Introduction
Breast cancer is the major cause of morbidity and mortality among malignant tumors in women [
1‐
3]. It is worth noting that triple-negative breast cancer (TNBC) accounts for approximately 10–15% of all breast cancers and its malignant is characterized by the worst prognosis, increased recurrence rates regardless of the stage of the disease and resistance to chemotherapy [
4,
5]. To some extent, the refractoriness and chemotherapeutic failure of TNBC may attribute to the higher basal levels of autophagy with self-healing in tumor cells than that in normal cells [
1,
6‐
8]. Meanwhile, in the progression of TNBC, autophagy contributes to tumor survival by providing nutrition, regulating oxidative stress and promoting drug resistance [
6,
9]. As an evolutionary conserved cellular process, autophagy is aroused by the recognition of disposable or potentially harmful cytoplasmic entities and is culminated in the lysosomal degradation [
10,
11]. Basal autophagy generally promotes cell survival as a protective process. Paradoxically, excessive or sustained autophagy leads to extreme levels of autophagic flux and subsequently excessive consumption of organelles and cytoplasmic contents, then promoting cell death. In this sense, autophagy is considered to be a type II programming cell death or autophagic death [
12‐
14]. Given that, we propose a hypothesis that ingeniously designed synergistic therapy might emerge as an efficient strategy for interfering with the multistep process of autophagy to induce autophagic death on tumor cell itself and escaping the degradation on drugs.
Currently, new targeted therapies on TNBC have been developed, including immune checkpoint inhibitors, androgen receptor or poly ADP-ribose polymerase, but treatment options were still limited and cytotoxic chemotherapy remained the dominant treatment [
7,
15]. Doxorubicin (DOX) is a conventional chemotherapeutic drug for TNBC, whose mechanism is mainly to inhibit tumor proliferation by interfering with mRNA and DNA synthesis of tumor cells [
16,
17]. Although the anti-tumor activity of DOX is quite robust, its clinical applications are greatly limited due to its irreversible tissue toxicity and drug resistance aroused off-target [
18‐
20]. In the present study, nano-scaled polymeric micelles were designed for targeted delivery of DOX to reduce toxicity and interference on autophagic process [
10,
21,
22].
Nano-engineered delivery systems have received extensive attention due to their promotion of tumor tissue accumulation and retention through enhanced permeability and retention (EPR) effects and receptor-mediated ligand targeting to tumor cells, whereas the endo/lysosomal barrier is a major challenge for delivering and releasing drugs into cytoplasm [
19,
23]. Chitosan (CG), a natural polysaccharide, is widely developed in drug delivery system owning to its biocompatibility, biodegradability, cell adhesion and tumor inhibition [
23‐
25]. In addition, 2,3-dimethylmaleic anhydride (DMMA) appropriately modified to CG could capture protons from the outside of lysosome, and the concomitantly entered chloride ions and water into lysosome might induce potential rupture (proton sponge effect).
Based on the above, a conjugate methotrexate-polyethylene glycol (MTX-PEG)-modified CG/DMMA polymeric micelles were designed and synthesized. Specifically, CG was connected by DMMA to form a polymer with negative charge, and DOX was loaded into the delivery system by electrostatic interactions [
24,
26]. To improve the stability and targeting of the system, in this design, the surface of the micelles was modified with polyethylene glycol (PEG) connected to methotrexate (MTX) at one end. PEG can shield protein adsorption and avoid becoming a protein crown in the blood circulation [
27,
28]. MTX can specifically bind to the folic acid receptor overexpressed on the surface of tumor cells to promote tumor targeting and reduce the toxicity of DOX [
29]. The effect of the synthesized micelles on autophagy and its potential mechanism was detected by MDA-MB-231 cells in vitro, and its anti-tumor effect and selectivity were measured in tumor-bearing mice. On the one hand, tumor-targeting ability can effectively promote DOX aggregation in tumor tissue to exert therapeutic effect; on the other hand, DOX and the micelles-induced autophagosomes lead to autophagic death of tumor cells due to lysosomal damage and autophagic flux blockage [
18,
30]. All the results showed that the CG/DMMA polymeric micelles based on MTX-PEG modification could effectively improve the targeting and therapeutic effect of DOX, while the autophagic flux blockage effect of the micelles could also exert synergistic anti-tumor effect.
Materials and methods
Reagents and materials
Doxorubicin (DOX) and methotrexate (MTX) were obtained from Beijing Solarbio Science & Technology Co., Ltd. 2,3-Dimethylmaleic anhydride (DMMA) was sourced from Shanghai Yuanye Bio-Technology Co., Ltd. MTX-PEG was obtained from Yarebio. Chitosan (CG), 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI) and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (Shanghai) Trading Co. Ltd. Cell Counting Kit-8 (CCK-8) was sourced from Dojindo Laboratories. Fetal bovine serum (FBS) was sourced from Gibco Laboratories. 1% antibiotics (streptomycin 100 mg/mL and penicillin 100 U/mL), DMEM-H/F-12 medium and DMEM/HIGH Glucose medium were obtained from HyClone Laboratories. Antibodies for Beclin-1 (D40C5), LC3A/B (D3U4C), SQSTM1/p62 (D5L7G) and GAPDH (D16H11) were sourced from Cell Signaling Technology.
Preparation of CG-MTX-PEG
CG-MTX-PEG was synthesized through the catalysis of EDC and NHS according to previous reports. In brief, CG (120 mg), HOOC-MTX-PEG (76 mg), NHS (33.2 mg) and EDC (55.2 mg) were stirred in 12 mL of deionized water at room temperature. After 48 h, the reaction solution was dialyzed in deionized water for 3 days to separate excess catalyst (MWCO = 3500 Da) and then lyophilized to obtain a yellow flocculent product CG-MTX-PEG. The sample was stored at − 20 °C.
Preparation of CG/DMMA-MTX-PEG (CDPM)
The amidation reaction of CG-MTX-PEG and DMMA was completed under triethylamine (TEA) catalysis according to the previous report [
31]. In short, 50 mg of CG-MTX-PEG and 40 mg of DMMA were dissolved in 10 mL of DMSO, and then, 50 μL of TEA was added, and the reaction was stirred for 24 h at room temperature. Subsequently, the reaction solution was transferred into a dialysis tube (MWCO = 3500 Da) and dialyzed in deionized water with pH (8–9) adjusted by NaOH solution to remove catalyst and excess DMMA. The solution was lyophilized to obtain the final product CG/DMMA-MTX-PEG (CDPM), and the sample was stored at − 20 °C.
Preparation of DOX-loaded micelles
In brief, CDPM and DOX were dissolved in 30 mL DMSO under dark conditions. After vigorous stirring for 1 h, the mixture was slowly dropped into 10 mL deionized water and stirred. Finally, the mixture solution was transferred to a dialysis tube (MWCO = 3500 Da) and dialyzed to form micelles.
Characterizations
The infrared spectra of different samples were measured by Fourier infrared spectroscopy (FT-IR) with the KBr method over the wavenumber range of 4000–400 cm−1. The Zeta potential and size distribution of the micelles were obtained by Zetasizer. The morphology of the micelles was observed by a transmission electron microscope (TEM).
Protein adsorption
Bovine serum albumin (BSA) was used as plasma protein model to evaluate the protein adsorption effect of the micelles in this experiment. The sample solutions were separately incubated with BSA solution in ddH2O, and the final micelle and protein concentrations were 0.5 and 0.3 mg/mL, respectively. The mixture solutions were incubated at 37 °C for 2 h and then centrifuged for 10 min at 12,000 rpm to precipitate the protein adsorption aggregates completely. The supernatant was separated, and the concentration of BSA was determined and calculated according to the BCA protein assay kit.
In vitro drug release
The method of dialysis was used to study in vitro release behavior of drugs. The PBS solution with different pH value was used to simulate the microenvironment of tumor and normal tissue. Briefly, the micelles were dissolved in 5 mL of PBS solution and transferred to dialysis tubes (MWCO = 3500 Da). The dialysis tube was immersed in 25 mL PBS buffer, and the entire dialysis system was maintained at 37 °C, 120 rpm. At desired time intervals, 2 mL of dialysate was taken out and then replaced with the same volume of PBS solution. In order to evaluate the release of MTX, 1 mg/mL protease was added to simulate the lysosomal environment. The concentration of DOX was detected by fluorescence intensity at 590 nm (λex: 490 nm), and the concentration of MTX was obtained by measuring absorbance (305 nm).
Cell culture
MDA-MB-231 cells were purchased from BeNa Culture Collection (BNCC) and cultured in CM1-1 medium (90% DMEM/HIGH containing 10% (V/V) FBS) in an atmosphere of 5% CO2 (V/V) at 37 °C. HK-2 cells were purchased from American Type Culture Collection (ATCC) and cultured in CM9-1 medium (90% DMEMH/F-12 containing 10% (V/V) FBS) in an atmosphere of 5% CO2 (V/V) at 37 °C.
Cellular uptake
The cellular uptake behavior of the micelles was verified by confocal laser scanning microscopy (CLSM). MDA-MB-231 cells (1 × 104 cells/well) and HK-2 cells (1 × 104 cells/well) were incubated in confocal glass bottom dishes. After 24 h, the medium was replaced with a medium containing the micelles or free drug and cultured for another 6 h. Then, the cells were treated with 4% paraformaldehyde for 20 min and stained with 2 mL DAPI solution for another 15 min. Finally, the cells were washed three times with 2 mL PBS solution and visualized with CLSM.
In vitro cytotoxicity
The in vitro cytotoxicity and selectivity of the micelles were investigated by tumor cells (MDA-MB-231 cells) and normal cells (HK-2 cells) through the CCK-8 assay. In brief, MDA-MB-231 cells (1 × 104 cells/well) and HK-2 cells (1 × 105 cells/well) were cultured in 96-well plates for 24 h. Then, the cells were treated with the free drug mixture (the mixture of DOX and MTX) and the micelles with different concentrations. After 24 h, 10 μL CCK-8 solution was added to each well. The cells were incubated into the incubator for another 2 h, and then, the relative cell viability was calculated by measuring the absorbance at 450 nm with a microplate reader.
Fluorescent probes staining
AO/EB staining was used to detect apoptosis of MDA-MB-231 cells. Briefly, MDA-MB-231 cells were planted in 12-well plates (5 × 105 cells/well). After treatment, AO/EB mixture was added to each well for staining according to the protocol and then observed and photographed under a fluorescence microscope. DCFH-DA staining kit was used to detect the formation of reactive oxygen species (ROS). Briefly, the ROS was assayed with a DCFH-DA staining kit in accordance with the instructions provided by the manufacturer. MDA-MB-231 cells were seeded into six-well plate (2 × 105 cells/well), after drug treatment, incubated in the dark for 30 min at 37 °C with 2 mL of working solution. Then, the cells were washed three times by PBS solution and visualized using the fluorescence microscope. The lysosomal escape capacity of the micelles was assessed by fluorescence microscope. MDA-MB-231 were plated in six-well plate (2 × 105 cells/well). After drug treatment, LysoTracker Green staining was performed for 30 min, and the medium was aspirated and rinsed three times with ice-cold PBS solution, and then observed by fluorescence microscope.
Western blotting
Briefly, MDA-MB-231 cells were plated in dishes overnight, and then, the medium was replaced with the free drug and the micelles solution for further incubation. After 24 h, the cells were washed three times with PBS solution, and total protein was isolated with RIPA lysis buffer. Subsequently, the protein was fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the PVDF membrane. The PVDF membrane was sealed with BSA solution and then incubated with corresponding primary antibody overnight at 4 °C. Finally, the protein band was incubated with the secondary antibody, imaged by ECL and observed by the Bio Imaging system.
Biological transmission electron microscopy
MDA-MB-231 cells were planted into a 6-well plate and cultured for 12 h at a density of 1 × 106 cells/mL. Then, the cells were treated with free drug and the micelles and cultured for further 24 h. Subsequently, the culture medium was removed and added 2.5% glutaraldehyde to stabilize the cells. After 1 h, the cells were scraped off and centrifuged to remove the supernatant. Then slowly add 2.5% glutaraldehyde and store at 4 °C. The cells were prepared according to the procedure and observed using a biological transmission electron microscope.
In vivo anti-tumor efficacy
Female Balb/c mice (18 ± 2 g, 5–6 weeks) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. The MDA-MB-231 tumor-bearing mice model was constructed by subcutaneous injection of MDA-MB-231 cells at a density of 2 × 106 cells/mL into the axilla. After the tumor volume reached 100 mm3, the mice were randomly divided into three groups and treated with saline, free drug and the micelles on 0, 3, 6, 9, 12 and 15 days. The tumor volume and body weight were recorded every three days. The tumor volume was measured with a vernier caliper, and the volume (V, mm3) was calculated by the following formula: V = A × B2/2. (A represents the maximum diameter, and B represents the minimum diameter.)
Biodistribution
The MDA-MB-231 tumor-bearing mice model was used to evaluate the biodistribution behavior of the drugs. After the tumor volume reached about 100mm3, the mice were intravenously injected with saline, the free drug and the micelles, respectively, through the tail vein at a dose equivalent to 5 mg/kg of DOX. At the scheduled time, the mice were killed, and the liver, heart, kidney, lung, spleen and tumor of the mice were taken to analyze the fluorescence distribution through the in vivo imaging system (excitation filter 475 nm, emission fluorescence 500–750 nm).
Histological examination
At the end of the efficacy study in mice, the main organs (liver, heart, kidney, lung, spleen) and tumor tissues were collected for histopathological examination. All tissues were fixed with 4% paraformaldehyde and then dehydrated with gradient ethanol, embedded in paraffin, sectioned and baked. After deparaffinization and dehydration with xylene and absolute ethanol, all the slices were stained with hematoxylin and eosin and observed under a microscope.
Statistical analysis
All results were represented as the mean ± standard deviation (SD), and statistical analysis was analyzed by Origin 8.5 software and SPSS 16.0. Statistical analysis of group differences was performed by Student’s t test. p < 0.05 was considered statistically significant (***p < 0.001, **p < 0.01 and *p < 0.05).
Conclusion
In conclusion, the MTX-PEG-modified CG/DMMA polymeric micelles were designed and synthesized for targeted delivery of DOX to induce synergistic autophagic regulatory effects against TNBC. On the one hand, the MTX-PEG encapsulation improves stability and selectivity of the micelles, which effectively promotes specific aggregation of the micelles in tumor tissues, while reduces systemic toxicity and synergizes the anti-tumor efficiency of DOX. On the other hand, ROS under micellar therapy promotes the production of autophagosomes in tumor cells, while the lysosomal damage based on proton sponge effect blocks the autophagosome flux, leading to the accumulation of autophagosomes. Excessive accumulation of autophagosomes depletes normal cellular components and induces autophagic death of the stubborn tumor cells. Although the preliminary mechanism of autophagy has been verified, the deeper mechanism of micelles on autophagy flux has not been clearly explored. In addition, single cell lines and ectopic model were the limitations of this study.
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