,000 Da for 48 h using deionized water as the dialysis medium. The retention solution was transferred to a beaker and dried in an oven to obtain SF. The retained solution was transferred to a beaker and dried in an oven (50 °C ) to obtain a lumpy solid, which was gently ground to obtain SF flowable powder.

Preparation of SF-AGN Nanospheres
Hydrophobic amino acid residues in SF molecules, such as tyrosine, glycine, and alanine, can effectively allow the entrapment of hydrophobic drug AGN through hydrophobic interaction and p-p stacking [17]. SF nanospheres were prepared by the nanoprecipitation method. Briefly, an appropriate amount of AGN was weighed and dissolved in DMSO. The resulting solution was added dropwise to SF aqueous solution (2 mg/mL), stirred and mixed well and then left for 4 h in a refrigerator at 4 °C and stirred for 12 h in a water bath at 40 °C ; the solution was centrifuged at 3000 r/min for 30 min, and the precipitate was washed three times with purified water and dried in a vacuum drying oven at 40 °C , and SF-AGN nanospheres were obtained.

Analysis of the Secondary Structure of SF
The secondary structure analysis was carried out by scanning the infrared spectra of silk fibroin. A small amount of silk fibroin was taken, mixed well with potassium bromide in proportion, and pressed into a translucent sheet of 1 mm thickness by a press. The infrared spectrum of silk fibroin was obtained by Fourier infrared spectroscopy with wave number 4000-400 cm −1 and a resolution of 4 cm −1 .

Preparation of SF-AGN Nanospheres
Hydrophobic amino acid residues in SF molecules, such as tyrosine, glycine, and alanine, can effectively allow the entrapment of hydrophobic drug AGN through hydrophobic interaction and p-p stacking [17]. SF nanospheres were prepared by the nanoprecipitation method. Briefly, an appropriate amount of AGN was weighed and dissolved in DMSO. The resulting solution was added dropwise to SF aqueous solution (2 mg/mL), stirred and mixed well and then left for 4 h in a refrigerator at 4 • C and stirred for 12 h in a water bath at 40 • C; the solution was centrifuged at 3000 r/min for 30 min, and the precipitate was washed three times with purified water and dried in a vacuum drying oven at 40 • C, and SF-AGN nanospheres were obtained. The secondary structure analysis was carried out by scanning the infrared spectra of silk fibroin. A small amount of silk fibroin was taken, mixed well with potassium bromide in proportion, and pressed into a translucent sheet of 1 mm thickness by a press. The infrared spectrum of silk fibroin was obtained by Fourier infrared spectroscopy with wave number 4000-400 cm −1 and a resolution of 4 cm −1 .

Particle Size, Zeta Potential, and Surface Morphology of SF-AGN
The SF-AGN nanospheres were diluted with a small amount of water to create a suspension of 0.2 mg/mL concentration. Their particle size and potential were determined using a particle size analyzer (Brookhaven 90Plus PALS, Suffolk, NY, USA). The morphological characteristics of SF-AGN were investigated using transmission electron microscopy (TEM, FEI Talos F200X, FEI Corporation, Hillsboro, OR, USA). Sample preparation for TEM was performed with a 0.2 mg/mL suspension of nanospheres in pure water followed by sonication for 3 min with an amplitude of 30% [20]. A drop of this suspension was placed on a 200-mesh copper mesh coated with carbon. After natural drying at room temperature, the copper mesh was stained with a 1% phosphotungstic acid solution for 60 s. The copper mesh was imaged by TEM at a voltage of 80 kV.

Drug Loading (DL) of SF-AGN Nanospheres
The content of AGN was determined by UV-visible spectrophotometry [21]. The standard curve obtained is y = 0.0218x + 0.009, R 2 = 0.9992 ( Figure S3). The amount of SF-AGN was weighed precisely, added to 30% ethanol-DMSO mixture, sonicated to make it fully dissolved, filtered using 0.22 µm microporous membrane, and its absorbance was measured by UV to calculate its AGN content and the drug loading of SF-AGN; the experiment was repeated three times. The DL was calculated using the following formula: SF-AGN was taken appropriately, mixed with potassium bromide in proportion, and then pressed. The transmittance spectra of the silk fibroins were measured by FTIR spectroscopy (WQF-510A, Beijing Ruili, Beijing, China) with a wave number of 4000-400 cm −1 and a resolution of 4 cm −1 . Another appropriate amount of SF blank carrier, AGN, SF, and AGN mixture was taken and analyzed by infrared spectroscopy according to the above conditions.

Thermal Stability Test of SF-AGN
The SF-AGN nanospheres were weighed appropriately and tested by a simultaneous thermal analyzer (TGA, TGA/DSC Type 3+, Mettler-Toledo, Greifensee, Switzerland). The heating rate was set to 10 • C/min, the heating range was 25-400 • C, and the controlled N 2 flow rate was 50 mL/min.

Cell Culture
Two cell models, the human breast cancer cell line (MDA-MB-231) and murine breast cancer cell line (4T1), were selected for this experiment. The MDA-MB-231 was grown according to the culture conditions recommended in the literature [22]. MDA-MB-231 was cultured using DMEM medium containing 10% fetal bovine serum in a 37 • C, 5% CO 2 incubator. Fresh medium was changed twice a week and passaged at 1:3, with trypsin digestion at room temperature for 1-2 min. 4T1 was grown using RPMI 1640 medium containing 10% fetal bovine serum in a 37 • C, 5% CO 2 incubator. The medium was changed every 2-3 days and passaged at 1:4 at 80% confluence.

In Vitro Antitumor Activity
Cells were seeded into 96-well plates at a density of 10 5 cells/well and incubated in 95% air and 5% CO 2 incubator at 37 • C for 24 h. Then, different concentrations (3.75-60 µg/mL) of AGN and SF-AGN nanospheres were added to the plates, and after 24 h of treatment, cells were washed twice with PBS, and serum-free medium without phenol red was replaced in all wells. Next, 5 mg/mL MTT solution was added to incubate the cells for 4 h. The MTT solution was discarded and washed twice with PBS, and 100 µL of DMSO was added and shaken to dissolve the formazan fully. The optical density (OD) of each well was measured at 490 nm using an enzyme marker, and the cell survival rate was calculated [cell survival rate (%) = OD administration group/OD blank control group × 100%]. Cell viability was expressed as the percentage of viable cells compared to the control group. A growth medium without the treatment drug was used as a control in each experiment. The column used in this experiment was a C18 column (4.6 × 250 mm, 5 µm) [23]. The mobile phase was acetonitrile-0.2% phosphoric acid aqueous solution (50:50) at a flow rate of 1 mL/min, the detection wavelength was 270 nm, the column temperature was 30 • C, isocratic elution was used, and the injection volume was 20 µL each time.

Plasma Processing
Briefly, 100 microliters of plasma was taken in a 1.5 mL centrifuge tube, to which 1 mL of methanol was added to precipitate the protein. The plasma proteins were precipitated by vortex mixing for 1 min, followed by centrifugation at 12,000 rpm for 3 min. The supernatant was passed through a 0.22 µm microporous membrane, and the drug concentration was calculated according to the above chromatographic conditions.

Dosing and Sampling Protocols
Six mice were randomly divided into two groups of three mice each and fasted for 12 h before the experiment but were allowed to drink freely. Blood was collected by orbital blood sampling at the 1st, 2nd, 3rd, 4th, 5th, 6th, and 7th hours after administration, and 0.1 mL of blood was placed in a centrifuge tube with sodium heparin solution, treated in the same way as above, and stored in a refrigerator at −80 • C. An equal amount of saline was also injected intraperitoneally to maintain a constant blood volume in the animals.

In Vivo Toxicity Study
To assess the chronic toxicity of SF-AGN, mice were treated with SF-AGN for two weeks at a dose of 25 mg/kg. After 2 weeks, the mice were sacrificed, and various organs (heart, liver, spleen, lungs, and kidneys) were removed by autopsy and fixed in 4% paraformaldehyde. These tissues were sectioned, stained with hematoxylin-eosin (H&E), and photographed and observed [24].

Statistical Analysis
Microsoft Excel calculated the mean and standard deviation (SD). SD analysis was performed on all samples in triplicate. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc comparison test. A confidence level of p < 0.05 was considered significant.

Results and Discussion
3.1. Characterization of SF and SF-AGN Nanospheres 3.1.1. Analysis of the Secondary Structure of SF Protein secondary structure is the structure of a polypeptide chain formed by hydrogen bonding and coiling, including α-helix, β-folding, β-turning, and irregular coiling. When the external conditions change, the protein secondary structure changes accordingly. Currently, Fourier infrared spectroscopy is the main method to study protein secondary structure. According to the literature, the secondary structure of the silk fibroin is different, and the wave numbers of the characteristic peaks corresponding to the amide bonds in its molecular structure are also different. The main secondary structure in the structure can be judged [25,26]. The wave numbers of the characteristic peaks corresponding to the amide bonds in the molecular structure are shown in Table 1. According to the results in Figure 2 and Table 1, it can be found that the FTIR spectra of SF exhibit typical peaks of crystalline β-sheet at 1530 cm −1 (amide II), 1625 cm −1 (amide-I), and 1263 cm −1 (amide-III), demonstrating that SF was in its stable conformation [17]. The secondary structure of the extracted silk protein in this experiment was mainly β-folded, and its crystal structure was a more stable type, which was consistent with the previously reported results [17,27] and indicated that we successfully extracted SF. different, and the wave numbers of the characteristic peaks corresponding to the amide bonds in its molecular structure are also different. The main secondary structure in the structure can be judged [25,26]. The wave numbers of the characteristic peaks corresponding to the amide bonds in the molecular structure are shown in Table 1. According to the results in Figure 2 and Table 1, it can be found that the FTIR spectra of SF exhibit typical peaks of crystalline β-sheet at 1530 cm −1 (amide II), 1625 cm −1 (amide-I), and 1263 cm −1 (amide-III), demonstrating that SF was in its stable conformation [17]. The secondary structure of the extracted silk protein in this experiment was mainly β-folded, and its crystal structure was a more stable type, which was consistent with the previously reported results [17,27] and indicated that we successfully extracted SF.  The DLS particle size analysis of SF-AGN nanospheres is shown in Figure 3A. The particle size of SF-AGN is uniformly distributed with a "bell-shaped curve", and the average particle size is 163.35 nm ±1.22 nm. The morphology of SF-AGN nanospheres is shown in Figure 3B. It can be seen that the nanospheres are oval or spherical in shape, the particle size is mainly distributed around 140 nm, and no apparent adhesion was observed. This result is slightly smaller than the DLS result, which may be because the particles were dry in TEM. In contrast, under DLS conditions, on the one hand, the particles were dispersed in water, and the swelling effect was caused by protein-water interaction; on the other hand, the DLS size included the diffusion layer around the particles [25]. The zeta potential of the nanospheres was also measured to be −18.5 mV. Zeta potential is a  Figure 3A. The particle size of SF-AGN is uniformly distributed with a "bell-shaped curve", and the average particle size is 163.35 nm ±1.22 nm. The morphology of SF-AGN nanospheres is shown in Figure 3B. It can be seen that the nanospheres are oval or spherical in shape, the particle size is mainly distributed around 140 nm, and no apparent adhesion was observed. This result is slightly smaller than the DLS result, which may be because the particles were dry in TEM. In contrast, under DLS conditions, on the one hand, the particles were dispersed in water, and the swelling effect was caused by protein-water interaction; on the other hand, the DLS size included the diffusion layer around the particles [25]. The zeta potential of the nanospheres was also measured to be −18.5 mV. Zeta potential is a crucial determinant of colloidal dispersion stability. Zeta potential describes the degree of electrostatic repulsion between neighboring, similarly charged particles [28]. The zeta potential of SF-AGN nanospheres was greater than 15 mV in absolute value, indicating good colloidal stability [27]. The UV test results showed that the drug loading capacity of SF was 6.20% ± 0.25%, which could meet the needs of subsequent formulation studies. crucial determinant of colloidal dispersion stability. Zeta potential describes the degree of electrostatic repulsion between neighboring, similarly charged particles [28]. The zeta potential of SF-AGN nanospheres was greater than 15 mV in absolute value, indicating good colloidal stability [27]. The UV test results showed that the drug loading capacity of SF was 6.20% ± 0.25%, which could meet the needs of subsequent formulation studies.

FTIR of SF-AGN
The results of IR analysis of the SF blank carrier, AGN, SF, AGN mixture, and SF-AGN are shown in Figure 4. From the figure, it can be seen that SF-AGN has characteristic peaks at 1630 cm −1 , 1517 cm −1 , 1243 cm −1 , and 691 cm −1 , from which it can be inferred that the secondary structure of SF nanospheres is still dominated by β-folding after loading AGN, indicating that the loading of AGN does not affect the secondary structure of SF and still has specific stability. Meanwhile, compared with SF nanospheres, new characteristic absorption peaks appeared at 2815 cm −1 , 2715 cm −1 , 1350 cm −1 , and 829 cm −1 , which corresponded to the absorption peaks of AGN, indicating that AGN was successfully loaded.

FTIR of SF-AGN
The results of IR analysis of the SF blank carrier, AGN, SF, AGN mixture, and SF-AGN are shown in Figure 4. From the figure, it can be seen that SF-AGN has characteristic peaks at 1630 cm −1 , 1517 cm −1 , 1243 cm −1 , and 691 cm −1 , from which it can be inferred that the secondary structure of SF nanospheres is still dominated by β-folding after loading AGN, indicating that the loading of AGN does not affect the secondary structure of SF and still has specific stability. Meanwhile, compared with SF nanospheres, new characteristic absorption peaks appeared at 2815 cm −1 , 2715 cm −1 , 1350 cm −1 , and 829 cm −1 , which corresponded to the absorption peaks of AGN, indicating that AGN was successfully loaded.
colloidal stability [27]. The UV test results showed that the drug loading capacity of S was 6.20% ± 0.25%, which could meet the needs of subsequent formulation studies.

FTIR of SF-AGN
The results of IR analysis of the SF blank carrier, AGN, SF, AGN mixture, and SF AGN are shown in Figure 4. From the figure, it can be seen that SF-AGN has characteristi peaks at 1630 cm −1 , 1517 cm −1 , 1243 cm −1 , and 691 cm −1 , from which it can be inferred tha the secondary structure of SF nanospheres is still dominated by β-folding after loadin AGN, indicating that the loading of AGN does not affect the secondary structure of S and still has specific stability. Meanwhile, compared with SF nanospheres, new character istic absorption peaks appeared at 2815 cm −1 , 2715 cm −1 , 1350 cm −1 , and 829 cm −1 , whic corresponded to the absorption peaks of AGN, indicating that AGN was successfull loaded.

Thermal Stability of SF-AGN
The thermogravimetric curves of SF-AGN nanospheres are shown in Figure 5. It can be learned from the figure that when the temperature was less than 260 • C, the weight loss rate of SF and SF-AGN was about 5%; their thermal stability was good and the difference was not obvious, which was mainly caused by the loss of adsorbed water and structural water on the surface and in the pore channel of SF nanospheres by heat [29], and the degradation of macromolecules did not occur. Furthermore, when the temperature rises to the range of 260-400 • C, the weight loss rates of SF and SF-AGN were about 40% and 50%, respectively, and the rate of mass loss began to accelerate, and the amount of loss increased, indicating that the organic matter and macromolecules in the nanospheres began to decompose in this temperature interval. When the temperature was 400 • C, the residual mass of SF was 55%, while the residual mass of SF-AGN was 45%, mainly because when the temperature was greater than 300 • C, the AGN in SF-AGN also underwent thermal decomposition, resulting in a rapid weight percentage decrease, and the weight loss was eventually higher than that of SF. In summary, SF-AGN nanospheres have good thermal stability.
The thermogravimetric curves of SF-AGN nanospheres are shown in Figure be learned from the figure that when the temperature was less than 260 °C , the loss rate of SF and SF-AGN was about 5%; their thermal stability was good and th ence was not obvious, which was mainly caused by the loss of adsorbed water an tural water on the surface and in the pore channel of SF nanospheres by heat [29] degradation of macromolecules did not occur. Furthermore, when the temperat to the range of 260-400 °C , the weight loss rates of SF and SF-AGN were about 4 50%, respectively, and the rate of mass loss began to accelerate, and the amoun increased, indicating that the organic matter and macromolecules in the nanosph gan to decompose in this temperature interval. When the temperature was 400 residual mass of SF was 55%, while the residual mass of SF-AGN was 45%, mainly when the temperature was greater than 300 °C , the AGN in SF-AGN also underw mal decomposition, resulting in a rapid weight percentage decrease, and the we was eventually higher than that of SF. In summary, SF-AGN nanospheres have go mal stability.

In Vitro Cytotoxicity
To assess the cytotoxicity of free AGN and SF-AGN, MTT assays were pe using breast cancer cell lines (4T1 and MDA-MB-231). Cells were incubated wi concentrations (3.75-60 μg/mL) of free AGN or SF-AGN for 24 h. Both free AGN AGN inhibited 4T1 ( Figure 6A) and MDA-MB-231 ( Figure 6B) breast cancer cells centration-dependent manner. However, SF-AGN showed better cytotoxic effects breast cancer cell lines compared to free AGN in the concentration range of 7 μg/mL (p < 0.05 vs. AGN), suggesting that encapsulation of AGN in SF enhances bition of AGN on breast cancer cells in vitro and therefore that SF-AGN nano have sound anti-breast-cancer effects. These results are consistent with previous highlighting the increased toxicity of common drugs when encapsulated in nano systems by a mechanism that may be attributed to the preferential uptake of nano carriers followed by efficient drug release within tumor cells [27,30].

In Vitro Cytotoxicity
To assess the cytotoxicity of free AGN and SF-AGN, MTT assays were performed using breast cancer cell lines (4T1 and MDA-MB-231). Cells were incubated with serial concentrations (3.75-60 µg/mL) of free AGN or SF-AGN for 24 h. Both free AGN and SF-AGN inhibited 4T1 ( Figure 6A) and MDA-MB-231 ( Figure 6B) breast cancer cells in a concentration-dependent manner. However, SF-AGN showed better cytotoxic effects on both breast cancer cell lines compared to free AGN in the concentration range of 7.5 to 60 µg/mL (p < 0.05 vs. AGN), suggesting that encapsulation of AGN in SF enhances the inhibition of AGN on breast cancer cells in vitro and therefore that SF-AGN nanoparticles have sound anti-breast-cancer effects. These results are consistent with previous reports highlighting the increased toxicity of common drugs when encapsulated in nanoparticle systems by a mechanism that may be attributed to the preferential uptake of nanoparticle carriers followed by efficient drug release within tumor cells [27,30].

Pharmacokinetic Studies
Using HPLC (UV230II, Dalian Elite Analytical Instruments, China) analysis, the AGN chromatogram is shown in Figure S1, and the results show that the peak time of AGN was about 6.15 min with good peak shape. The blood concentration of the samples was calculated, and the mean blood concentration-time curve was plotted using the mean blood concentration as the vertical coordinate and the sampling time as the horizontal coordinate ( Figure 7). From the drug-time curves, it can be seen that the blood concentration of each period of the SF-AGN group was significantly higher than that of the AGN group, indicating that SF-AGN nanospheres can be absorbed into the blood faster and the metabolism rate in the body is slowed down, which can improve the bioavailability of AGN.
Relevant pharmacokinetic parameters were calculated using the PK Slover software, and the non-atrial model non-pulsatile delivery mode was chosen for analysis. The pharmacokinetic parameters are shown in Table 2. Compared with free AGN, SF-AGN effectively prolonged the half-life and increased the relative bioavailability by 1.47 times, while the Cmax, AUC0-t, and MRTt were all raised to different degrees. The improved bioavailability may be attributed to the direct uptake of SF through the cell membrane by the lymphatic system, which facilitates drug absorption [31].

Pharmacokinetic Studies
Using HPLC (UV230II, Dalian Elite Analytical Instruments, China) analysis, the AGN chromatogram is shown in Figure S1, and the results show that the peak time of AGN was about 6.15 min with good peak shape. The blood concentration of the samples was calculated, and the mean blood concentration-time curve was plotted using the mean blood concentration as the vertical coordinate and the sampling time as the horizontal coordinate ( Figure 7). From the drug-time curves, it can be seen that the blood concentration of each period of the SF-AGN group was significantly higher than that of the AGN group, indicating that SF-AGN nanospheres can be absorbed into the blood faster and the metabolism rate in the body is slowed down, which can improve the bioavailability of AGN.

Pharmacokinetic Studies
Using HPLC (UV230II, Dalian Elite Analytical Instruments, China) analysis, the AGN chromatogram is shown in Figure S1, and the results show that the peak time of AGN was about 6.15 min with good peak shape. The blood concentration of the samples was calculated, and the mean blood concentration-time curve was plotted using the mean blood concentration as the vertical coordinate and the sampling time as the horizontal coordinate (Figure 7). From the drug-time curves, it can be seen that the blood concentration of each period of the SF-AGN group was significantly higher than that of the AGN group, indicating that SF-AGN nanospheres can be absorbed into the blood faster and the metabolism rate in the body is slowed down, which can improve the bioavailability of AGN.
Relevant pharmacokinetic parameters were calculated using the PK Slover software, and the non-atrial model non-pulsatile delivery mode was chosen for analysis. The pharmacokinetic parameters are shown in Table 2. Compared with free AGN, SF-AGN effectively prolonged the half-life and increased the relative bioavailability by 1.47 times, while the Cmax, AUC0-t, and MRTt were all raised to different degrees. The improved bioavailability may be attributed to the direct uptake of SF through the cell membrane by the lymphatic system, which facilitates drug absorption [31].  Relevant pharmacokinetic parameters were calculated using the PK Slover software, and the non-atrial model non-pulsatile delivery mode was chosen for analysis. The pharmacokinetic parameters are shown in Table 2. Compared with free AGN, SF-AGN effectively prolonged the half-life and increased the relative bioavailability by 1.47 times, while the C max , AUC 0-t , and MRT t were all raised to different degrees. The improved bioavailability may be attributed to the direct uptake of SF through the cell membrane by the lymphatic system, which facilitates drug absorption [31].

In Vivo Toxicity Study
We evaluated the biosafety of SF-AGN, as this property is a crucial parameter for using nanotherapeutics in cancer treatment [32]. Histological analysis (H&E staining) of major organs (heart, liver, spleen, lung, and kidney) showed negligible damage in the SF-AGN treatment group (Figure 8). No histopathological changes were found in the major organs. Cardiac myocytes were clear and well-arranged, and no necrosis, hemorrhage, or inflammatory exudates were observed. Therefore, the above results suggest that SF-AGN has a perfect safety profile after administration [24].

In Vivo Toxicity Study
We evaluated the biosafety of SF-AGN, as this property is a crucial parameter for using nanotherapeutics in cancer treatment [32]. Histological analysis (H&E staining) of major organs (heart, liver, spleen, lung, and kidney) showed negligible damage in the SF-AGN treatment group (Figure 8). No histopathological changes were found in the major organs. Cardiac myocytes were clear and well-arranged, and no necrosis, hemorrhage, or inflammatory exudates were observed. Therefore, the above results suggest that SF-AGN has a perfect safety profile after administration [24].

Conclusions
In this study, the nanoprecipitation method was successfully used to prepare an apigenin-loaded silk fibroin nanodelivery system (SF-AGN) with silk fibroin nanospheres as drug carriers. It exhibited nanoscale size (163.35 nm), spherical or oval shape, good thermal stability, and high drug loading. MTT experiments showed that SF-AGN showed significantly increased cytotoxicity against MDA-MB-231 and 4T1 cells compared to AGN. In vivo pharmacokinetic studies revealed that SF-AGN significantly prolonged the halflife of AGN and improved its bioavailability compared to free AGN. In addition, in vivo toxicity experiments showed that SF-AGN had good biocompatibility and no organ toxicity in mice. The above results suggest that SF could be a promising nanocarrier, and SF-AGN may be an effective agent for the treatment of breast cancer.
Supplementary Materials: The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Figure S1: High performance liquid chromatogram of apigenin; Figure S2: Chemical structure of silk fibroin; Figure S3: The obtain standard curve.

Conclusions
In this study, the nanoprecipitation method was successfully used to prepare an apigenin-loaded silk fibroin nanodelivery system (SF-AGN) with silk fibroin nanospheres as drug carriers. It exhibited nanoscale size (163.35 nm), spherical or oval shape, good thermal stability, and high drug loading. MTT experiments showed that SF-AGN showed significantly increased cytotoxicity against MDA-MB-231 and 4T1 cells compared to AGN. In vivo pharmacokinetic studies revealed that SF-AGN significantly prolonged the halflife of AGN and improved its bioavailability compared to free AGN. In addition, in vivo toxicity experiments showed that SF-AGN had good biocompatibility and no organ toxicity in mice. The above results suggest that SF could be a promising nanocarrier, and SF-AGN may be an effective agent for the treatment of breast cancer.