Bletilla striata polysaccharide (BSP, purity > 95%, MW≈50000) was purchased from Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd. (Shanghai, China). Vitamin E succinate (VES, purity 98%), andrographolide (AG, purity 98%), 1-yl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, purity 98%), 4-dimethylpyridine (DMAP, purity 99%), dimethyl sulfoxide (DMSO, purity 99%) and thiazolyl blue tetrazolium bromide (MTT, purity 98%) are all purchased from Shanghai Aladdin biochemical Technology Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS) was purchased from Zhejiang Tianhang Biotechnology Co.,Ltd. (Hangzhou, China). RPMI. 1640 medium and PBS were purchased from Zhejiang Senrui Biotechnology Co., Ltd. (Hangzhou, China). 0.25% trypsin and penicillin-streptomycin solution were purchased from Gibco Company in the United States. Anhydrous ethanol with 99% purity was purchased from Chengdu Jinshan Chemical Reagent Co., Ltd. (Sichuan, China). Chromatographic methanol was purchased from Anhui Tiandi High Purity Solvent Co., Ltd. (Anhui, China); water is ultra-pure water; other reagents are analytical purity.

The NCM460 cell (normal human colon epithelial cells), CT26-Luc (The luciferase-expressing mouse colon cancer cells) and CT26 (mouse colon cancer cells) were obtained from American Type Culture Collection (ATCC, Manassas, USA). These cell lines were cultured in RPMI- 1640, 10% FBS and 1% penicillin-streptomycin. BALB/c mice, SPF class, weighing 20 to 25 g, female, were purchased from Beijing Huafukang Biotechnology Co., Ltd. (certificate number: SCXK (Beijing) 2019–0008). Feeding conditions: the temperature is 22–25°C and the humidity is 40–70%. During the experiment, the animals were free to eat and drink, and the circadian rhythm was normal. The animal experiment was approved by the Animal Experimental Ethics Committee of Guizhou University of Traditional Chinese Medicine (Grant No.20220061).

Firstly, BSP-VES polymer was synthesized by esterification reaction in the presence of DMAP and EDC. Briefly, 202 mg of VES, 46.5 mg of DMAP, and 86.8 mg of EDC were dissolved in 15 mL of DMSO according to the ratio (N (VES):N(DMAP): N (EDC)= 1:1:1.2), then activated at 38°Cfor 1.5 h by stirring. Afterwards, 101 mg of BSP were dissolved in 25 mL of DMSO, and added into the mixture, which continued to react for 24 h. After reaction, 10 times the amount of ice anhydrous ethanol was added to the reaction solution, centrifuged and the acquired VES-BSP polymer was dried at 40°C. Then, the prepared VES-BSP polymer was dissolved in 8 mL of DMSO and transferred into a dialysis bag (Molecular weight cut-off, MWCO 3500) against deionized water for 48 h. The final BSP-VES polymer was obtained by freeze-drying.

BSP, VES, and BSP-VES structure were analyzed by 1H NMR on a Bruker Avance 500 (Billerica, MA, USA) with deuterated water (D2O) or DMSO. The BSP-VES polymer was diluted to 1 mg/mL with distilled water, and their particle size, PDI and zeta potential were measured using a DelsaMaxPRO Beckman nanoparticle size analyzer (Beckman, USA). The morphology of BSP-VES polymer was observed by transmission electron microscopy (TEM; JEOL JEM- 1230, Japan).

The critical micelle concentration (CMC) of BSP-VES polymer was determined by fluorescence spectrometry, as described in the literature (29). Briefly, a pyrene solution with a concentration of 1.2 × 10–5 mmol/mL was prepared with methanol and placed in a brown volumetric flask, and the methanol was quickly evaporated by nitrogen. Various concentrations of BSP-VES solution (2.5, 10, 25, 100, 200, 400 µg/mL) were added to a pyrene methanol solution. Then, the resulting mixture was balanced for 24 h at 37°C. The emission spectra of a series of pyrene-containing BSP-VES solutions at 300–500 nm were scanned by fluorescence spectrophotometry. The first peak (I=374 nm) and the third peak (I=385 nm) of the fluorescence emission spectrum were recorded, and the ratio of the two peaks was calculated. Taking the logarithm of the BSP-VES concentration Lg C as the abscissa and I374/I385 as the ordinate, the experimental data were analyzed and fitted, and the BSP-VES concentration corresponding to the intersection of the curves was the CMC value.

AG@BSP-VES was prepared by the dialysis method. Briefly, 29 mg of BSP-VES micelles were dissolved in ultrapure water. Then, 2.9 mg of AG was dissolved in absolute ethanol and added into the BSP-VES micelle solution. After stirring for 8 h, the micelle solution was transferred into a dialysis bag (3500 MWCO) against deionized water for 8 h. Finally, AG@BSP-VES was obtained through 0.45 microporous filter membrane.

Furthermore, High-performance Liquid Chromatography was used to evaluate the AG loading content and encapsulation efficiency in BSP-VES (HPLC, Agilent, China). Chromatographic conditions: C18 column (250 mm x 4.6 mm, 5 μm); The mobile phase was methanol-water solution (60:40) at a flow rate of 0.8 mL/min. The column temperature was 30°C. Detection wavelength was 226 nm and sample volume was 10 µL.

To draw the standard curve of AG, AG reference substance 10.1 mg was precisely weighed and diluted with methanol to the reference substance reserve solution of 0.202 mg/mL. Furthermore, 0.1, 0.5, 1, 2, 4, 6, 8 and 10 mL of the above solution were taken and supplemented with methanol to 10 mL, and then determined by HPLC. AG was extracted by organic solvent, and the content of AG was quantified by high-performance liquid chromatography. Encapsulation efficiency (EE) and drug loading (DL) were calculated by Equations 1 and 2, respectively.

In the formula, W₁ and W₂ represent the added mass of AG (mg) and the total mass of AG@BSP-VES (mg), respectively. C₁ represents the drug concentration in methanol (μg/mL). V represents the added methanol volume (mL) when disrupt the nanomicelles.

The stability of AG@BSP-VES polymer was further investigated by monitoring the changes in particle size, PDI and Zeta potential for 7 consecutive days using a Beckman nanoparticle size analyzer. During the experiment, the samples were stored at room temperature.

The hemocompatibility of the AG@BSP-VES micelle was evaluated by the erythrocyte hemolysis assay described in the literature (30, 31). Briefly, 2% (v/v) erythrocyte suspension obtained from the blood of rat was prepared by adding a certain amount of physiological saline. The AG@BSP-VES polymer was diluted with physiological saline to obtain solutions with concentrations of 0.172, 0.344, 0.685, 1.375, 2.750, 3.300, 4.240 and 5.500 mg/mL, respectively. Add 0.5 mL of AG@BSP-VES solution at various concentrations to a 2 mL EP tube containing 0.5 mL of erythrocyte suspension. The sample was shaken 30 min at 37°C, and centrifuged at 100 rpm for 15 min. Then, the supernatants of the samples in each group were collected. After that, the absorbance of supernatant at 540 nm was measured by the microplate reader. Physiological saline was used as a negative control group, and distilled water was used as a positive control group. The percentage of hemolysis was calculated through the formula below.

The sample was transferred into a dialysis bag (MWCO 3.5 kDa), and then submerged into phosphate buffer PBS medium (pH=7.4) under the stirring (100 rpm) at 37°C. At the pre-designed time point (0, 0.5, 2, 4, 8, 12 and 24 h), 1 mL of release medium was taken out, and the fresh release medium of the same volume was supplemented. The released AG in the medium was determined by HPLC, and the cumulative release of the AG was calculated according to the standard curve.

NCM460 or CT26 cells in the logarithmic growth phase, were digested with 0.25% trypsin into a single cell suspension. The suspension was then diluted to a concentration of 2×105 cells/mL using RPMI- 1640. Then, a 1-mL cell suspension was seeded into a 24-well cell plate and placed in an incubator at 37°C with 5% CO2 for 24 h. After that, the Coumarin 6 (C6)-labeled AG@BSP-VES (AG, 2μg/mL) was diluted to a concentration of 1 mg/mL and added to a 24-well plate in a time gradient 1, 2, 4, 12 and 24 h. For investigating the targeted cellular uptake of AG@BSP-VES, CT26 and NCM460 cells were incubated with AG@BSP-VES (AG, 2μg/mL) for 2 h. At the end of the incubation period, the supernatant was removed and washed twice with PBS to eliminate any residual serum and free drug interfering substances (32). Finally, the cellular fluorescence was examined using a fluorescence microscope (Leica DMIL LED; Leica, Solms, Germany). Furthermore, the cellular fluorescence intensity was determined through the Image J Software.

The in vitro biosafety of BSP-VES was evaluated using the thiazolyl blue tetrazolium bromide (MTT) assay. NCM460 or CT26 cells were seeded in 96-well plates and treated with BSP-VES at concentrations of 0.125, 0.25, 0.5, 0.7, 0.8, and 1 mg/mL for 24 or 48 h. Untreated blank cells were used as the control. Then, 20 µL of MTT (5 mg/mL) was added to each well and cultured for 4 h in the incubator. Afterward, the supernatants were removed, and 100 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The absorbance of cells at 570 nm was measured using a Microplate Reader (Thermo, Multiskan Sky, USA), and the cell proliferation rate was calculated using the provided formula.

The in vitro antitumor activity of AG@BSP-VES was evaluated by MTT assay, the fluorescent live/dead (33) and the scratch wound assay (34). For the MTT assay, CT26 cells were initially inoculated into a 96-well plate at a density of 5×104/mL. Following a 24-hour incubation period, AG and AG@BSP-VES were added to the plate at various concentration gradients from 0.09 to 22 µg/mL, while untreated cells were used as a control. The absorbance was then measured at 570 nm on a Microplate Reader. For the fluorescent live/dead assay, NCM460 or CT26 cells in logarithmic growth phase were inoculated on 96-well plate and cultured for 24 h. Then, AG and AG@BSP-VES solutions were added at a same drug concentration of 15 µg/mL. Besides, the untreated blank cells were used as control. Post-culture, cells were washed twice with 100 µL PBS, and LIVE/DEAD Near-IR Fixable dye was added, cultivated continuously for 20 minutes, and then imaged under a fluorescence microscope. Calcein AM and ethidium homodimer- 1 were used to stain living and dead cells, respectively. The number of live and dead cells was measured by Image J software. For the scratch wound assay, CT26 cells were divided into 24-well plates at a cell density of 10× 105 cells/mL, and cultured for 24 h to form a monolayer of cells. When the density reached 90%, a 10 µL pipette tip was adopted to draw a line, and washed with PBS for two times to remove cell debris. Appropriate concentrations of AG and AG@BSP-VES were added to a 24-well plate. The migration and invasion of CT26 cells were observed under a microscope at four different time points (0 h, 6 h, 12 h, 24 h). The scratch area was determined using Image J software, and the average value was measured three times simultaneously. The cell migration rate was calculated using the formula (%) = (0 h scratch width − 24 h scratch width)/0 h scratch width × 100%.

All animal studies were conducted under Institutional Animal Care and Use Committee-approved protocols of Guizhou University of Traditional Chinese Medicine. In order to investigate the in vivo distribution, AG@BSP-VES micelles (AG, 10 mg/kg) were further labeled with DIR (a near infrared fluorescent fuel). The subcutaneous (CT26 cells) and in situ (CT26-Luc cells with the expression of insect luciferase) tumor models were established based on previous studies (35). Briefly, BABL/C mouse were anesthetized and its abdominal cavity was cut longitudinally to expose colon tissues. CT26-luc cells (5×10⁶ cells in 20 μL PBS) were injected to the colorectal membrane. The wound of mouse was closed by biodegradable stitches. Four days later, the growth of the tumors was monitored by the IVIS Lumina II in vivo imaging system (Caliper Life Science, USA) after an injection of luciferin. To further confirm the establishment of orthotopic colon tumors, mice were killed, and colon tissues bearing tumors were stripped and immediately fixed in 4% (vol/vol) formaldehyde, which were evaluated by hematoxylin and eosin (H&E) and Ki67 staining. AG@BSP-VES were injected into CT26 tumor-bearing mice by intravenous injection. The mice were observed by the IVIS Lumina II in vivo imaging system at the predetermined time (1 h, 2 h, 4 h, 8 h, 12 h, and 24 h) after the injection. After that, the mice were euthanized by vein air injection. Various tissues, including tumors, were collected, weighted and observed by the in vivo imaging system. The accumulation of AG@BSP-VES micelles in various tissues was calculated as %ID/g (the percentage of the injected dose per gram of tissue).

Student’s t-test was used to determine the statistical significance of differences between groups. For all analyses, statistical significance was set at P < 0.05 for each paired experiment.

Amphiphilic BSP-VES polymer was synthesized by esterification and confirmed by 1H NMR ( Figure 1 ). The 1H NMR spectra of BSP, VES and BSP-VES were presented in this order from top to bottom. As shown in Figure 1 , BSP has two characteristic peaks at approximately 3.74–3.8 ppm and 4.66–4.80 ppm, which corresponds to the methine and hydroxyl proton peak of the simple sugar molecule in BSP. VES has a single peak at 1.21- 1.26 ppm, which can be attributed to the proton peak of methylene on the long chain of VES. All different characteristic peaks from a-c in BSP and VES appeared in the 1H NMR spectrum of BSP-VES, which indicates that BSP-VES polymer has been successfully synthesized.

The average diameter and zeta potential of the BSP-VES polymer micelle were 65.34 ± 1.03 nm and -9.12 ± 0.61 mV, respectively. As shown in Figure 2B , the average diameter of BSP-VES was nearly consistent with the TEM results. In addition, BSP-VES polymer showed excellent dispersity and ability to self-assemble into nano micelles in aqueous environments with the critical micelle concentration (CMC) of 48.3 μg/mL ( Figure 2C ).

AG@BSP-VES micelles were successfully prepared by the dialysis method, presenting a circular specific core-shell structure. The average diameter of AG@BSP-VES micelles increased from 65.34 ± 1.03 nm to 84.68 ± 2.08 nm because of the loading of AG, as determined using dynamic light scattering ( Table 1 ). In addition, AG@BSP-VES micelles showed good stability. As shown in Figure 2D , after AG@BSP-VES micelles were placed at room temperature for 7 days, no significant changes were observed in particle diameter, Zeta Potential and PD Index value. Hemolysis is an essential preliminary assessment of the toxic effects of biological materials. As shown in Figure 2E , the hemolysis rate is 1%-3% (less than 5%), which has good blood compatibility and was coincident with the criteria of the AG@BSP-VES micelles could easily disperse in the aqueous medium to form the micelles again, which indicated that AG@BSP-VES micelles have higher physical-chemical stability before the injection for the administration ( Figure 2A ).

According to the above chromatographic conditions, linear regression was performed on the peak area (Y) and concentration (X), and the linear regression equation was obtained as Y=20. 133X+14. 153 (R2 = 0. 9999). The results showed that AG had a good linear relationship in the range of 2.02–202 μg/mL. Based on the linear regression equation, the entrapment efficiency of AG@BSP-VES was found to be 91. 15 ± 0.05%, with a drug loading of 6.84 ± 0.01%. These results suggest that the synthesized micelles exhibit a high drug-loading capacity. Figure 2F shows the characteristics of AG release from AG@BSP-VES micelles. As shown in Figure 2F , almost all of the free AG was released within 8 h. However, AG release from the micelles was significantly slow, with only about 47.6% AG accumulative release after 24 h, defined as the percentage of released AG to total entrapped AG. The result showed that AG@BSP-VES micelles had a good sustained release effect. (PS. The initial rapid release of AG@BSP-VES may be attributed to the quick release of drugs that are embedded or adsorbed on or near the surface of the particles within a short periodof time).

As shown in Figure 3A , significantly more AG@BSP-VES micelles were internalized into CT26 cells compared with NCM460 cells during whole incubation time (i.e. 24 h), which was confirmed quantitatively by Image J software ( Figure 3B ). As shown by further targeted cellular uptake result, the average fluorescent intensity of AG@BSP-VES micelles in CT26 cells after 2 h incubation, was significantly higher than that in NCM460 cells ( Figures 3C, D ).

These data demonstrated that AG@BSP-VES micelles had potential colon cancer cell specificity. The in vitro bio-safety of BSP-VES micelles against CT26 and NCM460 cells was investigated by MTT assay. As shown in Figures 4A, B , blank BSP-VES micelles showed good biosafety during the period of incubation. The cell viabilities of CT26 and NCM460ncells were almost all over 80% after 24 or 48 h of incubation.

Figure 5A shows the cytotoxic effects of free AG and AG@BSP-VES in CT26 cells. The significantly higher cell-killing efficiency of AG@BSP-VES micelles than that of free AG alone was presented (P<0.001). Over 75% of CT26 cells were killed after 24 h incubation with AG@BSP-VES micelles (17.6 µg/mL). However, under the same AG concentration, only 23.9% of CT26 cells were killed. IC50 values were respectively 11.69 and 31.16 µg/mL for CT26 cells treated with AG@BSP-VES micelles and free AG. It was mainly attributed to the excellent internalization and tumor specificity of AG@BSP-VES micelles, resulting in increased AG concentration in CT26 cells. In addition, significantly more CT26 cells were killed (stained red by EthD- 1) due to the enhanced chemotherapeutical effect of AG from AG@BSP-VES micelles (the cell death percentage of 52.78%), compared with free AG group (the cell death percentage of 24.46%), which was attributed to more AG@BSP-VES micelles were internalized into CT26 cells than the free AG group ( Figures 5B, C ). Due to lack of selectivity, free AG group had induced similar cell death in both CT26 (24.46%) and NCM460 (26.25%) cells. The wound healing assay was used to investigate anti-invasion and anti-migration effects of AG@BSP-VES micelles. As shown in Figures 5D, E , the scratch area of AG@BSP-VES micelles group (71.40 ± 0.43%) measured by ImageJ software was significantly larger than that of free AG group (49.8 ± 0.56%). In addition, the percentage of scratch area was significantly decreased by 12.6 ± 3.95% in control group. These results showed that AG@BSP-VES micelles exhibited an excellent inhibitory effect on CT26 cells, compared with free AG, which was a direct result of BSP-VES micelles effectively increased cellular uptake of AG into CT26 cells and enhanced sensibility of CT26 cells to AG.

In order to further investigate the targeted accumulation of AG@BSP-VES micelles in CT26 tumors, we simultaneously established the subcutaneous and in situ tumor models. As shown in Figure 6A , the dividing line tumor and colon tissue is clearly visible, and tumor cells are marked by the tendency to spread, especially into normal colon tissue ( Figure 6B , cells in proliferation was stained (brown) by Ki67 stain). With the increase of the experiment time, the accumulation of AG@BSP-VES micelles in subcutaneous CT26 tumors showed an obvious increase tendency ( Figure 6B ). The fluorescent intensity of various tissues after 24 h injection was observed ( Figure 6C ), and quantitated. There was the highest accumulation of AG@BSP-VES micelles at 24 h after injection in subcutaneous CT26 tumors (55.37%, ID/g) and normal colon tissues (20.44%, ID/g) tumors ( Figure 6D ). Surprisingly, the targeted accumulation effect of AG@BSP-VES micelles was also observed in in situ CT26-Luc tumors ( Figures 6E–H ). There was the highest accumulation of AG@BSP-VES micelles at 24 h after injection in CT26-Luc tumors (17.31%, ID/g) and normal colon tissues (5.66%, ID/g). Interestedly, the bioluminescence signal of CT26-Luc was almost merged with the fluorescent signal of AG@BSP-VES micelles ( Figure 6F ). These results indicated that AG@BSP-VES could effectively and specifically internalize into colon tumors.