Dodecanol (1.86 g, 10 mmol) was dried under vacuum at 50 °C for 5 h, followed by the addition of Sn (Oct)2 and continued drying for 0.5 h. Then the dried $\epsilon$-caprolactone (16.8 g, 15 mmol) was added and reacted at 105 °C for 24 h. After cooling, the reaction system was added with a small amount of ethanol and dissolved in tetrahydrofuran. After precipitation in cold diethyl ether, the mixture was filtered and dried to obtain PCL-OH.

PCL-OH (6.0 g, 3 mmol) was dried under vacuum at 50 °C for 8 h, dissolved in 30mL of trichloromethane, added with succinic anhydride (1.5 g, 15 mmol) at 70 °C for 48 h. After cooling, the precipitated succinic anhydride was removed by filtration, precipitated in cold ethanol, and dried by filtration to obtain PCL-COOH.

PCL-COOH (4.0 g, 2 mmol) was dried under vacuum for 5 h, dissolved in 20 mL dichloromethane, and supplemented with NHS (0.46 g, 4 mmol). The mixture was placed in an ice bath, after which, DCC (0.82 g, 4 mmol) was added, and the reaction continued with stirring for 24 h. Then the insoluble DCU was removed by filtration, and the filtrate was precipitated in cold ether. After filtration, the filtrate was washed once with ether and isopropanol, and dried to obtain PCL-NHS.

PEG (2.0 g, 1 mmol) was dried under vacuum at 90 °C for 8 h. After dissolving in 10 mL dimethyl sulfoxide (DMSO), PEG was added with 10 mL THF solution containing PCL-NHS (2.0 g, 1 mmol) and stirred for 24 h. The resulting solution was dialyzed in deionized water for 3 d to obtain the PEG-PCL. In this study, the PEG-PCL micelle materials were provided by Tanshui Technology Co., Ltd. (Guangzhou, China) as a technical service.

For ${}^1$H NMR, the polymers were dissolved with deuterated chloroform, and the chemical shifts of each proton were determined on a Varian Mercury-Plus 400 instrument. Tetramethylsilane (TMS) was used as an internal standard. Solid samples were mixed with dry KBr powder, finely ground and pressed. IR test was conducted on an infrared spectrometer (Nicolet/Nexus 670) with the wavelength of 400–4000 cm${}^{-1}$.

40 mg of PEG-PCL was dissolved in 2 mL of chloroform, to which 20 mL of ultrapure water was slowly added under ultrasonication, using a sonicator (Scientz, Ningbo, China), to form an emulsion. The aqueous phase solution of blank micelles was obtained by rotary evaporator to remove chloroform at 30 ℃. The blank micelles were concentrated by ultrafiltration three times, aggregates were removed by filtration using a 0.22 µm filter, and the product was stored at 4 °C. 3 mg PTX and 3 mg DGL (Selleck Chemicals, USA) were dissolved in 1 mL DMSO, which were added to 1.5 mL chloroform containing 40 mg polymer. Then 20 mL ultrapure water was added slowly under ultrasonication to form an emulsion, and the chloroform was removed by rotary evaporation. The product was added to a dialysis bag with a molecular retention capacity of 14000 Da for more than 24 h. After ultrafiltration concentration and filtration, the product was stored at 4 °C.

The micelle particle size and surface potential were measured using a Brookhaven Instruments BI-200 SM DLS system at 25 °C. The scattered light was detected at 90${}^o$ and collected on an automatic accelerator. The surface potential was tested with a micro-detection device (Brookhaven Instruments, BI Zetaplus), as Zhong et al. [28] described.

10 µL of DGL and PTX-loaded PEG-PCL micelles (0.5 mg/mL) were deposited onto TEM copper grids and then placed in a desiccator for 8 h. The samples were stained with a 2% solution of acetyldioxymethylcellulose for 1 min, and the excess solution was then blotted with filter paper. After drying overnight at 60 °C, the samples were examined under a Transmission Electron Microscope (TEM) with an operating voltage of 120 kV.

Blood samples were centrifuged at 1500 $\times$g for 10 min to obtain erythrocytes. 300 µL of erythrocyte suspension was added to 1.5 mL deionized water (positive control) and 1.5 mL solution containing 0 ppm, 100 ppm, 200 ppm, 400 ppm, and 800 ppm PEG-PCL micelles, respectively. After being left to stand for 3 hours at 37 °C, the absorbance of the supernatant was tested at 541 nm using a UV spectrophotometer to analyze the release of hemoglobin. The percentage of hemolysis for each sample was calculated by the formula (A${}_{\text{sample}}$–A${}_{\text{negative}}$)/(A${}_{\text{positive}}$–A${}_{\text{negative}}$). A${}_{\text{sample}}$ is the absorbance in the sample group, and A${}_{\text{positive}}$ is the absorbance in the positive group, and A${}_{\text{negative}}$ is the absorbance in the negative group. Solution without micelles (0 ppm) was set as a control solution in this experiment.

Both MDA-MB-231 and MDA-MB-468 cells were all purchased from the ATCC and then induced then into PTX-resistant cells. Both types of cells were grown in L-15 medium (GIBCO, Thermo Fisher Scientific, MA, USA) including 10% fetal bovine serum (Gibco, USA) at 37 °C with 5% CO${}_2$. The breast cancer cells used in our experiments were meticulously cultured under stringent conditions. They underwent rigorous mycoplasma testing to ensure their contamination-free status. To establish PTX-resistant cell lines, breast cancer cells were subjected to a treatment involving a gradual escalation in the concentrations of PTX [29]. For non-nanoparticle encapsulated PTX and/or DGL, DMSO (0.1%) was used for dissolution to treat cells. In this study, the concentrations (or equivalent concentrations) of PTX and DGL used were 50 ng/mL and 60 ng/mL, respectively. All cell lines were authenticated shortly before use by the PCR technique, carried out by Zhongqiaoxinzhou Biotech (Shanghai, China).

EdU solution (Solarbio, Beijing, China) was diluted with culture medium to prepare 50 µM EdU medium. MDA-MB-231 and MDA-MB-468 cells (1 $\times$ 10${}^4$ cells) were collected, washed, and counted. Then the cells were equably inoculated in a 96-well plate and treated in the light of experimental purpose. The treated BC cells were supplemented with 100 µL 50µM EdU medium for 2 h. After washing, cells were fixed by 50 µL 4% paraformaldehyde for 30 min and permeabilized by 0.5% Triton X-100. Subsequently, the cells were exposed to 50 µL of 2 mg/mL glycine for 5 min. the results were obtained with a fluorescence microscopy (Excitation wavelength: 555 nm; Emission wavelength: 565 nm).

Each group of cells were digested using trypsin (0.25%) and suspended in culture medium. After that, cells in each well were addressed with 10 µL of CCK-8 (Dojindo, Kumamoto, Japan) and incubated for another 3 h. The OD value was tested using a microplate reader at 450 nm.

The cells of each group were fixed using 4% paraformaldehyde for 15 min after removing the culture medium, then incubated with 0.1% TritonX-100 in ice bath for 2 min. After washing, the cells were exposed to 0.3% hydrogen peroxide in methanol for 20 min. Subsequently, appropriate TUNEL solution (Beyotime, Shanghai, China) was added to each well of cells and incubated for 1 h at 37 °C protected from light. After washing, the cells were treated with TUNEL working solution for 10 min, ethanol (dehydration) for 2 min, and xylene (transparency) for 2 times. After sealing, TUNEL positive cells were observed under a fluorescence microscope.

Annexin-FITC/PI apoptosis kit (BD Biosciences, NJ, USA) was utilized for apoptosis analysis. After processing, each group of cells (5 $\times$ 10${}^5$, 200 µL) was collected and treated with AnnexinV-FITC (5 µL) and PI (5 µL) for 30 min in the dark. Then the cells were washed using 190 µL binding buffer, centrifuged, and resuspended with phosphate buffered saline (PBS). Flow cytometry (FACSCalibur; BD Biosciences, NJ, USA) was used to analyze cell apoptosis.

BC cells from each group were added with 100 µL protein lysate. After ice bath and centrifugation, the supernatant was harvested. Protein concentration was confirmed by applying BCA kit (Beyotime, Shanghai, China). 20 µg of protein was mixed with 5$\times$ loading buffer and denatured. Then groups of samples were applied for SDS-PAGE electrophoresis, and electrotransferred onto PVDF membranes. After incubation with 5% skim milk powder for 2 h, the membranes were treated with the diluted primary antibodies including Caspase 3 (Abcam, 1:500) and GAPDH (Abcam, 1:8000) overnight at 4 °C, and the secondary antibody (Abcam, 1:10,000) for 2 h. After treatment with Pierce™ ECL substrate (Thermo Fisher Scientific, MA, USA), the blots were obtained using an ECL system (Thermo Fisher Scientific, MA, USA). A GAPDH was used as an internal reference.

All experiments were independently repeated 3 times and the data were denoted as mean $\pm$ SD. Data were analyzed using SPSS 23.0 software (IBM Inc., Chicago, IL, USA). A one-way ANOVA, followed by a Tukey’s test, was used to analyze the difference between groups. p 0.05 was regarded as a significant difference.

The structure of PEG-PCL was confirmed by 1H NMR (nuclear magnetic resonance) (Fig. 1A). The molecular weight of PCL was calculated as 2054 Da in the same way, which is consistent with our designed molecular weight of PEG2000-PC2000. Therefore, results showed that PEG-PCL has been successfully synthesized. The characterization of FTIR (fourier transform infra-red) spectra showed that 1740 cm${}^{-1}$ is the C=O absorption peak in the ester bond, and 1650 cm${}^{-1}$ is the C=O absorption peak in the amide bond (Fig. 1B). These characteristic peaks further demonstrate the successful synthesis of PEG-PCL.

Fig. 1.

nuclear magnetic resonance (NMR) spectrum and fourier transform infra-red (FTIR) spectra of PEG-PCL. (A) ${}^1$H NMR spectra (CDCl3, 400MHz) of PEG-PCL. The proton characteristic chemical shifts are attributed to $\delta$3.38ppm CH${}_{\mathrm{3}}$(OCH${}_2$CH${}_2$)-, a; $\delta$3.65ppm CH${}_3$(OCH${}_{\mathrm{2}}$CH${}_{\mathrm{2}}$)-, b; $\delta$4.05ppm -NH-(COCH${}_{\mathrm{2}}$CH${}_2$CH${}_2$CH${}_2$CH${}_2$O) CH${}_3$, c; $\delta$1.66ppm -NH-(COCH${}_2$CH${}_{\mathrm{2}}$CH${}_2$CH${}_{\mathrm{2}}$CH${}_2$O) CH${}_3$, d; $\delta$1.41ppm -NH-(COCH${}_2$CH${}_2$CH${}_{\mathrm{2}}$CH${}_2$CH${}_2$O) CH${}_3$, e; $\delta$2.33ppm -NH-(COCH${}_2$CH${}_2$CH${}_2$CH${}_2$CH${}_{\mathrm{2}}$O) CH${}_3$, f. The number of hydrogen atoms in the repeating unit of the PEG backbone was 194, based on the methoxy peak of PEG. The molecular weight of PEG was calculated to be 2138 Da based on the molecular weight of each repeating unit (Mw = 44 Da). (B) FTIR spectra of PEG-PCL. NMR, nuclear magnetic resonance; FTIR, fourier transform infra-red; PEG-PCL, polyethylene glycol- polycaprolactone.

Next, the data of DLS signified that the average particle size and surface potential of micelles formed by blank carriers were 30.86 $\pm$ 2.1 nm and –3.55 $\pm$ 0.27 mV, respectively. The average particle size and surface potential of micelles loaded with DGL and PTX were 35.78 $\pm$ 0.35 nm and –2.84 $\pm$ 0.27 mV. Compared with the control micelles, the particle size of micelle loaded with DGL and PTX increased and the potential was slightly reduced (Fig. 2A). Besides, TEM was applied to determine the morphological structure of nanomicelles loaded with DGL and PTX at pH 7.4. The results denoted that the nanomicelles exhibited a uniform spherical structure with a particle size around 30 nm, which was similar to the DLS results (Fig. 2B). In this manner, we determined the physicochemical properties of DGL and PTX-loaded PEG-PCL.

Fig. 2.

Particle size and morphology characterization of deguelin (DGL) and paclitaxel (PTX)-loaded polyethylene glycol-polycaprolactone (PEG-PCL) nano-micelle. (A) Particle sizes of blank micelle (left) and drug-loaded micelle (right) through dynamic light scattering assay. (B) Drug-loaded micelles were observed using TEM with uranyl acetate. DGL, deguelin.

Specifically, the acetonitrile solution of PTX showed the maximum absorption at 227 nm, while the DMSO solution of DGL showed the maximum absorption at 272 nm. The standard curve concerning concentration for PTX was: A = 0.02951c + 0.00173, R2 = 0.99627; whereas, for DGL with respect to concentration, it was: A = 0.05549c - 0.02276, R2 = 0.99829. Having obtained the absorbance of PTX and DGL in the micelles, their loading capacities were calculated to be 5.3 $\pm$ 0.17% and 6.1 $\pm$ 0.21%, respectively, based on the standard curves (Supplementary Fig. 1). Using the pyrene fluorescence probe method, the critical micelle concentration (CMC) of the sample was measured. The results indicated that the CMC for PEG${}_{\text{2k}}$-PCL${}_{\text{2k}}$ was 19.95 µg/mL (4.99 $\times$ 10${}^{-6}$ mol/L), as shown in Supplementary Fig. 2. Serum Stability Test showed that drug-loaded micelles displayed excellent size stability in the PBS solution with 10% FBS, maintaining a particle size of around 35 nm over a week (Supplementary Fig. 3).

Crucially, we further validated the blood biocompatibility of the micelles. Once hemolysis occurs, the hemoglobin in the red blood cells will be released into the solution. The redness of this solution, indicating hemolytic activity, can be estimated by measuring the UV absorbance of the supernatant at 541 nm. As represented in Fig. 3, there was no significant hemolysis in micelles at the experimental concentrations (100–800 ppm). Even at a concentration of 800 ppm, only about 4% hemolysis occurred in micelles at 3 h exposure time. Therefore, the hemolytic activity of micelles was negligible.

Fig. 3.

Hemolysis caused by micelles at different concentrations. Hemolysis assay was conducted after processing with different concentrations of micelles. Deionized water was served as a positive control (+) and phosphate buffered saline (PBS) as a negative control (-). PBS, phosphate buffered saline.

Subsequently, we validated the impact of DGL and PTX-loaded PEG-PCL nano-micelles on the proliferation of BC cells. MDA-MB-231 and MDA-MB-468 cells were treated with PEG-PCL, PTX, DGL, PTX+DGL, DGL/PEG-PCL, PTX/PEG-PCL, and DGL-PTX/PEG-PCL. EdU staining results displayed that PEG-PCL, PTX, and DGL treatment alone did not significantly affect the proliferative activity of the BC cells; Four combined treatments including PTX+DGL, DGL/PEG-PCL, PTX/PEG-PCL, and DGL-PTX/PEG-PCL could observably reduce BC cell proliferation activity, especially DGL-PTX/PEG-PCL (Fig. 4A). Similarly, CCK-8 data showed that treatment of PEG-PCL, PTX, and DGL alone also failed to induce changes in BC cell viability; Combined treatments (PTX+DGL, DGL/PEG-PCL, PTX/PEG-PCL, and DGL-PTX/PEG-PCL) also could markedly restrain BC cell proliferation viability, and this suppression effect was strongest in the DGL-PTX/PEG-PCL group (Fig. 4B). Overall, these data suggest that DGL and PTX-loaded PEG-PCL nano-micelles can cause a remarkable reduction in BC cell proliferation.

Fig. 4.

DGL and PTX-loaded PEG-PCL nano-micelles suppresses the proliferation of breast cancer (BC) cells. MDA-MB-231 and MDA-MB-468 cells were dealt with DMSO, PEG-PCL, PTX, DGL, PTX+DGL, DGL/PEG-PCL, PTX/PEG-PCL, and DGL-PTX/PEG-PCL, respectively. (A) Cell proliferative activity was confirmed with Edu staining. Magnification, 200$\times$. (B) Cell viability was monitored through CCK-8. NS means no significance; * means p 0.05; ** means p 0.01.

We also further confirmed the changes in apoptosis of BC cells after treatment with DGL and PTX-loaded PEG-PCL nano-micelles. Data from TUNEL indicated that PEG-PCL, PTX, DGL alone did not lead to prominent changes in the apoptotic capacity of BC cells; relative to the control (DMSO) group, the apoptotic capacity of BC cells was significantly increased in PTX+DGL, DGL/PEG-PCL, PTX/PEG-PCL, and DGL-PTX/PEG-PCL groups, especially in DGL-PTX/PEG-PCL group (Fig. 5A). Flow cytometer data also indicated that PTX+DGL, DGL/PEG-PCL, PTX/PEG-PCL, and DGL-PTX/PEG-PCL could dramatically induce BC cell apoptosis, and relative to other groups, DGL and PTX-loaded PEG-PCL nano-micelles (DGL-PTX/PEG-PCL) had the strongest induction of apoptosis in BC cells (Fig. 5B). Additionally, western blot data showed that Caspase 3 expression was notably upregulated in PTX+DGL, DGL/PEG-PCL, PTX/PEG-PCL, and DGL-PTX/PEG-PCL groups versus that in DMSO group; and the degree of upregulation of Caspase 3 expression was most significant in DGL-PTX/PEG-PCL group (Fig. 5C). Thus, we testified that DGL and PTX-loaded PEG-PCL nano-micelles can result in a noteworthy elevation in apoptotic capacity of BC cells.

Fig. 5.

DGL and PTX-loaded PEG-PCL nano-micelles induces the apoptosis of BC cells. MDA-MB-231 and MDA-MB-468 cells were also processed with dimethyl sulfoxide (DMSO), PEG-PCL, PTX, DGL, PTX+DGL, DGL/PEG-PCL, PTX/PEG-PCL, and DGL-PTX/PEG-PCL, respectively. (A) Cell apoptosis was determined with TUNEL staining. Magnification, 200$\times$. (B) Flow cytometer was utilized to monitor the role of DGL and PTX-loaded PEG-PCL nano-micelles on cell apoptosis. (C) Western blot exhibited the alteration of Caspase 3 expression in each group. NS means no significance; * means p 0.05; ** means p 0.01.