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ROS-Switchable Polymeric Nanoplatform with Stimuli-Responsive Release for Active Targeted Drug Delivery to Breast Cancer Yu Zhang,†,‡ Qin Guo,†,‡ Sai An,†,‡ Yifei Lu,†,‡ Jianfeng Li,†,‡ Xi He,† Lisha Liu,†,‡ Yujie Zhang,†,‡ Tao Sun,†,‡ and Chen Jiang*,†,‡ †

Key Laboratory of Smart Drug Delivery, Ministry of Education and Department of Pharmaceutics, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China ‡ State Key Laboratory of Medical Neurobiology, Fudan University, 138 Yixueyuan Road, Shanghai 200032, China S Supporting Information *

ABSTRACT: Tumor microenvironment plays a vital role in the process of tumor development, proliferation, invasion, and metastasis. It is well acknowledged that reduction in pH, reactive oxygen species (ROS), and increased level of glucose transporter 1 (GLUT1) have become featured intracellular and extracellular biochemical markers of cancer owing to oncogenic transformation and abnormal metabolism. To establish a distinctive drug delivery system directed against the tumor microenvironment features, we develop a newly engineered polymeric nanoplatform for efficient doxorubicin (DOX) delivery with reduced systemic toxicity and high antitumor efficiency. A thioketal cross-linker is used to improve the formulation’s stability during circulation and to foster quick intracellular drug release in response to tumor’s ROS potential. Furthermore, the low drug loading efficiency of conventional micelles is ameliorated in this polymeric nanoplatform via a drugconjugation strategy with an acid-labile chemical bond. The optimized formulation, MPLs-sB-DOX micelles, possesses a high drug-loading efficiency (31%) within nanosize diameter (37.8 nm). In addition, this formulation shows significant improvement in the pharmacokinetics and biodistribution profiles with a 2.69-fold increase of tumor accumulation, while with largely reduced systemic toxicity in comparison with free DOX. With advantages of efficient cellular uptake, preferential tumor accumulation, and controlled release behaviors, MPLs-sB-DOX micelles demonstrate good tumor-targeting ability with reduced systemic toxicity, proving to be a promising formulation for breast cancer therapy. KEYWORDS: ROS responsive, tumor microenvironment, controlled release, drug delivery, polymeric nanoplatform

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INTRODUCTION

compatible for encapsulation of various insoluble drugs, whereas the hydrophilic shell enables the nanoplatform to be stealth to the opsonic effect. However, a micellar delivery system should simultaneously have the ability of both resistance to leakage in plasma and rapid release in the cancerous site. While circulating in the blood, the micelles demand considerable stability against plasma dilution. After the accumulation of micelles in the cancerous site, quick drug release turns out to be the lynchpin. What is more, the micelles in clinical application are also limited by the low drug loading efficiency. Other than the traditional optimizing methods on formulations and polymer structure control, drug−polymer conjugation gradually turns out to be an efficient strategy for high drug loading and stimuli-responsive release.14−17 Depending on the intricate biological features, a series of factors

Polymeric micelles have been explored as a promising nanoplatform for chemo-drug delivery for their innate superiority of core−shell structures.1−3 They could provide a platform to overcome drawbacks of free drugs, such as poor solubility, nonselectivity, nonspecific biodistribution and pharmacokinetics (PK) property, and even multidrug resistance.1,4−6 On the one hand, the nanoscale (10−100 nm) selfassembly signature of micelles makes it facile to prepare and boosts the preferential accumulation in the cancerous site through leaky blood vessels and defective lymphatic drainage, which is defined as the enhanced permeability and retention (EPR) effect.7,8 Moreover, a lot of targeting ligands have been screened to improve internalization in the tumor region by receptor-mediated endocytosis, such as folate acid, hyaluronic acid, peptides, aptamers, and so forth.9−13 Active targeting strategy increases the uptake efficiency of micelles in the tumor region, thus improving the therapeutic effect of chemo drugs. On the other hand, the hydrophobic core of micelles is © XXXX American Chemical Society

Received: December 30, 2016 Accepted: March 22, 2017

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DOI: 10.1021/acsami.6b16815 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

glucose.38−40 With this specific cellular characteristic, a series of applications of 18-fluorodeoxyglucose (FDG) derivatives that targeted GLUT1 have been developed for positron emission tomography imaging of tumors in clinics, indicating a potential tumor marker for accurate drug delivery.41−43 Mannose, a natural substrate of GLUT1, is an important monosaccharide in human metabolism as a key factor in the glycosylation of functional proteins on the cell membrane and in the signaling pathway. Therefore, mannose-modified micelles’ endocytosis is anticipated to be enhanced by GLUT1-mediated transportation. To improve the general antitumor efficacy of drug-loaded micelles, we elaborately designed a micellar nanoplatform with high drug load as well as rapid drug unpacking in response to tumor microenvironmental stimuli. On the basis of the biochemical features of the inflammatory microenvironment, in this study, the thioketal bond was explored as a unique responsive moiety for intracellular ROS stimulus to foster the fast release of chemo drugs. A copolymer of PEG and poly(amino acids), poly(ethylene glycol)−poly(N6-carbobenzyloxy- L-lysine)−poly(β-benzyl-L-aspartate) (PEG−pLys− pBla), was synthesized to achieve biocompatibility and biodegradability. The primary-amine-rich pLys block would provide interlace sites for the ROS cleavable cross-linker, whereas the pBla block would form the hydrophobic core by acid-labile covalent conjugation of DOX via a hydrazine bond. In vivo stability was supposed to be enhanced by the crosslinking strategy, whereas high drug loading efficiency was expected to be achieved through the drug-conjugation strategy. To further fuel the endocytosis efficiency of the micelles, mannose was chosen as the monosaccharide-targeting ligand for surface modification. In this article, the stability, kinetic behaviors, and antitumor efficacy of these micelles were investigated in vitro and in vivo.

influence the in vivo PK and uptake efficiency of the micelles. An ideal polymeric micellar nanoplatform for in vivo drug delivery should optimize the miscellaneous aspects of the micelles’ characteristics to be applicable to the intricate physiological and pathological environment of the diseases. Overall, for chemo-drug delivery to tumors, increased drug loading capacity, enhanced micelle stability, controlled drug release, and specific cell targeting pave the road for precise drug delivery and enhanced therapeutic effect. Over the last few decades, many pathological features of tumor have been unfolded to be involved in the disease development, including, but not limited to, the inflammatory symptoms, abnormal metabolism, significant heterogeneity, altered biochemical microenvironment, anomalous angiogenesis, lymph defect, and so forth. Various studies demonstrate that tumor development and metastasis are in correlation with chronic inflammatory symptoms.17−19 Cancerrelated inflammation is therefore stamped as the seventh hallmark of cancer, as found to be involved in the proliferation of lung cancer, colorectal cancer, breast cancer, brain cancer, and so forth.20−22 Particularly, breast cancer is the second leading cause of death from cancer in women; inflammatory breast cancer tends to be aggressive and malignant with poor prognosis.23 In addition, the inflammation features seem to be correlated with distant metastasis through lymph nodes.24,25 Because of the involvement of numerous immune cells, such as macrophages, granulocytes, dendritic cells, and natural killer cells, the inflammatory tumor microenvironment is featured by biochemical signatures, such as the presence of oxidative stress (oxidants include •OH, ONOO−, H2O2, etc.) and reduction of pH.26−28 Mounting evidence suggests that many tumor types, including breast cancer, have an increased level of reactive oxygen species (ROS) compared with their counterparts, which provide a new target for tumor therapy and inspire the intelligent design of drug-delivery systems.29 Many chemical compounds have been reported to be oxidation sensitive, such as thioketal, propylene sulfide, selenium-containing bond, and so forth.30−33 Poly-(1,4-phenyleneacetone dimethylene thioketal) (PPADT), a thioketal-containing condensation polymer, has been explored to pack TNF-α-siRNA and encapsulate curcumin for the ROS-responsive intracellular delivery of therapeutic to the inflammatory site of ankle inflammation and intestinal inflammation. 34,35 As cancer cells constantly generated high levels of intracellular ROS owing to oncogenic transformation, herein, we renovated the thioketal bond as a small molecular cross-linker for a biocompatible drug-loading nanoplatform to reduce the drug preleakage by fastening the micellar structure. Furthermore, tumor is widely acknowledged by the high metabolic burden due to the insufficient metabolism of carbohydrate in the hypoxia microenvironment, known as the Warburg effect.36 To survive under these ischemic conditions, tumor cells therefore modulate the expression level of certain functional transporters and receptors against the hypoxia microenvironment, which provide promising targeting sites for a drug-delivery system. Because of the less efficient glycolytic pathway, the tumor cells demand tremendous amounts of saccharide supplement as extra energy source. Increased sugar uptake is accomplished by upregulation of glucose transporter expression.37 As the leading member of the glucose transporter family, facilitative glucose transporter isoform 1 (GLUT1) is highly expressed on malignant tumor cells catering to their unusual excessive consumption of

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RESULTS AND DISCUSSION For efficient tumor-targeting therapy, it required stable long circulation in plasma and quick drug release in the cancerous site. In this work, we prepared a multifunctional polymeric micellar nanoplatform for breast cancer targeting and controlled drug release. With the thioketal-contained crosslinking and drug-conjugation strategies, the micelles could avoid preleakage in plasma as the cross-linking interlayer provided a barrier against blood dilution. After accumulation in the cancerous sites via the EPR effect, the mannose-targeting moiety could actively facilitate the endocytosis of the formulation via GLUT1 binding, which enhanced the targeting efficiency. After entering the tumor cells, the drug would undergo quick release triggered by both acidity and ROS stimuli. The following results demonstrated that MPLs-sBDOX micelles worked as an efficient targeting drug delivery nanoplatform for breast cancer. Preparation and Characterization of Polymers and Micelles. The triblock amphiphilic copolymer was synthesized via a one-pot ring-opening polymerization (ROP) reaction with N3-PEG-NH2 and CH3O-PEG-NH2 as initiators, respectively, for targeting the ligand-modified polymer and the nonmodified one. The amino acids were activated by triphosgene as previously reported and extensively purified by repeated precipitation,46,47 as shown in the 1H NMR spectra (Figure S2A,B). The molar composition ratio of PEG, pLys block, and pBla block was 1:10:10 (Figure S2E,F). Each block was anticipated to play different roles in the formulations: the PEG B


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Figure 1. Size distribution of (A) MPLs-sB-DOX and (B) MPLB-DOX micelles measured by dynamic light scattering (DLS). AFM/TEM images of (C, E) MPLs-sB-DOX and (D, F) MPLB-DOX micelles.

ness and targeting efficiency. Mannose-modified copolymers were added as functional portion to improve the internalization efficiency of the micelles. Miscellaneous properties of MPLs-sBDOX and MPLB-DOX micelles are shown in Figure 1A,B and Table 1. A sharp decrease in the zeta-potential from MPLB-

block would form the hydrophilic corona of the micelles for prolonging circulation in plasma and conjugation of targeting ligands; the pLys block would work as the interlayer for crosslinking to enhance the stability of the formulations; the pBla block was designed for drug conjugation, therefore forming the hydrophobic core of the micelles for drug-loading activity. The small molecular thioketal-containing cross-linker was prepared as N-hydroxysuccinimide ester for cross-linking (Figure S2C) inspired by the poly-(1,4-phenyleneactone dimethylene thioketal polymer used in the anti-inflammation drug-delivery system.33,34 The alkynyl mannose was synthesized via an esterification reaction, as previously reported (Figure S2D).45 Conjugation of alkynyl mannose to N3-PEG−pLys− pBla was performed via a click reaction. The IR spectrum (Figure S3) showed the disappearance of the azide group peak at 2100 cm−1, indicating successful conjugation. The micelles were prepared via a facile method of thin-film hydration, whereas cross-linking was accomplished by adding cross-linkers after the micellar structure formed. Four kinds of micelles, with or without the cross-linking or targeting modification, were prepared to evaluate the stimuli responsive-

Table 1. Summary of Properties of Major Micellar Formulations formulations size d (nm) PDI zeta-potential (mV) DOX loading efficiency (%)

MPLs-sB-DOX 37.8 0.203 3.72 31.32

± ± ± ±

2.3 0.002 0.17 0.14

MPLB-DOX 39.2 0.255 11.7 31.87

± ± ± ±

4.1 0.004 1.83 0.25

DOX to MPLs-sB-DOX indicated effective cross-linking. The average hydrodynamic diameter of MPLs-sB-DOX micelles was 37.8 nm, which showed slight shrinkage in comparison to the non-cross-linked ones possibly due to the reduced repellant effect among the primary amine groups on pLys blocks. The micelles’ stability against dilution was tested by measuring the C


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Figure 2. (A) Cellular uptake of (a) PLs-sB-DOX micelles and MPLs-sB-DOX micelles with different mannose/PEG−pLys−pAsp ratios: (b) 10%, (c) 20%, and (d) 40% and (e) free DOX in MDA-MB-231 tumor cells 60 min after incubation. Possible pathway of MPLs-sB-DOX micelle internalization into MDA-MB-231 tumor cells. The cells were blocked by different inhibitors (f) 0.5 μg/mL filipin; (g) 0.25 μg/mL PhAsO; (h) 1 μg/mL colchicine; (i) 10 mM D-glucose. Original magnification = 200×. (B) Flow cytometry analysis results of (a)−(i).

the innate ongoing proliferation property of tumor cells. The minute difference between 20 and 40% modified micelles may result from the saturation of transport capacity of GLUT1 to mannose. In consideration of systemic long circulation, 20% modified MPLs-sB-DOX micelles were chosen in the subsequent studies. To further elucidate the internalization mechanism of MPLssB-DOX micelles, several possible internalizing pathways were inhibited individually to evaluate the effect on endocytosis efficiency. Inhibitors including D-glucose (blocking GLUT1), PhAsO (blocking clathrin-dependent pathway), filipin complex (blocking caveolae-mediated pathway), and colchicine (blocking macropinocytosis) were applied to treat the cells (Figure 2A,f−i). Fluorescence signals of DOX and flow cytometry results demonstrated that a high concentration of D-glucose (10 mM) and filipin complex exhibited notable inhibiting effect on the uptake of MPLs-sB-DOX micelles. Colchicine and PhAsO also showed potential inhibiting ability for internalization. According to the above results, MPLs-sB-DOX micelles were internalized into tumor cells principally relying on the caveolaemediated endocytosis, whereas the clathrin pathway also partially contributed. The small size of MPLs-sB-DOX micelles may also exert an effect on the endocytosis efficiency. In Vitro DOX Release Behaviors. To verify our design about the dual responsive property of MPLs-sB-DOX (Figure 3A), we investigated the in vitro releasing behaviors of MPLBDOX and MPLs-sB-DOX under various artificial milieu, mimicking the tumor microenvironment. In addition, the releasing profile of MPLB-DOX, which is not presented in the data figure, showed 37% release at pH 7.4 (24 h) and 65% at pH 5.0 (24 h). The MPLs-sB-DOX underwent fast release within 6 h in the presence of H2O2 at pH 5.0 with over 60% drug released within 48 h in a sustained profile (Figure 3B).

size and PDI with different dilution ratios. Formulations diluted to different concentrations could maintain the size within 37− 42 nm, PDI < 0.26. Atom force microscopy (AFM) (Figure 1C,D) and transmission electron microscopy (TEM) (Figure 1E,F) images showed uniform spherical morphology with moderate dispersity of the micelles. Because the cross-linking reaction was conducted after the formation of the micellar structure, no obvious aggregation was observed according to the TEM results. The DOX loading efficiency was significantly improved due to the covalent conjugation strategy (Table 1), reaching as high as approximately 31%, which made the micelles efficient for in vivo drug administration. Cellular Uptake and Internalization Mechanism of Micelles. To optimize the delivery efficiency of the dual stimuli-responsive micellar drug delivery nanoplatform, mannose was introduced to modify the copolymer for enhanced uptake. We investigated the cellular uptake signatures and the possible internalization pathway of micelles in MDA-MB-231 cell line. The DOX uptake was observed using a fluorescence microscope (FL3 channel, Figure 2A, a−i), whereas quantitative data were obtained from flow cytometry (Figure 2B). To evaluate the optimized ligand-modified ratio, 10, 20, and 40% mannose-modified PEG−pLys−pBla copolymers were applied to prepare MPLs-sB-DOX micelles. In comparison to the nontargeting PLs-sB-DOX micelles, the uptake increased as the modified ratio increased, whereas there was no significant difference in uptake between the 20 and 40% ones (Figure 2A,c,d). Such results may be due to the boosted demand of tumor cells mediated by GLUT1 transportation. Mannose is an essential monosaccharide required for glycosylation of many important functional glycoproteins on cell membranes and intracellular signaling pathways. GLUT1 appeared as an important transporter for monosaccharide uptake, catering to D


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Figure 3. (A) Illustration of the release mechanism of MPLs-sB-DOX micelles. (B) Release profile of MPLs-sB-DOX micelles in different artificial milieu. (C) Drug release of MPLs-sB-DOX micelles on MDA-MB-231 cells at 0.5 and 1.5 h.

reaching the accumulative release platform. However, compared to the non-cross-linked micelles (pH 5.0, 65%, 24 h), the cross-linked ones could achieve similar accumulative release (pH 5.0, 58%, 24 h) in the presence of H2O2, while remaining relatively stable at pH 7.4 without H2O2 (20%, 24 h). Therefore, we could conclude that the cross-linking layer worked more like an indirect lock instead of a decisive switch. From this point, although the cross-linker may not contribute to an expedited drug release to a great extent, we can see that it

This result validated the dual stimuli-responsive signature of MPLs-sB-DOX micelles. Under the physiological condition at pH 7.4 without H2O2, which mimicked blood circulation, the micelles remained relatively stable with little drug released. The cross-linking layer might work as an interlace lock preventing the disassembling process against blood dilution. When treated with H2O2 at pH 5.0, the interlace cross-linking moieties got cleaved before the H+-rich releasing medium quickly hydrolyzed the hydrazine bond and unleashed the free drug, finally E


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Figure 4. (A) Pharmacokinetic profiles of DOX in SD rats after i.v. injection with different DOX formulations at a dose of 5 mg DOX/kg. Data are presented as mean ± SD (n = 5). (B) Distribution profiles of DOX in main organs and tumor after i.v. injection with different DOX formulations at a dose of 5 mg DOX/kg. Data are presented as mean ± SD (n = 4).

still worked extraordinarily as an anti-preleakage lock that provided the micelles with controlled release behavior. To further investigate the release behaviors of MPLs-sBDOX in biological milieu, we performed confocal imaging experiments to follow the micelles’ trace after being internalized by tumor cells (Figure 3C). The yellow and orange spots in the merged images indicated the co-localization of DOX and late endosome. The DOX co-localized with the late endosomes at 0.5 h, whereas at 1.5 h, according to the results, obvious DOX release was observed, indicating the burst release from MPLssB-DOX in the intracellular microenvironment. Therefore, the MPLs-sB-DOX micelles showed stimuli-responsive release behaviors in tumor cells, while retaining good stability in the physiological environment. Enhanced Tumor Accumulation and Reduced Systemic Toxicity in Vivo. We assumed that the cross-linking strategy endowed the micellar structures with enhanced stability, high integrity, and antileakage signature against blood dilution. The kinetic behaviors of MPLs-sB-DOX on animals were then studied to test the micelles in vivo. Pharmacokinetic profiles of MPLs-sB-DOX micelles and the non-cross-linked ones were tested on healthy SD rats, and the DOX concentrations in plasma at various time points were recorded (Figure 4A and Table 2). There was a significant 4.6fold improvement in the AUC value between the cross-linked and non-cross-linked formulations, indicating that the cross-

Table 2. Pharmacokinetic Parameters of DOX in SD Rats after i.v. Injection with Different DOX Formulations at a Dose of 5 mg DOX/kga formulations free DOX PLB-DOX PLs-sB-DOX MPLB-DOX MPLs-sB-DOX a

MRT (h)

AUC (mg/L h)

± ± ± ± ±

1.611± 0.053 83.030 ± 5.842 383.959 ± 35.728 62.066 ± 2.744 287.91 ± 21.481

3.083 7.741 8.927 8.028 8.485

0.325 0.483 0.986 0.504 0.821

Data are presented as mean ± SD (n = 5).

linking strategy also exerted efficacious stabilizing function in vivo. The general increase in AUC by micellar formulation compared with that with free DOX also showed the improvement in long circulation. Because of encapsulation in the MPLs-sB-DOX micelles, the clearance of DOX from blood was reduced, thus resulting in a higher chance of accumulation in the cancerous site. A biodistribtion study further validated this hypothesis by analyzing the DOX accumulated in principal organs and tissues on MDA-MB-231 orthodox breast cancer xenograft models 12 h after being injected with different formulations. According to biodistribution profiles (Figure 4B), the cross-linked micelles showed approximately 3-fold accumulation in tumor sites in comparison with the non-cross-linked ones, whrease only a F


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Figure 5. (A) In vivo noninvasive NIR fluorescence imaging of MDA-MB-231 orthodox breast cancer xenograft models at (a) 2 h, (b) 4 h, (c) 8 h, and (d) 12 h at Ex/Em 650/665 nm after being injected with BODIPY-labeled MPLB-DOX micelles (left) and MPLs-sB-DOX micelles (right). (B) Main organs and tumor imaging 12 h after injection. From top to bottom, heart, liver, spleen, kidney, lung, and tumor of MPLB-DOX micelle-treated mouse (left) and MPLs-sB-DOX micelle-treated one (right) are displayed. (C) In vivo 3D fluorescence imaging of MDA-MB-231 orthodox breast cancer xenograft models 12 h after injection with BODIPY-labeled MPLs-sB-DOX micelles.

drug concentration in liver. It is undisputed that RES phagocytic cells would capture some of the micelles because liver is reported to be an organ with high blood perfusion and the main metabolic pathway of nanoparticle formulations. To some extent, as the cross-linked micelles remained stable during circulation, they maintained the same possibility of flowing through liver and tumor. Therefore, we deduced that the DOX concentration in liver indicated a successful elongation of the micelles’ circulating time in vivo. To further confirm the potential liver and systemic toxicity, HE staining of the principal organs was performed after the subsequent in vivo

trace amount was seen in hearts. In vivo imaging of the BODIPY-labeled micelles also verified notable accumulation in the cancerous site at the right flank of the mice after 8 h (Figure 5A). Main organs and tumor were eviscerated 12 h after injection for imaging (Figure 5B). It showed that there was significant accumulation in the tumor tissues and livers by all of the active targeting micelles, whereas the cross-linked ones went beyond the non-cross-linked ones. In vivo threedimensional (3D) fluorescence imaging of MPLs-sB-DOX micelle-treated mouse also verified this result (Figure 5C). However, there remained a problem about the unexpected high G


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Figure 6. HE staining results of main organs of mice treated with different DOX formulations. A saline-treated group was used as the control.

that such a strategy mainly contributed to the micellar stability in vivo. To further elucidate the antitumor efficacy of MPLs-sB-DOX micelles, we evaluated in vitro apoptosis on the MDA-MB-231 cell line by the Annexin V-FITC and PI assay. Phosphatidylserine (PS) is found only on the intracellular leaflet of the plasma membrane in normal cells. Annexin V could specifically bind to PS when it’s turning out of the cell membrane during early apoptosis of cells. FITC-labeled annexin V could be visualized using a fluorescence microscope as green signals. PI could specifically bind to intracellular DNA/RNA through damaged cell membranes, which appeared in the late apoptosis of cells, because the normal cells and even the early apoptosis ones still maintained complete membranes. The cells treated by different DOX formulations and Annexin V/PI staining presented the general apoptosis status, as shown in the fluorescence pictures (Figure 7B). Single green FITC staining indicated the cells in early apoptosis, whereas the dual staining of FITC/PI shown as yellow spots in the merged images indicated those in late apoptosis. Co-localization of the green and red signals (as marked by the arrows) showed MPLs-sBDOX micelle induced efficient apoptosis in MDA-MB-231 cells. The results of apoptosis experiments are consistent with the MTT data on cytotoxicity, indicating the good antitumor efficacy of MPLs-sB-DOX micelles in vitro.

antitumor experiments to evaluate repeated dose toxicity (Figure 6). Obvious inflammatory symptoms in the portal area of liver and lung and slight hyperemia in myocardium were observed in free DOX-treated mice, whereas those of micellar formulation treated ones remained in the healthy state. Consequently, MPLs-sB-DOX micelles could serve as an efficient DOX delivery system reducing systemic toxicity, while improving drug-delivery efficiency in cancerous sites. Detection of in Vitro Antitumor Efficacy. In vitro cytotoxicity of free DOX and different micellar formulations was studied on MDA-MB-231 cell lines. MTT assay was performed to determine the IC50 values and general antitumor efficacy. In vitro results revealed that the mannose-modified micelles exhibited higher inhibiting ability against breast cancer cells than free DOX mainly due to the coefficient transportation of GLUT1 with IC50 values of 0.504 and 0.462 μg/mL for cross-linked ones and non-cross-linked ones, respectively (Figure 7A). Moreover, the micelles without ligand modification showed retarded cytotoxicity compared to free DOX. This phenomenon possibly resulted from their different uptake pathways. Free DOX could enter cells via passive diffusion with its hydrophobicity assisting to cross the lipoid double-layer cell membrane. In contrast, the micelles without ligand modification mainly depended on endocytosis. Cross-linking seemed to have no role in the conspicuous improvement in antitumor efficacy at the cell level because previous data demonstrated H


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Figure 7. (A) Cell viability and IC50 of MDA-MB-231 cells after being treated with cascade concentrations of different DOX formulations for 48 h. (B) In vitro apoptosis assay of MDA-MB-231 cells with pretreatment of different DOX formulations at a normalized DOX concentration of 1 μg/ mL (green signals: early apoptosis cells, red signals: late apoptosis cells, original magnification = 100×).

Detection of in Vivo Antitumor Efficacy. The in vivo antitumor efficacy of various DOX formulations was evaluated by the tumor volume changes during 3-dose administrations, which were calculated by the luminescence signals from the luciferase-transfected cells. Figure 8B,C shows that MPLs-sBDOX micelle-treated mice exhibited remarkably smaller tumor volume compared to other groups. Various degrees of limited inhibition were seen in the free DOX-, MPLB-DOX micelle-, and PLs-sB-DOX micelle-treated groups due to rapid clearance, in vivo instability, or the absence of targeting ligand. In addition, the mice body weight was recorded every 2 days to evaluate the general toxicity (Figure 8A). The free DOX-treated groups showed significant decrease in body weight, whereas MPLs-sB-DOX micelle-treated mice showed no significant influence on the body weight increase with age. Furthermore, after the 3-dose course of therapy, the mice were sacrificed and the tumors were excised to carry out the TUNEL immunohistological assay for the detection of apoptosis at the tissue level (Figure 8D). The green stained signals of FITC-labeled dUTP stained area indicated an apoptosis site in tumor. Samples from the MPLs-sB-DOX micelle-treated mice showed the most extensive apoptotic cells,

indicating the considerable antitumor efficacy at the tissue level, which was consistent with the previous results of tumor volumes. In comparison to the in vitro antitumor results, crosslinked micelles presented better efficiency than the non-crosslinked ones, possibly due to enhanced accumulation in the cancerous site depending on the in vivo stability and long circulation of the formulations.

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CONCLUSIONS

In brief, dual stimuli-responsive cross-linked DOX-conjugated MPLs-sB-DOX micelles, with monosaccharide as the targeting ligand, were developed for efficient therapy of breast cancer with a controlled release profile. Such formulations provided a promising micellar nanoplatform with considerable antitumor efficacy and low systemic toxicity by integrated formulation design catering to the whole in vivo course of drug-delivery systems. The drug loading efficiency was elevated by covalently conjugated DOX via pH-sensitive hydrazine bonds on the polymer as the hydrophobic block. The micelles were stabilized via the cross-linking strategy in the amine-rich interlayer during in vivo circulation. Owing to the PEG corona and monosaccharide modification, the micelles were accurately I


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Figure 8. Body weight (A) and tumor volume changes (B) of MDA-MB-231 orthodox breast cancer xenograft models after 3-dose i.v. injection of different DOX formulations at a dose of 5 mg DOX/kg on day 0, 7, and 14. A saline-treated group served as the control. Data are presented as mean ± SD (n = 6). (C) Bioluminescence images of MDA-MB-231 orthodox breast cancer xenograft models within the 3-dose administration of different DOX formulations on days 12, 16, and 20. (D) Representative histological images of MDA-MB-231 tumor xenografts excised from mice models used in the in vivo antitumor efficacy experiments; original magnification = 100×. J


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protection. After 48 h, the solution was added to Bla-NCA (250 mg, 1 mmol) in 5 mL anhydrous DMSO solution and maintained for another 48 h. The polymer was precipitated into diethyl ether and was dried under vacuum to yield a white solid. Next, the polymer underwent 2-step deprotection. After being treated with TFA (10 mL/ g) and HBr/HOAc (0.5 mL/g), the purified intermediate polymer was then removed the benzyl esters of pBla blocks through an aminolysis reaction by reacting with hydrazide (500 μL/g). For each step of deprotection, the product polymer was purified by dialysis using a membrane (molecular weight cutoff (MWCO: 5000) for 24 h before lyophilization to obtain a white flocculent solid. Finally, the polymer was conjugated with DOX via hydrazine bonds. Desalted with triethylamine, DOX was added to the hydrazide groups of PEG− pLys−pBla in DMSO in a molar ratio of 1:1 at 30 °C for 24 h before being purified by dialysis and Sephadex LH20 gel separation followed by lyophilization. N3-PEG-NH2-initiated polymer for ligand modification was synthesized by the same procedure. For the synthesis of thioketal cross-linker, to a mixture of anhydrous 2,2-dimethoxypropane (0.49 g, 4.7 mmol) and 4-methylbenzenesulfonic acid (40 mg, 0.235 mmol) was added anhydrous 3-mercaptopropionic acid (1 g, 9.4 mmol) in an ice bath and reacted for 48 h at ambient temperature. Then, a THF solution of N-hydroxysuccinimide (1.1 g, 9.4 mmol) and trimethylamine (0.95 g, 9.4 mmol) were then added and reacted for another 24 h at ambient temperature.44 The product was purified by column chromatography. Alkynyl mannose was prepared as follows: to a DMF solution of D-mannose (2 g, 11.1 mmol) was added 6heptynoic acid (1.39 g, 11.1 mmol) and pyridine for 48 h at 60 °C. The product was repetitively purified by silica gel plates.45 All synthesis routes are illustrated in Figure S1 in the Supporting Information. Preparation of Micelles. The PLB-DOX micelles were prepared by the thin-film hydration method. For preparation, 1 mg mPEG-pLyspBla was dissolved in DCM and the solvent was retrieved to form a thin film on the round bottom flask by a rotatory evaporator. Then, 1 mL water (or PBS 7.4) was added and stirred vigorously for 12 h in the dark. The DOX-loaded non-cross-linked micelles PLB-DOX were collected after filtration using a 0.22 μm filter membrane. As for crosslinked micelles, the thioketal-containing cross-linker was then added to the as-prepared solution at a molar ratio of [cross-linker]/[Lys] = 1:5. The reaction was maintained for 3 h at pH 8.0 before being dialyzed against deionized water for 24 h to remove extra cross-linkers. The cross-linked micelles PLs-sB-DOX were then collected after filtration using a 0.22 μm filter membrane. As for mannose-modified micelles, mPEG−pLys−pBla and mannose−PEG−pLys−pBla polymers were mixed at a molar ratio of 4:1 followed by the same preparation procedure as above. As for micelles applied in pharmacodynamic experiments, to ensure that the experimental animals receive the same concentration of DOX, DOX loading of each formulation was calibrated after preparation and diluted to a certain concentration (equivalent to 1 mg DOX/mL) for injection. The general drug loading efficiency of all four formulations was within an acceptable range of 30−32%. So the dilution would not bring in significant difference in number density. The near-infrared fluorescent probe, BODIPY, labeled micelles were prepared according to the procedure mentioned above with addition of 10 wt % BODIPY in the cross-linking step. All of the prepared micelles were stored in 4 °C in the dark before use. Characterization. Synthetic monomers and copolymers were characterized by 1H NMR spectra measured using an NMR spectrometer (400 MHz, Bruker, Coventry, U.K.) with tetramethylsilane as the internal reference and chloroform-d or DMSO-d6 as the solvent. The size/PDI and zeta-potential of the prepared micelles were measured by DLS (Zetasizer Nano-ZS, Malvern 3600, Worcestershire, U.K.). The morphological examination of micelles was performed using AFM (Multimode NanoscopeIIIa, Bruker, Coventry, U.K.) and Biology Transmission Electron Microscopy (B-TEM, Tecnai G2 spirit Biotwin, FEI, Hillsboro). The DOX loading efficiency was measured using a microplate spectrophotometer (BioTek, Winooski). Cellular Uptake and Internalization Mechanism Studies. MDA-MB-231 cells were seeded in 24-well plates (Corning, New York) at a density of 1 × 104 cells/well and incubated at 37 °C for 24 h

accumulated in breast cancer cells, relying on both EPR effect and active cell targeting. Then, the drug release was hastened in response to the intracellular ROS stimulus. In addition to these, the nontoxic PEG and biodegradable poly(amino acid) blocks endowed the micelles with favorable biocompatibility. Therefore, these micelles provided a prospective attempt in enhanced chemotherapy for breast cancer.

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MATERIALS AND METHODS

Materials. Triphosgene was purchased from TCI (Japan). N6Carbobenzyloxy-L-lysine (Lys), β-benzyl-L-aspartic acid (Bla), 3mercaptopropionic acid, N-hydroxysuccinimide (NHS), 2,2-dimethoxypropane, 6-heptynoic acid, and D-mannose were purchased from Aladdin (Shanghai, China). Anhydrous hydrazine, 33% hydrobromic acid in acetic acid (HBr/HOAc), trifluoroacetic acid (TFA), anhydrous tetrahydrofuran (THF), anhydrous hexane, anhydrous dimethylsulfoxide (DMSO), and anhydrous dichloromethane (DCM) were purchased from Acros Organics (Brussels, Belgium). α-Methoxyω-amino-poly(ethylene glycol) (mPEG-NH2, MW 5000) was purchased from Seebio Biotech (Shanghai, China). α-Azide-ωamino-poly(ethylene glycol) (N3-PEG-NH2, MW 5000) was purchased from Jenkem Technology (Beijing, China). Doxorubicin hydrochloride (DOX·HCl) was purchased from Meilun Biotech (Dalian, China). All of the chemical reagents were used as received without further purification. High-concentration Matrigel Matrix, phenol red-free, was purchased from Corning BioCoat (New York). Phenylarsine oxide, filipin complex, colchicine, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis). 4,6-Diamidino-2-phenylindole (DAPI), Hoechst 33342 trihydrochloride, LysoTracker Deep Red, and BODIPY FL were purchased from Molecular Probes (Eugene). One-Step TUNEL Apoptosis Assay Kit and Annexin V-FITC/PI Apoptosis Detection Kit were purchased from KeyGEN BioTECH (Nanjing, China). Other reagents without specific explanation were purchased from Sinopharm Chemical Reagent (Shanghai, China). MDA-MB-231 human breast cancer cell lines were kindly provided by Perkin Elmer. MDA-MB-231 cells were carefully maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L glucose supplemented with 10% heat-inactivated fetal bovine serum and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 100 U/mL penicillin, and 100 μg/mL streptomycin cultured at 37 °C under a humidified atmosphere containing 5% CO2. Female Balb/c nude mice of about 20 g body weight and male SD rats of about 220 g body weight were purchased from the Department of Experimental Animals, Fudan University, and carefully maintained under standard SPF laboratory conditions. All animal experiments were performed in accordance with guidelines evaluated and approved by the ethics committee of Fudan University. For construction of the orthodox breast cancer xenograft model, 1 × 106 MDA-MB-231 cells in a suspension solution of 25% Matrigel in 100 μL serum-free DMEM medium were subcutaneously inoculated into the right flank of the mice under the fat pad. Synthesis of Monomers and Copolymers. The N-carboxyanhydride (NCA) monomers of N6-carbobenzyloxy-L-lysine and L-aspartic acid-benzyl ester were synthesized according to the Fuchs−Farthing method by triphosgene in anhydrous THF solution for 4 h at 40 °C under a nitrogen atmosphere. The products were precipitated and purified by slowly adding into anhydrous hexane. The solution was recrystallized at −20 °C overnight until white needlelike crystals appeared. The NCA monomers were further washed with anhydrous hexane and dried under vacuum for their subsequent use in copolymer synthesis. ROP of Lys-NCA and Bla-NCA was conducted by mPEG-NH2 as a high molecular weight initiator to prepare PEG−pLys−pBla block copolymers. The polymerization reaction was carried out in anhydrous DMSO at 55 °C for 2 days for each block. In brief, to a stirred anhydrous DMSO (10 mL) solution was added mPEG-NH2 (500 mg, 0.1 mmol) and Lys-NCA (310 mg, 1 mmol) at 55 °C with nitrogen K


Research Article

ACS Applied Materials & Interfaces before reaching a confluence of 80−90%. The cells were washed and preincubated with D-glucose-free Hank’s solution at 37 °C for 15 min before the subsequent experiments without further explanation. The cells were then incubated with free DOX, PLs-sB-DOX, and MPLs-sBDOX with different ligand modification ratios (10, 20, and 40 wt %) at a normalized DOX concentration of 10 μg/mL in D-glucose-free medium. For internalization mechanism studies, various inhibitor solutions in D-glucose-free Hank’s solution were used instead. 10 mM D-glucose, 1 μg/mL colchicine, 0.3 μg/mL phenylarine oxide (PhAsO), and 0.5 μg/mL filipin complex were applied to the cells as endocytic inhibitors. After incubation for 30 min at 37 °C, the medium was removed and cells were carefully washed three times with PBS (pH 7.2) before being observed and photographed using a fluorescence microscope (Leica, Solms, Germany). For flow cytometry analysis, the cells were seeded in 6-well plates (Corning, New York) at a density of 1 × 105 cells/well and incubated at 37 °C for 24 h before reaching a confluence of 90%. The cells were digested and harvested after the same incubating procedure mentioned above before analysis using a flow cytometer (BD Bioscience, Franklin Lakes) at 488 nm excitation with 1 × 104 events recorded for each assay. Cells without any treatment were used as the control. In Vitro DOX Release Study. The in vitro DOX release profiles of MPLB-DOX and MPLs-sB-DOX micelles under different external stimulations were obtained and measured through a dialysis method (n = 3). To a dialysis bag (MWCO: 1000), with both ends sealed, was added 400 μL micelle solution before suspending it in a centrifuge tube containing 10 mL PBS (pH 7.4). An aliquot of solution (0.5 mL) was withdrawn from the outside tube with equal volume replenishment at various time points when the tube was shaken at 100 rpm at 37 °C. The concentration of DOX was measured using a microplate spectrophotometer at Ex/Em 480/525 nm. The pH-sensitive and ROS-responsive properties were evaluated by the same method with different external media. PBS (pH 5.0) and 10 mM peroxidecontaining PBS (pH 5.0/pH 7.4) were used. Intracellular release behaviors of MPLs-sB-DOX micelles were studied through confocal imaging. MDA-MB-231 cells were seeded in a confocal culture dish (Corning, New York) at a density of 1 × 104 cells/dish and incubated at 37 °C for 24 h before reaching a confluence of 50−60%. The cells were treated with MPLs-sB-DOX micelles at a normalized DOX concentration of 10 μg/mL. Hoechst 33342 (1 μg/mL, 20 min) and LysoTracker Deep Red (50 nM, 10 min) were applied to stain the nuclei and late endosome, respectively. After different incubating times, the solutions were removed and cells were washed twice with PBS before being observed and photographed using the confocal fluorescence microscope (Carl Zeiss LSM710, Oberkochen, Germany) by a 63× oil immersion lens. PK and Biodistribution Studies. PK study was performed on SD rats intravenously injected with free DOX, PLB-DOX, MPLB-DOX, PLs-sB-DOX, and MPLs-sB-DOX micelles at a dose of 5 mg DOX/kg (n = 5). Blood samples of each group were collected at various time points in heparin-treated tubes. Biodistribution study was performed on MDA-MB-231 orthodox breast cancer xenograft models intravenously injected with free DOX, PLB-DOX, MPLB-DOX, PLs-sBDOX, and MPLs-sB-DOX micelles at a dose of 5 mg DOX/kg (n = 4). The mice were sacrificed after 24 h, and tissues and main organs (heart, liver, spleen, lung, kidney, and tumor) were excised and collected after fully rinsing in saline and were weighed and homogenized. DOX concentration in blood or tissues was determined by highperformance liquid chromatography (HPLC) according to a previously reported method. The biological samples (blood or tissue homogenate) were centrifuged twice (3500 rpm, 10 min), and the supernatant was collected. Methanol (200 μL) was added to 100 μL of the supernatant to precipitate proteins before being centrifuged twice (12 000 rpm, 5 min) to collect the supernatant. Each sample (20 μL) was injected into HPLC (Agilent ODS C18 column, 4.6 × 250 mm2, 5 μm particle size), 10 mM KH2PO4/acetonitrile/acetic acid = 70:30:0.3 (v/v/v), 1.0 mL/min, 25 °C, Ex/Em = 480/560 nm) for DOX quantification.

In vivo imaging study of the micelle distribution was performed on MDA-MB-231 orthodox breast cancer xenograft models intravenously injected with BODIPY-labeled MPLB-DOX and MPLs-sB-DOX micelles at a dose of 1 mg micelles per mouse. The mice were anesthetized, visualized, and photographed under an IVIS Spectrum in vivo imaging system (Caliper) at Ex/Em 650/665 nm at 2, 4, 8, and 12 h after injection. The tissues and main organs (heart, liver, spleen, lung, kidney, and tumor) were collected and photographed after rinsing them in saline. In Vitro Antitumor Efficacy Study. In vitro antitumor efficacy study was evaluated by MTT assay (n = 4) and cell apoptosis assay. As for the MTT assay, MDA-MB-231 cells were seeded evenly in 96-well plates at a density of 1 × 103 cells/well and incubated at 37 °C for 24 h until a confluence of 70% was reached. The cells were treated with free DOX, PLB-DOX, MPLB-DOX, PLs-sB-DOX, and MPLs-sB-DOX micelles at various concentrations for 48 h. Afterward, the medium was removed and cells were washed three times before 100 μL MTT solution (0.5 mg/mL) was added in each well followed by incubation at 37 °C for 4 h. The solution was then removed and 100 μL DMSO was added and shaken for 10 min to dissolve the crystal of formazan. The absorbance of formazan was read at 570 nm using a microplate spectrophotometer. Cells without drug treatment served as the control. As for cell apoptosis assay, MDA-MB-231 cells were seeded evenly in 24-well plates at a density of 1 × 104 cells/well and incubated at 37 °C for 24 h until a confluence of 70−80% was reached. The cells were treated with free DOX, PLB-DOX, MPLB-DOX, PLs-sB-DOX, and MPLs-sB-DOX micelles at a normalized DOX concentration of 1 μg/mL for 4 h at 37 °C. The drug solutions were then removed and cells were incubated for 48 h. The cells were then treated with Annexin V-FITC and propidium iodide working solutions for 5 min before being observed and photographed using a fluorescence microscope (FL1 channel for Annexin V-FITC, FL3 channel for PI, Leica, Solms, Germany). In Vivo Antitumor Efficacy Study. MDA-MB-231 orthodox breast cancer xenograft models were firstly randomized into groups (n = 6). In vivo antitumor efficacy study was performed by intravenously injecting with saline, free DOX, PLB-DOX, MPLB-DOX, PLs-sBDOX, and MPLs-sB-DOX micelles on days 0, 7, and 14 at a dose of 5 mg DOX/kg. The body weight of mice was recorded every other day. The tumor volume was evaluated by the luminescence signals’ intensity of luciferase-containing tumor cells. Tumors excised from the tumor model on day 22 were dehydrated in 15 and 30% sucrose solution for 48 h, respectively, before being fixed with 4% polyoxymethylene for 48 h. The tumor tissues were then sliced into 10 μm thick slices at −20 °C, collected on cationic polylysine-coated glass slides. The as-prepared samples were then stained with TUNEL and DAPI before being observed and photographed using a fluorescence microscope, with an in situ apoptotic cell detection kit according to the manufacturer’s instructions. Immunohistological Staining Study. To further investigate the reduced potential toxic effects of MPLs-sB-DOX, the MDA-MB-231 orthodox breast cancer xenograft models were sacrificed on day 22 with main tissue (heart, liver, spleen, lung, and kidney) excised and fixed in 4% neutral buffered formalin solution and embedded in paraffin. Sections of 5 μm were stained with hematoxylin and eosin (HE) and observed and photographed. Statistical Analysis. Results are presented as mean ± standard error (SD). Analysis was performed using the GraphPad Prism software. The pharmacokinetic profile was calculated and fitted using the DAS software based on noncompartmental analysis. Statistical comparisons were assessed by one-way ANOVA. The accepted level of significance was P < 0.05.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b16815. L


Research Article


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Synthesis route and 1H NMR and IR characterization of polymers (PDF)

(15) Joralemon, M. J.; McRae, S.; Emrick, T. PEGylated Polymers for Medicine: From Conjugation to Self-Assembled Systems. Chem. Commun. 2010, 46, 1377−1393. (16) Hrubý, M.; Konák, C.; Ulbrich, K. Polymeric Micellar pHSensitive Drug Delivery System for Doxorubicin. J. Controlled Release 2005, 103, 137−148. (17) Chambers, A. F.; Groom, A. C.; MacDonald, I. C. Dissemination and Growth of Cancer Cells in Metastatic Sites. Nat. Rev. Cancer 2002, 2, 563−572. (18) Folkman, J. What Is the Evidence That Tumors Are Angiogenesis Dependent? J. Natl. Cancer Inst. 1990, 82, 4−6. (19) Mardekian, S. K.; Bombonati, A.; Palazzo, J. P. Ductal Carcinoma in Situ of the Breast: the Importance of Morphologic and Molecular Interactions. Hum. Pathol. 2016, 49, 114−123. (20) O’Byrne, K. J.; Dalgleish, A. G.; Browning, M. J.; Steward, W. P.; Harris, A. L. The Relationship Between Angiogenesis and the Immune Response in Carcinogenesis and the Progression of Malignant Disease. Eur. J. Cancer 2000, 36, 151−169. (21) Bonomi, M.; Patsias, A.; Posner, M.; Sikora, A. The Role of Inflammation in Head and Neck Cancer. Adv. Exp. Med. Biol. 2014, 816, 107−127. (22) Goswami, B.; Rajappa, M.; Sharma, M.; Sharma, A. Inflammation: Its Role and Interplay in the Development of Cancer, with Special Focus on Gynecological Malignancies. Int. J. Gynecol. Cancer 2008, 18, 591−599. (23) Fouad, T. M.; Kogawa, T.; Reuben, J. M.; Ueno, N. T. The Role of Inflammation in Inflammatory Breast Cancer. Adv. Exp. Med. Biol. 2014, 816, 53−73. (24) Watanabe, M. A.; Oda, J. M.; Amarante, M. K.; Cesar, V. J. Regulatory T Cells and Breast Cancer: Implications for Immunopathogenesis. Cancer Metastasis Rev. 2010, 29, 569−579. (25) Thiery, J. P.; Acloque, H.; Huang, R. Y.; Nieto, M. A. EpithelialMesenchymal Transitions in Development and Disease. Cell 2009, 139, 871−890. (26) Mariotto, S.; de Prati, A. C.; Cavalieri, E.; Amelio, E.; Marlinghaus, E.; Suzuki, H. Extracorporeal Shock Wave Therapy in Inflammatory Diseases: Molecular Mechanism that Triggers AntiInflammatory Action. Curr. Med. Chem. 2009, 16, 2366−2372. (27) Trachootham, D.; Alexandre, J.; Huang, P. Targeting Cancer Cells by ROS-Mediated Mechanisms: A Radical Therapeutic Approach? Nat. Rev. Drug Discovery 2009, 8, 579−591. (28) Steen, K. H.; Steen, A. E.; Reeh, P. W. A Dominant Role of Acid pH in Inflammatory Excitation and Sensitization of Nociceptors in Rat Skin, in vitro. J. Neurosci. 1995, 15, 3982−3989. (29) Pelicano, H.; Carney, D.; Huang, P. ROS Stress in Cancer Cells and Therapeutic Implications. Drug Resist. Updates 2004, 7, 97−110. (30) Smith, R. J.; Capaldi, R. A.; Muchmore, D.; Dahlquist, F. CrossLinking of Ubiquinone Cytochrome C Reductase (Complex III) with Periodate-Cleavable Bifunctional Reagents. Biochemistry 1978, 17, 3719−3723. (31) Hu, P.; Tirelli, N. Scavenging ROS: Superoxide Dismutase/ Catalase Mimetics by the Use of An Oxidation-Sensitive Nanocarrier/ Enzyme Conjugate. Bioconjugate Chem. 2012, 23, 438−449. (32) Gupta, M. K.; Meyer, T. A.; Nelson, C. E.; Duvall, C. L. Poly(PS-b-DMA) Micelles for Reactive Oxygen Species Triggered Drug Release. J. Controlled Release 2012, 162, 591−598. (33) Shim, M. S.; Xia, Y. A Reactive Oxygen Species (ROS)Responsive Polymer for Safe, Efficient, and Targeted Gene Delivery in Cancer Cells. Angew. Chem., Int. Ed. Engl. 2013, 52, 6926−6929. (34) Pu, H. L.; Chiang, W. L.; Maiti, B.; Liao, Z. X.; Ho, Y. C.; Shim, M. S.; Chuang, E. Y.; Xia, Y.; Sung, H. W. Nanoparticles with Dual Responses to Oxidative Stress and Reduced pH for Drug Release and Anti-Inflammatory Applications. ACS Nano 2014, 8, 1213−1221. (35) Wilson, D. S.; Dalmasso, G.; Wang, L.; Sitaraman, S. V.; Merlin, D.; Murthy, N. Orally Delivered Thioketal Nanoparticles Loaded with TNF-α-siRNA Target Inflammation and Inhibit Gene Expression in the Intestines. Nat. Mater. 2010, 9, 923−928. (36) Warburg, O. On the Origin of Cancer Cells. Science 1956, 123, 309−314.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-21-5198-0079. Fax: +86-21-5198-0079. ORCID

Chen Jiang: 0000-0002-4833-9121 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the grant from the National Basic Research Program of China (973 Program, 2013CB932500), National Science Fund for Distinguished Young Scholars (81425023), and National Natural Science Foundation of China (81373355). The MDA-MB-231 cell line was kindly gifted by Perkin Elmer.

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