The Synthesis of Glutamine-Functionalized Block Polymer and Its Application in Triple-Negative Breast Cancer Treatment

Triple-negative breast cancer (TNBC) is a highly malignant tumor. At present, there are still no targeted drugs for TNBC. Clinical chemotherapeutic drugs, such as doxorubicin (DOX), have the characteristic of nontargeted distribution in treatment of TNBC, causing severe side effects. Therefore, new target treatment strategies for TNBC are of urgent need. It was speculated that glutamine could be a potential target because it is in high demand by TNBC. In this study, we found that the transporter for glutamine, ASCT2 (solute carrier family 1 member 5 (SLC1A5)), is highly expressed in TNBC by analysis of data from The Cancer Genome Atlas (TCGA) and experiments in vitro. Based on this, glutamine was grafted onto a polymeric drug carrier in order to develop a tumor-targeting drug delivery system for treatment of TNBC. Firstly, pH-responsive glutamine-PEG5000-b-PAE10000 (Gln-PEG-b-PAE) copolymers were synthesized using Fmoc-PEG5000-b-PAE10000 (Fmoc-PEG-b-PAE) copolymers. Then, Gln-PEG-b-PAE@DOX micelles were prepared by loading DOX to Gln-PEG-b-PAE copolymer using a solvent casting technology. In vitro, Gln-PEG-b-PAE@DOX micelles exhibited pH-dependent micellization-decellularization behavior; namely, they can rapidly release DOX in acidic environment of pH 6.0 but release very slowly in physiological condition. Moreover, glutamine competition experiment showed that Gln-PEG-b-PAE@DOX micelles had the ability to target MDA-MB-231 cells. Compared to free DOX, Gln-PEG-b-PAE@DOX micelles had significantly greater cytotoxic effect and antiproliferative activity against MDA-MB-231 cells. In vivo, compared to free DOX and mPEG-b-PAE@DOX micelles, Gln-PEG-b-PAE@DOX micelles significantly inhibited tumor growth in tumor-bearing mice. Therefore, Gln-PEG-b-PAE@DOX micelles, as a tumor-targeting drug delivery system, may provide a new method for the treatment of TNBC.


Introduction
Triple-negative breast cancer (TNBC), lacking immunohistochemical expression of estrogen receptor (ER), progesterone receptor (PgR), and human epithelial growth factor 2 (HER2), is the notorious subtype of breast cancer with highly aggressive and lethal behavior. Doxorubicin (DOX) remains one of the most standard first-line chemotherapy agents, usually used alone or in combination with other drugs for the treatment of advanced or metastatic breast cancer. However, DOX is always used limitedly due to the toxicity and some severe side effects, especially cardiotoxicity [1,2]. Therefore, tumor-targeting drug delivery methods were studied to address this deficiency.
In recent years, more and more attention has been paid to tumor-targeting drug delivery methods to increase the availability of chemotherapeutic drugs targeting on reducing toxic side effects [3,4]. Polymer drug carrier is a new drug delivery technology based on pharmacology, biomaterials, and clinical medicine [5][6][7]. Polymeric micelles are a series of the most promising carriers to realize the precise tumor-targeted therapy and have attracted much attention in drug delivery research [8]. Polymeric micelles, formed by amphiphilic block copolymers in aqueous conditions, can form a stable nanoparticle micelle structure to encapsulate antitumor drugs and subsequently be disintegrated by specific enzyme or by varying the pH [9,10]. The unique pH-dependent structure of the pH-responsive polymeric micelles is disrupted due to the tumoral acidic pH, making the loaded drug more easily accumulate in tumor tissue than normal tissue [9,11].
In addition to pH-responsive polymer, the most popular tumor-targeting drug delivery strategy is to conjugate micelles with small molecule ligands, which could facilitate specific uptake by tumor cells overexpressing corresponding receptors, so as to achieve high intracellular drug dosage while minimizing nonspecific biodistribution [12,13]. The molecule ligands include antibodies, peptides, aptamers, and phage, such as epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), arginyl-glycylaspartic acid (RGD), polypeptides, and mucin 11 (MUC11) [14]. Amino acids play an important role in the biological process of living cells, especially in the proliferation and maintenance of tumor cells. Glutamine, highly taken up by TNBC cells, is a vital kind of nutrient in tumor metabolism by providing nitrogen for protein and nucleotide synthesis [15][16][17]. ASCT2 (solute carrier family 1 member 5 (SLC1A5)), a Na +coupled transporter for amino acid, is overexpressed in TNBC [17]. It has a high affinity for glutamine, especially at acidic pH [18]. Additionally, folic acid and methionine are frequently chosen as the targeting ligands for improving tumor-specific delivery; their comediated micelles presented higher affinity for cancer cells, compared with micelles containing folic acid or methionine alone [19]. Due to the overexpression of SLC1A5 in tumor cells, SLC1A5 was used as a target for tumor treatment. A previous study reported a competitive small molecule antagonist of transmembrane glutamine flux that selectively targets SLC1A5 to contribute to antitumor responses in vitro and in vivo [20]. Moreover, a research team discovered a targeting strategy based on polyglutamines with high affinity for SLC1A5 which can be used as a drug/gene delivery system [21].
In this study, we have prepared Gln-PEG-b-PAE, pH-responsive, and glutamine-grafted copolymers as drug carriers. Then, taking advantage of high uptake of glutamine by TNBC cells, we used pH-responsive Gln-PEG-b-PAE copolymer to load free DOX to achieve the purpose of targeted transport of DOX to TNBC cells. Gln-PEG-b-PAE@-DOX micelles can target tumor cells and release DOX in tumor tissues, making it a new tumor-targeting drug delivery system for TNBC.  (FPKM). The unpaired Student's t-test was used to calculate the statistical significance of difference between two independent groups, and p < 0:05 was considered statistically significant using GraphPad Prism (La Jolla, CA, USA). Patients were stratified into a high SLC1A5 expression group and a low SLC1A5 expression group based on the cut-off value of Kaplan-Meier analysis with the log-rank test between the two groups. The analysis was performed in GraphPad Prism (La Jolla, CA, USA). The hazard ratio (HR) with 95% confidence interval (CI) was applied to report the results, and p < 0:05 was considered statistically significant.

Gene Expression Analysis.
Total RNA from cell lines MDA-MB-231, MCF-7, SKBR3, ZR-75-1, and BT549 was extracted using an RNAsimple Total RNA Kit (DD419, Tiangen Biotech, Beijing, China) according to the manufacturer's instruction. The concentration and quality of the RNA were measured using the UV absorbance at 260 and 280 nm (260/280 nm) on a OneDrop OD-1000+ spectrophotometer (OD-1000, OneDrop, Nanjing, China). 500 ng of total RNA was reverse transcribed using HiScript II Q RT SuperMix for qPCR (R222-01, Vazyme, Nanjing, China) following the manufacturer's instruction. For determination of mRNA expression levels, RT-qPCR was then performed on the StepOnePlus Real-Time PCR System (4376600, Thermo Fisher Scientific, Waltham, MA, USA) with AceQ qPCR SYBR Green Master Mix (high ROX premixed) (Q141-02, Vazyme, Nanjing, China). Cycling parameters, which include a complete melt curve stage, were set up as follows: 95°C for 10 min followed by 40 cycles at 95°C for 10 s and 60°C for 30 s. Primers used to amplify SLC1A5 are as follows: forward: 5 ′ -TATTTTGGCGGCTGTAGTTGTG-3 ′ ; reverse: 5′-CCTGGGAGTGTTTCTGTTATTGTG-3′. GAPDH was used as an internal control to normalize mRNA. All the primers were purchased from RiboBio (Guangzhou, China). All the samples were analyzed in triplicate for each specific gene. The Ct values for each gene were normalized to the endogenous control, and the relative expression levels were calculated by the ΔΔCt method.

Synthesis of Gln-PEG-b-PAE Copolymer.
For the purpose of designing a TNBC-targeting and pH-responsive drug delivery system, Fmoc-PEG-b-PAE copolymer was synthesized by Michael addition reaction. Then, glutamine was modified to NH 2 -PEG-b-PAE copolymer through DCC/DMAP coupling chemistry. To acquire the Gln-PEG-b-PAE copolymer, Fmoc-PEG-b-PAE copolymer was synthesized as the literature described [22]. Typically, Fmoc-protected PEG 5000 (0.2 mmol, RuiXi, China) was dissolved in anhydrous dichloromethane (20 mL, Aladdin, Shanghai, China), 2 times of equivalent triethylamine was added, and the solution was placed in the reaction bottle in 0°C cold hydrazine (DC-3006, Shanghai Sunny Hengping Scientific Instrument, Shanghai, China). Then, acryloyl chloride (0.4 mmol, Aladdin, Shanghai, China) was slowly added to the reaction bottle. After 2 h of stirring at 0°C, the solution was recovered to room temperature and stirred for another 24 h. The Fmocprotected poly(ethylene glycol) methyl ether acrylate (monoacylated Fmoc-PEG) was obtained by vacuuming the solution and precipitating in hexane solution. The Fmoc-PEG poly(β-amino ester) (Fmoc-PEG-b-PAE) copolymer was prepared by Michael addition reaction. One equiv. of 1,6-hexanediol diacrylate (HDD, Aladdin, Shanghai, China) and 1.1 equiv. of 3-aminopropanol (TDP, Aladdin, Shanghai, China) were dissolved in 2 mL of chloroform; then, 0.1 equiv. of monoacylated Fmoc-PEG 5000 was added and stirred at 50°C for 48 h. The Fmoc-PEG-poly(β-amino ester) copolymer was precipitated by adding an amount of methyl tertbutyl ether solution. The NH 2 -PEG-b-PAE copolymer was obtained by deprotection reaction. Briefly, Fmoc-PEG-b-PAE copolymer (0.1 mmol) was dissolved in 8 mL of dichloromethane; then, 2 mL of piperidine was added and stirred at room temperature for 1 h. The solution was decompressed and concentrated after being washed by diluted hydrochloric acid solution, and NH 2 -PEG-b-PAE copolymer was obtained by precipitation. NH 2 -PEG-b-PAE copolymer (0.1 mmol) and Fmoc-glutamine (0.2 mmol) were dissolved in 15 mL of chloroform; then, 0.2 mmol of N,N ′ -dicyclohexylcarbodiimide (DDC, Aladdin, Shanghai, China) and 0.05 mmol of 4-dimethylaminopyridine (DMAP, Aladdin, Shanghai, China) were added into the solution to react overnight at room temperature. The Fmoc-protected Gln-PEG-b-PAE copolymer was collected after precipitating it in methyl tert-butyl ether solution. The Gln-PEG-b-PAE (0.05 mmol) copolymer was obtained by deprotection reaction and characterized by a 1 H NMR and a gel permeation chromatography (GPC): 1

Preparation and Characterization.
Gln-PEG-b-PAE@-DOX micelles were prepared by loading DOX to Gln-PEGb-PAE copolymer using a solvent casting technology. Firstly, the dissolving DOX and the Gln-PEG-b-PAE copolymer were sufficiently stirred in the solution, followed by evaporation of the solvent and dissolution of the dried thin film with phosphate-buffered solution (PBS). Then, the DOX-encapsulated Gln-PEG-b-PAE micelles were manufactured from the solution by sonicating, and the encapsulation efficiency was up to 75.3%.
The Gln-PEG-b-PAE copolymer and DOX-loaded micelles were prepared by organic solvent evaporated method. Typically, 10 mg of Gln-PEG-b-PAE copolymer and 1 mg of DOX were dissolved in chloroform in a round-bottom flask, and then, the solvent was removed by rotary evaporators. The DOX-loaded micelles were obtained by adding 5 mL of PBS (10 mM, pH 7.4) in the round-bottom flask which was rotated for 3 h. After dispersing the micelles in a probe-type sonifier, the free DOX in micelle solution was removed by washing it with size exclusion centrifugal filters (0.5 centrifugal filters, Merck Millipore, Darmstadt, Germany, 5000 rcf, 10 min) for 3 times in PBS. The empty micelles were obtained without addition of DOX. The average particle size and morphology of Gln-PEG-b-PAE copolymer and DOX-loaded micelles were measured by dynamic light scattering (DLS) with a particle sizing analyzer (Zeta Plus, Brookhaven, Suffolk, NY, USA) and transmission electron microscopy (TEM, FEI Tecnai F-20, USA).
The DOX loading ability of Gln-PEG-b-PAE copolymer was quantified by UV-Vis spectroscopy (UV-3600, Shimadzu, Kyoto, Japan) analysis. The freeze-dried DOXloaded micelles were weighed and dissolved in chloroform. The absorbance of chloroform solution at 483 nm was measured to determine DOX content in the micelles using an established free DOX calibration curve.
The critical micelle concentration (CMC) was measured by a fluorescence reader (Infinite 200 PRO, Tecan, Männedorf, Switzerland) using pyrene as a fluorescence probe. Briefly, the Gln-PEG-b-PAE copolymer was dissolved in chloroform and the concentration of the copolymer varied from 0.00187 mg/mL to 1 mg/mL. Then, 100 μL of pyrene (1 mg/L) was added in copolymer solution, which makes 3 Journal of Nanomaterials the volume of the mixture 1 mL in PBS after evaporating the organic solvent. The fluorescence intensity of copolymer was performed at 373 nm and 384 nm. The CMC was estimated from the onset of a decrease in the intensity ratio of peaks at 373 nm to peaks at 384 nm plotted versus the polymer concentrations.   [23]. A FACScan flow cytometer (FACSVerse, BD Biosciences, NJ, USA) was applied to analyze cellular apoptosis.

Antitumor Efficacy of Gln-PEG-b-PAE@DOX Micelles In
Vivo. Subcutaneous tumors were established by inoculating 1:0 × 10 5 luciferase-labeled MDA-MB-231 (MDA-MB-231luc) cells into the right armpit of female BLAB/c nu/nu mice and allowing them to grow for 14 days. When the tumor volume reached about 100 mm 3 , the mice were randomly assigned into 4 groups; subsequently, tumor-bearing mice (6 per group) were injected in the tail veins with the following: group 1, saline; group 2, free DOX solution (2 mg/kg); group 3, mPEG-b-PAE@DOX micelles (DOX 2 mg/kg); and group 4, Gln-PEG-b-PAE@DOX micelles (2 mg DOX/kg) once every 4 days for 4 times. Every day, the tumor volume was calculated by a digital calliper as a × b 2 /2, where a was the largest diameter and b was the smallest diameter. Five days after the last injection, the fluorescence signals of tumors were recorded by an in vivo imaging system (IVIS Spectrum, PerkinElmer, Waltham, MA, USA), using 20 mg/mL D-Luciferin Firefly potassium salt (luc001, Sciencelight Biology Science&Technology, Shanghai, China). The experiment had previously been approved by Animal Ethical and Welfare Committee of Nanjing Medical University; the ethical approval code was IACUC-1906003.

Expression of SLC1A5 in Different Subtypes of Human
Breast Cancer. Survival analysis showed that the overall survival (OS) of patients with high SLC1A5 expression was significantly lower compared to that of patients with low SLC1A5 expression (p = 0:048) (see Figure 1(a)), indicating that high expression of SLC1A5 is a poor prognostic factor for breast cancer patients, making SLC1A5 a potential therapeutic target in the treatment of breast cancer. In order to investigate the expression of SLC1A5 in different subtypes of breast cancer, we firstly obtained SLC1A5 expression data and clinical information of breast cancer patients from TCGA database. SLC1A5 expression was found to be the highest in TNBC patients and significantly higher compared to normal tissues and patients with luminal A breast cancer (see Figure 1 1(e)). Consistent with PCR results, Western blotting analyses also revealed that the protein expression level of SLC1A5 in TNBC cell lines, especially in MDA-MB-231, was significantly higher than other subtypes.
A previous study revealed that SLC1A5 not only is overexpressed but also has a marked reliance on its activity for sustained cellular proliferation transporter activity in breast cancer [17]. In this study, SLC1A5 was confirmed to be overexpressed in TNBC cell lines as well as in human TNBC 4 Journal of Nanomaterials

Journal of Nanomaterials
tissue samples compared to other subtypes by analysis of data from TCGA database. We further verified that TNBC cells highly express SLC1A5, because TNBC cells may require higher glutamine supply to provide energy for highly aggressive biological behavior. TNBC cells have a high demand for glutamine, and the receptor of glutamine on TNBC cells may be a potential target for treatment. A previous study has used glutamine analogue-modified therapeutic compound delivery systems to target glutamine-addictive cancer cells [21]. Therefore, we further explore a TNBC target therapy strategy using glutamine-grafted block copolymer as drug carrier.

Characterizations of Gln-PEG-b-PAE Copolymer.
The chemical structure and molecular weight of Gln-PEG-b-PAE copolymer were confirmed by 1 H NMR (Figure 2(a)) and GPC methods, respectively. The results showed that the Gln-PEG-b-PAE copolymer was successfully synthesized, and the Mw of the Gln-PEG-b-PAE copolymer was 16452, and the polymer dispersity index (PDI) was 1.25.
In order to determine the micelle forming ability of Gln-PEG-b-PAE copolymer, the critical micelle concentration (CMC) of the copolymer was assessed by pyrene fluorescein experiment [24]. The CMC of Gln-PEG-b-PAE copolymer was calculated according to the intensity ratio of the first emission peak to the third emission peak I1/I3 (the fluorescence intensity at 373 nm and at 384 nm) plotted against the base 10 logarithm of the tested concentrations [25,26]. The result showed that at physiological pH (7.4), the CMC of Gln-PEG-b-PAE copolymer was about 0.016 mg/mL (see Figure 2(b)), which means that thermodynamically structural stable polymeric micelles had formed under physiological conditions. The low CMC value enables the drug to be stably encapsulated in the micellar systems, so as to improve the bioavailability of the drug [27]. In this study, under physiological environment (pH 7.4), with the CMC value being of ca. 0.016 mg/mL, Gln-PEG-b-PAE copolymer could not only effectively carry DOX but also maintain the stability of the micellar structure. These results indicated that Gln-PEG-b-PAE copolymer was successfully prepared and can be used as drug carrier due to the ability of keeping structural stability under physiological conditions.

Characterizations of DOX-Loaded Polymeric Micelles.
The morphology of Gln-PEG-b-PAE copolymer and DOX-loaded micelles was characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The result of the light scattering experiments showed that the diameter of Gln-PEG-b-PAE@DOX micelles was 75.04 nm, which was 10 nm more than empty Gln-PEG-b-PAE copolymer (see Figures 2(d) and 2(e)), indicating that the average size of DOX-loaded polymeric micelles increased due to the entrapped DOX molecules. TEM also showed that the DOX-loaded polymeric micelles were in a spherical morphology (see Figure 2(c)), with an average size of about 45 nm.
These results demonstrated that we have successfully prepared DOX-loaded polymeric micelles and formed a uniformly distributed nanoscaled micelles structure in an aqueous medium. A previous study reported that the size of nanoparticles is one of the keys to determine whether to have an enhanced permeability and retention (EPR) effect, which also could be explained as the ability to accumulate in tumor tissues [28]. In this study, with the average size of about 75.04 nm, Gln-PEG-b-PAE@DOX micelles may show EPR-mediated passive targeting ability to accumulate in tumor tissues.

Stability and Drug Release of Gln-PEG-b-PAE@DOX.
The pH-responsive release of DOX from Gln-PEG-b-PAE@-DOX micelles was detected by the dialysis method in different simulated buffer solutions, including pH 7.4 of the physiological environment and pH 6.0 of tumor tissues. The pH-dependent DOX release profile is shown in Figure 3(a). In the initial 8 hours, DOX was explosively released under pH of 6.0, with about 80% release burst. The fast release rate of DOX indicated that DOX-loaded micelles were rapidly demicellized to release the drug in acidic environments. However, DOX was slowly released from the micelles at pH 7.4. After the initial burst of about 30%, the cumulative release grew slowly and reached the plateau gradually. Gln-PEG-b-PAE@DOX micelles exhibited significant pHdependent micellization-decellularization behavior, and micelles released DOX rapidly in an acidic environment of pH 6.0 but very slowly under physiological conditions. Moreover, as shown in Figure 3(b), Gln-PEG-b-PAE copolymer and Gln-PEG-b-PAE@DOX micelles retained steady sizes in pH 7.4 buffer solutions within 24 h. It is reasonable to believe that the Gln-PEG-b-PAE@DOX micelles can maintain structural integrity for a sufficient time before being distributed to the tumor tissues.
The key to the successful design of drug-loaded polymer micelles is to be able to control the location of drug release. Previous studies have revealed that pH-responsive polymer micelles allow the drug to be efficiently released in the acidic environment of tumor tissues [29,30]. Consistently, in this study, Gln-PEG-b-PAE@DOX micelles effectively released DOX under acidic conditions, showing significant pH-dependent micellization-decellularization behavior. Importantly, Gln-PEG-b-PAE@DOX micelles could reduce the release of DOX at physiological pH conditions, so as to reduce the side effects. Moreover, in this study, Gln-PEG-b-PAE@DOX micelles retained steady sizes in pH 7.4 buffer solutions within 24 h. It is reasonable to believe that Gln-PEG-b-PAE@DOX micelles could maintain structural stability in physiological environment during drug release, ensuring the feasibility of Gln-PEG-b-PAE copolymer as drug carrier.
3.5. In Vitro Cellular Affections. MTT assay and flow cytometry were carried out, in order to verify the antiproliferative and apoptosis effects of Gln-PEG-b-PAE@DOX micelles on MDA-MB-231 cells correspondingly. As shown in Figure 4(a), Gln-PEG-b-PAE@DOX micelles showed greater antiproliferative activity compared to free DOX. It could be explained that compared to cells treated with free DOX, cells treated with Gln-PEG-b-PAE@DOX micelles were effectively targeted, which led to stronger antiproliferative activity. Additionally, Gln-PEG-b-PAE copolymer 6 Journal of Nanomaterials     Journal of Nanomaterials of both pH-responsive release of DOX and glutaminemediated active targeting ability, Gln-PEG-b-PAE@DOX micelles could be a potential TNBC-targeted drug delivery system.

In Vivo Antitumor Efficacy of Gln-PEG-b-PAE@DOX
Micelles. The antitumor efficacy of free DOX, mPEG-b-PAE@DOX, and Gln-PEG-b-PAE@DOX micelles in vivo was evaluated in tumor-bearing BLAB/c nu/nu mice (see Figure 5(a)). When the mice were treated with Gln-PEG-b-PAE@DOX micelles, the tumor growth rate was significantly slower compared to those treated with free DOX and saline (see Figure 5(b)). After 17 days, free DOX at 2 mg/kg suppressed tumor volumes by 45.6%, compared to the control group, while mPEG-b-PAE@DOX micelles at 2 mg DOX/kg inhibited tumor sizes by 63.7%. Importantly, Gln-PEG-b-PAE@DOX micelles at 2 mg DOX/kg significantly inhibited tumor sizes by 78.3% (see Figures 5(d)-5(f)). Additionally, compared with mice treated with saline and free DOX, mice treated with Gln-PEG-b-PAE@DOX micelles had significantly lower fluorescence intensity (see Figures 5(c) and 5(g)). Tumor volumes in mice treated with mPEG-b-PAE@DOX micelles were significantly smaller than those in mice treated with free DOX. This result could be explained by the EPR effect which makes mPEG-b-PAE@-DOX micelles more likely to be accumulated in tumors. Previous studies have also reported this passive accumulation of polymer conjugate in various solid tumor models due to the EPR effect [28,31,32]. Generally, in this study, Gln-PEG-b-PAE@DOX micelles showed the highest antitumor effect on transplanted tumors compared with mPEG-b-PAE@DOX micelles and free DOX. The pHdependent release of DOX and glutamine-mediated targeting ability make Gln-PEG-b-PAE@DOX micelles show excellent antitumor effect not only in vitro but also in vivo. Therefore, it is reasonable to believe Gln-PEG-b-PAE@DOX micelles are an ideal tumor-targeting drug delivery system for TNBC.

Conclusions
In this study, glutamine was conjugated to the amphiphilic PEG-b-PAE copolymer, in which glutamine was used for active targeting of TNBC cells. The pH-triggered Gln-PEGb-PAE@DOX copolymeric micelles showed a spherical morphology with an average size about 75.04 nm, being able to maintain structural stability in physiological environment and release DOX in tumoral acidic environment effectively. In vitro, Gln-PEG-b-PAE copolymer showed low cytotoxicity, while Gln-PEG-b-PAE@DOX micelles showed significantly stronger cytotoxicity. Compared to free DOX and mPEG-b-PAE@DOX, Gln-PEG-b-PAE@DOX micelles exhibited significantly higher ability to inhibit tumor growth in vivo due to pH-responsive release of DOX and glutaminemediated active tumor-targeting ability. Therefore, the amphiphilic Gln-PEG-b-PAE@DOX micelles may be a new target therapy strategy for TNBC, and also, Gln-PEG-b-PAE copolymer could be a promising carrier of antitumor drugs for TNBC.