pH-sensitive pluronic micelles combined with oxidative stress amplification for enhancing multidrug resistance breast cancer therapy

Journal of Colloid and Interface Science 565 (2020) 254–269

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pH-sensitive pluronic micelles combined with oxidative stress amplification for enhancing multidrug resistance breast cancer therapy Xu Cheng 1, Xiaoli Zeng 1, Yan Zheng, Qin Fang, Xin Wang, Jun Wang, Rupei Tang ⇑ Engineering Research Center for Biomedical Materials, Anhui Key Laboratory of Modern Biomanufacturing, School of Life Sciences, Anhui University, 111 Jiulong Road, Hefei, Anhui Province 230601, PR China

g r a p h i c a l a b s t r a c t

a r t i c l e

i n f o

Article history: Received 30 September 2019 Revised 11 January 2020 Accepted 11 January 2020 Available online 14 January 2020 Keywords: Multidrug resistance Pluronic Oxidative therapy Ortho ester Drug delivery

⇑ Corresponding author. 1

E-mail address: [email protected] (R. Tang). Authors contributed equally to this work.

https://doi.org/10.1016/j.jcis.2020.01.029 0021-9797/Ó 2020 Elsevier Inc. All rights reserved.

a b s t r a c t Multidrug resistance (MDR) is one of the major obstacles to clinical cancer chemotherapy. Herein, we designed new pH-sensitive pluronic micelles with the synergistic effects of oxidative therapy and MDR reversal. Pluronic (P123) was modified with a-tocopheryl succinate (a-TOS) via an acid-labile ortho ester (OE) linkage to give a pH-sensitive copolymer (POT). Self-assembled POT micelles exhibited desirable size (~80 nm), excellent anti-dilution ability, high drug loading (~85%), acid-triggered degradation and drug release behaviours. In vitro cell experiments verified that POT micelles could significantly reverse MDR through suppressing the function of drug effluxs mediated by P123 and induce more reactive oxygen species (ROS) generation mediated by a-TOS, resulting in enhanced cytotoxicity and apoptosis in MDR cells. In vivo studies further revealed that DOX-loaded POT micelles (POT-DOX) possessed the highest drug accumulation (3.03% ID/g at 24 h) and the strongest tumour growth inhibition (TGI 83.48%). Pathological analysis also indicated that POT-DOX could induce more apoptosis or necrosis at the site of tumour without distinct damage to normal tissues. Overall, these smart POT micelles have great potential as promising nano-carriers for MDR reversal and cancer treatment. Ó 2020 Elsevier Inc. All rights reserved.

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1. Introduction Nowadays, chemotherapy remains the primary option for clinical cancer treatment. However, efficient treatment is often hindered with the occurrence of multidrug resistance (MDR) during chemotherapy [1–3]. The activated MDR greatly reduce the accumulation of drugs in cancer cells by the superfamily of adenosine-triphosphate (ATP) binding cassette proteins such as P-glycoprotein (P-gp) and MDR-associated proteins (MRPs), eventually leading to a failed chemotherapy [4,5]. In order to reverse MDR, considerable efforts have been made to develop small molecular MDR inhibitors that either evade efflux or inhibit the function of efflux transporters [6–8]. However, little success has been achieved in clinical trials, which is mainly due to the low efficacy and nonspecific side effects [9–11]. Therefore, it is extremely urgent to develop an efficient and safe strategy for overcoming tumour MDR. Nano-drug delivery systems (NDDS) including polymeric nanoparticles, liposomes, nanogels and micelles have emerged as innovative and promising strategies for cancer therapeutics [12,13]. In particular, pluronic copolymers-based NDDS have received widespread attention because of their excellent antiMDR properties. As amphiphilic block copolymers, pluronics can self-assemble into nano-sized micelles to improve drug solubility and stability, prolong circulation time in vivo, and control drug release behaviour in cells or at the site of a tumour [14,15]. More importantly, it has been reported that pluronic copolymers are capable of reversing tumour MDR via multiple pathways, including inhibiting P-gp mediated efflux, blocking glutathione (GSH)/ glutathione-S transferase (GST) detoxification system, increasing intracellular ROS levels, and destroying anti/pro-apoptotic balance in MDR cells [16–19]. Furthermore, low cytotoxicity and immunogenicity make pluronics suitable for local or systemic administration, for example, a mixed micelle (SP1049C) consisting of pluronic L61 and F127 has been used in clinical trials [20,21]. Therefore, pluronic copolymers have been considered a very promising material in the design of intelligent NDDS for anticancer drugs delivery. However, several technical challenges remain to be solved. The low micellization and solubilization ability to hydrophobic drugs of pluronic copolymers make it necessary to use a high dose of carrier materials and may cause toxicity [22]. The poor dilution stability in bloodstream of traditional pluronic micelles may cause premature and nonspecific drug release in vivo due to their relatively high CMC (critical micelle concentration) values [23,24]. Therefore, a simple and effective modification is worth studying to improve pluronic copolymer-based NDDS. pH-stimulus-responsive strategy has been considered an effective method for reconstruction of the polymer structure and function, which can improve targeted drug delivery and control drug release at the tumour site [25,26]. As is well known, there is a distinct pH gradient from blood vessels (~7.4) to most solid tumour micro-environments (6.5–7.2 in tumour matrix, 5.5–6.0 in endosomes and 4.0–5.0 in lysosomes) [27]. In this work, pluronic polymer (P123) was chosen as the initial material and modified by ortho ester end-capping (P123-OE), and then grafted with atocopheryl succinate (a-TOS), as shown in Scheme 1. The obtained copolymer (POT) could self-assemble into pH-sensitive pluronic micelles (POT-M, Fig. S5). Compared to other acid-labile linkages such as ketal and acetal, ortho ester bond is more sensitive in response to mildly acid conditions, which may facilitate rapid drug release in tumour cells [28,29]. Besides, ortho ester also has good biocompatibility and biostability in normal tissue, which has been widely used in various nano-carriers, such as nanospheres, micelles and nanogels [30–32]. a-TOS as a class of vitamin E derivative was imported to increase drug solubilization and encapsulation ability due to its relatively bulky lipophilic portion [33].

Scheme 1. The synthetic route of functionalized pluronic copolymer (POT); Reaction conditions: (I) Py-PTSA, 130 °C; (II) 50 mL tetrahydrofuran containing 1 M sodium hydroxide; (III) EDC/NHS.

Furthermore, a-TOS could increase intracellular ROS levels and induce oxidative damage through disrupting mitochondrial function and in turn triggering apoptosis in tumour cells, but displayed less toxic or non-toxic effects in normal cells [34–36]. Hence, these self-assembled POT micelles were expected to realize MDR reversal and have desirable drug delivery process: (i) remaining stable and long-circulating in blood vessels; (ii) triggering drug release in tumour acidic organelles; (iii) increasing intracellular drug accumulation by reversing tumour MDR; (iv) amplifying intracellular ROS levels which enhanced tumour-killing; (v) breaking the balance of anti/pro-apoptotic mechanisms which induced more apoptosis. In order to verify the potential effects above, POT micelles and single P123 micelles (P123-M, as the control) were prepared by the thin-film dispersion method [37]. The stability and pHresponsive characteristics (particle size, morphology, zeta potential, transition of micellization/demicellization and drug release profile, etc.) of these pluronic-based micelles were assessed under different pH conditions in vitro. The synergistic effect of ROS and reverse mechanisms of MDR such as cellular uptake, cytotoxicity, mitochondria damage and apoptosis proteins expression were carefully studied in vitro by using MCF-7 and MCF-7/ ADR cells. Finally, in vivo drug metabolism, antitumor efficiency, and histopathology evaluation were performed using mice with MCF7/ADR xenograft tumours. As expected, these pH-sensitive POT micelles would achieve more effective tumour growth inhibition by reversing MDR and amplifying ROS-mediated cell-killing ability. 2. Materials and methods 2.1. Materials Pluronic P123 (98%) was purchased from Energy Chemical Co., Ltd. (Shanghai, China). a-Tocopheryl acid succinate (a-TOS, 98%) was obatined from Mackin Biochemical Co., Ltd. Ortho ester monomer (OE) was synthesized based on previous works [38]. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

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(EDC
HCl, 98.5%), N-hydroxysuccinimide (NHS, 98%), 1-(4,5-dime thylthiazol-2-yl)-3,5-diphenyl-formazan (MTT, 98%) was obtained from Mackin Biochemical Co., Ltd. Doxorubicin hydrochloride (DOX
HCl, 98%) was purchased from TCI Development Co.,Ltd. (Shanghai, China). Micro BCA (bicinchoninic acid) protein assay kit, Annexin V-FITC apoptosis detection kit, ATP assay kit, GSH assay kit and ROS assay kit were purchased from Beyotime Biotechnology Co.,Ltd. (Nanjing, China). Primary antibodies for Bcell lymphoma-2 (Bcl-2), Bcl-2-associated X (Bax), caspase-3, survivin and streptavidin-perosidase (SP) immunohistochemistry Kit were obtained from Solarbio Science & Technology Co.,Ltd. (Beijing, China). Human breast cancer cells (MCF-7) and drug-resistant human breast cancer cells (MCF-7/ADR) were obtained from KeyGen Biotech (Nanjing, China). Female BALB/c nude mice at the age of 5–6 weeks were purchased from the Cavens Laboratory Animal Limited Company (Changzhou, China). All other chemicals were analytical reagent and used without further purification. 2.2. Characterization 1

H NMR spectra were recorded by a Bruker Aavance 400 NMR spectrometer at 400 MHz using deuterated chloroform (CDCl3) or hexadeuterodimethyl sulfoxide (DMSO) as the solvent. Fourier transform infrared (FT-IR, NEXUS-870, Nicolet, USA) was further used for detecting the chemical structure of products. Samples were mixed with potassium bromide (KBr) powder at weight ratio of 1/100, and scanned from 4000 to 500 cm1 for 32 times with 2 cm1 resolution to average signal at 25 °C. The size and zeta potential of micelles were determined using a Zetasizer dynamic light scattering detector (DLS, Malvern Zetasizer Nano ZS, United Kingdom). The measurements were carried out at 25 °C under 633 nm incident beam and 173° scattering angle. Transmission electron microscopy (TEM, JEM-2100, Japan) was performed at an accelerating voltage of 80 kV. Briefly, 10 lL micelles solution (0.1 mg/mL) were dropped onto a copper grid, then allowed to air-dry at room temperature and observed by TEM. The amount of DOX was measured by a microplate reader (Molecule Devices, USA) at an excitation wavelength of 480 nm and an emission wavelength of 590 nm. All measurements were performed three times.

room temperature for 30 min to give activated a-TOS. After that, 2.92 g P123-OE (0.49 mmol) was added into the above solution, and slowly stirred for 48 h. Finally, the mixture solution was dialyzed (MWCO 3500 Da) against 80% ethanol solution to remove any small molecular residues. The product was collected by vacuum distillation and freeze-drying, and named as POT. The chemical structure of POT was determined by 1H NMR and FT-IR. 2.5. Preparation and characterization of micelles P123 and POT micelles were prepared by thin-film hydration method [37]. In brief, 100 mg P123 or POT was dissolved in 5 mL acetonitrile in a round-bottom flask, and then the solvent was evaporated by rotary evaporation at 50 °C for 1 h to obtain a thin copolymer film. Residual acetonitrile remaining in the film was removed under vacuum overnight at room temperature. The resultant thin film was hydrated with 10 mL pH 7.4 phosphate (PB) buffer (dropwise) at 60 °C for 30 min and sonicated for 5 min to obtain the micelle solution. These micelles solution were filtered through 0.22 lm filters to remove possible large aggregates formed by pluronic polymer. The obtained micelles were named P123-M and POT-M, respectively. The zeta potentials, hydrodynamic diameter and polydispersity index of micelles were measured by a DLS detector. The morphology of micelles was observed by TEM. The critical micelle concentration (CMC) of pluronic copolymer was determined by fluorescence measurements using nile red as a fluorescence probe [40]. Briefly, 20 lL nile red solution in acetone (1.0  104 mol/L) was added into brown glass vials, then acetone was evaporated at room temperature. After that, 2 mL micelles solution (concentration ranging from 1.0  108 to 1 mg/L) was added into each brown glass vials. These samples were incubated overnight in the dark, and the fluorescence was measured by a spectrofluorometer (Japan, Shimadzu RF-5301PC) at an excitation wavelength of 553 nm. The emission spectra were recorded from 590 to 720 nm. The CMC was determined as the inflection point on the plots representing the maximum emission wavelength as a function of P123 or POT concentration. 2.6. Stability and pH-responsive behavior analysis

2.3. Synthesis of ortho ester terminated pluronic (P123-OE) Ortho ester-terminated pluronic (P123-OE) was synthesized by an ester exchange reaction as previous reports [39]. Briefly, 10 g pluronic (P123, 1.72 mmol) was added into 100 mL glass bottle, then OE monomer and pyridinium-p-TSA (Py-PTSA) were added at a molar ratio of P123:OE:Py-PTSA = 1:6:0.02. The above system was heated at 120 °C for 8 h. After cooling to room temperature, the residue was dissolved in dichloromethane (200 mL), washed with 0.5% (w/v) sodium bicarbonate (NaHCO3) and saturated sodium chloride (NaCl), then evaporated to obtain crude product. The crude product was dissolved in tetrahydrofuran (50 mL) and sodium hydroxide (1 M, 50 mL) was added. The mixture was vigorously stirred overnight at room temperature. After that, the solvent was removed, then the collected product was dialyzed by a dialysis bag (molecular weight cut off (MWCO) 3500 Da) against 80% ethanol solution for 48 h, and finally dried by lyophilization to give P123-OE. The chemical structures of P123 and P123-OE were analyzed by 1H NMR using CDCl3 as solvent.

To assess anti-dilution stability of micelles, P123 and POT micelles were diluted 1, 10, 20, 40, 100 fold with pH 7.4 PB buffer, then the change of size and count rate were measured by DLS at 25 °C. Meanwhile, the storage and physiological environments stability (including saline, PBS (phosphate buffered saline), FBS (fatal bovine serun), and cell culture medium (RPMI 1640)) of both micelles at room temperature were monitored by DLS. Besides, the anti-dilution ability of micelles were also tested in culture medium containing 10% FBS by DLS and scanning electron microscopy (SEM, HITACHI S-4800, Japan). All data were obtained by three testing. Besides, the pH-responsive behavior of POT micelles was assessed. Briefly, 2 mL POT-M solution (5 mg/mL) was dispersed in 0.01 M PB buffer with pH 5.5 and incubated at 37 °C with a shaken speed of 100 rpm. The diameter and light intensities were monitored at predetermined time by DLS. Meanwhile, the morphology of acid-treated POT-M was observed by TEM. In addition, the degradation product was further detected by 1H NMR analysis using DMSO d6 as solvent.

2.4. Synthesis of tocopheryl succinate conjugated pluronic (POT) 0.67 g a-tocopheryl acid succinate (a-TOS, 1.25 mmol) was dissolved in anhydrous dimethyl sulfoxide (DMSO), then EDC, NHS and (triethylamine) TEA were added at a molar ratio of a-TOS:ED C:NHS:TEA = 1:1.5:1.5:0.05. The mixture solution was stirred at

2.7. DOX loading and release DOX-loaded micelles were prepared using the similar above method. First, DOX
HCl (5 mg) was stirred overnight with TEA (1:3) in acetonitrile (2 mL) to obtain DOX base. Then, the DOX base

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and 100 mg P123 or POT copolymer were co-dissolved in 5 mL acetonitrile and sonicated for 30 min. The next operations were the same as blank micelles. The obtained DOX-loaded micelles were named as P123-DOX and POT-DOX, respectively. In order to determine drug loading content (DLC) and drug loading efficiency (DLE), DOX-loaded micelles solution was freeze-dried to measure total weight of DOX-incorporated micelles. These dried samples were dissolved in DMSO and total weight of DOX in micelles was measured thrice by a microplate reader. Finally, DLC and DLE were calculated as formula (1) and (2) [41]:

Drug loading content ð%Þ ¼

Weight of DOX in micelles Weight of DOX - loaded micelles  100% ð1Þ

Drug loading efficiency ð%Þ ¼

Weight of DOX in micelles Weight of the feeding DOX  100%

ð2Þ

In vitro DOX release was performed by the dialysis method. Briefly, 1.0 mL P123-DOX or POT-DOX (DOX concentration: 450 lg/mL) was placed into a dialysis bag (MWCO 1.4 KDa), immersed in 10 mL PB solution with pH 5.5 or 7.4, and then shaken (100 rpm) at 37 °C. At predetermined time intervals, 10 mL release medium was withdrawn and 10 mL fresh PB solution was added. Finally, the released DOX was measured by a microplate system at an excitation wavelength of 480 nm and an emission wavelength of 590 nm. All in vitro drug release experiments were conducted in triplicate, and the release profiles were plotted with cumulative drug release as a function of time.

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2.10. Flow cytometry In order to further distinguish the differences of intracellular DOX level in both cell lines, flow cytometry (FCM, Becton Dickinson, USA) was performed. Briefly, MCF-7 or MCF-7/ADR cells were seeded at a density of 1  105 cells/well in 6-well plates and cultured for 24 h. Then, these cells were incubated with tested formulations (free DOX, P123-DOX and POT-DOX). After incubation 4 h, each cell was washed with PBS twice, digested with 1 mL trypsin and collected by centrifugation (1000 rpm). Finally, intracellular fluorescence intensity of DOX was measured thrice by FCM.

2.11. MTT assays In vitro cytotoxicities of DOX-loaded micelles were evaluated via MTT assay [43]. Briefly, MCF-7 or MCF-7/ADR cells (5  103 per well) were seeded on 96-well plates and cultured for 24 h. Then, the growth medium was replaced with fresh medium containing a series of gradient concentration samples (free DOX, P123-DOX and POT-DOX), and incubated for another 24 h. After that, the old culture medium was removed and 200 lL fresh medium containing 0.5 mg/mL MTT were added into each well. After incubation for 4 h, the medium was completely removed and 150 lL DMSO was added to dissolve the purple formazan crystals. Finally, the absorbance of each well was measured using SpectraMax M2e Molecular devices at 570 nm. The half maximal inhibitory concentration (IC50) values were calculated using nonlinear regression analysis, and the MDR reversal effect was assessed according to previous literature [44]. Besides, the cytotoxicity of free a-TOS, blank P123-M and POT-M were also evaluated using the same method at concentration ranging from 3.125 to 100 lg/ ml. All tests were performed for six times.

2.12. Intracellular ROS production 2.8. In vitro cellular uptake MCF-7 and MCF-7/ADR cells lines were cultured as previous work [42]. Then, in vitro cellular uptake experiment was carried out. Briefly, MCF-7 or MCF-7/ADR cells were seeded into 6-well plate containing a cover glass and cultured for 24 h. After that, free DOX, P123-DOX and POT-DOX were added and incubated for 4 h, final DOX concentration was 4 lg/mL. Then, cells were washed twice with cold PBS, fixed with 4% paraformaldehyde, and stained with Hoechst 33,258 to visualize the nuclei. Finally, the fluorescence images were collected and processed by the confocal laser scanning microscope (CLSM, FluoView TM FV1000, Olympus). Besides, MCF-7/ADR cells were also incubated with free rhodamine 123 (R-123), P123-M + R-123, POT-M + R-123 for 2 h, and then the old medium was replaced with fresh medium without any agents. Cells were cultured for another 0, 4 and 8 h, then intracellular R123 accumulation was also evaluated by CLSM.

2.9. Subcellular co-localization MCF-7 or MCF-7/ADR cells were seeded into a 6-well plate containing a cover glass and cultured for 24 h. Then cells were incubated with 100 nM Lyso-Tracker Green for 30 min at 37 °C. Next, cells were incubated with 0.2 mL of P123-DOX (4 lg/mL) and POT-DOX (4 lg/mL) for 2 h, 4 h and 8 h. After that, cells were washed with ice PBS three times, and then fixed with 4% paraformaldehyde and stained with Hoechst 33,258. Finally, cells were observed by CLSM at an excitation wavelength of 504 nm for lysosome and 594 nm for DOX.

Next, intracellular ROS generation was measured according to the manufacturer’s instruction (ROS assay kit, Beyotime Biotechnology, Shanghai). Briefly, cells were seeded in a 6-well plate containing the cover glass and cultured for 24 h. Then, cells were treated with free a-TOS, P123 -M and POT-M (sample concentration: 100 mg/mL) for 2 h. After that, cells were washed with PBS three times and incubated with 2,7-Dichlorodi-hydrofluorescein diacetate (DCFH-DA) in the dark for 20 min. Cells were washed with PBS again and fixed with 4% paraformaldehyde for 5 min. Finally, the fluorescence signal was observed and collected by a fluorescence inverted microscope (Leica, DMIL) with the excitation (488 nm) and emission (500–550 nm) wavelength, and the fluorescence intensity was also measured by Image J. Z-stack.

2.13. Mitochondrial transmembrane potential The change of mitochondrial membrane potential was assessed using the tetrechloro-tetraethylbenzimidazol carbocyanine iodide (JC-1) probe (JC-1 detection Kit, Beyotime Biotechnology, Shanghai). In brief, cells were seeded in a 6-well plate containing a cover glass and cultured for 24 h. Then cells were co-cultured with free a-TOS, P123-M and POT-M at the dosage of 10 mg/mL. After incubation for 2 h, cells were washed with cold PBS three times and incubated with JC-1 staining solution for 20 min. Finally, cells were washed, fixed with 4% paraformaldehyde for 5 min and observed by a fluorescence inverted microscope. JC-1 monomer was detected at 490 nm excitation and 530 nm emission, and JC-1 aggregates signals was acquired at 425 and 590 nm. The rate of red/green was calculated by fluorescence intensity statistics.

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2.14. Intracellular ATP assays Intracellular ATP levels were studied as described in previous reports [38]. Briefly, 5  103 number of cells were seeded in a 6well plate and cultured for 24 h to allow cells attachment. Then, cells were incubated with free a-TOS, P123-M and POT-M for 2 h. Next, cells were washed twice with cold PBS and solubilized in lysis buffer, followed by centrifugation (1.2  104 rpm) at 4 °C for 10 min. The supernatant was collected and ATP content was measured thrice based on luciferin/luciferase assay. Besides, ATP contents were normalized by detecting protein content in each sample (BCA kit, Beyotime Biotechnology, Shanghai). RPMI 1640 culture medium was used as control. 2.15. Immunochemistry MCF-7 or MCF-7/ADR cells were seeded in acid-treated coverslips and cultured for 24 h at 37 °C. Then, these cells were incubated with free a-TOS, P123-M and POT-M for 4 h. After that, the old medium was replaced with fresh medium and cultured for 12 h. Then, each cover-slip were washed with PBS, fixed with ice acetone, incubated with 3% H2O2 and blocking solution to inactivate endogenous enzymes and suppress non-specific binding. Next, these samples were incubated with the antibodies of caspase-3, survivin, Bax, and Bcl-2 for 1 h at room temperature. Cells were rinsed with PBS for 5 min (three times), and then incubated with the goat anti-mouse IgG (IgG/Bio) for 30 min at 37 °C. Cells were rinsed again and incubated with the S-A/HRP at 37 °C for 30 min. After that, cells were stained with 3,3diaminobenzidin (DAB) and hematoxylin. Finally, cells were washed with deionized water and observed by an optical microscope. Besides, the level of proteins was quantified by Image-Pro Plus 6.0 software, and the average optical density (AOD) was calculated thrice according to the equation: AOD = Sum integrated optical density (IOD)/sum area. 2.16. Apoptosis analysis To further evaluate the growth inhibition of tumour cells induced by DOX formulations, cell apoptosis experiment was measured by FCM. In brief, 1  105 number of cells were seeded in 6well plate and cultured for 24 h. Then, the old medium was replaced with fresh medium containing free DOX and DOXloaded micelles with final dosage of 10 mg/mL, or free a-TOS, blank P123 and POT micelles with final dosage of 100 mg/mL. After incubation for 24 h, cells were washed with PBS three times and digested with trypsin solution. Then, cells were collected by centrifugation, dispersed in binding buffer, stained with Annexin Vfluorescein and propidium iodide (PI) according to the manufacturer’s protocol, Finally, cells apoptosis were measured by FCM.

major organs were collected and washed. These tissues were weighted and immersed in 4 mL of 70% ethanol with 0.3 M HCl, then thoroughly homogenized and extracted for 48 h in dark. Finally, the above samples were centrifuged (3500 rpm) for 10 min, and DOX content in the supernatant was measured for three times by a microplate reader at an excitation wavelength of 480 nm and emission wavelength of 590 nm. Besides, tumour tissue were fixed with 4% paraformaldehyde and embedded into paraffin, then cut into 5 mm and stained with 2-(4-amidinophe nyl)-6-indolecarbamidine dihydrochloride (DAPI). Finally, DOX signal in these samples were observed and imaged by CLSM. 2.18. In vivo anti-tumor evaluation MCF-7/ADR tumour-bearing mice model was established as described above. All mice were randomly divided into six groups (5 mice per group), and administered with the following regimens via tail vein: (a) saline; (b) P123-M (100 mg/kg); (c) POT-M (100 mg/kg); (c) free DOX (5 mg/kg eq.); (d) P123-DOX (5 mg/kg eq.) and (e) POT-DOX (5 mg/kg eq.). Tumour size was measured every day with a vernier caliper and tumour volume was calculated as mentioned above. The body weight of each mouse was also recorded every day. At the last day, mice were sacrificed by cervical dislocation, then tumour mass were imaged, collected and weighted. In addition, MDR tumour-bearing mice were also treated with free DOX, P123-DOX and POT-DOX for 17 days (n = 7). The injection was performed at day 1, 5 and 10, and the next operations were the same as above. 2.19. Histological analysis Tumour-bearing mice were treated with the above formulations for 48 h. After that, major organs including heart, liver, spleen, lung, kidney, and tumor were collected. Each sample was fixed with paraformaldehyde and embedded with inparaffin. Each section was cut into 5 um, and then stained with hematoxylin & eosin (H&E). Finally, these tissues were visualized by a fluorescent microscope. Besides, the cell apoptosis in tumour tissues were also evaluated using TUNEL method according to the manufacturer’s instructions. 2.20. Statistical analysis Data are presented as mean ± SD. The statistical comparisons was determined by using Student’s t-test; significant differences between groups were indicated *P < 0.05, **P < 0.01 and ***P < 0.001, respectively. 3. Results and discussion

2.17. In vivo drug distribution

3.1. Pluronic functionalization with ortho ester and a-TOS

All animal experiments were done in accordance with the guidelines of the Use of Laboratory Animals and with the approval of Institutional Authority for Laboratory Animal Care of Anhui University. Briefly, MCF-7/ADR cells (5  105) in 0.1 mL saline were inoculated into the backs of nude Balb/c female mice (immunodeficiency). When tumour volume reached 60–70 mm3, in vivo experiments were performed. The volume was calculated by the formula: d2  D/2 (‘d’ and ‘D’ represented the minimum and maximum diameter, respectively). In order to detect the drug distribution in vivo, tumour-bearing mice were randomly divided into three groups and intravenously injected with free DOX, P123DOX and POT-DOX at the dosage of 5 mg/kg. At predetermined time points, mice were sacrificed, and blood, tumour tissue and

The pH-sensitive pluronic copolymer was synthesized through a two-step modification process (Scheme 1). First, pluronic P123 was terminated with ortho ester monomer (OE). As shown in Fig. S1a and b (Supporting Information), the characteristic peaks of P123 were contributed at d 1.0–1.2 ppm (ACH3 of PPO) and d 3.2–3.75 ppm (ACH2CHO of PPO and ACH2CH2OA of PEO), and the typical signal of ortho ester from OE was observed at d 5.8. Besides, FT-IR spectrum confirmed the CACAO stretching vibration from ortho ester rings appearing in the P123-OE copolymer chain (1194 cm1) but not in P123 (Fig. S2a, Supporting Information). These results indicated that the synthesis of P123-OE was successful. P123-OE was further conjugated with hydrophobic a-TOS by the EDC/NHS catalytic system under TEA conditions. The main

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signals of a-TOS were observed at d 0.75–3.0, and the characteristic chemical shifts corresponding to P123-OE (d 1.0–1.3, 3.2–4.3 and 5.82) were still found in the POT copolymer (Fig. S1c, Supporting Information). Particularly, the typical proton peak ratio of ortho ester against methyl groups from substituted benzene was 1:9.04, which agreed well with the theoretical values (1:9). This result fully demonstrated that the terminal NH2 of P123-OE was completely replaced by a-TOS. Besides, the C@O stretching vibration of a-TOS were found at 1704 and 1735 cm1 (Fig. S2b, Supporting Information), while the new adsorption peak of C@O (1714 and 1745 cm1) appeared in the POT copolymer. The amide I and II were observed at 1612 and 1653 cm1, suggesting that aTOS grafted pluronic was successfully synthesized. 3.2. Characterization of micelles Blank micelles were prepared by the thin-film hydration method [37]. As shown in Fig. 1a and b, DLS measurement indicated that both P123 and POT micelles had a desirable diameter (50–85 nm), and with good polydispersity indexes (0.16–0.18). Besides, TEM images showed that two micelles had a spherical morphology, and the size was correlated well with the results of DLS (Fig. 1c and d). As is well known, the CMC of micelles is closely related to the hydrophilicity/hydrophobicity of polymers. Higher hydrophobicity resulted in smaller CMC, higher stability and more drug loading [45,46]. As shown in Fig. S3 (Supporting Information), CMC of P123 micelles was 30.4 lg/mL (4.67  106 mol/L), which was similar to the previous reports (4.4  106 mol/L) [47]. However, POT micelles displayed lower CMC and the value just was 6.3 lg/mL (9.7  107 mol/L), because the introduced a-TOS increased the hydrophobicity of P123 copolymer. Then, the stability of two micelles was further evaluated via monitoring the change of particle size. Fig. S4a and b (Supporting Information) evaluated the dilution tolerance of micelles in pH 7.4 PB solution. It could be seen that the size of P123-M remarkably increased at dilution ratio from 1:1 to 1:20, suggesting particles gradually dis-

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integrated and became unstable. When the dilution rate was 1:100, micellar diameter quickly decreased and the Tyndall effect disappeared, which meant particles were completely dissociated into free pluronic copolymer. Generally, when the concentration of pluronic copolymers was equal to or lower than its corresponding CMC, the structure of micelles would be disrupted and its diameter increased [21]. Surprisingly, POT-M possessed a stronger anti-dilution capacity owing to its lower CMC. Even if it was diluted 100 fold (10 lg/mL), the Tyndall beam was still observed, and micellar size remained at about 230 nm. A similar phenomenon was observed in the culture medium containing 10% FBS (Fig. S4c, Supporting Information), suggesting the superior anti-dilution property of POT-M. SEM images further confirmed this result. POT micelles wtih dilution 100 times remained relatively complete spherical shape with size 150–200 nm, but hardly P123 particles appeared under the same conditions (Fig. S4d, Supporting Information). These results fully explained the high dilution tolerance of POT micelles, which might reduce drug leakage in vivo and facilitate the blood circulation. Besides, the storage stability was further measured and as shown in Fig. S4e (Supporting Information). It was found that the size of particles had slightly increased during the storage period, but the 75–100 nm diameter of polymeric micelles was still in the optimal range for the EPR effect [48]. Fig. S4f (Supporting Information) revealed that two micelles both had good stability in different buffer solutions. This phenomenon might be explained due to the pluronic PEO chains maintaining steric hindrance and avoiding the adsorption of serum proteins by the production of a hydrophilic shell layer [44]. 3.3. pH-sensitive evaluation In our previous studies, ortho ester as an acid-labile bond has been widely used in various nano-carriers, because it holds good stability in a neutral environment while rapidly hydrolyzing under mildly acidic conditions [38,49]. Therefore, we further evaluated the pH responsiveness of POT-M. After incubation in a pH 5.5 PB

Fig. 1. Size of P123 (a) and POT micelles (b); TEM images of P123 (c) and POT micelles (d).

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solution, the size of micelles gradually increased with time (Fig. 2a) and was accompanied by a non-uniform distribution. When treated for 24 h, micelles displayed a small size distribution, suggesting particle disintegration. Fig. 2b further indicated that the count rate of micelles continued to decrease and the final concentration was below 40 kcps compared to the initial 320 kcps. The above result was further verified by TEM images, and presented in Fig. 2c. The micelles firstly exhibited a disorganized distribution and its size increased at 0.5–6 h, and most of the particles were disintegrated at 12 h, eventually disappeared at 24 h. The possible degradation processes were presented in Fig. S5 (Supporting Information). Clearly, micelles eventually broke into free pluronic or a-TOS. Furthermore, the degradation products of micelles were analyzed by 1 H NMR. At pH 5.5, the characteristic proton peak of ortho ester at (red labelled, d 5.80 ppm) almost disappeared while new proton peaks of formate (blue labelled, degradation product) appeared at d 8.23 ppm (Fig. S6a, (Supporting Information). On the contrary, there was only a proton peak at d 5.80 ppm after treatment with pH 7.4 for 12 h. As a result, the hydrolysis of cyclic ortho ester in copolymer was the same as previous reports [30], which also followed the exocyclic pathway exclusively (Fig. S6b, Supporting Information). All above results indicated that the pH-sensitive

POT micelles could maintain stability in neutral conditions while responding to a mildly acidic environment, which was beneficial for drug delivery and release in vivo.

3.4. DOX loading and release DOX as an anticancer model drug was successfully loaded into polymeric micelles, and the drug loading capacities of micelles were evaluated in Table S1 (Supporting Information). The drug loading efficiency (DLE) was 67.28 ± 1.71% and 82.59 ± 2.34% for P123-M and POT-M, respectively. Notably, a-TOS grafting improved the drug encapsulation owing to the stronger hydrophobic interaction between poor-soluble DOX and micellar aTOS + PPO core compared with single PPO core. Besides, we also found that DOX loading did not significantly affect particle size and polydispersity index, and these values were still within an acceptable range. Furthermore, the surface charge analysis showed that either blank micelles or DOX-loaded micelles had a nearly neutral surface potential (Fig. 2d), which reduced proteins absorption in blood circulation. As a result, the low absorption mediated by neutral charge and high stability provided by steric repulsion

Fig. 2. Acid-triggered degradation property under pH 5.0 of POT micelles included the diameter change (a), count rate change (b), morphology change (c), scale bar = 100 nm; Zeta potential of blank and DOX-loaded micelles (d); In vitro drug release at pH 5.5 and 7.4 buffer solution (e); Data are represented as mean ± SD (n = 3).

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(PEO chains) could prolong circulation time in vivo, thereby improve the bioavailability of drug [50]. In vitro drug release of DOX-loaded micelles was evaluated by the dialysis method. As expected, DOX release was timedependent (Fig. 2e). For P123-DOX micelles, the release profile displayed a biphasic manner with the initial fast (first 12 h) followed by a slow release. Besides, the pH change of solution had no significant effect on drug release, and the cumulative release amount were both over 60% within 48 h at pH 7.4 or 5.5. However, in vitro DOX release from POT-DOX micelles showed an obviously pH-dependent pattern. At pH 5.5, the released DOX reached 81.5% within 24 h, while only a few DOX (~20%) was detected at pH 7.4. This difference was attributed to the hydrolysis of ortho ester under a mildly acidic environment, resulting in the breakage of the hydrophilic-lipophilic balance of POT copolymer, thereby triggering pH-dependent disintegration of micelles and drug release. So, it could be suspected that POT-M had great potential for drug delivery in vivo, because it could maintain stability in blood circulation while degrading in acidic tumour tissue. 3.5. Cellular uptake and co-localization Our past studies had shown that the expression of MDRrelated proteins (e.g. MRPs and P-gp) in MCF-7/ADR cells were much higher than that of MCF-7 cells [51,52], thus the cells experiments of anti-MDR were performed using MCF-7 and MCF-7/ADR cells. To evaluate cellular uptake, MCF-7 and MCF7/ADR cells were incubated with DOX-loaded micelles for 4 h. Then these cells were stained with DAPI and observed by CLSM.

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In MCF-7 cells, the strong red fluorescence of DOX were found in all groups (Fig. 3a), which meant three DOX formulations both could be internalized by tumour cells. Besides, free DOX was mainly located in the nucleus owing to the fast diffusion after entering into cells. DOX-loaded micelles were widely distributed in cytoplasm and nucleus, which was due to the sustained release of DOX after endocytosis by cells [32]. On the other hand, MCF-7/ADR cells images showed a significant difference in the intracellular DOX level after incubation with the above formulations (Fig. 3b). Obviously, the red fluorescence intensity in P123-DOX and POT-DOX groups were much higher than that of free DOX group. This result indicated that MCF-7/ADR cells had strong resistance to DOX because of the high expression of efflux transporters (such as P-gp and MRPs), while P123 or POT micelles could suppress this effect and improve DOX accumulation in drug-resistant tumour cells [53,54]. In order to further evaluate the intracellular DOX level, the quantitative analysis was performed by FCM. As shown in Fig. 3c–e, there was no significant difference between free DOX and DOXloaded micelles. But in MCF-7/ADR cells, DOX fluorescence intensity in the free DOX group was much lower than that of P123-DOX and POT-DOX groups, and the mean intensity was 19.4, 53.8 and 60.2, respectively. This result was consistent with the CLSM images, suggesting that pluronic-based micelles were capable of inhibiting drug efflux in MCF-7/ADR cells. In order to further verify the anti-MDR ability of micelles, the cellular uptake of R-123 (a P-gp substrate) was performed using MCF7/ADR cells. As shown in Fig. S7 (Supporting Information), P123-M and POT-M groups displayed a bright green fluores-

Fig. 3. Cellular uptake of free DOX, P123-DOX and POT-DOX by CLSM images for MCF-7 cells (a) and MCF-7/ADR cells (b), scale bar = 10 lm; Intracellular DOX assessment by flow cytometry for MCF-7 cells (c) and MCF-7/ADR cells (d); Average DOX content in MCF-7 and MCF-7/ADR cells (e), Data are represented as mean ± SD (n = 3).

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cence, indicating R-123 accumulation in MDR cells. With prolonged time, the fluorescence intensity remained at a relatively high level, but the R-123 signal in the control group was barely visible. This result once again demonstrated that pluronic micelles could enhance intracellular drug concentration and retention in MDR tumour cells. In addition, the distribution of DOX-loaded micelles in tumour cells was further observed by subcellular co-localization. As shown in Fig. S8a and b (Supporting Information), DOX-loaded micelles were both ingested into cell, and mainly distributed in lysosome at 2 h (seen merge). A large number of drug molecules escaped the lysosome and diffused into the nucleus as an acid-triggered drug release with the prolonged time. Particularly at 8 h, most particles were dissociated, and DOX staining was mainly located in nucleus. This results might be because pluronic copolymer was capable of forming pores on the bio-membrane, thereby abolishing drug sequestration in lysosome [16,18]. In other words, POT micelles had significant advantages for in vivo usage, because it could be kept stable in blood circulation as mentioned above while exhibiting accelerated drug release and escape at the acidic organelles.

3.6. In vitro cytotoxicity assay In vitro cytotoxicities of blank and drug-loaded micelles were evaluated using MCF-7 and MCF-7/ADR cells. As shown in Fig. 4a and b, blank P123 micelles displayed slight cytotoxicity as the increasing concentration, and this effect might be attributed to the cytostatic action of pluronics such as impeding cell membrane fluidity, inducing apoptosis factors and selective energy depletion [55,56]. Under the same concentration, lower cell viability was observed in blank POT-M owing to the introduction of a-TOS. This result was verified by free a-TOS, and the viabilities were 60–65% at the maximum concentration. According to Neuzil and coworkers reports, a-TOS could interfere with the ubiquinonebinding site of the mitochondrial complex II, cause rapid generation of reactive oxygen species (ROS), and in turn triggering apoptosis [57]. In addition, DOX-loaded micelles also displayed remarkably dose-dependent cytotoxicity in two cells lines. After incubation 24 h, the final viabilities of MCF-7 cells were 30.5%, 26.4% and 15.31% for free DOX, P123-DOX and POT-DOX, respectively (Fig. 4c). The lowest IC50 appeared in POT-DOX group (2.14 lg/mL), which was attributed to the combination therapy

Fig. 4. In vitro cytotoxicity of a-TOS and blank micelles in MCF-7 cells (a) and MCF-7/ADR cells (b); In vitro cytotoxicity of free DOX and DOX-loaded micelles in MCF-7 cells (c) and MCF-7/ADR cells (d); Data are represented as mean ± SD (n = 6).

Table 1 IC50 values, resistance and reversal index of different formulations against MCF-7 cells and MCF-7/ADR cells. Formulations

Free DOX P123-DOX POT-DOX

IC50 (mg/mL) MCF-7

MCF-7/ADR

5.5 ± 0.5 3.7 ± 0.7 2.1 ± 0.3

104.7 ± 3.8 8.3 ± 1.1 3.6 ± 0.6

Data are represented as mean ± SD (n = 3). Resistance index: the ratio of IC50MCF-7/ADR to IC50MCF-7. Reversal index: the ratio of IC50free drug to IC50drug-loaded nanoparticles against MCF-7/ADR cells.

Resistance index

Reversal index

19.1 ± 1.8 2.2 ± 0.2 1.7 ± 0.1

— 12.7 28.9

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between DOX and a-TOS (Table 1). But in MCF-7/ADR cells, free DOX-induced cytotoxicity was greatly suppressed and its viability reached to 71.23%, which was 1.7-fold and 2.92-fold for P123-DOX and POT-DOX, respectively (Fig. 4d). Particularly, the IC50 of free DOX in drug-resistant cells (104.73 lg/mL) was much higher than that of drug-sensitive cells (5.49 lg/mL). Thus, the resistance index of MCF-7/ADR cells was calculated as 19.07. However, P123-DOX could remarkably reverse tumour MDR and its IC50 value just was 8.25 lg/mL, because the pluronic could interfere with the function of drug efflux pumps, increase intracellular drug concentration, and kill more tumour cells. Furthermore, POT-DOX showed the strongest tumour cells growth inhibition, its IC50 value just was 3.62 lg/mL and reversal index reached to 28.93. This result was probably attributed to the multiple synergistic effects: one was pH-triggered drug release by the cleaving of ortho ester; another was the modulation of MDR by pluronic copolymers; the last was ROS-mediated cell-killing by a-TOS. 3.7. Intracellular ROS level assay In order to verify the above result, DCFH-DA was used as a fluorescent probe to detect intracellular ROS level, and cells were imaged by fluorescence microscope. As shown in Fig. 5a, the control group showed a weak green fluorescence, which was connected with the intrinsic ROS inside cells. After incubation with different samples, intracellular ROS level was remarkably improved. Among them, P123-M exhibited a moderate regulation on ROS generation, and the fluorescence intensity reached 8.77% in MCF-7 cells and 11.2% in MCF-7/ADR cells (Fig. 5b). However, a-TOS and POT-M groups showed stronger green fluorescence signal, and the fluorescence intensity were both over 20% in two

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tumour cells. This result fully demonstrated that the grafting of a-TOS could induce more ROS production and then accelerate cell death, which confirmed MTT results. 3.8. Mitochondrial transmembrane potential and ATP assay As is well known, most drug efflux pumps (P-gp and MRPs) are ATP-dependent and involve mitochondrial metabolism to provide energy [58]. Therefore, we further evaluated the effect of these formulations on mitochondrial transmembrane potential (DW) and ATP level. First, we used a JC-1 probe to detect mitochondrial depolarization, and red fluorescence represented normal mitochondria while green fluorescence meant mitochondria damage. As shown in Fig. 5c, the control group displayed a homogeneous red fluorescence signal without obvious green fluorescence, while red signal transformed into green signal in three samples groups. Furthermore, the average JC-1 red/green fluorescence intensity ratio was calculated, the values were 1.57–1.68 for P123-M, 1.07–1.47 for a-TOS and 0.38–0.56 for POT-M, respectively (Fig. 5d). This result demonstrated the capacity of pluronic micelles to interfere with the membrane potential and potentially trigger a series of events that damage cells. Typically, since mitochondria are responsible for ATP synthesis, their damage directly down-regulates intracellular ATP levels [38,59]. Therefore, ATP level in cells was determined using luciferin/luciferase assay and the result was presented in Fig. 5e. Compared to the control, ATP level was significantly reduced after treatment with P123-M, a-TOS and POT-M. In particular, the total ATP content in the POT-M group decreased to 12.74 and 11.41 lmol/gprot for MCF-7 and MCF-7/ADR cells, respectively. Based on above data, we knew that pluronic or their derivatives played a vital role on ATP depletion, which was beneficial to

Fig. 5. Intracellular ROS level after incubation with a-TOS and blank micelles (a); ROS fluorescence intensity evaluation (b); Mitochondrial depolarization was detected by JC1 probe (c); The ratio of red/green fluorescence intensity was calculated by Image J. Z-stack (d); Intracellular ATP level after incubation with a-TOS and blank micelles in MCF7 and MCF-7/ADR cells (e). ‘P’ means as compared to control group in the same tumor cells; Data are represented as mean ± SD (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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reverse MDR, because various drug-resistance mechanisms were energy-dependent such as drug efflux and detoxification systems [15]. 3.9. Immunochemistry and apoptosis Besides, DW loss also triggers pro-apoptotic factors release or anti-apoptotic defence system destruction that induce cellular dysfunction and cell death [60,61]. Herein, the expression of the apoptotic-related proteins in tumour cells was assessed by immunochemical staining. Compared to the control, the proapoptotic proteins levels (Bax and caspase-3) were remarkably increased in all samples, while the anti-apoptotic proteins expression (Bcl-2 and survivin) were obviously suppressed and only a few brown proteins were found in MCF-7 and MCF-7/ADR cells (Fig. 6a and 6b). Notably, the disruption of anti/pro-apoptotic system bal-

ance might modify cell growth processes, and in turn causing cell apoptosis. Furthermore, the semi-quantitative optical densities of proteins concentrations were calculated by Image-Pro Plus. Fig. 6c clearly exhibited that POT-M could induce more bax protein expression than single P123-M, which was because a-TOS had a stronger ability to induce mitochondrial damage. Similar results appeared in Fig. 6d, and caspase-3 protein level in tumour cells treated with POT-M were 1.3–1.6 fold higher than that of P123M. Besides, two pluronic-based micelles also significantly downregulated bal-2 and survivin proteins level in tumour cells, especially in POT-M, suggesting that they could efficiently prevent the activation of anti-apoptotic cellular defence (Fig. 6e and f). These results were similar to previous reports, which were closely related to the mitochondrial damage [62]. In order to evaluate the apoptotic effect of different formulations, cells apoptosis experiment was performed by flow cytome-

Fig. 6. Immunohistochemical (i.e., Bax, Bcl-2, caspase-3 and survivin) analyses of tumor cells slides after treatments with a-TOS, P123-M and POT-M in MCF-7 cells (a) and MCF-7/ADR cells (b); Relative optical densities of tumor cells slides for bax (c), caspase-3 (d), bcl-2 (e) and survivin (f). Data are represented as mean ± SD (n = 3). ‘P’ means as compared to control group in the same tumor cells.

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try. As shown in Fig. 7, P123-M had weak toxicity while higher cellkilling appeared in POT-M. However, after treatment with different DOX formulations, there was a great difference in MCF-7 and MCF7/ADR cells. For MCF-7 cells, the total apoptotic rate (early and late) induced by free DOX, P123-DOX and POT-DOX were 61.6%, 72.7% and 79.1%, respectively (Fig. 7a). For MCF-7/ADR cells, free DOX mediated apoptosis (30.4%) was significantly limited (Fig. 7b), while P123-DOX and POT-DOX kept high apoptosis rate (52.8% and 73.5%). These findings were consistent with MTT results, which indicated that pluronic P123 could reverse MDR and enhance DOX toxicity, and the a-TOS grafting could further induce more cell apoptosis. 3.10. In vivo drug distribution In order to evaluate the tumour targeting of DOX-loaded micelles in vivo, the mice bearing MCF-7/ADR tumour were intravenously injected with free DOX and DOX-loaded micelles at 5 mg/ kg DOX equivalent. At the setting time point, tumour tissues were collected and observed using a fluorescence microscope. As shown in Fig. 8a, free DOX group displayed a weak red fluorescence at the

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initial time, and then the fluorescence intensity rapidly decreased as time prolonging and was hardly visible at 24 h. However, the DOX-loaded micelles groups showed long residence at tumour tissue, DOX signal could be observed even at the end time. This difference was attributed to the passive targeting property of nanoparticles by the EPR effect. In addition, the DOX fluorescence intensity in POT-DOX group was higher than that of P123-DOX group at the same sampling time (Fig. 8b). A reasonable explanation was that POT micelles had higher stability and longer blood circulation time, while single P123 micelles had poor serum dilution resistance owing to its high CMC value. In order to verify this point, DOX concentration in plasma was detected using fluorescence spectroscopy. As shown in Fig. 8c, POT-DOX displayed the highest plasma DOX content at all points in time, followed by P123-DOX and free DOX. Even at 24 h, the drug level from POT-DOX group was 3.03% ID/g, which was 2.26 and 10.8-fold for P123-DOX and free DOX, respectively. These data fully demonstrated that POT micelles could efficiently prolong blood circulation time in vivo and improve drug accumulation in tumour tissue. In addition, it was worth noting that DOX content in heart tissue of DOXloaded micelles was much lower than that of free DOX at the same

Fig. 7. Apoptotic evaluation after incubation with different formulations in MCF-7 (a) and MCF-7/ADR cells (b).

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dose of 5 mg/kg, which meant pluronic micelles could reduce the cardiotoxicity of DOX (Fig. S9a, Supporting Information). Besides, P123-DOX and POT-DOX possessed a relatively high distribution in the liver, which was owing to the capture by the reticuloendothelial system (Fig. S9b–e, Supporting Information). 3.11. In vivo antitumor effcacy In vivo antitumor efficacy of DOX-loaded micelles was evaluated using MCF-7/ADR xenografted nude mice, and saline was used as the control. Tumour volume and body weight of each mouse were measured every day. As shown in Fig. 9a, tumour volume in saline group rapidly increased from day 1 to day 7, and the final volume reached to 263.7 mm3 owing to the unlimited reproduction of malignant cancer cells. Besides, there was no tumour growth inhibition (TGI) appearing in blank P123-M, while POT-M displayed a certain anti-proliferation, and final tumour volume was 118.62 mm3. This difference was attributed to POT micelles having had more accumulation at tumour tissue owing to its higher stability, thereby improving a-TOS mediated tumor cell killing. Besides, free DOX also showed an inadequate antitumor effect, and its TGI against saline just was 34.62%, which was due to the powerful efflux ability of MCF-7/ADR cells. However, DOX-loaded micelles exhibited superior antitumor efficiency, TGI were 68.37%

and 85.36% for P123-DOX and POT-DOX, respectively. Furthermore, Fig. 9b visually presented the tumour tissue in each group, and the smallest tumour mass size appeared in the POT-DOX group. Besides, the tumour weight of mice treated with POT-DOX was 3.23-fold and 5.22-fold lower than mice treated with P123DOX and free DOX, respectively (Fig. 9c). These results demonstrated that the combination of chemo-oxidative therapy by POTDOX contributed higher antitumor effect compared to single chemotherapy by P123-DOX. Furthermore, TUNEL staining was used to detect cell apoptosis in tumour tissues. As shown in Fig. 9d, there was little green fluorescence (dead cell) observed in saline, P123-M and free DOX groups, indicated that these samples had no or weak therapeutic effects. However, DOX-loaded micelles groups presented a broad green fluorescence signal, especially in the POT-DOX group, which meant drug-loaded pluronic micelles could induce severe cell apoptosis and the synergistic therapy by a-TOS led to better therapeutic efficiency. The similar result was observed by H&E staining (Fig. 9e). Tumour tissue was extensively destroyed and the number of tumour cells significantly decreased, indicating the excellent antitumor effect of POT-DOX. In order to further verify the above results, in vivo long-term antitumor experiments were conducted for 17 days. After injection three times, two DOX formulations obviously restrained tumour growth compared to the control and free DOX, and POT-DOX

Fig. 8. DOX staining in tumor site after treatment with free DOX, P123-DOX and POT-DOX by fluorescence microscope (a), scale bar = 100 lm; DOX content in solid tumor was detected (b); DOX content in blood was detected (c); Data are represented as mean ± SD (n = 3).

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achieved the best tumour inhibition (Fig. S10a–c, Supporting Information). And final TGI against control were 22.74%, 53.43% and 80.68% for free DOX, P123-DOX and POT-DOX, respectively. Undoubtedly, the efficient anti-MDR effect mediated by P123 and ROS killing ability mediated by a-TOS led to the best tumour treatment. Besides, the body weight change of the different mice groups was shown in Fig. S11 (Supporting Information). Mice treated with free DOX exhibited obvious body weight loss, suggesting the severe systemic toxicity of free DOX. Heart staining further proved this result, and serious pathological damage and necrosis of muscle fibers were found in cardiac tissues of free DOX treated mice (Fig. S12, Supporting Information). Compared to free DOX, mice treated with blank or DOX-loaded micelles did not show obvious weight loss or pathological damage in heart tissue. Meanwhile, major organs such as liver, spleen, lung and kidney also showed no noticeable morphological changes in these groups. As a result, pluronic-based micelles were safe and suitable for use in vivo, which was closely related to phase II clinical studies of SP1049C [63,64].

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4. Conclusion In summary, we developed the pH stimuli-responsive pluronic micelles (POT-M) by ortho ester linkage that was able to overcome tumour MDR and amplify intracellular ROS level, resulting in enhanced antitumor efficiency. Compared to traditional pluronic or its derivative formulations [15,22], POT micelles possessed high storage stability and anti-dilution in blood circulation owing to its lower CMC, while rapidly triggering drug release in the acidic environment of tumour tissue by the cleaving of ortho ester. Besides, DOX-loaded POT micelles exhibited the enhanced drug accumulation and retention in MDR cells by blocking the function of efflux transporters, thereby increasing cytotoxicity and apoptosis. Furthermore, the grafting of a-TOS remarkably induced cytotoxic ROS generation, leading to more cell damage or death. In vivo experiments further confirmed the outstanding inhibitory effect on tumour growth of POT-DOX, and TGI were both more than 80% (single or three administration times). As expected, longterm treatment or further optimization of POT-M (such as introducing targeting molecules) may lead to more advanced therapeu-

Fig. 9. In vivo antitumor performance of P123-DOX and POT-DOX; (a) the change of tumor volume after treatment various formulations for 7 days; (b) tumor mass images; (c) mean tumor weight was calculated; (d) TUNEL staining of tumor sections, green and blue means dead signal and nucleus; (e) H&E staining of tumor sections; Scale bar = 100 lm; Data are represented as mean ± SD (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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tic effect. Overall, this combined strategy with chemo-oxidative therapy is potentially useful against MDR breast cancer and could be tried to treat other malignancies. CRediT authorship contribution statement Xu Cheng: Data curation, Writing - original draft. Xiaoli Zeng: Visualization, Investigation. Yan Zheng: Project administration. Qin Fang: Writing - review & editing. Xin Wang: Supervision. Jun Wang: Software, Validation. Rupei Tang: Conceptualization, Methodology. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 51803001, 51603001, and 51503001), the Research Foundation of Education Department of Anhui Province of China (No. KJ2018ZD003, and KJ2018A0006), and the Academic and Technology Introduction Project of Anhui University (AU02303203). Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.jcis.2020.01.029. References [1] O. Yasuhiko, E. Yuki, C.J. Rui, K. Takashi, O. Masayasu, M. Masaaki, Y. Jun, K. Naoji, Supermolecular drug challenge to overcome drug resistance in cancer cells, Drug Discov. Today. 23 (2018) 1556–1563. [2] L. Zhou, H. Wang, Y. Li, Stimuli-responsive nanomedicines for overcoming cancer multidrug resistance, Theranostics 8 (2018) 1059–1074. [3] A.M.E. Abdalla, L. Xiao, M.W. Ullah, M. Yu, C. Ouyang, G. Yang, Current challenges of cancer anti-angiogenic therapy and the promise of nanotherapeutics, Theranostics 8 (2018) 533–548. [4] Y. Chen, W. Zhang, Y. Huang, F. Gao, X. Sha, X. Fang, Pluronic-based functional polymeric mixed micelles for co-delivery of doxorubicin and paclitaxel to multidrug resistant tumor, Int. J. Pharm. 488 (2015) 44–58. [5] J.Y. Lee, U. Termsarasab, M.Y. Lee, D.H. Kim, S.Y. Lee, J.S. Kim, D.D. Kim, Chemosensitizing indomethacin-conjugated chitosan oligosaccharide nanoparticles for tumor-targeted drug delivery, Acta Biomater. 57 (2017) 262–273. [6] Z.Z. Laura, C. Elena, C. Mariangela, C. Marialessandra, L. Marcello, A.C. Nicola, Small and innovative molecules as new strategy to revert MDR, Front. Oncol. 4 (2014) 1–11. [7] F. Marco, K.J. Linton, Investigational ABC transporter inhibitors, Expert Opin. Inv. Drug. 21 (2012) 657–666. [8] S. Shukla, C.P. Wu, S.V. Ambudkar, Development of inhibitors of ATP-binding cassette drug transporters–present status and challenges, Expert Opin. Drug Metab. Toxicol. 4 (2008) 205–223. [9] L. Lv, K. Qiu, X. Yu, C. Chen, F. Qin, Y. Shi, G. Li, Amphiphilic copolymeric micelles for doxorubicin and curcumin co-delivery to reverse multidrug resistance in breast cancer, J. Biomed. Nanotechn. 12 (2016) 973–985. [10] A. Pan, H. Zhang, Y. Li, T. Lin, F. Wang, J. Lee, K.S. Lam, Disulfide-crosslinked nanomicelles confer cancer-specific drug delivery and improve efficacy of paclitaxel in bladder cancer, Nanotechnology 27 (2016) 425103. [11] J. Huang, Q. Duan, P. Fan, C. Ji, Y. Lv, X. Lin, X. Yu, Clinical evaluation of targeted arterial infusion of verapamil in the interventional chemotherapy of primary hepatocellular carcinoma, Cell Biochem. Bioph. 59 (2011) 127–132. [12] M. Elsabahy, K.L. Wooley, Design of polymeric nanoparticles for biomedical delivery applications, Che. Soc. Rev. 41 (2012) 2545–2561. [13] B. Jiang, C. Li, J. Tang, T. Takei, J.H. Kim, Y. Ide, Y. Yamauchi, Tunable-sized polymeric micelles and their assembly for the preparation of large mesoporous platinum nanoparticles, Angew. Chem. Int. Ed. 55 (2016) 10037–10041. [14] L.Y. Zhao, W.M. Zhang, Recent progress in drug delivery of pluronic P123: pharmaceutical perspectives, J. Drug Target. 25 (2017) 471–484.

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