Facile Fabrication of Oxidation-Responsive Polymeric Nanoparticles

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Facile Fabrication of Oxidation-Responsive Polymeric Nanoparticles for Effective Anticancer Drug Delivery Yamei Huang, Qiubing Chen, Panpan Ma, Heliang Song, Xiaoqian Ma, Ya Ma, Xin Zhou, Shuangquan Gou, Zhigang Xu, Jiucun Chen, and Bo Xiao Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00634 • Publication Date (Web): 28 Nov 2018 Downloaded from http://pubs.acs.org on November 30, 2018

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Molecular Pharmaceutics

Facile Fabrication of Oxidation-Responsive Polymeric Nanoparticles for Effective Anticancer Drug Delivery

Yamei Huang,† Qiubing Chen,† Panpan Ma,‡ Heliang Song,ǁ Xiaoqian Ma,† Ya Ma,† Xin Zhou,† Shuangquan Gou,† Zhigang Xu,*,† Jiucun Chen,† and Bo Xiao*,†

†Institute

for Clean Energy and Advanced Materials, Faculty for Materials and Energy, Southwest

University, Beibei, Chongqing 400715, P. R. China ‡National

Engineering Research Center for Healthcare Devices, Guangdong Key Lab of Medical

Electronic Instruments and Polymer Material Products, Guangdong Institute of Medical Instruments, Guangzhou, Guangdong 510500, P. R. China ǁInstitute

for Biomedical Sciences, Center for Diagnostics and Therapeutics, Georgia State

University, Atlanta, GA 30302, United States

Corresponding Authors Zhigang Xu: Institute for Clean Energy and Advanced Materials, Faculty for Materials and Energy, Southwest University, Beibei, Chongqing 400715, P. R. China; Tel: +86-23-6825-3792; Fax: +86-23-6825-3204; E-mail: [email protected]. Bo Xiao: Institute for Clean Energy and Advanced Materials, Faculty for Materials and Energy, Southwest University, Beibei, Chongqing 400715, P. R. China; Tel: +86-23-6825-4762; Fax: +8623-6825-3204; E-mail: [email protected] or [email protected].

1 ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table of Contents Graphic


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ABSTRACT Reactive oxygen species (ROS) are highly overproduced in cancerous tissues, and thus oxidationresponsive nanoparticles (NPs) have emerged as a promising drug carrier for cancer-targeted drug delivery. In this study, we successfully synthesized poly(vanillyl alcohol-co-oxalate) (PVAX) polymer with an excellent ROS-responsive capacity. A well-established emulsion-solvent evaporation method was used to fabricate PVAX-based curcumin (CUR)-loaded NPs (PVAX-NPs) and their counterparts (poly(lactic-co-glycolic acid)-based CUR-loaded NPs, PLGA-NPs). It was found that these NPs had a hydrodynamic particle size of approximately 245 nm, narrow size distribution (polydispersity index less than 0.1), negative zeta potential (around -18 mV), smooth surface appearance and high drug encapsulation efficiency. Moreover, we found that the CUR release rate of PVAX-NPs was greatly increased in the presence of hydrogen peroxide-rich environment due to the cleavage of polyoxalate ester bonds in PVAX polymer, resulting in the evenly distribution of CUR within the whole cancer cells. More importantly, PVAX-NPs exhibited much stronger anticancer activities and pro-apoptotic capacities than PLGA-NPs both in vitro and in vivo. These results clearly demonstrate that these ROS-responsive PVAX-NPs can be exploited as a robust anticancer drug delivery platform in chemotherapy.

KEYWORDS: Reactive oxygen species, stimuli-responsive polymer, nanoparticle, drug delivery, chemotherapy



INTRODUCTON Cancer has been recognized as one of the most challenging and devastating diseases, accounting for 13% of all deaths worldwide.1,

2

More seriously, its morbidity and mortality continue to

increase sharply in recent decades. Although various therapeutic approaches (e.g., surgery, radiotherapy and immunotherapy) are available for cancer treatment, chemotherapy is the most common strategy.3 Nowadays, a number of chemotherapeutic drugs have been used in chemotherapy, including curcumin (CUR), paclitaxel, doxorubicin and camptothecin.4, 5 Among these drugs, CUR, extracted from rhizomes of turmeric, has attracted increasing attention due to its unique advantages of anti-proliferation and induction of apoptosis in cancer cells through the regulation of various cellular signaling pathways.6, 7 To date, CUR has already demonstrated its striking therapeutic potentials against numerous types of cancers, such as colorectal cancer, pancreatic cancer and non-small cell lung cancer.8 Nevertheless, several inherent limitations of CUR (e.g., low water solubility, rapid metabolization rate and poor bioavailability) have largely impeded its clinical translations.9,

10

To overcome these obstacles, nanocarriers, especially

polymeric nanoparticles (NPs), have been employed as CUR delivery systems, which are liable to improve the water solubility of CUR, prevent its degradation and deliver CUR to tumor tissues driven by the enhanced permeability and retention (EPR) effect.3 In recent years, stimuli-responsive NPs have already been extensively explored in anticancer drug delivery, as they are capable of delivering and releasing drugs to tumor tissues in response to disease-associated physiological factors that differentiate cancerous tissues from healthy tissues, such as pH, oxidation and enzyme as well as hypoxia.11, 12 Among them, oxidation-responsive NPs have been developed very recently, as the tumor cells were accompanied by elevated concentrations of reactive oxygen species (ROS) with the treatment of anticancer agents.13, 14 As


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reported, cellular ROS are a collective term of highly reactive chemical species mainly generated by mitochondria due to the incomplete reduction of molecular oxygen, including hydrogen peroxide (H2O2), hydroxyl radical, superoxide, hypochlorite ion and singlet oxygen.11 Additionally, H2O2 is the most abundant and stable non-radical ROS in cells. Its concentration in healthy tissue is strictly controlled to around 20 nM, whereas that in cancerous tissue is as high as 50-100 µM owing to the excess of H2O2 production.15, 16 Considering these tremendous differences in H2O2 concentrations, H2O2 is believed to be one of the most promising physiological stimuli for achieving tumor-specific drug delivery.17 So far, a variety of ROS-responsive materials, such as those containing thioether, elenium/tellurium, thioketal, boronic ester, peroxalate ester, polyproline, polysaccharide and aminoacrylate, have been explored, and they often required multistep chemical synthesis.18-20 Very recently, poly(vanillyl alcohol-co-oxalate) (PVAX) was synthesized by Lee et al., which had the advantages of excellent biocompatibility, facile synthesis, good ROS responsibility and high drug encapsulation efficiency.21 Thus, it has been employed in the treatment of acute liver failure,22 doxorubicin-induced cardiomyopathy

23

and peripheral

arterial disease.24 However, PVAX has never been used as a drug carrier matrix for cancer therapy. Poly(lactic-co-glycolic acid) (PLGA) is an FDA-approved biocompatible polymer, which has no ROS responsibility. And it has been extensively utilized as drug carrier matrix in our group and others.25, 26 To investigate the beneficial features of oxidation-responsive PVAX in anticancer drug delivery, PLGA was thus used as a control in the present study. Initially, we physically encapsulated CUR into PLGA or PVAX matrix, and obtained PLGA-based CUR-loaded NPs (PLGA-NPs) and PVAX-based CUR-loaded NPs (PVAX-NPs), respectively. Subsequently, we characterized their physicochemical properties, CUR release behaviors in the presence or absence



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of H2O2 and cellular uptake profiles. Finally, in vitro and in vivo anti-tumor activities of PLGANPs and PVAX-NPs were comparatively investigated. EXPERIMENTAL SECTION Materials PLGA with the molecular weight (MW) of 38–54 kg/mol, camptothecin (CPT), poly(vinyl alcohol) (PVA, 86–89% hydrolyzed), trimethylamine (TEA), anhydrous methylene dichloride (DCM), oxalyl chloride, chloroform-d, tetramethylsilane, Triton X-100, dimethyl sulfoxide (DMSO) and buffered formalin (10%) were purchased from Sigma-Aldrich (St. Louis, United States). 1,4Cyclohexanedimethanol (1,4-CHDM), 4-vanillyl alcohol (4-VA) and CUR was obtained from Adamas-beta® (Shanghai, P. R. China). 4ʹ, 6-Diamidino-2-phenyl-indole dihydrochloride (DAPI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl

tetrazolium

bromide

(MTT),

paraformaldehyde

solution (16%), Alexa Fluor 633 phalloidin, ROS Assay Kit, hematoxylin and eosin were from Beyotime Institute of Biotechnology (Shanghai, P. R. China). Alexa Fluor 633 phalloidin was obtained from Invitrogen (Eugene, United States). Terminal deoxynucleotide end labeling (TUNEL) kit was purchased from Roche Diagnostics (Indianapolis, United States). Synthesis and Characterization of PVAX Polymer PVAX polymer was synthesized as described previously.22 Briefly, under the protection of argon, 1,4-CHDM (22 mmol, 3.2 g) and 4-VA (5.5 mmol, 0.86 g) were co-dissolved in 20 mL of anhydrous DCM. Subsequently, TEA (71.4 mmol, 9.9 mL) was added dropwise with a dropping funnel, and the resulting mixture was placed in dark and stirred for 30 min at 4 °C. Thereafter, oxalyl chloride (27.5 mmol, 3.49 g) were poured into the above mixture, and stirred at 4 °C for another 30 min. After a further overnight reaction, the crude product was extracted by DCM, and obtained by precipitation in pre-cold hexane.


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Fabrication of NPs PLGA-NPs and PVAX-NPs were fabricated using an oil-in-water (O/W) single-emulsion solvent evaporation method, which has been commonly utilized for producing polymeric NPs with diameters between 200 to 300 nm.27, 28 In brief, PLGA or PVAX (100 mg) and CUR (7 mg) were co-dissolved in 2 mL of DCM. The obtained oil phase was transferred to 4 mL of PVA solution (5%, w/v). Subsequently, the mixture solution was sonicated with a sonicator (S-450D, Branson, United States) at 40% amplitude for 50 s in an ice bath. The resultant O/W emulsion solution was immediately poured into 100 mL of diluted PVA solution (0.5%, w/v), and the organic solvent was further removed using a rotary evaporator. The larger particles in the product were eliminated by centrifugation at a lower centrifugation rate (4 000 g, 5 min), and the resultant NPs were further centrifuged at 13 000 g for 15 min. Finally, these NPs were washed in deionized water, freezedrying with trehalose as a cryoprotectant and kept at -20C. Physicochemical Characterization of PVAX Polymer and NPs The chemical structure of PVAX was verified by a BRUKER Avance 600 NMR spectrometer. The adopted deuterated solvent was chloroform-d and the internal was tetramethylsilane (δ = 0). The MW and MW distribution (MWD) of PVAX were studied by an Agilent 1260 gel permeation chromatography (GPC, Agilent Technologies, Santa Clara, United States). The average particle size, polydispersity index (PDI) and zeta potential of PLGA-NPs and PVAXNPs were examined using a dynamic light scattering (DLS) technique (Malvern Zetasizer Nano S90, Worcesterhire, United Kingdom). Their average values were calculated on the basis of measurements assigned to 3 batches of NPs. The loading amount and encapsulation efficiency of CUR in PLGA-NPs and PVAX-NPs were measured using a method as described in our previous study.29 NPs were completely dissolved in



DMSO. Subsequently, the obtained solutions were transferred into a black 96-well plate. The fluorescence intensities were determined using a fluorescence spectrophotometer (RF-5301 PC, Shimadzu, Japan) at em=530 nm and ex= 425 nm. The morphologies of PLGA-NPs and PVAX-NPs were characterized using a JEOL JSM-6510LV scanning electron microscope (SEM, Tokyo, Japan). NP suspensions were dropped onto a cleaned silicon chip, and further dried overnight at room temperature. The dried NPs were coated with platinum before SEM observation. XRD spectra of pristine CUR, pristine PLGA, pristine PVAX, PLGA-NPs and PVAX-NPs were studied on an XRD-7000 instrument (Shimadzu, Japan) by scanning samples in the range between 10 and 80° (2). Drug Release Profiles of NPs The drug release behaviors of NPs were investigated in the presence and absence of H2O2 (100 µM) in different buffers (pH 7.4 or 5.5). The fluorescence intensity of CUR was unstable under an oxidation environment, whereas CPT was relatively stable in solution with H2O2. Thus, CPT was encapsulated into NPs instead of CUR in this experiment. NP suspensions containing an equivalent CPT amount of 150 μg were introduced into dialysis bags (MWCO, 10 000 Da), and these bags were immersed in centrifuge tubes (50 mL) supplemented with 20 mL of releasing medium with stirring at 150 rpm and 37 C. 1 mL of releasing medium was withdrawn and replenished with the same volume of fresh medium at pre-determined time points. The CPT concentrations in the releasing medium were determined using a microplate reader (Biotek Instruments, United States). Cytotoxicity Assay Human breast cancer MCF-7 cells or human umbilical vein endothelial cells (HUVECs) were cultured in 96-well culture plates, and the cell densities of these two types of cells were set as 1 ×


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104 and 4 × 104 cells/well, respectively. After overnight incubation, cells were incubated with PLGA-NPs or PVAX-NPs for 24 h and 48 h, respectively. MTT solution (0.5 mg/mL) was added to each well, and incubated with cells in CO2 incubator for 4 h. Thereafter, DMSO (50 μL) was added into each well to dissolve the formazan crystals, and the wavelength for measuring absorbance of the formed formazn was at 570 nm. Cells treated with Triton X-100 solution (1%, w/v) were utilized as a positive control, whereas untreated cells were utilized as a negative control. Cell Internalization Imaging of NPs MCF-7 cells were cultured in 8-well culture plates (BD Falcon, Bedford, United States), and the cell density was set as 5×104 cells/well. PLGA-NPs and PVAX-NPs were dispersed in serum-free medium to form NP suspensions (equivalent CUR concentration: 50 μM), and the resulting suspensions were then added into wells. After 3 h of incubation, cells were washed with PBS solution to eliminate un-phagocytized NPs. Thereafter, cells were fixed in a paraformaldehyde solution (4%, v/v) for 15 min, and further stained with DAPI and Alexa Fluor 633 phalloidin solution. The cellular uptake images of NPs were observed by a confocal laser scanning microscope (CLSM, Zeiss-800, Jena, Germany). Quantification of Cell Internalization Efficiency of NPs MCF-7 cells were cultured in 12-well culture plates, and the cell density was set as 2×105 cells/well. PLGA-NPs and PVAX-NPs were dispersed in serum-free medium to form NP suspensions (equivalent CUR concentration: 16 μM), and the resulting suspensions were then added into wells. After 1, 3 or 5 h of incubation, cells were washed 3 times with PBS solution to eliminate unphagocytized NPs. Cells were digested using trypsin, and were re-suspended in flow cytometric buffer. The obtained cell suspensions were analyzed using an ACEA NovoCyteTM Flow Cytometry System (ACEA Biosciences, San Diego, United States).



Blood Compatibility Studies In vitro blood compatibility was examined following our reported studies.3, 30 Briefly, red blood cells (RBCs) from mice blood were gathered at 1 400 g for 12 min. Subsequently, they were washed 3 times and re-suspended in PBS. The suspensions of PLGA-NPs and PVAX-NPs were mixed with RBCs, and further incubated at 37 °C for 1 h. After centrifugation (4 oC, 4 000 g, 12 min), the absorption intensity of hemoglobin in the supernatant were determined using spectrophotometric measurements at 570 nm. RBC suspensions treated with Triton X-100 (1%, w/v) were utilized as a positive control, and RBC suspensions (no treatment) were treated as 100%. For in vivo blood compatibility studies, suspensions of PLGA-NPs and PVAX-NPs (10 mg CUR/kg mouse) were intravenously injected into female Kunming mice (8 weeks old; Chongqing Tengxin Biotechnologied Company, Chongqing, P. R. China). After administration for 6 h or 24 h, blood parameters were analyzed using a hematology analyzer (BC-3200, Mindray, Shenzhen, P. R. China). In Vivo Antitumor Activity of NPs BALB/c nude mice (6 weeks old; Chongqing Tengxin Biotechnologied Company, Chongqing, P. R. China) were used to establish a mouse model of subcutaneous tumor. All mice procedures were approved by the Institutional Animal Care and Use Committee at Southwest University. Mice were implanted subcutaneously with 1×106 MCF-7 cells in the left flank. After implantation with tumor cells for 12 days, mice were divided into the following 3 groups: (i) PBS-treated group; (ii) PLGANP-treated group (CUR, 10 mg/kg) and (iii) PVAX-NP-treated group (CUR, 10 mg/kg). These mice were intravenous injected with different formulations every other day for a total of 3 doses. Their body weight and tumor size were recorded during the treatment process. Tumor inhibition


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ratio has been commonly used to evaluate the inhibitory effect of drug formulations on tumor growth.31 Its equation was described as follows: Tumor inhibition ratio 

Tumor weight in control group - Tumor weight in treated group 100% Tumor weight in control group

(1)

In the context of histological analysis, tissues were fixed using 10% buffered formalin solution, and further embedded in paraffin. Subsequently, tissue sections (5 µm) were stained using hematoxylin and eosin (H&E). The apoptosis profiles of tumor cells were studied with an apoptosis detection kit (Roche Diagnostics, Indianapolis, United States). RESULTS AND DISCUSSION Synthesis of PVAX The PVAX polymer was synthesized by one-step condensation between two diols (1,4-CHDM and 4-VA) and oxalyl chloride. The combined application of two diols in the present study was due to the fact that they had the capacity to improve the stability and degradability of the overall polymer.32 The synthetic route and H2O2-triggered degradation process were presented in Figure 1a. 1H NMR results in Figure 1b showed that the methylene signals of 1,4-CHDM emerged at 4.21 ppm (peak g) and 2.08-0.67 ppm (peak h), while the signals of 4-VA located at 4.11 ppm (peak f) and 3.01 ppm (peak e). In addition, the peak at 3.45 pm originated to the methyl hydrogen of 4-VA. According to peak d (3.45 ppm) and peak h (2.08-0.67 ppm) signals, the polymerization ratio of 1,4-CHDM and 4-VA was calculated to be 1:4. Furthermore, the multiple aromatic protons of 4-VA registered at 7.07 and 6.77 ppm, indicating the successful synthesis of PVAX. As seen in Figure 1c, the MW and MWD of PVAX were 4 100 and 1.77, respectively. After the treatment with H2O2 (10 mM) for 0.5 h, the peak was obviously shifted to the high elution time, indicating the ROS-responsible degradation profile of PVAX. Physicochemical Characterization of NPs 11 ACS Paragon Plus Environment


Hydrodynamic particle size, particle size distribution and zeta potential of NPs are critically correlated to their stability, drug release behavior, cell internalization efficiency and in vivo biodistribution.33-35 Hence, these parameters were characterized, and the relevant results were presented in Table 1. PLGA-NPs had an average hydrodynamic diameter of 254.5 nm, and their PDI was relatively low (0.091), suggesting that they had a narrow size distribution. Furthermore, no obvious differences in diameter and PDI were found between PLGA-NPs and PVAX-NPs. Recently, it was reported that PLGA-based polymeric NPs with hydrodynamic diameter around 200 nm exhibited obvious EPR effect.36 Thus, these two types of NPs were expected to avoid passing through normal blood vessels, and facilitate the accumulation of chemical drugs into the tumor tissues because of EPR effect. In addition, both types of NPs had slightly negative zeta potentials. It was found that the loading amount and encapsulation efficiency of CUR in PVAXNPs were similar to that in PLGA-NPs. The representative morphological images of PLGA-NPs (Figure 2a) and PVAX-NPs (Figure 2b) indicated that both types of NPs were spherical in shape with a smooth surface. As reported, the crystallinity of hydrophobic agents loaded in NPs has an important impact on the drug release pattern of NPs,37 and thus the crystalline forms of CUR in PLGA-NPs and PVAXNPs were investigated. As can be seen in Figure S1, pristine CUR had numerous sharp and intense peaks, revealing their highly crystalline nature. Conversely, pristine PLGA and pristine PVAX showed no obvious peaks. Interestingly, with the introduction of CUR into NPs, PLGA-NPs and PVAX-NPs exhibited pretty smooth curves and had no characteristic peaks. These results clearly demonstrate that CUR has been uniformly distributed throughout the polymeric matrix (PLGA or PVAX), and forms an amorphous complex with matrix molecules, which would be favorable for achieving a constant drug release rate.


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To investigate the oxidation-responsive properties of NPs, we initially studied the morphology changes of PLGA-NPs and PVAX-NPs under H2O2 condition. Owing to the resolution limitation of SEM instrument, we did not detect any obvious morphology change from the SEM images of NPs (data not shown). In an attempt to make visual comparisons, we further prepared two types of microparticles (MPs), i.e., PLGA-MPs and PVAX-MPs following the same preparation methods described in Experimental Section, except that sonication was not applied to the fabrication processes of MPs. The typical SEM images of MPs were showed in Figure 2c. It was obvious that PLGA-MPs kept their spherical shape and smooth surface morphology after incubation in H2O2 solution (10 mM) for 0.5 or 1 h. In terms of PVAX-MPs, they had relatively smooth surfaces prior to incubation. However, numerous surface pores and structure damages were observed on the surface of these MPs, suggesting their oxidation-responsible capacity. Drug Release patterns of NPs To quantitatively investigate the ROS-responsive drug release patterns of PVAX-based NPs, we comparatively investigated the drug release rates of PLGA-based NPs and PVAX-based NPs in solutions with different pH values (7.4 or 5.5) and H2O2 (0 or 100 µM). Figure 2d and Figure 2e elucidated that both types of NPs had a slightly fast initial release and a subsequently slower constant release. The initial burst release of drug molecules can be attributed to their rapid diffusion cross the surface layer of NPs, and the subsequently sustained release can be ascribed to drugs in the interior of NPs. Moreover, the drug release rate of PLGA-CPT-NPs in H2O2 solution had no obvious difference with that in the solution without H2O2. On the contrary, the drug release rate of PVAX-CPT-NPs increased observably in the presence of H2O2, which can be due to the accelerated degradation of NP matrixes.



As reported, healthy cells had low levels of ROS to facilitate their growth adaptation and survival, whereas ROS amounts in cancer cells were over a thousand times more than that in healthy cells.15, 16, 38

Furthermore, we investigated where ROS were generated in cells. As seen in Figure S2, MCF-

7 cells without treatment (negative control) showed ROS signal. Interestingly, the ROS amount increased sharply in the PVAX-NP-treated cells, and these ROS were dispersed in the whole cells. These results obviously suggest that the increased amount of ROS is expected to facilitate the release of drug from PVAX-NPs into cells, resulting in the improved anticancer activity. Intracellular Uptake Profiles of NPs Cell internalization is a fundamental requirement for the application of chemotherapeutic drugs in breast cancer therapy because they take their antitumor effect mainly within cells.39 Thus, we qualitatively and quantitatively studied the cellular uptake properties of NPs using CLSM and flow cytometry, respectively. MCF-7 cells were treated with PLGA-NPs or PVAX-NPs for 3 h to make qualitative comparisons. As shown in Figure 3a-b, the control cells without NP treatment had no green fluorescence signals. As expected, cells with the treatment of PLGA-NPs or PVAX-NPs showed obvious CUR accumulation in their cytoplasm. Moreover, it is rather surprising for us that green fluorescence signal was distributed evenly throughout the cytoplasm with the treatment of PVAX-NPs compared to PLGA-NP-treated cells. The possible reason is that much more CUR molecules released from the ROS-responsive PVAX-NPs can enter into intracellular environment than that of PLGA-NPs. In the quantitative experiments, MCF-7 cells were incubated with PLGA-NPs or PVAX-NPs at an equivalent CUR concentration of 16 μM for 1, 3 and 5 h, respectively. Figure 3c showed the typical flow cytometric histograms for the cells treated with NPs for 5 h. These results revealed that PVAX-NPs showed remarkably stronger green fluorescence intensities inside the cells compared


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with that for PLGA-NPs. Furthermore, cellular uptake amounts (Figure 3d) and green fluorescence intensities (Figure 3e) of PVAX-NPs was more than that of PLGA-NPs, even though there was no statistically significantly difference between these two types of NPs. In addition, as presented in Figure 3d and Figure S3a, after 5 h incubation, the percentages of PLGA-NP- or PVAX-NP-treated cells with green fluorescence were respective 41.5% and 61.6%, which were much higher than that of pristine CUR-treated cells (34.1%). It was also found that green fluorescence intensities in cells exhibited the similar trend, as shown in Figure 3e and Figure S3b. Although certain percentage of pristine CUR-treated cells showed green fluorescence, two critical points had to be mentioned: (1) Pristine CUR, a hydrophobic drug, was dissolved in medium containing DMSO (5%, v/v) in this study. It was reported that DMSO could greatly enhance the diffusion of hydrophobic drugs into cells;40 (2) Pristine CUR-treated cells were exposed to CUR solution directly, whereas NP-treated cells were grown in the medium containing very low concentrations of extracellular CUR released from NPs, which were due to the slow release of CUR from NPs (less than 10% and 20% of the total CUR for respective PLGA-NPs and PVAX-NPs within the initial 5 h). Therefore, we can conclude that green fluorescence signals in MCF-7 cells are mainly attributable to intracellular uptake of NPs. In Vitro Antitumor Activities of NPs To investigate in vitro antitumor activity of PLGA-NPs and PVAX-NPs, we treated MCF-7 cells with these NPs for 24 h and 48 h, respectively. It can be seen from Figure 4a and Figure 4b that both types of NPs inhibited the cell viability depending on the drug concentration and the incubation time. Notably, the viability differences between cells treated with PLGA-NPs or PVAX-NPs appeared relatively weaker under low concentration of CUR (less than 2 µM). However, when CUR concentration increased to over 2 µM, the antitumor activities of PVAX-



NPs was significantly stronger than that of PLGA-NPs, which might be attributed to the oxidationresponsive drug release features of PVAX-NPs. To quantitatively characterize the cytotoxicity of PLGA-NPs and PVAX-NPs, we calculated IC50 values of these two types of NPs. As depicted in Table 2, the IC50 values for PLGA-NPs were 47.2 µM and 32.3 µM at 24 h and 48 h, respectively, which was much higher than the corresponding IC50 values in PVAX-NPs. These results clearly suggest that PVAX-NPs have much stronger antitumor activities than PLGA-NPs. To attempt to determine the potential adverse effects of NPs, we evaluated their cytotoxicity against HUVECs, which were normal cells. As can be seen in Figure S4 and Table S1, although PLGA-NPs and PVAX-NPs inhibited the viabilities of HUVECs to certain extent, their IC50 values were significantly higher than that against MCF-7 cells. Therefore, we can conclude that PLGA-NPs and PVAX-NPs have relatively low adverse effects. Hemolysis Assay The hemocompatibility of NPs is an important parameter for in vivo application. As shown in Figure 5a-b, there was no distinguishable evidence of hemolysis in tubes treated with PLGA-NPs or PVAX-NPs under CUR concentration of 100 µM, which was much higher than the concentrations applied in the in vitro experiments. In addition, in vivo blood compatibility results (Figure 5c) revealed that all of the blood components, such as white blood cells (WBC), red blood cells (RBC), platelets (PLT), lymphocytes (LYM), monouclear cells (MON) and granulocyte cells (Gran), were in the normal range. These results collectively indicate the excellent blood biocompatibility of both types of NPs. In Vivo Antitumor Activity of NPs To verify the in vivo antitumor efficacy, PBS, PLGA-NPs and PVAX-NPs were intravenously injected into mice bearing a subcutaneous MCF-7 tumor. Body weight change is a common


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parameter for assessing the systemic adverse effects of nanotherapeutics. It could be noticed from Figure 6a that injecting both types of NPs resulted in no obvious body weight loss compared with PBS control group, indicating the comparable safety of PLGA-NPs and PVAX-NPs. The tumor volumes for the different treatment groups are displayed in Figure 6b. The volume of MCF-7 tumor treated with PBS increased throughout the experiment phase. However, the increase trend of tumor volume was reversed by intravenous injection of NPs. Furthermore, intravenous injection of PLGA-NPs suppressed the volume of tumor by 73.1% at day 21, whereas administration of PVAX-NPs with the same dose of CUR (10 mg/kg) decreased tumor volumes by 87.2%. The remarkable suppression of tumor volume in PVAX-NP-treated group could be attributed to the beneficial property of their ROS sensitivity. In addition, we found that tumor weights (Figure 6c) and tumor inhibition ratios (Figure 6d) correlated well with tumor sizes. Histological examinations of tumor tissue sections (Figure 6e) were carried out to compare the morphology of tumor tissue between PBS control group and NP-treated groups. It was obvious that tumor cells in the PBS control group were observed with large nucleus. Conversely, NPtreated groups showed obviously decreased in tumor cell numbers and nucleus shrinkage. It was worth noting that a large necrotic area was detected in the PVAX-NP-treated group. The proapoptotic effects of PVAX-NPs were further studied using TUNEL assays. Figure 6f showed that TUNEL-stained cells (green fluorescence) was barely observed in the tumor tissue sections from PBS control group, but was obviously detected in tumor tissue sections of mice treated with PLGA-NPs. Furthermore, PVAX-NP-treated group showed much more green fluorescence than PLGA-NPs, indicating that PVAX-NPs had much stronger pro-apoptotic activity than PLGA-NPs in vivo. In addition, we performed histological analysis of the main organs to examine the systemic toxicity. We found that no clear evidence of organ or tissue impairments was observed in H&E-



stained slices from all the treatment groups (Figure S5), demonstrating the excellent biocompatibility of PLGA-NPs and PVAX-NPs. CONCLUSION In summary, an oxidation-responsive polymer, termed as PVAX, was successfully synthesized and exploited to fabricate curcumin (CUR)-loaded nanoparticles (NPs). The resulting PVAX-NPs had a mean hydrodynamic diameter (about 242 nm) and a negative-charged surface (around -17.3 mV) that effectively facilitated their cell internalization. Moreover, these NPs could particularly respond to reactive oxygen species and accelerate release of drugs in the tumor cells. We further found that PVAX-NPs showed much stronger antitumor activity against breast cancer cells compared with their counterparts (PLGA-NPs). In vivo mice experiments clearly indicated that PVAX-NPs exhibited much higher antitumor activities than PLGA-NPs. Collectively, our findings indicate that PVAX-NPs might hold a promising potential as a nanotherapeutic platform for cancer chemotherapy.

ASSOCIATED CONTENT The authors declare no competing financial interest. Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut. xxxxxxx. Experimental description, X-ray diffraction patterns of NPs, cellular uptake profiles of pristine CUR in MCF-7 cells, ROS levels in MCF-7 cells, toxicity of NPs against HUVECs; H&E staining of sections of heart, liver, spleen, lung, and kidney. AUTHOR INFORMATION


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Corresponding Authors E-mail: [email protected] E-mail: [email protected] or [email protected] ORCID Zhigang Xu: 0000-0003-1805-5061 Bo Xiao: 0000-0002-2992-6435 Author Contributions Y.H. and B.X. designed experiments, supervised the project, and wrote the draft of manuscript. Y.H., Q.C., P.M., H.S., X.M., Y.M., X.Z., S.G. and J.C. performed the experiments. X.Z. and B.X. edited and revised the manuscript. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (81571807 and 51503172), the Fundamental Research Funds for the Central Universities (XDJK2017B058), the Young Core Teacher Program of the Municipal Higher Educational Institution of Chongqing, the China Postdoctoral Science Foundation (2016M602627), the Chongqing Postdoctoral Science Special Foundation (Xm2016032) and the State Key Laboratory of Silkworm Genome Biology. REFERENCES (1) Sud, A.; Kinnersley, B.; Houlston, R. S. Genome-wide association studies of cancer: current insights and future perspectives. Nat. Rev. Cancer 2017, 17 (11), 692‒4. (2) Yoo, W.; Yoo, D.; Hong, E.; Jung, E.; Go, Y.; Singh, S. V. B.; Khang, G.; Lee, D. Acidactivatable oxidative stress-inducing polysaccharide nanoparticles for anticancer therapy. J. Control. Release 2018, 269, 235‒44.



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Figure Captions Figure 1. (a) Schematic illustrations of synthesis and H2O2-triggered degradation of PVAX. (b) 1H

NMR spectrum and (c) GPC trace of PVAX and its degraded products.

Figure 2. Representative SEM images and corresponding size distributions of (a) PLGA-NPs and (b) PVAX-NPs. Scale bar represents 200 nm. (c) Representative SEM images of PLGA-MPs and PVAX- MPs as a function of incubation time in H2O2 solution (10 mM). Scale bar represents 1 µm. In vitro release profiles of CPT from PLGA-CPT-NPs and PVAX-CPT-NPs with or without H2O2 in (d) buffer (pH = 7.4) and (e) buffer (pH = 5.5) at 37 oC. Each point represents the mean ± S.E.M. (n = 3). Figure 3. Representative (a) flat-field and (b) 3-dimensional fluorescence images showing cellular uptake of PLGA-NPs and PVAX-NPs at an equal CUR concentration of 50 μM in MCF-7 cells for 3 h. Fixed cells were stained with Alexa Fluor 633 phalloidin and DAPI for visualization of actin (red) and nuclei (purple), respectively. Scale bar represents 20 µm. (b) Flow cytometric histogram profiles of fluorescence intensities for cells treated with PLGA-NPs or PVAX-NPs (CUR, 16 µM) for 5 h. (c) Percentage of CUR-containing MCF-7 cells and (d) quantification of CUR fluorescent intensity in MCF-7 cells after treatment with PLGA-NPs or PVAX-NPs (CUR, 16 mM) at different time points (1, 3, and 5 h). Each point represents the mean ± S.E.M. (n= 3). Statistical significance was assessed using Student's t-test (*P < 0.05 and **P < 0.01). Figure 4. In vitro antitumor activities of PLGA-NPs and PVAX-NPs against MCF-7 cells after incubation for (a) 24 h and (b) 48 h based on MTT assays. Triton X-100 (1%) was used as a positive control to produce a maximum cell death rate (100%), whereas cell culture medium was used as a negative control (death rate defined as 0%). Cytotoxicity is given as the percentage of viable cells remaining after treatment. Each point represents the mean ± S.E.M. (n = 5).



Figure 5. The hemocompatibility of PLGA-NPs and PVAX-NPs. (a) Photograph and (b) hemolytic analysis of erythrocyte suspensions treated with various NPs at different CUR concentrations. Triton X-100 (1%) was used as a positive control (100%), whereas PBS was used as a negative control (0%). Each point represents the mean ± S.E.M. (n = 5). (c) Blood test of Kunming mice injected with PBS (control), PLGA-NPs or PVAX-NPs for 6 or 24 h. Each point represents the mean ± S.E.M. (n = 3). Figure 6. In vivo antitumor activities of PLGA-NPs and PVAX-NPs. (a) Changes in body weight in different treatment groups. Mouse body weight was normalized to day 0 body weight (expressed as a percentage). (b) Tumor growth profiles, (c) tumor weights and (d) tumor inhibition ratios in different groups with the treatment of PLGA-NPs or PVAX-NPs. Statistical significance was assessed using ANOVA followed by a Bonferroni post-hoc test (*P < 0.05 and **P < 0.01). Each point represents the mean ± S.E.M. (n = 5). Measurement of apoptosis. (e) H&E staining of tumor tissues from mice treated with PLGA-NPs or PVAX-NPs. Scale bar represents 200 µm. (f) Representative images of double-fluorescence labeling with DAPI (nuclei, blue) and TUNEL (green) in breast tumors from different mouse groups. Scale bar represents 200 µm. Table 1. Characteristics of NPs (mean ± S.E.M.; n = 3). Table 2. IC50 (μM) of NPs against MCF-7 cells.


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Table 1. Characteristics of NPs (mean ± S.E.M.; n = 3). NPs

Size (nm)

PDI

Zeta potential (mV)

Drug loading (%)

Encapsulation efficiency (%)

PLGA-NPs 254.5 ± 1.1

0.091

-19.1 ± 0.5

5.8 ± 0.2

40.1 ± 1.2

TPOX-NPs 242.3 ± 4.4

0.034

-17.3 ± 1.2

6.0 ± 0.2

44.0 ± 1.7



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Table 2. IC50 (μM) of NPs against MCF-7 cells. Incubation time

PLGA-NPs

TPOX-NPs

24 h

47.2

22.4

48 h

32.3

12.3


Facile Fabrication of Oxidation-Responsive Polymeric Nanoparticles

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