ROS and GSH-responsive S-nitrosoglutathione functionalized polymeric nanoparticles to overcome multidrug resistance in cancer

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ROS and GSH-responsive S-nitrosoglutathione functionalized polymeric nanoparticles to overcome multidrug resistance in cancer Wen Wu , Min Chen , Tingrong Luo , Ying Fan , Jinqiang Zhang , Yan Zhang , Qianyu Zhang , Anne Sapin-Minet , Caroline Gaucher , Xuefeng Xia PII: DOI: Reference:

S1742-7061(19)30845-1 https://doi.org/10.1016/j.actbio.2019.12.016 ACTBIO 6499

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Acta Biomaterialia

Received date: Revised date: Accepted date:

8 September 2019 11 December 2019 12 December 2019

Please cite this article as: Wen Wu , Min Chen , Tingrong Luo , Ying Fan , Jinqiang Zhang , Yan Zhang , Qianyu Zhang , Anne Sapin-Minet , Caroline Gaucher , Xuefeng Xia , ROS and GSHresponsive S-nitrosoglutathione functionalized polymeric nanoparticles to overcome multidrug resistance in cancer, Acta Biomaterialia (2019), doi: https://doi.org/10.1016/j.actbio.2019.12.016

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ROS and GSH-responsive S-nitrosoglutathione functionalized polymeric nanoparticles to overcome multidrug resistance in cancer Wen WU1*, Min CHEN1, Tingrong LUO1, Ying FAN1, Jinqiang ZHANG1, Yan Zhang1, Qianyu Zhang1, Anne SAPIN-MINET2, Caroline GAUCHER2, Xuefeng XIA1* 1 Chongqing Key Laboratory of Natural Product Synthesis and Drug Research, School of Pharmaceutical Sciences, Chongqing University, Chongqing 401331, China 2

Université de Lorraine, CITHEFOR, F-54000 Nancy, France

Wen WU and Min CHEN made equivalent contributions to this work *corresponding author. Email address: [email protected] (Wen Wu); [email protected] (Xuefeng Xia)

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Abstract

Multidrug resistance of cancer cells is one of the major obstacle for chemotherapeutic efficiency. Nitric oxide (NO) has raised the potential to overcome multidrug resistance (MDR) with low side effects. Herein, we report a reactive oxygen species (ROS) and glutathione (GSH) responsive nanoparticle for the delivery of NO prodrug such as S-nitrosoglutathione (GSNO), which was chemically conjugated to an amphiphilic block copolymer. The GSNO functionalized nanoparticles show high NO loading capacity, good stability and sustained NO release with specific GSH activated NO-releasing kinetics. Such GSNO functionalized nanoparticles delivered doxorubicin (DOX) in a ROS triggered manner and increased the intracellular accumulation of DOX. However, in normal healthy cells, showing physiological concentrations of ROS, these nanoparticles presented good biocompatibility. The present work indicated that these multifunctional nanoparticles can serve as effective co-delivery platforms of NO and DOX to selectively kill chemo-resistant cancer cells through increasing chemo-sensitivity.

Keywords: Nitric oxide, Multidrug resistance of cancer, Reactive oxygen species-responsive, Glutathione-responsive, S-nitrosoglutathione

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Statement of significance In this work, we constructed nitric oxide donor (S-nitrosoglutathione, GSNO) functionalized amphiphilic copolymer (PEG-PPS-GSNO) to deliver doxorubicin (DOX). The developed PEG-PPS-GSNO@DOX nanoparticles presented high NO capacity, ROS triggered DOX release and GSH triggered NO release. Thus NO reversed the chemo-resistance in HepG2/ADR cells increasing intrcellular accumulation of DOX. Furthermore, these PEG-PPS-GSNO@DOX nanoparticles exhibited biocompatibility to healthy cells and toxicity to cancer cells, due to elevated ROS.

1. Introduction

Multidrug resistance (MDR) leading to high recurrence rates and treatment failures, remains a tremendous challenge in cancer chemotherapy [1]. As widely studied, the abnormal expression of P-glycoprotein (P-gp) plays predominate role in pumping chemotherapeutics out of cancer cells, thus inducing MDR [2,3]. This results in low accumulation of therapeutics and low treatment efficiency.

Nitric oxide (NO) is an important bioactive molecule exerting multiple functions in many physiological and pathological processes, such as cardiovascular homeostasis 3

[4], immune responses [5], neurotransmission, and cell apoptosis. Besides, NO is also an effective molecule to accelerate the depletion of intracellular glutathione (GSH) [6-9]. Moreover, NO can react with ROS to generate reactive nitrogen species (RNS), which boosted the destructive activity and exacerbates the overall damage by triggering free radical peroxidation [10-12].

Recent studies have indicated that NO presented promising potentiality in inhibiting tumor growth. The bioactivity of NO is highly dependent on its concentration, the duration of its production and the location. At low concentrations (pM~nM), NO acts as a vasodilator to promote cancer cell proliferation and infiltration [13]. However, at high concentrations (μM~mM) it induces cell apoptosis and death [14], as well as reduces the P-gp expressions and reverses MDR. Therefore, the tumor-targeted delivery of NO with spatiotemporal control are extremely important. Currently two strategies were reported for NO-mediated anticancer therapy: ⅰ direct killing of cancer cells through delivering high NO concentrations [15] to the tumor site; ⅱ combinational application, in wide ranges of NO concentrations, with chemotherapeutics through chemosensitization and/or synergistic effect [16.17]. Different kinds of NO donors, like N-diazeniumdiolates (NONOates) and S-nitrosothiols (RSNO) have been previously conjugated to linear polymers or inorganic nanoparticles [18-22]. However NONOates showed direct potential toxicity as well as secondary carcinogenesis induced by amine by-products generation along the release of NO [21,23]. In contrast, RSNO, as a physiological-based class of NO 4

donors, are more attractive. Previously Davis et al. synthesized a block copolymer containing thiol groups in its hydrophobic segments and then modified the thiol groups of the block copolymer with S-nitrosothiols [24,25]. Although the amount of NO released from the micelles formed by block copolymer was not enough to present cell killing effect, neuroblastoma cells pretreatment with these NO-releasing micelles reduced the half maximal inhibitory concentration (IC50) of cisplatin by 5-fold compared to free cisplatin [24,25]. However, NO released spontaneously from these nanoparticles and cannot be controlled. Therefore, the development of intelligent NO-releasing nanomaterials with controllable release property is urgent need.

Herein, we presented an alternative advanced strategy for developing NO-releasing nanoparticles to convey chemotherapeutics to cancer cells. As described in scheme 1, the lipophilic end of the amphiphilic copolymer, methoxy poly(ethylene glycol)-block-poly(propylene sulfide) (PEG-PPS) was functionalized with GSNO, and further explored as a drug delivery system for the encapsulation and on-demand delivery of doxorubicin (DOX) at tumor site. The PPS block could undergo an oxidative conversion from hydrophobic to hydrophilic, which enabled on-demand delivery of therapeutics [26]. PEG-PPS has been widely explored for engineering polymersomes for vaccine delivery and nanoparticles for hydrophobic drug loading [26-28]. However, to the best of our knowledge, the application of this system to overcome multidrug resistance in cancer has not yet been explored. Herein this study aimed at developing GSNO functionalized PEG-PPS nanoparticles for the delivery of 5

DOX, which promoted the accumulation of NO and DOX in cancer cells and released them in controllable way.

2. Materials and methods

2.1 Materials.

Poly(ethylene glycol) monomethyl ether (Mn 2000 g/mol), propylene sulfide, thionyl bromide, methyl acrylate, N-Hydroxysuccinimide (HOSU), N,N-Dicyclohexylcarbodiimide, pluronic F68 and Doxorubicin hydrochloride (DOXHCl) were purchased from Sigma-Aldrich (Beijing, China). Cell counting kit-8 (CCK8), Annexin V-APC/7-AAD apoptosis detection kit, 3-amino,4-aminomethyl-2`,7`-difluorofluorescein (DAF-FM DA), 4,6-diamino-2-phenyl indole (DAPI) were purchased from Beyotime (Jiangsu, China). Dulbecco's modified eagle medium (DMEM) was purchased from Gibco (Suzhou, China), fetal bovine serum (FBS) was purchased from Gibco (Auckland, New Zealand), penicillin-streptomycin were purchased from Solarbio (Beijing, China). All the reagents were of analytical grade. SpectraMax i3x (San Francisco, United States), CytoFLEX Beckman Coulter Biotechnology Co., Ltd. (Suzhou, China), confocal laser scanning microscopy Leica TCS SP8, (Heidelberg, Germany)，ultrasonic processor, Sonics & Materials (Newtown, United States), Transmission Electron Microscope Zeiss Libra 200FE, (Oberkochen, Germany).

2.2 Synthesis of GSNO functionalized PEG-PPS (PEG-PPS-GSNO) 6

Block copolymer, PEG-PPS, Poly(ethylene glycol)-block-poly(propylene sulfide) (PEG-PPS) was synthesized as previously published [29,30]. Briefly, commercially available methyl ether PEG (Mn 2000) was functionalized with mesylate and subsequently thioacetate groups. Deprotection, under basic condition, of the PEG-thioacetate afforded a thiolate anion, which was used to perform living ring-opening polymerization of 20 molar equivalents of propylene sulfide, which was end capped with methyl propionate. The terminal propionate was hydrolyzed under NaOH condition, and further react with HoSu to increase the activity. Finally, GSNO functionalized PEG-PPS polymer was obtained by conjugation of GSNO to the activated hydroxyl.

2.3 Preparation and characterization of DOX loaded nanoparticles

DOX loaded PGE-PPS-GSNO nanoparticles (PEG-PPS-GSNO@DOX) were prepared by emulsion-solvent evaporation method. Briefly, 20 mg of PEG-PPS-GSNO was dissolved in 500 μL of dichloromethane to obtain a solution 1, then 2 mg of DOXHCl was dissolved in 200 μL of F68 aqueous solution (0.01%) to obtain a solution 2. Then, the solution 1 was added dropwisely to the solution 2. The obtained mixed system was emulsified by Ultrasonic Processor (300 W, 23%) for 30 s to obtain primary emulsion. Then the colostrum was added dropwisely to 2 mL of F68 aqueous solution (0.01%), and the mixture was further emulsified by Ultrasonic Processor (300 W, 20%) for 60 s to obtained double emulsion. The organic phase was removed by rotary evaporation (50 rpm, 20 min) to obtain PEG-PPS-GSNO@DOX 7

nanoparticles. All the processes were performed in ice bath and carefully keep out of the light.

2.4 In vitro release property assay

In vitro release study was performed in PBS (0.1 M, pH 7.4) solution containing GSH (5 mM) or various concentrations of H2O2 (0, 5, 50 mM) at 37°C. Typically, PEG-PPS-GSNO nanoparticles (2 mg) were dispersed into PBS (1 mL) and transferred to a dialysis bag, which was then immersed into 50 mL of PBS containing GSH or mentioned concentrations of H2O2. Fixed volume of supernatant (0.6 mL) were iteratively removed for GSNO and DOX quantifications and replaced by fresh medium with the same volume. The cumulative release of GSNO or DOX was analyzed by Griess-Saville assay [31~33] or UV spectrometer [34], respectively.

2.6 cell culture and biocompatibility evaluation

Human embryonic kidney cells (293T cells) were purchased from American Type Culture Collection (ATCC) (Manassas, USA) and cultivated with 1640 medium containing fetal bovine serum (FBS, 10%), penicillin (100 U/mL), and streptomycin (100 μg/mL). Adriamycin resistance of human hepatocellular carcinoma HepG2/ADR cells were purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China) and incubated with DMEM containing FBS (10%), penicillin (100U/mL), streptomycin (100 μg/mL) and DOXHCl (2 μg/mL) under a humid atmosphere containing 5% CO2 at 37°C. 8

To evaluate the biocompatibility of PEG-PPS-GSNO nanoparticles in vitro, 293T cells (10000 cells/well) were incubated with PEG-PPS nanoparticles and PEG-PPS-GSNO nanoparticles (final concentration of polymer corresponds to 37~925 μg/mL) in 96-well plates for 24 h at 37°C. Then, the culture medium was replaced with 100 μL of fresh medium and 10 μL of CCK8 reagent. The plates were further incubated for 1 h, and absorbance was measured at 450 nm with a spectrophotometric microplate reader.

2.7 Estimation the cell cytotoxicity of PEG-PPS-GSNO@DOX nanoparticles and the enhancement of chemosensitivity of DOX in vitro

To evaluate the cytotoxicity of PEG-PPS-GSNO@DOX nanoparticles, HepG2/ADR cells were incubated with PEG-PPS-GSNO@DOX nanoparticles (final concentration of DOX and PEG-PPS-GSNO polymer corresponds to 1~30 μg/mL and 37~925 μg/mL, respectively) in 96-well (10000 cells/ well) plates for 24 h at at 37°C. Then, the culture medium was replaced with 100 μL of fresh medium and 10 μL of CCK8 reagent for each well. The plates were further incubated for 1 h, and absorbance was measured at 450 nm with a spectrophotometric microplate reader.

2.8 Cell uptake of PEG-PPS-GSNO@DOX nanoparticles by confocal microscopy

The visualization of cell morphology and intracellular trafficking of PEG-PPS-GSNO@DOX nanoparticles in HepG2/ADR cells were determined by confocal laser scanning microscopy (CLSM). HepG2/ADR cells were seeded in 35 9

mm2 confocal dishes at a density of 2×104 cells per well and incubated at37 °C for 24 h . The cells were then incubated with PEG-PPS@DOX nanoparticles (PEG-PPS@DOX NP), PEG-PPS-GSNO nanoparticles (PEG-PPS-GSNO NP) and PEG-PPS-GSNO@DOX nanoparticles (PEG-PPS-GSNO@DOX NP) and further cultivated for 8 h. Then, cells were washed twice with PBS and incubated at 37 °C for 30 min with 5 μM DAF-FM DA in DMEM. Then, after PBS washing, the cells were fixed with 4% (m/v) paraformaldehyde for 15 min at room temperature. DAPI was used to counterstained cell nucleus with an excitation of 405 nm before imaged on fluorescence microscopy. The fluorescence of NO adducts of DAF-FM DA and DOX were observed under an excitation of 515 nm and 575 nm respectively.

2.9 Cell uptake of PEG-PPS-GSNO@DOX nanoparticles by flow cytometry HepG2/ADR cells were seeded in 12-well plates at a density of 5×104 cells per well at 37 °C for 24 h. Cells were incubated with PEG-PPS@DOX NP, PEG-PPS-GSNO NP and PEG-PPS-GSNO@DOX NP (10 μg/mL equivalent DOX concentration) for 8 h. Then, the cells were then washed twice with PBS, and replaced by fresh DMEM medium containing 5 μM DAF-FM DA and further incubated at 37 °C for 30 min. The cells were then washed and harvested with trypsine and fixed with 4% (m/v) paraformaldehyde solution. The cells were collected by centrifuging (120g, 5 min) and analyzed using flow cytometry (FCM, DAPI, 405 nm; NO, 515 nm; DOX, 575 nm).

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2.10 Flow cytometry analysis of cells apoptosis induced by PEG-PPS-GSNO@DOX nanoparticles Typically, HepG2/ADR cells were seeded in 12-well plate at a density of 5×104 cells per well. Then the cells were treated with PEG-PPS@DOX NP, PEG-PPS-GSNO NP and PEG-PPS-GSNO@DOX NP (10 μg/mL equivalent DOX concentration). After incubation for 8 h, HepG2/ADR cells were harvested by centrifugation (200 g×10 min, 4°C). The collected cells were then suspended into cell binding solution (Annexin V-7AAD kit) added with Annexin V (5 μL) and 7-AAD (10 μL) in the dark for 10 min. Finally, the samples were analyzed by FCM.

2.11 Statistical analysis

Statistical analysis was performed using GraphPad Prism (version 6.01) software through Student's t-test and one-way analysis of variance (ANOVA). All data were expressed as mean ± standard deviation. The confidence levels of 95% and 99% were regarded as the threshold value for the determination of significant difference.

3. Results and discussion

3.1 Polymer synthesis and preparation of nanoparticles

Propylene sulfide monomer was used for polymerization to yield hydrophobic PPS agent, which could transform from the native hydrophobic phase to the hydrophilic phase in the presence of ROS. The resulting amphiphilic polymer based on PEG-PPS could disassemble easily as previously described. The synthesis of amphiphilic 11

copolymer PEG-PPS-GSNO was described in details in Materials and methods section and supporting information. The successful synthesis of PEG-PPS-GSNO copolymer was confirmed by nuclear magnetic resonance spectroscopy (NMR, Fig. 2A & Fig. S9), Fourier transforms infrared spectroscopy (FTIR, Fig. 2B), gel permeation chromatography (GPC, Fig. 2C) respectively. The loading capacity of NO was further tested by Griess-Saville assay. As depicted in Fig. 2A, the typical resonances at 1.3-1.33, 2.52-2.58, and 2.59-3.10 ppm belong to the protons in propylene sulfide monomer of PPS. The characteristic peaks at 3.3, 3.5-3.58 ppm and 1.99 ppm were respective assigned to protons of PEG and GSNO. It clearly suggests the conjugation of PPS and GSNO into PEG. In addition, 1HNMR, 13CNMR and FTIR spectra further demonstrated the successful stepwise conjugation of copolymer (Figs. S1-S8). Moreover, we also found that the number-molecular weight (Mn) of PPS and PEG-PPS-GSNO calculated from 1HNMR and GPC are consistent with the theoretical values (Table S1). The loading capacity of NO was 24 ± 8% (mol/mol). In brief, all results suggested that amphiphilic PEG-PPS-GSNO copolymer was successfully synthesized.

The nanoparticles were formulated via emulsion- solvent evaporation method. The TEM images showed that these nanoparticles displayed uniform spherical morphology with good monodispersity (Fig. 2B). The size of GSNO functionalized nanoparticles were approximately 41 ± 2 nm, which was adapted to drug delivery for cancer treatment. The size of PEG-PPS nanoparticles was larger than that of 12

PEG-PPS-GSNO nanoparticles, probably because PEG-PPS nanoparticles were constructed as polymersomes, whereas the GSNO functionalized nanoparticles were as micelles (scheme 2). Due to the different assembly forms, the PEG-PPS-GSNO nanoparticles presented lower DOX loading capacity (20% vs 28%) than the PEG-PPS nanoparticles. Nanoparticles were slightly negatively charged with a zeta potential of -0.23 ± 0.79 mV, which is favorable for drug delivery in vivo.

3.2 ROS-responsive size and morphology modification

The PEG-PPS-GSNO nanoparticles showed a sensitivity to oxidative stress as their sizes increased significantly in the presence of H2O2. Higher concentration of H2O2 contributed to a quicker enlargement of NP size. Indeed, it tooks 2 h to reach the maximum size at 5 M H2O2, while 24 h were needed at 1 M H2O2 (Fig. 2A). Those modifications were further confirmed by TEM with nanoparticles presented spherical shapes and intact morphology without H2O2. However those nanoparticles were swelling and then displayed hollow sphere when exposed to H2O2 (Fig. 2B,C). It was attributed to the fact that sulfide moieties of hydrophobic PPS inside the nanoparticles were oxidized to hydrophilic poly-sulfoxides/sulfones in the presence of ROS [34,38,39], leading to the swelling and disassembly of the nanoparticles.

3.3 GSH-responsive NO release and ROS-responsive DOX release in vitro

As presented in Fig. 3A, PEG-PPS-GSNO as a novel NO donor, sustained the release of NO till 8 h. Moreover, this high molecular weight NO donor released NO in a GSH 13

responsive way (Fig. 3B). The maximum of NO release was attempted after 4 h of incubation with GSH. GSH probably displace NO from PEG-PPS-GSNO to form GSNO following a transnitrosation process [35~37].

In accordance with the size modification, the release of DOX was responsive to ROS as well. As shown in Fig. 3C and 3D, the release of DOX increased immediately after the addition of H2O2. The responsive rate increased regards to the concentration of H2O2. 3.4 Cytotoxicity of PEG-PPS-GSNO nanoparticles and the enhancement of chemosensitivity of DOX

To evaluate the biocompatibility of the nanoparticles, cell proliferation assay was performed with normal cells (293T cells). As shown in Fig. S10, the viability of 293T cells treated with PEG-PPS NP (37~925 μg/mL) displayed no abnormality, suggesting a good biocompatibility of the nanomaterial. On the contrary, the cells treated with PEG-PPS-GSNO NP only displayed slight toxicity at high concentration (925 μg/mL), probably resulted from the high amount of conjugated NO (185 μg/mL).

To investigate the potential of PEG-PPS-GSNO in overcoming multidrug resistance of cancer, DOX was encapsulated into PEG-PPS-GSNO nanoparticles. The toxicity of free DOX and PEG-PPS-GSNO@DOX toward HepG2/ADR cells were tested and compared. As presented in Fig. 4, free DOX decreased less the cell viability than 14

PEG-PPS-GSNO@DOX at the same concentration. At 1 μg/mL, 20% of cells were dead in free DOX group and 36% of cells were dead in PEG-PPS-GSNO@DOX group. The half maximal inhibitory concentrations (IC50), in terms of DOX concentration, were calculated. From these plots the IC50 values of free DOX toward HepG2/ADR cells was 13 ± 1 μg/mL, and that of PEG-PPS-GSNO@DOX was 4 ± 1 μg/mL (table 2). These results indicated that the application of GSNO functionalized nanoparticles increased the sensitivity of HepG2/ADR cells to DOX by 3 times. The enhanced cytotoxicity of DOX was due to the additive anticancer effect of GSNO. Indeed, NO derived from supplied GSNO functionalized nanoparticles, may react with innate ROS and/or produced by DOX metabolism to form reactive nitrogen species like ONOO-[39], causing damage in mitochondria membranes and DNA in the nuclei. However, the IC50 of PEG-PPS-GSNO@DOX nanoparticles toward 293T cells was 162 ± 3 μg/mL (table 2), which was 37-fold higher than that in HepG2/ADR cells, due to the lack of elevated intracellular ROS (figure S12). The developed PEG-PPS-GSNO@DOX presented biocompatibility to normal cells and selective toxicity against HepG2/ADR cells, thus they will hopefully decrease the unwanted side effects for in vivo applications.

3.5 Intracellular release of NO and cellular uptake of PEG-PPS-GSNO@DOX

To investigate the endocytosis and intracellular distribution of NO and DOX released from nanoparticles in HepG2/ADR cells, we employed confocal laser scanning microscopy (CLSM) . After treatments with PEG-PPS-GSNO@DOX nanoparticles 15

for 8 h, both strong intracellular green fluorescence signals corresponding to NO and red signals assigned to DOX were observed in HepG2/ADR cells. However only red fluorescence signal was observed in cells treated with PEG-PPS@DOX nanoparticles, and only green fluorescence signal was found in cells treated with PEG-PPS-GSNO nanoparticles (Fig. 5). Notably, DOX signals were much stronger in cells treated with PEG-PPS-GSNO@DOX nanoparticles compared to that with PEG-PPS@DOX nanoparticles. Probably NO released from PEG-PPS-GSNO@DOX nanoparticles reduced the expression of active P-gp [41,42], thus more DOX remained in the cytoplasm. There was not significant difference of NO signal in the cells treated with PEG-PPS-GSNO nanoparticles and PEG-PPS-GSNO@DOX nanoparticles. NO signals were localized in the cytoplasm and nucleus, while DOX signals were mainly localized in the cytoplasm. Probably NO has with smaller molecular weight than DOX enters easier and faster in the nucleus. Flow cytometer was used to further quantify the cellular uptake of DOX and/or NO. The mean fluorescence intensities of DOX exposed to PEG-PPS@DOX nanoparticles or PEG-PPS-GSNO@DOX nanoparticles were 1.46×105 and 2.02×106, showing 14-fold increase in the uptake of DOX (Fig. 6). In the meantime the mean fluorescence intensities of NO exposed to PEG-PPS-GSNO nanoparticles and PEG-PPS-GSNO@DOX nanoparticles were 5.61×105 and 1.66×106, showing 3-fold increase in the uptake of NO. These results indicate that GSNO functionalized nanoparticles efficiently enhance the cell internalization of NO and DOX. In accordance with Zhang et al, NO

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prodrug-conjugated nanoparticles could promote the intracellular co-delivery of NO and DOX for cancer therapy [43]. In the present work, the locoregional and sustained NO promoted the cellular accumulation of DOX, that is adapted to reverse the drug resistance in anticancer treatments.

To test the apoptotic potential of GSNO functionalized nanoparticles, annexin V-7AAD and PI (an indicator for early and late stage cell apoptosis, respectively) co-staining was performed and assessed by flow cytometry. The percentage of total apoptotic rate was respectively 10% and 34% when HepG2/ADR cells were treated with PEG-PPS-GSNO nanoparticles or PEG-PPS@DOX nanoparticles after 8 h, which was further increased to 100% by the co-delivery of NO and DOX with PEG-PPS-GSNO@DOX nanoparticles (Fig. 7). The combination strategy resulted in a notably higher cell apoptosis rate in contrast to NO or DOX treatment alone. We also found that cell apoptosis induced by PEG-PPS-GSNO@DOX nanoparticles was higher than the sum of NO and DOX treatment alone, which indicated NO might present indirect “cancer killer” by chemo-sensitization of DOX. These results were in accordance with the CLSM and FCM where the fluorescence signals of DOX were always much stronger in cells exposed to PEG-PPS-GSNO@DOX nanoparticles than in cells exposed to PEG-PPS@DOX nanoparticles.

4. Conclusion

In the present work, we have successfully conjugated GSNO into PEG-PPS backbone for the first time. The resultant GSNO functionalized nanoparticles are of elevated 17

NO capacity and stability in normal media including PBS and DMEM. These nanoparticles present GSH-responsive NO release properties. Besides, PEG-PPS-GSNO nanoparticles show biocompatibility to normal cells, which are adapted to anticancer application. These GSNO functionalized nanoparticles can efficiently co-deliver DOX with ROS triggered release behavior. PEG-PPS-GSNO@DOX nanoparticles were able to significantly inhibit the growth of HepG2/ADR cells through increasing the accumulation of intracellular DOX and inducing early and late stage cell apoptosis. Our findings provide a new strategy to construct GSNO functionalized nanomaterials and also new type of stimuli-sensitive NO delivery platform, which could be utilized to reverse chemo-resistance of hepatocellular carcinoma and selectively kill cancer cells. In the future, work including how PEG-PPS-GSNO@DOX nanoparticles deliver NO and DOX in vivo and overcome chemo-resistance would be performed.

Declaration of Competing Interest The authors have no conflicts of interest to declare.

Acknowledgements We want to acknowledge the financial support by the National Natural Science Fundation of China (No. 21602024) and the Fundamental Research Funds for the 18

Central Universities (No. 0903005203498, 2018CDYXYX0027 and 0247001104416). The authors are grateful to Mr. Jiangbei Yuan and Jiale Zhang (Chong Qing University) for their technical help and discussion.

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Fig.1. Characterization of PEG-PPS-GSNO. (A) 1HNMR spectra (400 MHz, 298 K) of PEG-PPS-GSNO (the arrows indicates the chemical shift of corresponding H); (B) ATR-FTIR spectra of mPEG2000 (green), GSNO (purple), PEG-PPS (brown) and PEG-PPS-GSNO (red) (the arrows indicates the infrared characteristic absorption peak position of corresponding chemical structure); (C) gel permeation chromatography curves of PEG-PPS and PEG-PPS-GSNO copolymer.

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Fig.2. ROS-responsive size changements (A) the average hydrodynamic diameters, measured by dynamic light scattering, of PEG-PPS-GSNO nanoparticles incubation with 1 M, 2.5 M or 5 M H2O2; (B) TEM images of PEG-PPS-GSNO nanoparticles, (C) after treatment with 2.5 M H2O2 for 4 h, scale bar: 100 nm. Data shown as mean ± sd, n=3.

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Fig.3. In vitro release profiles: (A) Release of NO from PEG-PPS-GSNO nanoparticles; (B) GSH-responsive release of NO from PEG-PPS-GSNO nanoparticles; (C) ROS-responsive release of DOX from PEG-PPS-GSNO@DOX nanoparticles incubed with 5 mM or 50 mM H2O2 within 12 h and (D) presented the enlargement of release profile in first 3 h. *p<0.05, ** p<0.001. Data shown as mean ± sd, n=3.

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Fig.4. (A) Cytotoxicity assay of HepG2/ADR cells incubated with PEG-PPS-GSNO@DOX nanoparticles (equivalent DOX concentration of 1-30 μg/mL) for 24 h; (B) dose-responsive curves of free DOX and PEG-PPS-GSNO@DOX nanoparticles to HepG2/ADR cells; (C) Cytotoxicity assay of 293T cells incubated with PEG-PPS-GSNO@DOX nanoparticles (equivalent DOX concentration of 1-500 μg/mL) for 24 h; (D) dose-responsive curves of free DOX and PEG-PPS-GSNO@DOX nanoparticles to 293T cells. presented as mean ± sem, n=3.

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*p<0.05, Results are

Fig.5. Cell uptake and intracellular DOX and/or NO release from nanoparticles. HepG2/ADR cells were incubated with PEG-PPS@DOX nanoparticles, PEG-PPS-GSNO nanoparticles and PEG-PPS-GSNO@DOX nanoparticles for 8 h. Cell nuclei were labeled with DAPI (blue), NO was stained with DAF-FM (green) and DOX exhibited red fluorescence, the scale length of each image is 25 μm.

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Fig. 6. Flow cytometer quantification of the intracellular fluorescence intensity of NO and/or DOX in HepG2/ADR cells incubated with PEG-PPS@DOX nanoparticles, PEG-PPS-GSNO nanoparticles or PEG-PPS-GSNO@DOX nanoparticles for 8 h. A presented DOX signal of HepG2/ADR cells treated with PEG-PPS@DOX nanoparticles; B presented DOX signal of HepG2/ADR cells treated with PEG-PPS-GSNO@DOX nanoparticles; C presented NO signal of HepG2/ADR cells treated with PEG-PPS-GSNO nanoparticles; D presented NO signal of HepG2/ADR cells treated with PEG-PPS-GSNO@DOX nanoparticles; E presented mean fluorescence intensity of intracellular NO and/or DOX. Data are expressed as mean± sd (n=3) *p<0.001.

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Fig.7. Annexin V-7AAD/PI staining for apoptosis in HepG2/ADR cells treated with PEG-PPS@DOX nanoparticles, PEG-PPS-GSNO nanoparticles and PEG-PPS-GSNO@DOX nanoparticles (25 μM NO, 18 μM DOX) for 8 h was assessed by flow cytometry analysis. The percentages of early or late apoptosis are presented in the bottom right and top right quadrants, respectively. Columns represent the average proportions of apoptotic cells. *p < 0.05, ** p < 0.001, n = 3.

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Scheme 1 Synthesis of GSNO functionalized polymeric nanoparticles for NO delivery by thiol-ene reaction (A). The obtained copolymers can self-assemble into nanoparticles in aqueous solutions and doxorubicin could be encapsulated into the nanoparticles by a co-assembly process (B). When incubated with chemo-resistant HepG2/ADR cells, PEG-PPS-GSNO@DOX nanoparticles were internalized. Elevated intracellular ROS triggered fast release of DOX and disassembly of nanoparticles, and then NO was released through transnitrosation by intracellular GSH. However, in the healthy cells, showing physiological concentrations of ROS, the PEG-PPS-GSNO@DOX nanoparticles could not be disassembled. Thus these nanoaprticles were transported to extracellular space through exocytosis (C).

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Scheme 2 (A) presented PEG-PPS nanoparticles constructed as bilayer polymersomes; (B) presented PEG-PPS-GSNO nanoparticles constructed as micelles.

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Table 1. Particle size, polydispersity index (PDI), surface charge (zeta potential), encapsulation efficiency (EE) and drug loading (DL) of nanoparticles. Data are shown as mean ± sd, n = 5. *p<0.001 vs PEG-PPS NP; # p<0.001 vs PEG-PPS@DOX NP.

nanoparticles

size(nm)

PDI

Zeta potential (mV)

EE (mg/mg)(%)

DL (mg/mg)(%)

PEG-PPS-GSNO NP

25.7 ± 0.7*

0.22 ± 0.01

-3.8 ± 0.7

-

-

40.7 ± 2.2#

0.27 ± 0.05

-0.2 ± 0.8

20.40 ± 0.03

1.61 ± 0.02

PEG-PPS NP

54.4 ± 0.4

0.23 ± 0.03

-12.3 ± 0.4

-

-

PEG-PPS@DOX NP

69.1 ± 0.5

0.26 ± 0.02

-2.7 ± 0.8

28.46 ± 0.03

2.44 ± 0.04

PEG-PPSGSNO@DOX NP

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Table 2. Mean half maximal inhibitory concentrations of HepG2/ADR cells and 293T cells treated with free DOX or PEG-PPS-GSNO@DOX nanoparticles for 24 h. Data are expressed as mean ± sd, n = 3. *p<0.001 vs free DOX; # p<0.001 vs 293T cells.

IC50 (μg/mL) HepG2/ADR cells

293T cells

Free DOX

13±1*#

0.8±0.1

PEG-PPS-GSNO@DOX NP

4±1#

162±3*

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Graphical abstract

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