Rational Design of Nanoparticles to Overcome Poor Tumor

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Biological and Medical Applications of Materials and Interfaces

Rational Design of Nanoparticles to Overcome Poor Tumor Penetration and Hypoxia-induced Chemotherapy Resistance: Combination of Optimizing Size and Self-inducing High Level of Reactive Oxygen Species Liandong Deng, Zujian Feng, Hongzhang Deng, Yujia Jiang, Kun Song, Yongli Shi, Shuangqing Liu, Jianhua Zhang, Su-Ping Bai, Zhihai Qin, and Anjie Dong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b12129 • Publication Date (Web): 07 Aug 2019 Downloaded from pubs.acs.org on August 10, 2019

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ACS Applied Materials & Interfaces

Rational Design of Nanoparticles to Overcome Poor Tumor Penetration and Hypoxia-induced Chemotherapy Resistance: Combination of Optimizing Size and Self-inducing High Level of Reactive Oxygen Species Liandong Deng, 1, †,§ Zujian Feng,1, †,§ Hongzhang Deng,* ‡,§, ‖ Yujia Jiang, § Kun Song, ‡ Yongli Shi, † Shuangqing Liu, ‡ Jianhua Zhang, § Suping Bai, † Zhihai Qin, *‡ Anjie Dong*†,§ , ‖ †

College of Pharmacy, Xinxiang Medical University, 453003, Xinxiang, P.R. China Laboratory of Protein and Peptide Pharmaceuticals, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China § Department of Polymer Science and Technology, Key Laboratory of Systems Bioengineering of the Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China ‖Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, China ‡Key

KEYWORDS: hypoxia, penetration, chemotherapy resistance, reactive oxygen species, polymer nanoparticles

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ABSTRACT One tough question induced by the hypoxia in cancer tissue is resistance to the anticancer drugs basing on reactive oxygen species (ROS) mechanism. Furthermore, the hypoxic regions locate in the center of tumor where tumor cells are easy to residual and survival due to the poor drug delivery efficiency even with nanocarriers. In this paper, these problems were well addressed through the rational combination of the enhanced penetration, self-inducing high level of intracellular ROS and synchronously pH-sensitive drug release, realized by a simple structural and accessible copolymer, poly(poly(ethylene glycol) methyl ether methacrylate-co-(2-methylpropenoic acid-glycerol-cinnamaldehyde) (PgEMC). For one thing, PgEMC could self-assemble into stable nanoparticles with PEG shell and optimizing diameters of 60 nm to simultaneously facilitate long blood circulation and deep tumor penetration. Second, cinnamylaldehyde moieties could detach from PgEMC NPs in intracellular acidic environment and trigger high level of ROS to allay the doxorubicin (DOX) resistance induced by hypoxia in solid malignancies. Other more, the DOX payload in PgEMC NPs could be synchronously released with the intracellular disassembly of PgEMC NPs due to the detaching of cinnamylaldehyde moieties. In 4T1 cells treated with PgEMC/DOX NPs, remarkably elevation of ROS level, and the enhanced DOX sensitivity in hypoxia environment were observed in vitro studies. Tumor spheroid penetration results certificated 60 nm-sized DOX loaded PgEMC NPs (PgEMC60/DOX) could distribute into deep site of tumor at a high intensity. In vivo studies using a 4T1 breast tumor model, PgEMC60/DOX NPs showed significant inhibition over 95.4% of the tumor growth. These results reveal that integrated optimizing size, self-inducing ROS and pH-sensitive drug release into one small-sized nanoparticle can efficiently overcome the poor tumor penetration and hypoxia-induced chemotherapy resistance.


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INTRODUCTION Conventional nanoparticles (NPs) have contribution to promising platforms of clinical cancer chemotherapy as drug delivery systems.1-6 NPs take the advantages of enhanced solubility, prolonged blood circulation and improved accumulation at the tumor site.7-9 While, the lack of delivery efficiency of nanoparticles (NPs) into tumor cells by intravenous administration is critical limitation for nanomedcine clinical translation because of the multistep delivery obstacles from the blood circulation system and the complex tumor microenvironments.4,5 The poor tumor penetration due to the pathological pressure gradients, such as abnormally high interstitial fluid pressure (IFP) and solid tissue pressure (STP) is one of critical challenges for nanotechnology mediated cancer therapy.5 Hypoxia, a condition deprived of adequate oxygen supply at the tissue level, is a character feature of solid tumor microenvironment due to rapid proliferation of tumor cells and the abnormalities in structure and functions of tumor blood vessels, and occurs in the core of the tumor that is far from the microvessels.10-15 Unfortunately, NPs normally can only reach to the periphery of the tumor and hard to penetrate into the center of the tumor. 4,5,16 It has been reported hypoxia induces fibrotic, stiff and aligned extracellular matrix (ECM),12 which may create more resistance to penetration. Therefore, enhancing the penetration of the nanoparticles into the deep of tumor tissues to kill the tumor cells in hypoxic region should be an efficient way to further suppress the tumor growth and relapse.17,18 Among several factors for nanoparticle penetration in tumor, including the shape, charge and size, etc., the particle size is the most important one. The diffusion distance of nanoparticles increases with decreasing the particle size.16,17 Higher permeability was generally observed on smaller sized NPs.16-23 Compared to 150 nm NPs, 50~60 nm NPs displayed significantly higher permeability in tumors.24, 25 Therefore, it’s necessary to design and prepare small size nanoparticles to overcome the penetration problem. Moreover, one of the serious consequences induced by hypoxia is the acquired resistance of cancer cells to the ROS-mediated treatments, such as photodynamic therapy, radiotherapy and some anticancer drugs.22, 2628

It had been reported tumor cells in the hypoxic region showed resistant to DOX treatment, because of the

intracellular metabolite of DOX produced less reactive oxygen species (ROS) in a hypoxic environment. 29-32 3 ACS Paragon Plus Environment


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A wide array of studies have confirmed that the cancer cells have abnormal redox balance with overproduction of ROS, as a result cancer cells under oxidative stress are more susceptible to further ROS than normal cells, by which some ROS-mediated anticancer strategies and drugs were developed. 32-38 While, intermediate levels of ROS facilitate repairing damage for resistance to chemotherapy and promoting proliferation of cells, but high levels of ROS exhibit cytotoxicity inhibiting cell proliferation and leading to apoptotic/necrotic cell death.32 Accordingly, it is necessary to increase the amount of intracellular ROS to solve the chemo-resistance problem resulted by hypoxia. Therefore, rational designed nanoparticles with the abilities to improve penetration and enhance the intracellular ROS generation may be a strategy to overcome the resistance to DOX treatment in hypoxia region. 33 In order to reverse the resistance to DOX in the hypoxia tumor region, one of the proposed methods is to provide additional oxygen to tumor.31, 34, 35 Hyperbaric oxygen therapy promoted oxygen translation to the hypoxic cancer tissues by raising the oxygen pressure in plasma. The enhanced sensitivity of tumor cells to anti-cancer drug was obtained by alleviating the hypoxia in cancer tissues. In other words, the hyperbaric oxygen is an indirect method to enhance the intracellular level of ROS by additional oxygen. However, the efficiency of hyperbaric oxygen therapy depended on the oxygen-generating ability from the depot. Furthermore, hyperbaric oxygen therapy was limited locally for intratumoral injection.29 In order to develop the method of enhancing ROS in tumor utility for clinical application, its suitability for systemic administration, biocompatibility and programmable ROS generation rate must be considered. Cinnamaldehyde, a major component of cinnamon, had widely been used as dietary factor and food additive approved by Food and Drug Administration (FDA). Containing α, β-unsaturated carbonyl moiety, known as an active Michael acceptor pharmacophore, cinnamaldehyde had been well certificated to induce intracellular reactive oxygen species (ROS), by which to mediate cell apoptosis and therefore present anticancer activity. 36-40 Cinnamaldehyde induce ROS generation mainly in the mitochondria and mediated mitochondrial permeability transition and caspase activation. 36 It had been reported cinnamaldehyde could inhibit growth of human cancer cells by ROS-mediated apoptosis with minimal cytotoxicity to normal cells.37-40 4 ACS Paragon Plus Environment

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In this study, a kind of nanoparticles was designed to overcome poor tumor penetration and hypoxia induced chemotherapy resistance assembled by poly(poly(ethylene glycol) methyl ether methacrylate-co-(2methylpropenoic acid-glycerol-cinnamaldehyde), P(PEG-co-(MAA-CQ)) (PgEMC). As shown in Scheme 1, firstly, by optimizing the size of the NPs about 60 nm, PgEMC NPs could present well penetration ability; Subsequently, by intracellular pH-sensitive detachment of cinnamyladehyde moieties, PgEMC NPs could self-induce intracellular high ROS level and thus reverse the hypoxia-induced DOX resistance; And finally, the intracellular pH-sensitive detachment of cinnamyladehyde moieties could synchronously enhance DOX intracellular release to significantly inhibit tumor growth. The structure of PgEMC NPs had been characterized. The intracellular ROS producing efficiency and the ability of deep tumor penetration of the 60 nm PgEMC NPs were certificated in vitro and in vivo. The enhanced DOX sensitivity in hypoxia environment was observed in vitro studies. In the studies in vivo by using a 4T1 breast tumor model, DOX loaded PgEMC NPs with optimizing size and self-inducing ROS shown significant inhibition over 95.4% of the tumor growth. Combination the function of excellent tumor penetration of small size NPs, the selfreversing DOX resistance and pH sensitive intracellular DOX release, the PgEMC NPs could significantly enhance the anticancer efficiency of DOX, which provides a new insight for drug delivery design. RESULTS AND DICUSSION Synthesis and characterization of P(PEG-co-(MAA-CQ)) (PgEMC) As shown in Scheme 2, the PgEMC was synthesized via the reversible addition-fragmentation chain transfer (RAFT) copolymerization and esterification reaction. P(PEG-co-MAA) (PgEM) copolymer was synthesized by a one-step RAFT copolymerization of poly(ethylene glycol) methyl ether methacrylate (mPEGMA) and methacrylic acid (MAA) (the detail shown in the supporting information). The structure and composition were characterized by 1H-NMR. As shown in Figure 1A, PgEM displayed the 1H NMR signals of both mPEGMA (d at 3.65 ppm) and MAA (g at 12.5 and f at 1.37 ppm). The monomer conversion and number molecular weight (Mn) of copolymers were summarized in Table S1, calculated by comparing the integrals of the peaks assigned to the hydrogen protons on the CH3 of MAA (at about 1.37 ppm) and mPEGMA (at about 3.65 ppm). The proportional increase in the ratio of MAA and PEG repeat units with the feed ratio 5 ACS Paragon Plus Environment


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demonstrated the living polymerization characteristics to some extent. Then, further esterification reaction between PgEM and cinnamaldehyde derivative (CQ) was conducted to obtain the final amphiphilic copolymer PgEMC. As shown in Figure 1B, the resonance at 7.3 ppm was assigned to the phenyl ring protons of CQ. The number molecular weight (Mn) of polymers and monomer conversion were summarized in Table S2. It could be seen the Mn calculated from 1H-NMR and Mn determined by GPC were coincident with theoretical values. We choose P(PEG20-co-(MAA-CQ)100) as a representative sample for further study. Preparation of PgEMC NPs and DOX loaded PgEMC NPs PgEMC NPs were prepared by adding dropwise PgEMC solution in DMSO to double distilled water followed by dialysis to remove the organic solvents.40 The core-shell structure of the PgEMC NPs was evidenced by 1H-NMR spectra with D2O as solvents (Figure S3). Only the peak at 3.65 ppm belonging to the methylene protons in the mPEG segments appeared without any peak for the protons in MAA-CQ moieties, indicating the shielded protonation in hydrophobic core in the core-shell structure of the NPs.41 Figure 1C, the results of laser particle size analyzer measurements revealed that PgEMC could selfassembled into monodisperse NPs with average size of 60 nm and low PDI (Table S3), named as PgEMC60. The small size of NPs with 60 nm was formed due to the organic solvent DMSO used in the dialysis method, which can lead to a more efficient solvent diffusion and polymer dispersion into water.42, 43 By the same method, DOX loaded PgEMC NPs (named as PgEMC60/DOX) with average sizes of 63.7 nm were prepared, as shown in Figure 1C and Table S3. The morphologies of PgEMC60 and PgEMC60/DOX observed by TEM in Figure 1D confirmed their size distribution within the range of around 20 to 60 nm, which was slightly smaller than that measured by laser particle size analyzer due to the shrinkage in the dehydration state. The same results were also obtained by atomic force microscope (AFM) as shown in Figure 1E and F. Furthermore, 150 nm sized PgEMC150 NPs and DOX loaded PgEMC150 NPs were also prepared by dialysis method using DMF as solvent. In addition, methoxy poly(ethylene glycol)-b-poly(ε-caprolactone)(PEG-PCL) NPs and DOX loaded PEG-PCL NPs with ~150nm size were used as control. The detail information of these NPs was shown in Figure S4 and Table S4.


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Compared with PEG-PCL NPs, PgEMC NPs showed a slightly higher DOX loading capacity and the DOX loading amount and encapsulation efficiency of PgEMC NPs increased with the content of MAA-CQ moiety increasing. These phenomena can be attributed to the π-π interaction between the benzene ring of CQ segments in the PgEMC NPs with DOX, leading to advantage drug encapsulation abilities. In acidic environment, the acetal groups on the hydrophobic CQ moieties of PgEMC60 can be selectively hydrolyzed to detach the cinnamylaldehyde and lead to hydrophilic transition of the hydrophobic backbone. The ability of pH-sensitive detachment of cinnamylaldehyde from the PgEMC60 NPs was evaluated as shown in Figure 1G. The rapid release of cinnamylaldehyde from PgEMC60 NPs was observed and the release rate increased with pH decreased. Furthermore, the pH-sensitive detachment of cinnamylaldehyde also resulted in the disassembly of PgEMC60 NPs due to the hydrophilic transition of the hydrophobic backbone, thus further accelerated the intracellular release of DOX in acidic endosomes. As shown in Figure 1H, the in vitro release of DOX from PgEMC60 NPs was largely enhanced at pH 5.0. The size and morphological changes of the PgEMC60 NPs nanostructure in Figure 1I and Figure S4D certificated the disassembly of PgEMC60 NPs. The average sizes of PgEMC60 remained unchanged throughout 24 h at pH 7.4, while significant size changes were observed for PgEMC60 NPs at pH 6.8. The ability of PgEMC in inducing ROS Confocal laser scanning microscope (CLSM) and flow cytometry were used to study the ability of PgEMC60 to induce high level of ROS in cancer cells. 4T1 cells were treated with PBS, DOX, PgEMC60 and PgEMC60/DOX (PgEMC 1 mg/mL, DOX 5 μg/mL). The intracellular ROS level was measured by dichlorodihydro-fluorescein diacetate (DCFH-DA) as a ROS fluorescent probe. As shown in Figure 2, after 24 h treatment, the PBS and DOX control groups showed very low level of ROS in 4T1 cells. However, PgEMC60 NPs and PgEMC60/DOX NPs significantly increased the fluorescence density of DCFH-DA, indicating the remarkably enhancement of the intracellular ROS accumulation, which was assigned to the function of the released cinnamaldehyde in ROS generation.38 While, as shown in Figure 2A, when the antioxidant n-acetylcystein (NAC) (5 mM) was used to entirely inhibit the ROS production, the 4T1 cells incubated with PgEMC60 and PgEMC60/DOX showed little ROS. In Figure 2B, the semiquantitative 7 ACS Paragon Plus Environment


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fluorescence intensities quantified using ImageJ software further confirmed above results.44 The flow cytometry evaluation results in Figure 2C also showed the induced ROS accumulations by PgEMC60 and PgEMC60/DOX. It had been known the cinnamaldehyde could deplete the intracellular glutathione (GSH) to introduce ROS.44 Therefore, we next evaluated the level of intracellular GSH as shown in Figure 3. The 4T1 cells incubated with DOX, PgEMC60, PgEMC150, PgEMC60/DOX and PgEMC150/DOX and the contents of GSH in the 4T1 cells were measured by GSH Assay Kit. Figure 3A showed that PgEMC and PgEMC/DOX NPs could largely inhibit the production of GSH compared with the control group. Furthermore, 1H-NMR approach was used to measure the GSH variation. As shown in Figure 3B, the peak from 3.00-3.33 represented the signal of GSH and GSSH.46 The amounts of GSH and GSSH were significantly decreased in the groups of PgEMC and PgEMC/DOX NPs compared with control groups. The relative peak areas of GSH and GSSH calculated in Figure 3C showed the corresponded results. The cellular uptake of PgEMC/DOX NPs Taking the red fluorescence signal of DOX as the fluorescence imaging of cells, we investigated the cellular uptake profiles of PgEMC60/DOX NPs and PgEMC150/DOX NPs in 4T1 cells using CLSM. The similar fluorescence intensities for PgEMC60/DOX NPs and PgEMC150/DOX NPs in Figure 4 indicated that PgEMC60 and PgEMC150 could be endocytosed into cells at the same uptake level, that is to say the nanoparticle size slightly influence the uptake into cells for PgEMC60/DOX NPs and PgEMC150/DOX NPs. The similar levels of co-location ratio of DOX with lysosome (shown in Figure S5A) for free DOX, PgEMC60/DOX NPs and PgEMC150/DOX NPs also confirmed above results. The CLSM images of MCF-7 cells incubated with DOX, PgEMC60/DOX and PgEMC150/DOX were shown Figure 4D, which indicated PgEMC60/DOX and PgEMC150/DOX also presented the similar levels of uptake in MCF-7 cells. Because free DOX molecules could be internalized into tumor cells through a passive diffusion mechanism and the internalization of DOX-loaded NPs need to be endocytosed,

47-49

DOX fluorescence for free DOX were

stronger than the DOX-loaded NPs in the cytoplasm and nucleus of 4T1 cells. To measure the DOX content in cells after incubated with free DOX, PgEMC60/DOX NPs and PgEMC150/DOX NPs, the DOX was 8 ACS Paragon Plus Environment

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extracted from the 4T1 cells and tested by high performance liquid chromatography (HPLC). The DOX contents (shown in Figure S5B) in 4T1 cells for all the test groups increased with time, and that incubated with free DOX was higher than those incubated with PgEMC60/DOX NPs and PgEMC150/DOX NPs either at 8h and 12h, but the DOX contents in 4T1 cells incubated with PgEMC60/DOX NPs kept the same levels with those incubated with PgEMC150/DOX NPs both at 8h and 12h. Furthermore, the cellular uptake of DOX in the hypoxia culture was evaluated on MCF-7 cells. From Figure 4E and Figure S5C, red fluorescence of DOX in the hypoxic culture were seen in MCF-7 cells, indicating that in hypoxic environment PgEMC60/DOX NPs and PgEMC150/DOX NPs also showed the similar cell uptake level. In vitro anti-tumor activity of the PgEMC/DOX NPs The cytotoxicities of free DOX, PgEMC60 and PgEMC60/DOX NPs were investigated in 4T1 cells by MTT assay. NAC was used as a ROS inhibitor. From Figure 5A and B, it could be noted that the IC50 (half inhibitory concentration) of PgEMC60/DOX NPs was 2.14 µg/mL lower than that of PgEMC60/DOX with NAC (15.06 µg/mL) and free DOX (5.515 µg/mL). The higher anti-tumor activity of PgEMC60/DOX NPs could be attributed to the higher ROS level enhanced by PgEMC60/DOX NPs. Furthermore, the cytotoxicity of blank PgEMC60 was evaluated and shown in Figure S6A and B, which certificated certain cytotoxicity with half inhibitory concentration of PgEMC60 at about 300µg/mL in 4T1 cells. In addition, the PgEMC60 showed low toxicity to 3T3 cells even under concentration of 250 μg/mL, guaranteeing the biosafety in normal tissues. The effects on cell apoptosis of DOX, PgEMC60 and PgEMC60/DOX (PgEMC: 1 mg/mL, DOX: 10 μg/mL) were investigated. Figure 5C indicated that very low apoptosis of 4T1 cells after 48 h incubation with PBS and PgEMC60. And free DOX and PgEMC60/DOX with NAC only induced 73.6% and 54.8% apoptotic cells, respectively. However, the total percentage of apoptosis of PgEMC60/DOX treated cells reached 79.2% with high to 66.7% of late apoptosis, which was much higher than DOX (15.4%) and PgEMC60/DOX with NAC (18.4%). The enhanced susceptibility of cancer cells incubated with PgEMC60/DOX was attributed to the enhancing in ROS generation, which improved the anticancer ability of DOX. Caspase 3 and p53 as the key apoptosis related proteins are normally used to evaluate the apoptotic properties.50 Figure 5D showed the levels of p53 in 4T1 cells incubated with DOX, PgEMC60, and 9 ACS Paragon Plus Environment


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PgEMC60/DOX with or without NAC. It could be seen that the p53 expression in cells incubated with PgEMC60/DOX was significantly higher than that of group PgEMC60/DOX with NAC. The obvious elevation in expression of cleaved caspase 3 protein was observed in cells treated with PgEMC60/DOX compared to the control groups of DOX and PgEMC60/DOX with NAC (Figure S7). Also, the semiquantitative results of the cleaved caspase 3 (shown in Figure S7) indicated that the amount of cleaved caspase 3 in the group of PgEMC60/DOX were obviously more than the total amount of the groups of DOX and PgEMC60 together. Therefore, the enhanced anticancer effect of PgEMC60/DOX compared with DOX and PgEMC60/DOX with NAC was not only the simple superposition effect of cinnamylaldehyde and DOX but also the enhanced anti-cancer ability of DOX by generating ROS induced by PgEMC60 NPs. To further confirm the effect of ROS on the enhanced anticancer effect, we used glutathione to scavenge ROS.51 Figure 5E showed the effects of DOX, PgEMC60/DOX with NAC, PgEMC60 and PgEMC60/DOX on DNA fragmentation, a hallmark of apoptosis. Nearly no fragmented DNA was induced by PBS. DOX, PgEMC60/DOX with NAC, PgEMC60 and PgEMC60/DOX treated cells showed nucleosomal DNA fragmentation, but PgEMC60/DOX showed the maximum fragmented DNA characteristics of apoptotic. These results indicated that generating ROS by intracellular delivery of PgEMC60 led to apoptosis of 4T1 cells. The 4T1 cells were pretreated by 100 µM CoCl2 about 24 h to simulate the hypoxia environment of cells for evaluating tumor cell sensitivity to PgEM, DOX, cinnamaldehyde and PgEMC60/DOX (shown in Figure S8(A-D)). It was observed that hypoxia did not affect the cytotoxicities of PgEMC and cinnamaldehyde, but led to obvious DOX resistance of 4T1 cells. Significantly, PgEMC60/DOX could overcome the hypoxiainduced DOX resistance and showed much higher cytotoxicity in hypoxia 4T1 cells. Then the cytotoxicities of free DOX, PgEMC60 and PgEMC60/DOX NPs with or without NAC were investigated in 4T1 cells by Annexin V-FITC staining and analyzed via flow cytometry. From Figure 6A, it was observed that the apoptotic cells induced by free DOX in hypoxia environment decreased to 41.8 % from 91.1% in normal 4T1 cells, further indicating the cytotoxicity of DOX was significantly inhibited in the hypoxia environment. Inspiringly, PgEMC60/DOX NPs led to about 89.2% cell apoptosis under hypoxia environment, which 10 ACS Paragon Plus Environment

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demonstrated the ability of PgEMC60/DOX NPs to reverse the hypoxia-induced chemotherapy resistance to DOX. The live or dead cell ratios after incubated with DOX, PgEMC60 with NAC, PgEMC60, and PgEMC60/DOX were measured by live-dead assay (Figure 6B). Correspondingly, PgEMC60/DOX induced more cells death compared with DOX in the hypoxia environment. In vitro and in vivo penetration of PgEMC60/DOX into the tumor The poor tumor penetration and inadequate concentration of oxygen was due to the impaired blood flow in hypoxia region.52 Due to hypoxia could attenuate the intracellular generation of ROS, tumor cells in the hypoxic region were resistant to DOX treatment.30, 31 Thus, in order to reverse the resistant to DOX of the cells in hypoxic region, the poor penetration of hypoxic region should be resolved firstly. Therefore, an efficient approach was developed with enhancing the penetrability of NPs by optimizing the size about 60 nm. First, we studied the diffusive transports of PgEMC60/DOX and PgEMC150 /DOX NPs in vitro tumor spheroid penetration with free DOX and DOX loaded PEG-polycaprolactone (PEG-PCL/DOX) NPs in size of about 150 nm as the controls. The fluorescent intensity of DOX was recorded to show the distribution of the DOX-loaded NPs. As shown in Figure 7A, the lower fluorescent intensities than free DOX in sections of spheroid both for PEG-PCL/DOX NPs and PgEMC150 /DOX NPs were observed, and dramatically decreased with the increase of depth in the section. Conversely, the higher fluorescent intensity for PgEMC60/DOX NPs showed in sections of spheroid especially in deep sections than free DOX. The semiquantitative fluorescence intensities of inner region at different sections of 4T1 tumor spheroids shown in Figure 6B also confirmed the results of Figure 7A. Therefore, the small size of PgEMC60/DOX NPs was much helpful to enhance the deep penetration into the tumor spheroids and the larger size hindered NPs to penetrate into tumor, which was agree with other literatures.53 Then, we examined the tumor accumulation and penetration of DOX, PEG-PCL/DOX, PgEMC150/DOX and PgEMC60/DOX in 4T1 tumor-bearing mice by intra-tumor injection at a fixed depth, as shown in Figure 7C. The CLSM images of tumors slice in the PgEMC60/DOX group indicated that stronger fluorescence signal of DOX could be seen in each tumor section especially at 2000 μm inside the tumor below the injection site. However, in the PEG-PCL/DOX and PgEMC150/DOX groups, weak DOX fluorescence was observed at the 11 ACS Paragon Plus Environment


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depth range of 2000 μm. These results further confirmed PgEMC60/DOX NPs have strong penetration ability in solid tumor. Furthermore, the interstitial penetration of PgEMC60/DOX, PgEMC150/DOX and PEG-PCL/DOX NPs were investigated. After intravenous injection of DOX, PEG-PCL/DOX, PgEMC60/DOX and PgEMC150/DOX, tumor was extracted 24 h post and stained with anti-CD31 antibody (red) for fluorescence image. As shown in Figure 8, in the groups of PEG-PCL/DOX and PgEMC150/DOX, DOX signals (green) were largely colocalized with tumor blood vessels (red) due to the limited intratumor penetration of large sized NPs (about 150 nm). However, in the group injected with PgEMC60/DOX, strong DOX fluorescence signals were found far from blood vessels, which indicated efficient intratumoral diffusion ability of PgEMC60/DOX NPs corresponding with the result in Figure 7. Meantime, for free DOX group, rather week DOX signals appeared inside the tumor, attributed to the poor blood circulation, avid binding to DNA and sequestration in acidic endosomes of cells near the vasculature.54 It had been recognized that the intercapillary distances of intratumoural hypoxia regions are out of the diffusion distance of oxygen (~100–200 μm). 55 The results showed that the penetration distance of PgEMC60/DOX NPs was greater than 200 μm from the vessels, where free DOX and the larger size particles, PEG-PCL/DOX, PgEMC150/DOX NPs did not reached. Therefore, we thought PgEMC60/DOX NPs could reach the hypoxia regions. PgEMC60/DOX with relatively small size (60 nm) had excellent intratumor penetrating ability and would thus be an ideal nanosystem for effective tumor therapy. In vivo bio-distribution Fluorescent dye DiR was used to evaluate tissue distributions. Free DiR, DiR loaded PgEMC60 and PgEMC150 NPs were injected into 4T1 xenografted tumor-bearing mice in vein with the same dose (DiR 1mg/kg). Figure 9A showed the time dependent tissue distribution via fluorescence imaging of DiR and the quantitative fluorescence intensity ex vivo after 24 h were shown in Figure S9 A and B. It was seen that PgEMC60/DiR NPs could definitely deliver payload in tumor tissue more efficiently than free DiR and PgEMC150/DiR NPs due to excellent stability and penetrating ability, and inhibition in DiR wastage during blood circulation. Further the in vivo DOX delivery efficiencies of different NPs was also evaluated by in vivo injection of free DOX, PgEMC60/DOX, PgEM60/DOX and PgEMC150/DOX (DOX 5 mg/kg), where 12 ACS Paragon Plus Environment

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PgEM60/DOX NPs prepared by copolymer PgEM without CQ moieties (Scheme 2) was used as one of controls. The total DOX amount in tumor and the DOX plasma concentration-time profiles were presented in Figure S9 B and C, respectively. The results revealed that PgEMC60 and PgEMC150/DOX could obviously prolong the blood circulation of DOX than free DOX. While, combing the advantages of good tumor penetration and long blood circulation, PgEMC60/DOX presented stronger capacity to deliver DOX into tumor with DOX concentration in tumor 3-times that of free DOX and 2-times of PgEMC150/DOX. By the way, PgEM60/DOX also showed similar tumor accumulation of DOX with PgEMC60/DOX NPs, certifying the superiority of small particle size in tumor targeted drug delivery. No hemolytic effect was observed with red blood cells after treated with PgEMC60 and PgEMC150 (shown in Figure 9B). In vivo antitumor ability Above results had well certificated the good penetration and long circulation properties of PgEMC60/DOX. Next, the in vivo antitumor ability of PgEMC60/DOX was further studied in 4T1 cells xenografted tumorbearing Balb-c mice, and PgEM (without CQ moieties) and PgEM 60/DOX NPs were used as control to evaluate contribution of the self-inducing ROS function to tumor inhibition. The tumor-bearing mice were randomly separated into four groups (n = 7) and were injected with PBS, free DOX, PgEMC60/DOX, PgEM60/DOX and PgEM150/DOX (DOX 5 mg/kg), respectively. As shown in Figure 9C, compared with PBS treatment, free DOX and PgEM60/DOX groups showed 69% and 78% antitumor efficacies on the day 20, respectively. While, PgEMC60/DOX group exhibited strong antitumor ability with 95.4% tumor growth inhibition. Meanwhile, PgEM150/DOX group slightly reduced the tumor growth (30% inhibition rate, shown in Figure S10) due to the poor drug delivery efficiency to tumor. The higher in vivo antitumor efficiency of PgEMC60/DOX NPs than PgEM60/DOX NPs certificated the generation of ROS by PgEMC60 NPs was an innovative approach for cancer treatment with enhanced cytotoxicity of DOX. As shown in Figure 9D, the body weight of mice treated with PgEMC60/DOX maintained steady after 20 days, indicating the related lower cytotoxicity of PgEMC60/DOX. But obvious decrease in body weight was occurred on free DOX groups. In Figure 9E, there were large necrotic areas in tumor and nuclear shrinkage fragmentation observed



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in the group of PgEMC60/DOX. Above results indicated PgEMC60 nanoparticles have potential to be exploited as an efficient drug delivery system.

CONCLUSION In summary, a pH sensitive copolymer with relatively simple structure, poly(poly(ethylene glycol) methyl ether methacrylate-co-poly(2-methylpropenoic acid-glycerol-cinnamaldehyde)) (PgEMC) was prepared to self-assemble into 60 nm sized NPs with DOX payload(PgEMC60/DOX NPs). PgEMC60/DOX NPs possessed multifunctions to overcome the hypoxia-induced DOX resistance in solid malignancies, including optimizing small diameter of 60 nm for deep tumor penetration, self-inducing ROS by intracellular pHsensitive release of cinnamylaldehyde moieties and synchronously trigger release of the DOX payload. The cytotoxicity results evaluated under normoxic and hypoxic conditions in 4T1 cells indicated PgEMC60/DOX NPs could pH-sensitively self-induce high level of ROS and hence led to a high cytotoxicity than free DOX under hypoxia conditions. Tumor spheroid penetration results showed the significant enhanced penetration efficiency of PgEMC60 NPs. In vivo studies on a 4T1 breast tumor model showed that PgEMC60/DOX NPs could distribute into tumor at high intensity and presented up to 95.4% tumor growth inhibition. Therefore, to address both tumor penetration and hypoxia-induced DOX resistance is an importance paythway to enhance cancer therapy of nanomedcines, and the PgEMC NPs with the simple structure and coordinating function had provided a new insight for drug design.

MATERIALS AND METHODS Materials. 4-(((ethylthio)carbonothioyl)thio)-4-methylpentanoic acid (ECMPA) and cinnamaldehyde derivative (CQ) were synthesized as reported56 and the detail information was given in supporting information. Poly(ethylene glycol) methyl ether methacrylate macromonome (mPEGMA, Mn=500 g/mol), methacrylic acid(MAA), azodiisobutyronitrile (AIBN), cinnamaldehyde N,N-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) were purchased form GL Biochem (Shanghai) Ltd.. Annexin V FITC was purchased from Beyotime Institute of Biotechnology, China. 7AAD was purchased from 14 ACS Paragon Plus Environment

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Sigma-Aldrich. Methoxy poly(ethylene glycol) block poly(ε-caprolactone)(PEG-PCL)( composition as mPEG113-b-PCL77) was prepared in our lab according to our previous work.41 Doxorubicin hydrochloride (DOX·HCl) was purchased from Wuhan Hezhong Biochemical in manufacturing co.,Ltd (China, Wuhan). Nanoparticles formation. Dialysis method was used to prepare PgEMC NPs. Typically, PgEMC solution in dimethylsulfoxide was added dropwise to double distilled water. Then the solution was added into a 3500 Da molecular weight cutoff dialysis bag and dialyzed against distilled water until the organic solvents was removed. Preparation of drug-loaded NPs. Typically, polymer and DOX were dissolved in dimethylsulfoxide, the mixture solution was dropwise added to the double distilled water. Then, the mixture was dialyzed to form DOX-loaded NPs. UV-Vis spectrophotometer was used to determine the amount of DOX. Drug loading content (DLC) and drug loading efficiency (DLE) were calculated from the following equations: DLC (%) = weight of loaded drug/(total wight of drug and polymer)-------[1] DLE (%) = weight of loaded drug/weight of drug in feed-------[2] Drug release. DOX release profiles from NPs in vitro were measured in the media of phosphate buffered solution (PBS) (10 mM, pH 7.4) and acetate buffer (pH 5.0), respectively. Briefly, 5 mL solution of PgEMC/DOX NPs (1 mg/mL) were sealed in a dialysis tube(cutoff Mn = 3500 Da) and incubated in 25mL release media under stiring at 37oC. At regular intervals, 5 mL of the supernatant liquid was extracted, The cumulative drug release percentage was calculated by the following equation: n 1

Er（ %（ =

Ve  Ci  V 0Cn 1

m

 100%------- 3

DOX

Where mDOX represents the amount of DOX in the NPs, V0 = 25 mL, Ve = 5 mL, and Cn represents the concentration of DOX in the sample. Hypoxic conditions. Chemical treatment was used to bulid the hypoxia conditions in 4T1 cells.57 The 4T1 cells were plated overnight. 100 µM cobalt chloride (CoCl2) was added to pretreat the cells. 4T1 cells were washed by PBS after 24 h. Hence, the hypoxia cells were obtained.



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Tumor sphere. 100 μL agarose (1 % in water) was added in 96-well plate. After cooling, 4T1 cells was plated in 96-well plate (1 × 103) for 5 day to form the tumor sphere. MTT assay. 4T1 cells were seeded in a 96 well plate (4 × 103 cells/well) using complete DMEM medium. After 24 h, the sulution of PgEMC NPs with different concentrations was added. After 24 h, 10 μL of 5 mg/mL MTT solution was added, followed by adding 100μL of DMSO after 4 h. The absobance at 590 nm was measured by using a microplate reader (Biotek instruments, Epoch 2-15073018). Cell uptake studies. 4T1 cells were plated on microscope slides (2 × 105 cells/well) using complete DMEM medium. Free DOX or DOX loaded PgEMC NPs was added for 4 h. Than the cells were rinsed with PBS. The endosome and the nuclei were dyed by using Lysotrakcer Deep Red and hoechst 33342, respectively. The CLSM images were observed by using OLYPUS FV1000. Cell apoptosis. The free DOX, PgEMC and PgEMC/DOX were added after the 4T1 cells were plated in 6well palte (2 × 105 cells/well). 48 h later, the cells were washed with PBS and collected in EP pipe, then stained with Annexin V FITC and 7AAD for 5 min. BD FACSCalibur Flow cytometer was used to measure the cells. The measurment of intracellualr GSH level. According to the introduction of GSH assy kit, we first measured the GSH. For NMR sample preparation, 106 4T1 cells were incubated with each groups for 12 h. Then washed by PBS and placed in -80 oC immediately. The extraction of intracellular metabolites was dissoved in 0.5 mL D2O. The 1H NMR data were obtained on 500 MHz Bruker (Bruker BioSpin Corp., MA, USA). DNA fragmentation. First, 4T1 cells were seeded in 6 well paltes (1 × 105 cells/well). Then free DOX, PgEMC and PgEMC/DOX were added. After 48 h, the cells were washed with PBS and the DNA was extracted from the cells by using DNA Ladder Detection Kit (Life Technologies, Frederick, MD). DNA was separated on agarose gel (1.8%) based on agarose gel electrophoresis, visualized by transillumination with UV light and photographed.


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Animals and Tumor Model. BALB/c mice (male, 4-6 weeks-old) were used as the tumor model, purchased from Vital River Laboratories (Beijing, China). All animal procedures were performed according to the guidelines of Administration of Experimental Animals (Tianjin, revised in June 2004). In vivo tumor inhibition study. 4T1 xenografted tumor-bearing BALB/c mice were intravenously injected via vein with PBS, free DOX, PgEM60/DOX and PgEMC60/DOX (DOX 5 mg/kg), respectively. Subsequently, the length and width of tumor were measured every 2 days and the tumor volume was calculated, as tumor volume (mm3) = (length × width2) × 1/2. Statistical analysis. The quantitative data collected were expressed as mean ± S.D. Statistical significance was analyzed by a three-sample Student’s t-test. Statistical significance is denoted by *p < 0.05, **p < 0.01 and ***p < 0.001. ACKNOWLEGEMENTS This research was financially supported by the National Natural Science Foundation of China (31671021, 31670881 and 81630068). ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.xxxxxxx. Structure and composition of P(PEG-co-MAA) (Table S1) and P(PEG-co(MAA-CQ)) (Table S2); Characteristics of blank and DOX-loaded PgEMC60 NPs (Table S3) and and DOX-loaded PgEMC150 NPs (Table S4); 1H-NMR spectra of ECMPA (Figure S1), cinnamaldehyde derivative (CQ) (Figure S2) and PgEMC NPs in D2O (Figure S3); Size stability of PgEMC60 and PgEMC150 (Figure S4); The co-location ratio and intracellular uptake of DOX-loaded NPs (Figure S5); Cytotoxicity of PgEMC60 in 4T1 cells and 3T3 cells (Figure S6); Gray value of each protein (Figure S7); The cytotoxicities in 4T1 cells at normal or hypoxia condition (Figure S8); The quantitative distribution and pharmacokinetic proifles evaluation (Figure S9); The relative tumor volumes in tumor xenograft models treated with PgEMC150/DOX (Figure S10). AUTHOR INFORMATION 17 ACS Paragon Plus Environment


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Corresponding Author 1Liangdong

Deng and Zujian Feng are equal contribution to this work.

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Scheme 1 Illustration of DOX loaded PgEMC NPs with optimizing size and self-generating ROS to enhance the tumor penetrability and overcome the hypoxia-induce resistance to anticancer drug.

Scheme 2. The synthesis route of P(PEG-co-(MAA-CQ)) (PgEMC). 23 ACS Paragon Plus Environment


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Figure 1. 1H-NMR characterization of PgEM and PgEMC (A and B). Size distributions (C) and TEM micrographs (D) of PgEMC60 and PgEMC60/DOX. The scale bar is 100 nm. Atomic Force Microscope (AFM) of PgEMC60 (E) and PgEMC60/DOX (F). pH-triggered cinnamylaldehyde release from CQ moieties of PgEMC60 NPs measured by HPLC at different pH (G). In vitro release of DOX from DOX loaded PgEMC60 NPs at pH 7.4 and pH 5.0 (H). pH-triggered size distribution changes of PgEMC60 NPs (I).

Figure 2. Confocal fluorescence images of 4T1 cells with ROS detection probes in different treatments: PBS, DOX, PgEMC60, and PgEMC60/DOX with or without antioxidant N-acetylcystein (NAC) (CQ: 250 μM, DOX: 10 μg/mL). Scale bar is 200 μm (A). Semi-quantitative intracellular fluorescence intensities of


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4T1 cells with different treatments (B). Flow cytometry analysis of ROS environment in 4T cells with different treatments (C).

Figure 3. Effects of cinnamaldehyde on the level of intracellular GSH. Cells were treated with PgEMC60, PgEMC60/DOX, PgEMC150, and PgEMC150/DOX (CQ: 250 μM, DOX: 10 μg/mL) for the indicated period and then the level of intracellular GSH were determined by GSH Assay Kit (A) and 1H-NMR (B). Relative peak area of GSH and GSSH in the 1H NMR was calculated compared with the standard substance (C).



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Figure 4. Intracellular uptake of DOX-loaded NPs, CLSM images of 4T1 cells incubated with DOX, PgEMC60/DOX, PgEMC150/DOX (DOX: 10 μg mL−1) for 4 h (scale bar, 20 µm) (A). Intracellular fluorescence intensities of DOX, PgEM60/DOX, and PgEMC150/DOX (10 μg mL−1) (B). Flow cytometric comparison of 4T1 cell uptake of NPs (C). Intracellular uptake of DOX loaded NPs, CLSM images of MCF7 cells incubated with DOX, PgEMC60/DOX and PgEMC150/DOX (D). Flow cytometric comparison of uptake of NPs in MCF-7 cells under hypoxia environment (E).


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Figure 5. Cytotoxicity of DOX-free, and PgEMC60/DOX with or without NAC in 4T1 cells (A). IC50 (half inhibitory concentration) values of DOX-free, and PgEMC60/DOX in 4T1 cells (B). The effect of DOX-free, and PgEMC60/DOX on apoptosis was investigated by Annexin V-FITC and 7AAD double staining and analyzed via flow cytometry (C). Western blot analysis of expression levels of the p53 and caspase family members after being treated by DOX, PgEMC60, PgEMC60/DOX+NAC and PgEMC60/DOX (D). DNA fragmentation assay of 4T1 cells treated with DOX, PgEMC60, PgEMC60/DOX+NAC and PgEMC60/DOX for 48 h (E).



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Figure 6. The effect of DOX, PgEMC60/DOX with NAC, PgEMC60, and PgEMC60/DOX on apoptosis under normal and hypoxia was investigated by Annexin V-FITC staining and analyzed via flow cytometry (A). Influence of PBS, DOX, PgEMC60/DOX and PgEMC60/DOX on the percentage of live cells determined by Live-Dead assay under normal and hypoxia environment (B), the red fluorescence in dead cells and green in live cells, the scale bar is 200 μm.


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Figure 7. Z-stack fluorescence distribution images of 4T1 tumor spheroids after incubated with DOX, PgEM60/DOX PgEMC150/DOX and PEG-PCL/DOX, the concentration of DOX was 10 g/mL and bar represents 200 μm (A). Semi-quantitative area fluorescence intensity of imaginary line region at different sections of 4T1 tumor spheroids incubated in PgEMC60/DOX, the imaginary line cycle indicates the center of spheroids (B). In vivo distribution of DOX into the tumors of the 4T1 tumor-bearing mice after intratumoral injection of DOX, PgEM60/DOX PgEMC150/DOX and PEG-PCL/DOX at DOX dosage of 1 mg/kg for 24 h. The frozen tumor sections were observed at different depths below the injection site using CLSM. The nuclei were stained by DAPI. Scale bar is 500 μm (C).



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Figure 8. Microdistribution of free DOX, PEG-PCL/DOX, PgEMC150/DOX and PgEMC60/DOX in 4T1 xenograft tumor after i.v. injection. CLSM images of immunofluorescence showing the microdistribution of green fluorescence (DOX) in tumor tissue at 4 h postinjection. Blood vessels were marked with platelet endothelial cell adhesion molecule CD31 and secondary antibody (red). (Scale bar, 200 μm).


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Figure 9. Fluorescence images of tissue distribution of DiR and PgEMC60/DiR (A). Hemolysis assay of empty micelles at concentrations of 1 mg/ml in PBS, using water as a positive control and PBS as a negative control (B). The relative tumor volumes in tumor xenograft models treated with PBS, DOX, DOX, PgEM/DOX and PgEMC60/DOX (DOX 5 mg/kg) (C) (Data represent averages of 5 biological replicates ± standard error of the mean. *, P < 0.05; **, P < 0.01; ***, P < 0.001). Body weight changes of mice bearing 4T1 metastatic tumor after treatment with PBS, DOX, PgEM60/DOX and PgEMC60/DOX (D). H&E and representative heart, liver, spleen, lung, kidney, tumor staining pictures from mice injected with PBS, DOX, PgEM60/DOX and PgEMC60/DOX (E).




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Rational Design of Nanoparticles to Overcome Poor Tumor

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