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Alleviating the Liver Toxicity of Chemotherapy via pH-Responsive Hepatoprotective Prodrug Micelles Ran Tao, Min Gao, Fang Liu, Xuliang Guo, Aiping Fan, Dan Ding, Deling Kong, Zheng Wang, and Yanjun Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b04192 • Publication Date (Web): 13 Jun 2018 Downloaded from http://pubs.acs.org on June 13, 2018

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ACS Applied Materials & Interfaces

Alleviating the Liver Toxicity of Chemotherapy via pHResponsive Hepatoprotective Prodrug Micelles Ran Tao, 1,† Min Gao, 1,† Fang Liu, 1 Xuliang Guo, 1 Aiping Fan, 1 Dan Ding, 2,3 Deling Kong, 2,3 Zheng Wang, 1,2 Yanjun Zhao*,1

1

School of Pharmaceutical Science & Technology, Tianjin Key Laboratory for Modern Drug Delivery

& High Efficiency, and Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin 300072, China 2

State Key Laboratory of Medicinal Chemical Biology (Nankai University), Tianjin 300071, China 3

Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, and

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin 300071, China.

∗ To whom correspondence should be addressed. Dr. Yanjun Zhao School of Pharmaceutical Science & Technology, Tianjin University 92 Weijin Road, Nankai District, Tianjin 300072, China Tel: +86-22-2740 7882, Fax: +86-22-2740 4018 Email: [email protected]

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ABSTRACT: Nanocarriers have been extensively utilized to enhance the anti-tumor performance of chemotherapy, but it is very challenging to eliminate the associated hepatotoxicity. This was due to the significant liver accumulation of cytotoxic drug-loaded nanocarriers as a consequence of systemic biodistribution. To address this, we report a novel type of nanocarrier that was made of hepatoprotective compound (oleanolic acid/OA) with a model drug (methotrexate/MTX) being physically encapsulated. OA was covalently connected with methoxy poly(ethylene glycol) (mPEG) via a hydrazone linker, generating amphiphilic mPEG-OA prodrug conjugate that could self-assemble into pH-responsive micelles (ca. 100 nm), wherein the MTX loading was ca. 5.1% (w/w). The micelles were stable at pH 7.4 with a critical micelle concentration of 10.5 µM. At acidic endosome/lysosome microenvironment, the breakdown of hydrazone induced the micelle collapse and fast release of payloads (OA and MTX). OA also showed adjunctive anti-tumor effect with a low potency, which was proved in 4T1 cells. In the mouse 4T1 breast tumor model, MTX-loaded mPEG-OA micelles demonstrated superior capability regarding in vivo tumor growth inhibition because of the passive tumor targeting of nanocarriers. Unsurprisingly, MTX induced significant liver toxicity, which was evidenced by the increased liver mass, increased levels of alanine transaminase, aspartate transaminase, and lactate dehydrogenase in serum as well as in liver homogenate. MTX-induced hepatotoxicity was also accompanied with augmented oxidative stress e.g. the increase of malondialdehyde level and the reduction of glutathione peroxidase and superoxide dismutase concentration in the liver. As expected, mPEG-OA micelles significantly reduced the liver toxicity induced by MTX due to the hepatoprotective action of OA, which was supported by the reversal of all the above biomarkers and qualitative histological analysis of liver tissue. This work offers an efficient approach for reducing the liver toxicity associated with chemotherapy, which can be applied to various antitumor drugs and hepatoprotective materials.

KEYWORDS: Micelles, hepatotoxicity, self-assembly, prodrug, oleanolic acid, methotrexate. 2 ACS Paragon Plus Environment

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INTRODUCTION Nanotechnology has revolutionized the anti-cancer chemotherapy.1-3 The employment of nanocarriers can bring multiple benefits including solubilization of hydrophobic drugs, enhanced cargo loading, extended systemic circulation, improved tumor targeting, facilitated cellular uptake, rapid endosomal escape and on-demand cargo release.4-9 Despite these, there is only quite a small portion (< 5%) of administered dose that eventually accumulates in the tumor tissue via the biodistribution process.10,11 The majority of nanomedicine goes to other healthy organs, particularly to the liver that is the major site of metabolism and a key part of the mononuclear phagocyte system.12,13 There is usually significant liver deposition of the drug due to nanomedicine sequestration by liver macrophages, which can induce substantial adverse effects to the liver, i.e. hepatotoxicity.14 This is particularly valid when the drug is potent and/or shows specific liver toxicity. Methotrexate (MTX) is one good example; it can induce the development of liver fibrosis and even cirrhosis, which are correlated to oxidative stress, and the downregulation of redox-sensitive transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2) together with other types of nuclear factors.15-17 The utilization of hepatoprotective agents is an attractive means to address the liver toxicity of nanoparticulate chemotherapy. Oleanolic acid (OA) is a natural pentacyclic triterpenoid that shows hepatoprotective effect for both chronic liver fibrosis and acute hepatic damage induced by various chemicals.18-23 Actually, there is a long history to use OA as a non-prescription medicine for managing human liver diseases in China.24 The hepatoprotective effect of OA usually involves multiple mechanisms, including the activation of Nrf2 and metallothionein that can regulate the oxidative damages induced by different triggers.25-28 Besides this, OA is also reported to have numerous additional pharmacological activities related to human health including antitumor and immunomodulatory effects.22,24 However, the poor aqueous solubility of OA (Log P = 7.5) and the low potency limit its



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pharmaceutical application and therapeutic efficacy because the payload loading in nanocarriers by conventional non-covalent approach is usually poor (< 5%).2,4,29

Scheme 1. Illustration of pH-responsive mPEG-oleanolic acid (OA) conjugate micelles for the reduction of hepatotoxicity of a model anticancer drug, methotrexate (MTX). The hydrazone-linked amphiphilic polymer can self-assemble into micelles to physically encapsulate MTX. Drugs release was triggered by endosomal pH when drug-loaded micelles entered cells by endocytosis. MTX can induce oxidative stress and hence liver damage. OA can protect liver injury against oxidative stress via activating Nrf2. Engineering amphiphilic prodrug micellar nanocarriers has been an appealing approach to enhance drug loading and manipulate the ratiometric ratios of different cargos.30-32 The design of stimulitriggered nanomedicine could further expedite intracellular payload release and hence enhance therapeutic efficacy.33 The acidic microenvironment of endosome/lysosomes provides an excellent pH trigger for on-demand drug release.34-37 It was postulated that the conjugates of OA and methoxy poly(ethylene glycol) (mPEG) would produce micellar nanocarriers via self-assembly due to the extremely high hydrophobicity of OA.38 Therefore, this work aimed to tailor-make pH-labile mPEG-OA conjugate micelles with MTX being physically loaded as a proof-of-concept model to address liver toxicity of cytotoxic agents (Scheme 1). Hydrazone was selected as the pH-sensitive linker to covalent link OA and PEG moiety, which would enable acid-responsive micelle disassembly and rapid cargo release in the cytosol. Regarding OA and MTX, their mechanisms match very well at the point of Nrf2 4 ACS Paragon Plus Environment

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manipulation, which was one major reason to select MTX as the model drug in the current study. It was expected that such nanoplatform would be a robust vehicle for reduction of liver toxicity of chemotherapy. RESULTS AND DISCUSSION Formulation and Characterization of Prodrug Conjugate Micelles. Hydrazone has been a popular linker to manufacture pH-sensitive nanomedicine due to the ease of synthesis.34,35 In the current work, OA was modified with levulinic acid (LA) via esterification to get an intermediate product, LA-OA (Figure S1-S3, Supporting Information). The reaction between LA-OA and hydrazine-bearing mPEG (2000 Da) produced the target conjugate, mPEG-OA with an average molecular weight (MW) of ca. 2600 Da (Scheme 2, Figure S4). The excess mPEG was removed by dialysis. The theoretical loading of OA was calculated to be ca. 17.6% (w/w). The ultraviolet-visible absorption spectra and fluorescent emission spectra of mPEG-OA, LA-OA, and OA were similar in methanol solution, which was mainly determined by the OA moiety (Figure 1A, 1B). The mPEG-OA conjugate could successfully selfassemble into micelles. Based on the probe-free approach,39 the corresponding critical micelle concentration (CMC) of mPEG-OA micellar nanocarrier at pH 7.4 was 27.3 ± 0.4 µg/mL (10.5 ± 0.2 µM) (Figure 1C). The obtained CMC value was much lower than PEGylated vitamin E (TPGS) at 132 µM.40 The CMC has been used an index of micelle stability with a lower CMC indicating a better stability.41 The good stability of mPEG-OA micelles at neutral conditions was because of the hydrophobic interaction between OA that was the driving force of micelle formation.38 A sufficient micelle stability is also a prerequisite to prevent premature drug release during systemic circulation post intravenous dosing. The MTX encapsulation in mPEG-OA micelles employed a typical dialysis method followed by lyophilization.42 The dialysis medium was a basic ammonium solution (pH 8.0) to maintain the stability



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of hydrazone bond that is subject to hydrolysis under acidic conditions. The ultraviolet-visible spectrum of aqueous micellar solution at the same concentration was almost identical pre- and post-dialysis, which demonstrated the integrity of ester bond and the stability of mPEG-OA conjugate (Figure S4). The MTX loading was determined at 5.1 ± 0.3% (w/w) by high performance liquid chromatography. The data were consistent with previous investigation into the loading ability of nanocarriers by physical method (< 5%).10,11 The decent MTX content in micelles was presumed as a result of hydrogen bonding between MTX and OA; both molecules contain the hydrogen donor and acceptor that can form hydrogen bonding, resulting in enhanced drug loading in micellar nanocarriers.43 The presence of drug also increased the micelle size. The hydrodynamic diameters were 69 ± 6 nm (placebo micelles) and 101 ± 10 nm (MTX-loaded micelles), respectively (Figure 1D, Figures S6). The solubilization-induced micelle core expansion and size increase was a well-reported phenomenon.38 Likewise, both micelles were spherical, which was evidenced by the transmission electron microscope (TEM) imaging. The TEM size was ca. 30 nm smaller than the matching hydrodynamic diameter, i.e. 35 ± 5 nm (placebo micelles) and 67 ± 8 nm (MTX-loaded micelles), (Figure 1E, 1F).

Scheme 2. Synthesis route of mPEG-hydrazone-OA. mPEG, OA and LA represent methoxyl poly(ethyl glycol), oleanolic acid and levulinic acid, respectively.


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Figure 1. (A) Ultraviolet-visible spectra and (B) fluorescence emission spectra of free OA, LA-OA and mPEG-OA in methanol; the OA concentration was fixed at 50 µg/mL. (C) CMC determination of mPEG-OA in PBS (100 mM, pH = 7.4). (D) Hydrodynamic sizes of placebo OA-NCs micelles and drug-loaded OA-NCs/MTX micelles in water (0.5 mg/mL, pH = 8). Transmission electron microscope images of OA-NCs (E) and OA-NCs/MTX (F), scale: 100 nm. The CMC data were presented as mean ± standard deviation (n = 3). Drug Release In Vitro and Cytotoxicity. The cargo release in aqueous buffers was investigated at the physiological temperature (37oC) under two pH conditions (pH 7.4 and pH 5.0). The former mimicked blood circulation environment and the latter mimicked that of endosome/lysosomes.44 The release profile of both LA-OA and MTX was pH-dependent; a lower pH resulted in increased release rate and extent (Figure 2A, 2B, and Figure S7). At a fixed pH, the release profile of LA-OA was analogous to that of MTX. Such discrepancy of release behavior was because of the pH-labile hydrazone stability and hence pH-dependent micelle stability. At pH 5.0, the hydrazone linker would experience a significant



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hydrolysis, leading to the breaking down of mPEG-OA conjugate, disassembly of micelles, and cargo release. Similar phenomena were observed for other types of hydrazone-linked conjugate micellar nanoformulations.34

Figure 2. Cumulative drug release of (A) LA-OA and (B) MTX in different buffer solutions contain 5% (w/v) sodium dodecyl sulfate. (C) Viability of 4T1 cells in response to free OA, free LA-OA, and OANCs (n = 4). (D) Viability of 4T1 cells upon treatment with free MTX and OA-NCs/MTX (n = 4). The cytotoxicity of OA, free LA-OA, and mPEG-OA micellar nanocarriers were evaluated in murine breast cancer cells (4T1) by the colorimetric MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assay. All three samples displayed a dose-dependent cytotoxicity, but the potency of all three samples was low and the cell viability was above 70% at the OA dose of 60 µM post 48 h’s incubation (Figure 2C). The corresponding half maximal inhibitory concentration (IC50) of free MTX and MTX-loaded mPEG-OA micelles was 29.4 ± 3.5 µM and 22.0 ± 2.6 µM (p < 0.05) (Figure 2D). In terms of IC50, the cytotoxic drug-loaded nanocarrier was usually less potent than the corresponding free drug since the drug had to be released first before exerting the toxic effect. However, 8 ACS Paragon Plus Environment

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the enhanced cytotoxicity of MTX-loaded mPEG-OA micelles in contrast to free MTX in the current study was a consequence of rapid drug release from micelles and the adjunctive cytotoxicity of OA at high doses.22,24 Cellular Uptake and Cell Cycle Assessment. The uptake of mPEG-OA micellar nanocarriers by 4T1 cells was assessed by an optical imaging technique, i.e. confocal laser scanning microscopy (CLSM). As the peak absorption of the emission spectrum of mPEG-OA conjugate located at the low wavelength (< 400 nm), the inherent fluorescence of OA could not be utilized for the purpose of tracking micelle localization. Because the encapsulation of fluorescent probe in micelles is complicated by the continuous release of probe, we employed a mixed micelle approach instead.45 The mixed micelles consisted of mPEG-OA and mPEG-FITC. The MW of mPEG in mPEG-FITC conjugate was 1000 Da instead of 2000 Da to ensure the self-assembly capacity. The formation of mixed micelles was verified by particle size analysis (Figure S8). The confocal images showed a kinetically increased internalization of micelles from 2 h to 6 h; this was revealed by the gradually elevated green FITC fluorescence intensity (Figure 3A and Figure S9).

Figure 3. (A) Cellular uptake of fluorescence (FITC) labeled mixed micelles upon inculcation with 4T1 cells for up to 6 h. The blue color indicates the site of nuclei and green color indicates that of micelles 9 ACS Paragon Plus Environment


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(scale bar: 20 µm). Cell cycle analysis of 4T1 cells treated by negative control (i.e. no treatment) (B), free MTX (C), OA-NCs (D) and OA-NCs/MTX (E). The concentration of MTX was constant at 50 µg/mL for both free MTX and OA-NCs/MTX formulations; the OA content of was 140 µg/mL for both OA-NCs and OA-NCs/MTX samples. MTX is a cell cycle-specific folate antagonist and it works by inhibiting dihydrofolate reductase and thymidylate synthetase, resulting in the inhibition of DNA and RNA synthesis.46 In contrast to the control cells receiving no treatment, the MTX-treated cells showed a significant cell cycle arrest at S phase (Figure 3B, 3C), which agreed well with the MTX’s mechanism of action. The placebo mPEGOA conjugate micelles exhibited a considerable G2/M phase arrest (Figure 3D), which concurred with previous investigations.47 Interestingly, only the S phase arrest was witnessed in the cells incubated with the MTX-loaded nanocarrier (Figure 3E), which might be because of the interplay between LA-OA and MTX. The more potent MTX determined the cell cycle arrest at the S phase. It should be noted that MTX also affect normal cells, which can induce significant side effects in the body, particularly in MTX-rich organs (e.g. liver). Pharmacokinetics and Biodistribution. To examine the kinetic biodistribution of conjugate micelles, a near infrared fluorescent probe (Cy5) together with MTX was co-encapsulated in the micelles. The Cy5 loading was 1.2% (w/w) based on the fluorescent probe quantification. The obtained Cy5-labelled micelles were intravenously administrated to BALB/c mice with free Cy5 as the control (probe dose: 0.36 mg/kg). The fluorescence intensity peaked at 6-8 hours post administration, which was a typical phenomenon of the enhanced permeability and retention (EPR) effect (Figure 4A, 4C).13 On the contrary, the intensity of free Cy5 control was significantly lower than that of micellar nanocarriers from 3 h to 24 h post dosing (p < 0.05). This was thought as a consequence of the extended blood circulation of micellar nanocarriers. At 24 h, the control formulation delivered significantly less Cy5 to the tumor compared to the micellar formulation (Figure 4). Besides deposition in tumor, Cy5 also accumulated at the major healthy organs (heart, liver, lung, kidney and spleen) 24 h post dosing. As fluorescence-based


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approach is semi-quantitative, we carried out additional pharmacokinetic and biodistribution study using the high performance liquid chromatography-mass spectrometry (HPLC-MS) technique for drug quantification.48,49 The micelle formulation can significantly extend the blood circulation of MTX compared to free MTX (Figure S10). The HPLC-MS approach also demonstrated that micelles could present significantly more MTX to the tumor tissue in contrast to free MTX (Figure S11), which coincided with the data produced by the fluorescence-based method. The fluorescence resonance energy transfer (FRET) analysis using Cy3/Cy5 pair showed that there was still a significant amount of cargos/probes within the micelle core 24 h post dosing, as demonstrated by the evident FRET phenomenon in tumor as well as kidney, liver and lung (Figure S12).

Figure 4. Biodistribution of Cy5-loaded mPEG-OA micelles in female BALB/c mice bearing 4T1 tumor (n = 3). (A) Kinetic biodistribution of free Cy5 and mPEG-OA micelles upon intravenous administration (Cy5 dose: 0.36 mg/kg). (B) Fluorescent quantification of Cy5 in tumor and major healthy organs ex vivo 24 h post formulation administration. (C) Analysis of kinetic fluorescence intensity of tumors (n = 3). (D) Analysis of fluorescence intensity of Cy5 in tumor and major organs 24 h post dose administration (n = 3). 11 ACS Paragon Plus Environment


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Figure 5. In vivo antitumor performance and hepatoprotection effect of OA-NCs/MTX. (A) The inhibition of tumor growth upon treatment with four formulations, PBS, free MTX, placebo micellar nanocarriers (OA-NCs), and drug-loaded micelles (OA-NCs/MTX); (B) Kinetic change of mice body weight; (C) The quantitative analysis of tumor weights, and (D) The relative liver weight of mice with reference to body weight in the end of efficacy experiment. Control group was normal mice without any treatment (n = 6). * p < 0.05, ** p < 0.01. (E) H&E staining (top) and TUNEL staining (bottom) of tumor tissue. Scale: 100 µm (H&E); 50 µm (TUNEL). In Vivo Efficacy Study. The in vivo performance of pH-responsive mPEG-OA conjugate micelles (OC-NCs/MTX) was investigated in the BALB/c mice bearing 4T1 tumor. The tumor growth inhibition capacity of different samples ranked as follows: OA-NCs/MTX > free MTX > OA-NCs > PBS (Figure 5A). Among all these samples, the dose of MTX and OA was maintained the same. For MTX-loaded micellar nanocarriers, both MTX and OA exerted anti-tumor effect and hence it exhibited the best tumor 12 ACS Paragon Plus Environment

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suppression outcome. The high degree of tumor accumulation of micelles also made a contribution. As MTX is much more potent than OA as an anti-tumor agent, free MTX was superior to placebo OA-NCs in terms of tumor inhibition. There was no dramatic change of mice body weight upon treatment by all four samples (Figure 5B). When the efficacy study was over, the size of collected tumor from the mice in four categories coincided well with the tumor inhibition curves (Figure 5C). Likewise, the liver was also collected from all groups and the weight percentage of liver with reference to the whole mice body weight was used as an index of hepatotoxicity. Free MTX induced an evident increase of liver weight; whereas the corresponding value was significantly lower for MTX-loaded OA-NCs at the same MTX dose (p < 0.01) (Figure 5D). This demonstrated that OA could indeed reduce MTX-induced liver toxicity. Tumor tissue analysis by hematoxylin and eosin (H&E) staining revealed optimal antitumor efficacy of OA-NCs/MTX nanoformulations, mainly as a consequence of passive targeting (i.e. EPR effect) (Figure 5E). The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay further proved the first-class apoptosis-initiation capability of OA-NCs/MTX (Figure 5E). Hepatoprotection Study. To examine the hepatoprotective performance of OA against MTX, we examined the serum level of two key clinical biomarkers of liver health, i.e. alanine transaminase (ALT) and aspartate transaminase (AST).50 The co-delivery of OA and MTX (i.e. OA-NCs/MTX) radically reduced the concentration of ALT and AST in serum compared to that treated by MTX alone (p < 0.01) (Figure 6A, 6B). There were no significant difference between OA-NCs/MTX and placebo OA-NCs (p > 0.05). These data revealed that OA could effectively reduce the MTX-induced liver toxicity. The lactate dehydrogenase (LDH) was used as another biomarker of liver function as it is released during tissue damage and cell injury. Similar to the other two indices, the presence of OA could substantially reduce the level of serum LDH caused by MTX (p < 0.01) (Figure 6C). The levels of ALT, AST, and LDH in liver homogenate concurred well with their corresponding concentration in serum; encapsulating MTX



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in mPEG-OA conjugate micelles significantly reduced the concentration of these three biomarkers in the liver tissue at the end of efficacy study (p < 0.05) (Figure 6D-6F). The hepatoprotective function of OA was presumed as a consequence of its ability of counteracting the action of MTX and maintaining the normal level of Nrf2.25-28

Figure 6. The influence of three formulations (PBS, placebo OA-NCs, and drug-loaded OA-NCs/MTX) on the key biomarkers of liver injury. Control group indicates no treatment. (A) Serum alanine transaminase (ALT); (B) Serum aspartate transaminase (AST); (C) Serum lactate dehydrogenase (LDH); (D) ALT in liver tissue; (E) AST in liver tissue; (F) LDH in liver tissue (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001, and n.s. indicates p > 0.05. As MTX-induced hepatotoxicity not only involves downregulation of Nrf2, but also implicates the induction of oxidative stress,15-17 we examined the levels of glutathione peroxidase (GSH-Px), malondialdehyde (MDA), and superoxide dismutase (SOD) in liver tissue in the end of efficacy study. Oxidative stress often involves reactive oxygen species (ROS) overproduction and hence disturbance of normal redox homeostasis, which can readily result in tissue damage and disease development. MDA is a typical marker of oxidative stress and its concentration in liver was boosted due to MTX treatment.


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However, the combinational delivery of OA and MTX significantly reduced the MDA level (p < 0.01) (Figure 7A). Previous work also reported the ability of OA to diminish MDA, which was consistent with the discovery in the current study.7 GSH-Px and SOD are two enzymes to protect the organism from oxidative damage by reducing different types of ROS. MTX treatment reduced the level of GSHPx and SOD in liver, but OA-NCs/MTX reversed this trend due to the protective action of OA (Figure 7B, 7C).19 The histological analysis of liver tissue of tumor-bearing mice after the MTX treatment showed an evident cell damage when compared to the liver of the tumor-free mice (i.e. control group); whereas the mPEG-OA micelles diminished the extent of such damage (Figure 7D). For all five groups, no apparent degree of apoptosis was observed. All these data supported that OA could reduce the MTXinduced hepatotoxicity by functioning against the oxidative stress.

Figure 7. Analysis of oxidative stress markers in liver homogenate following treatment by three formulations (PBS, placebo OA-NCs, and drug-loaded OA-NCs/MTX) (n = 6). Control group indicates no treatment. (A) malondialdhyde (MDA), (B) glutathione peroxidase (GSH-Px), (C) superoxide dismutase (SOD). * p < 0.05; ** p < 0.01; n.s. indicates no significant difference. (D) Histological and 15 ACS Paragon Plus Environment


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apoptosis analysis of liver tissue in the end of efficacy experiment. H&E staining (scale: 100 µm); TUNEL staining (scale: 50 µm). CONCLUSION In summary, we proposed a new concept to reduce the liver toxicity of chemotherapy using MTX as a model anti-tumor agent. This approach employed a unique molecule, OA that showed inherent hepatoprotective effect via the mechanism of Nrf2 activation and oxidative stress suppression. Hydrophobic OA was linked with PEG via a pH-responsive hydrazone linker, generating amphiphilic mPEG-OA conjugate that self-assembled into micellar nanocarriers featured with high OA loading and triggered micelle disassembly and on-demand cargo release. MTX was physically encapsulated in the micelles to match the potency of OA and MTX. Such nanosystem showed superior anti-tumor efficacy without the hassle of adverse effects in liver, which was demonstrated by quantitative analysis of the biomarkers of liver health both in the serum and liver tissue. The levels of key makers of redox homeostasis further supported the hepatoprotective function of OA. The current work proposed the idea of encapsulating antitumor drug by hepatoprotective agents, and integrated the concepts of selfassembly, prodrug, triggered release, and combinational delivery in one nanoplatform with additional benefits of ratiometric loading and potency matching. Such proof-of-concept can be extended to a broad variety of antitumor therapeutics and different potential agents that can reduce the adverse effects of chemotherapy. METERIALS AND METHOD Materials. Methoxypolyethylene glycol hydrazide (mPEG-NHNH2, 2000 Da), methoxy poly(ethylene glycol) amine (mPEG-NH2, 1000 Da) and methoxy poly(ethylene glycol) fluorescein (mPEG-FITC) were obtained from Ponsure Biotechnology Co., Ltd. (Shanghai, China). OA and MTX were purchased from Energy Chemical Co., Ltd. (Shanghai, China). Levulinic acid (LA) was sourced from Heowns Biochem Co., Ltd. (Tianjin, China). Anhydrous sodium sulfate was obtained from Baishi Chemical Co.,


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Ltd. (Tianjin, China). Dichloromethane (DCM), dimethyl sulfoxide (DMSO), trifluoroacetic acid (TFA), ethanol (EtOH), ethyl acetate (EA), methanol, acetonitrile and n-hexane were purchased from Concord Technology Co., Ltd. (Tianjin, China). Ammonia solution (25%, w/w), glacial acetic acid and phosphoric acid were sourced from Guangfu Fine Chemical Research Institute (Tianjin, China). 1-Ethyl3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDCI) and N,N-dimethylpyridin-4-amine (DMAP) were obtained from J&K Scientific Co., Ltd. (Beijing, China). Certified fetal bovine serum was purchased from Baibei Biotechnology Co., Ltd. (Biolnd, Cromwell, USA). RPMI 1640 medium, penicillin, streptomycin, and trypsin were from HyClone Inc. (Logan City, Utah, USA). MTT, saline and 4% paraformaldehyde were purchased from Baibei Biotechnology Co., Ltd. (Tianjin, China). Chloroform-d and dimethyl sulfoxide-d6 were sourced from Jinouxiang Science & Technology Co., Ltd. (Tianjin, China). Cy5 dye was obtained from InnoChem Science & Technology Co., Ltd. (Beijing, China). All other chemicals were purchased from Jiangtian Fine Chemical Research (Tianjin, China). Synthesis of LA-OA. Levulinic acid (4.4 g, 37.9 mmol), EDCI (6.0 g, 31.3 mmol) and DMAP (0.7 g, 1.6 mmol) were mixed in 20 mL anhydrous DCM under nitrogen atmosphere. The reactants were maintained at 30oC 40 min. OA (1.7 g, 3.8 mmol) was dissolved in 15 mL anhydrous DCM and combined with the above mixture under nitrogen atmosphere. The reaction was kept at 30oC for 24 h. Afterwards, the mixture was extracted by DCM and deionized water, dried against sodium sulfate (anhydrous), followed by concentration via rotary evaporation. Then the column chromatography was used to purify the crude product (n-hexane/EA = 5:1, v/v) and a light yellow solid was collected (yield: 82%). 1H NMR (400 MHz, CDCl3, ppm): 2.19 (3H, s, COCH3), 2.56-2.59 (2H, m, COOCH2), 2.75 (2H, t, COCH2), 2.82 (1H, dd, CH), 4.50 (1H, t, OCH), 5.27 (1H, t, CHC) (Figure S1). 13C NMR (100 MHz, CDCl3, ppm): 206.71, 184.44, 172.60, 143.59, 122.36, 81.06 (Figure S2). ESI-MS in methanol: m/z (expected) = 577.40, m/z (observed) = 577.80 [M+Na]+ (Figure S3).



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Synthesis of mPEG-OA conjugate. mPEG-hydrazide (0.2 g, 0.1 mmol) and LA-OA (0.8 g, 1.5 mmol) were mixed in 30 mL anhydrous DMSO under nitrogen protection. Then 60 µL TFA was added slowly with nitrogen protection. The reaction was maintained at 35oC for 24 h. Afterwards, TFA was removed by evaporation and the mixture was further dialyzed against ethanol (MWCO: 1000 Da) to obtain a slight yellow solid, mPEG-OA (yield: 76%). 1H NMR (400 MHz, DMSO-d6, ppm): 2.10 (3H, s, COCH3), 2.43-2.45 (2H, m, COOCH2), 2.61 (2H, t, COCH2), 2.74 (1H, dd, CH), 3.24 (3H, s, OCH3), 4.15 (2H, t, OCH2CO), 4.39 (1H, t, OCH), 5.16 (1H, t, CHC) (Figure S4). Micelle generation. The MTX-loaded micelles were manufactured using the standard dialysis approach. Concisely, mPEG-OA (200 mg) and MTX (20 mg) were combined in 10 mL DMF that was maintained at 25oC for one hour. The drug-loaded micelles were achieved by dialyzing against diluted ammonia water (pH = 8.0, 84 µM) at ambient temperature (25oC) using a regenerated cellulose membrane (MWCO = 1000 Da). The dialysis medium was regularly replaced and the process lasted for 24 h. Then the system was centrifuged followed by lyophilization to get OA-NCs/MTX. All the above process was performed with light protection. The blank micelles (OA-NCs) were prepared using the same method without MTX feeding. Micelle characterization. The UV-vis spectra and fluorescence spectra of free OA, LA-OA and mPEG-OA were recorded in methanol solution by the Hitachi U-3900 spectrophotometer and Tecan microplate reader, respectively. OA concentration of all samples was kept the same (50 µg/mL). Critical micelle concentration (CMC) was examined to indicate micelle stability by a FLS980 fluorescence spectrometer. Due to the intrinsic fluorescence of OA, an external probe was not utilized. A series of blank micelles were prepared in phosphate buffer saline (PBS) (pH = 7.4, 0.1 M) ranging from 1 to 100 µg/mL and the fluorescence of samples was measured (Ex = 237 nm, Em = 300-400 nm, slit = 8 nm). The OA’s maximum fluorescence was plotted against the concentration of mPEG-OA and the CMC was


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determined by calculating the inflection point (n = 3). The MTX content was analyzed using HPLC coupled with a UV detector (303 nm). The separation was achieved by a Dionex 3000 HPLC system together with a Phenomenex Gemini C18 column (250 mm × 4.6 mm, 5 µm) at 30°C. The sample was eluted by a mixed solvent, i.e. water containing 0.5% (v/v) phosphoric acid and acetonitrile (1:9, v/v). The flow rate was 1 mL/min and the injection volume was set at 20 µL. The hydrodynamic sizes of OANCs and OA-NCs/MTX (0.5 mg/mL) were studied in ammonia water (pH = 8.0) by dynamic light scattering (DLS) using the Malvern ZetaSizer Nano ZS90. The morphology of these two micelles was studied by a JEM-2100F transmission electron microscope. In vitro drug release. The in vitro drug release utilized our previously published method.43 The static Franz-type diffusion cells were employed and the temperature was 37oC (n = 3). The receiver fluid was phosphate buffer solution (pH 7.4/5.0, 0.18 M/0.15 M) containing 5% SDS (w/v). The donor phase was micellar suspension (2 mL) in the same buffer as the receiver fluid. Regenerated cellulose membrane (MWCO: 1000 Da) was used as diffusion barrier. The applied dose was ca. 594 µg MTX and ca. 2014 µg LA-OA for OA-NCs/MTX (5.9 mg/mL). A fixed amount of receiver fluid was withdrawn at predetermined time points for HPLC analysis of drug content (dual wavelength: 205 nm, 303 nm). Cell line and animal. Based on our previous work, the same cell line and animal model was used in the current study.43 In detail, Murine breast cancer cells (4T1) were provided by the State Key Laboratory of Medicinal Chemical Biology (Nankai University). The culture medium was RPMI 1640 containing 10% fetal bovine serum and 1% penicillin/streptomycin. The cell culture condition was 37°C with 5% CO2. Female BALB/c mice (6 weeks, 18-22 g) were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). The animal study was operated under protocols approved by the Animal Ethics Committee of Tianjin University.



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Cytotoxicity analysis. The viability of 4T1 cells in response to different samples were carried out using the MTT assay (n = 4).43 In brief, 4T1 cells were seeded on 96-well plates at a density of 4×103 cells per well. Three samples were selected to determine the cytotoxicity, including LA-OA, free OA, and OA-NCs. The OA dose of three samples was same ranging from 1 to 60 µg/mL. Cells were incubated with the formulations for 48 h prior to IC50 determination. Cytotoxicity of free MTX and MTX-loaded micelles were tested by same method. The MTX dose of two samples was also from 1 to 60 µg/mL. Cellular uptake study. To investigate into the uptake of mPEG-OA micelles by 4T1 cells, fluorescent probe-labelled micelles containing mPEG-FITC and mPEG-OA (molar ratio = 1:1) were manufactured via dialysis. The hydrodynamic diameters of mixed micelle and two control micelles (mPEG-OA and mPEG-FITC) were measured in ammonia water (pH = 8.0) by DLS (0.5 mg/mL) (Figure S5, Supporting Information). The 4T1 cells were seeded at a density of 1.0 × 105 cells per well containing 1.0 mL medium. After 24 h, the medium was replaced with fresh one containing mixed micelles (150 µg/ mL) and incubated for different time (2h, 4 h and 6 h). After cell washing by PBS in triplicate, 1 mL 4% paraformaldehyde was added. Twenty minutes later, the cells were washed again with PBS, followed by nucleus staining with DAPI (500 mL, 1 µg/ mL) for 10 min and PBS washing (10 mM). Finally, 1 mL PBS was added in every dish and stored at 4°C in dark. The intracellular location of FITC (green color) and nucleus (blue color) was recorded by a Carl Zeiss LSM710 confocal laser scanning microscope.43 Cell cycle assay. 4T1 cells were seeded in 6-well plates at a density of 5 × 105 cells per well, and then treated by MTX, OA-NCs and OA-NCs/MTX. The concentration of MTX was fixed at 50 µg/mL for MTX-containing samples, and the concentration of OA was 140 µg/mL for both OA-NCs and OANCs/MTX samples. The incubation time was set at 24 h. Then the cells were collected, washed by PBS,


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fixed in a mixed solvent containing 70% ethanol/PBS (3:1, v/v) and stored at -20oC. Fixed cells were centrifuged to remove the supernatant. Then the 4T1 cells were treated by 800 µL PBS solution containing 0.2% Triton X-100, 100 mg/ml RNAse A, and 10 mg/ml propidium iodide. After further incubation at 4oC for 30 min under dark, the samples were analyzed by a BD FACSCalibur flow cytometer. The data were analyzed by Modifit software. Pharmacokinetic and biodistribution study. In order to measure the biodistribution of OANCs/MTX micelles, Cy5-loaded micelles were prepared by dialysis method using OA-NCs, MTX and Cy5 (w/w/w, 20:2:1). The Cy5 content was quantified by a fluorescence method (Ex = 646 nm, Em = 662 nm). The animal study was carried out according to the guidelines set by the Tianjin Committee of Use and Care of Laboratory.43 BALB/c mice were subcutaneously inoculated with 4T1 cells on the axilla; the dose was 1×106 cells per mouse.43 When the tumor size reached 50-100 mm3, the mice were ready for the fluorescent imaging study. The formulations (120 µL) of Cy5 labeled OA-NCs/MTX (4.9 mg/mL) and free Cy5 (60 µg/mL) were intravenously (i.v.) administrated through the tail vein (n = 3); the Cy5 dose was set at 0.36 mg/kg for all formulations. A Cri Maestro in vivo imaging instrument was used to collect the fluorescence images at pre-determined time points (1 h, 3 h, 6 h, 8 h and 24 h).43 The excitation wavelength of Cy5 was 635 nm (30 ms) and the emission spectra were obtained from 670 to 900 nm (2 pixels binning and 10 nm bandwidth). The kinetic fluorescence intensity of Cy5 in the tumor site was plotted. In order to observe the ex vivo Cy5 distribution, two groups of animals were sacrificed 24 h post dosing (n = 3); the tumors together with major healthy organs (hearts, kidneys, livers, lungs, and spleens) were collected for fluorescence imaging. To investigate the extent of cargo release from micelles, a FRET approach was employed a wellknown FRET pair (Cy3/Cy5). Both Cy3 and Cy5 co-loaded OA-NCs micelles were obtained by the same approach and the supplement dose of both probes was 5% (w/w). The content of Cy3 and Cy5



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were quantified by the fluorescence method (Ex/Em = 545 nm/565 nm for Cy3; Ex/Em = 646 nm/662 nm for Cy5). Cy3/Cy5 co-loaded micelle formulation (150 µL, 3.4 mg/mL) was administrated to 4T1 tumor-bearing mice via i.v. route. The mixture of free Cy3 and Cy5 with identical dose was used as the control (Cy3: 37.8 µg/mL; Cy5: 100 µg/mL). The tumor and major healthy organs were collected 24 h post dosing, followed by FRET analysis at three different excitation/emission conditions (Ex/Em = 532 nm/560-750 nm; Ex/Em = 532 nm/670-820 nm; Ex/Em = 635 nm/670-820 nm). The pharmacokinetic and biodistribution of MTX was also performed with the aid of HPLC-MS. The same 4T1 tumor-bearing mice were used and randomly grouped prior to intravenous administration of either OA-NCs/MTX (11.8 mg/mL) or free MTX (0.6 mg/mL). The sample volume was 200 µL for each injection that corresponded to a MTX dose of 6 mg/kg. At pre-determined time points (5-30 min, and 1-24 h) post dose administration, the blood (30 µL) was collected in heparin-coated centrifuge tubes (n = 3). Aminopterin was selected as the internal standard of MTX. After centrifugation (3500 rpm) for 10 min, the plasma was obtained, followed by the supplement of aminopterin solution in methanol (20 µL, 65 µg/mL), formic acid aqueous solution (20 µL, 0.5% (v/v)), and methanol (200 µL). The mixture was fully vortexed before centrifugation (13300 rpm, 10 min) to get the supernatant. The MTX concentration in the supernatant was analyzed by Agilent 6420 triple quadrupole MS system coupled with an Agilent 1260 HPLC system. A Phenomenex Gemini C18 column was utilized for the elution. The mobile phase was a mixed solvent containing acetonitrile and 0.5% (v/v) formic acid aqueous solution (9:1, v/v); the flow rate was 1 mL/min and the injection volume was 10 µL. An electrospray ionization (ESI) source operated in the positive ionization mode was used for MTX quantification.48,49 Analyte analysis was achieved by the multiple reaction monitoring technique with the precursor-toproduct ion transitions at m/z 455.1→308.1 (MTX) and 441.2→294.0 (aminopterin), respectively. The collision energy was 20 V and the dwell time was 200 ms. At the end of pharmacokinetic experiment,


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the tumor and major organs were collected, weighed, and homogenized in saline (0.5 mL) using a dounce tissue homogenizer. Then the tissue homogenate (100 µL) was mixed with 90 µL aminopterin methanol solution (130 µg/mL), 100 µL formic acid aqueous solution (0.5%, v/v) and 2 mL methanol. After a vigorous extraction process, the mixture was centrifuged (3500 rpm) for 10 min, followed by supernatant dilution with mobile phase and MTX quantification by HPLC-MS. In vivo antitumor efficacy. Mice-bearing 4T1 tumor were randomly divided into four groups (n = 6).43 The formulations (200 µL) of OA-NCs/MTX (11.8 mg/mL), OA-NCs (9.5 mg/mL), free MTX (0.6 mg/mL) and PBS (0.01 M) were administered intravenously every 72 h for 5 times. The dose was 6 mg/kg (MTX) and 16.7 mg/kg (OA) for each injection. The tumor volume and mouse body weight were regularly measured during the whole medication course. The tumor volume of each mouse was calculated using the equation (width2 × length/2).51 Meanwhile, the healthy animal were used as control group under the same feeding condition as experiment groups during the treatment course (n = 6). All animals were sacrificed on the 17th day post the first dosing and the blood samples were withdrawn via the eye. Tumor together with liver tissues was collected followed by being washed in ice-cold saline. Then all liver tissues were dissected into two parts: the 1st part was fixed with 4% paraformaldehyde for prior to hematoxylin and eosin (H&E) staining; the 2nd part was used for biochemical analysis. The tumor tissues were analyzed by both H&E and TUNEL staining.43 Hepatotoxicity analysis. The obtained blood samples were kept to clot for 1 h prior to centrifugation (1800 g) for 10 min. The obtained serum was maintained at -20°C ready for biochemical analysis. Serum activities of ALT, AST and LDH were examined according to the protocol provided by commercial kits (Jiancheng Bioengineering Institute, Nanjing, China) using ELISA (SpectraMax M2, CA, USA). Similarly, the three biomarkers’ level in liver was also analyzed. In brief, liver sections (each of 0.4 g) were homogenized in 3.6 mL ice-cold saline to obtain 10% liver homogenate (w/v) using a



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dounce tissue homogenizer. The obtained homogenate maintained at 4°C underwent centrifugation (1800 g) for 15 min to collect the supernatant that was stored at -20°C for the assay of ALT, AST, and LDH. The makers of oxidative stress were also analyzed in liver tissues. The concentrations of glutathione peroxidase (GSH-Px), malondialdhyde (MDA), and superoxide dismutase (SOD) in liver homogenate were quantified based on the procedures of commercial assay kits (Jiancheng Bioengineering Institute, Nanjing, China). Statistical analysis. The data were shown as the mean ± standard deviation (SD). Statistical analysis of group differences was measured via Student’s t-test or analysis of variance (ANOVA) followed by appropriate Tukey’s post-hoc analysis.51 Statistical significant was determined at p < 0.05. ASSOCIATED CONTENT Supporting Information. The supporting information is available free of charge via the Internet at http://pubs.acs.org. NMR and MS spectra of LA-OA; NMR and UV-vis spectra of mPEG-OA; DLS size of placebo and drug-loaded micelles; Drug release profile at two pH conditions; DLS size of mixed micelles; Coassembly of fluorescent micelle and its cellular uptake; pharmacokinetic analysis of MTX; biodistribution of MTX by HPLC-MS; biodistribution of MTX by fluorescent approach. AUTHOR INFORMATION Corresponding Author *Email: [email protected] Author Contributions


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All authors have contributed to the manuscript writing and have given approval to the final version of the manuscript. † R. Tao and M. Gao made an equal contribution to this work. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We acknowledge the funding support from the National Basic Research Program of China (2015CB856500), National Natural Science Foundation of China (21650110447), and State Key Laboratory of Medicinal Chemical Biology (Nankai University) (2017030). ABBREVIATIONS ALT, alanine transaminase; AST, aspartate transaminase; CLSM, confocal laser scanning microscope; CMC, critical micelle concentration; DCM, dichloromethane; DMAP, N,N-dimethylpyridin-4-amine; DMSO, dimethyl sulfoxide; EDCI, 1-Ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride; EPR, enhanced permeability and retention; FRET, fluorescence resonance energy transfer; GSH-Px, glutathione peroxidase; HPLC-MS, high performance liquid chromatography-mass spectrometry; IC50, half maximal inhibitory concentration; LA, levulinic acid; LDH, lactate dehydrogenase; MDA, malondialdehyde; mPEG, methoxy poly(ethylene glycol); MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; MTX, methotrexate; MW, molecular weight; OA, oleanolic acid; PBS, phosphate buffer saline; ROS, reactive oxygen species; SOD, superoxide dismutase; TEM, transmission electron microscope; TFA, trifluoroacetic acid. REFERENCES (1) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nanocarriers as an Emerging Platform for Cancer. Nat. Nanotechnol. 2007, 2, 751-760. 25 ACS Paragon Plus Environment


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