Cargo-Free Nanomedicine with pH Sensitivity for Codelivery of DOX

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Cargo-free nano-medicine with pH-sensitivity for codelivery of DOX conjugated prodrug with SN38 to synergistically eradicate breast cancer stem cells Na Sun, Chenyang Zhao, Rui Cheng, Zerong Liu, Xian Li, Axin Lu, Zhongmin Tian, and Zhe Yang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00367 • Publication Date (Web): 20 Jun 2018 Downloaded from http://pubs.acs.org on June 21, 2018

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Molecular Pharmaceutics

Cargo-free nano-medicine with pH-sensitivity for co-delivery of DOX conjugated prodrug with SN38 to synergistically eradicate breast cancer stem cells

Na Sun†, 1, Chenyang Zhao†, 1, Rui Cheng1, Zerong Liu1, Xian Li1, Axin Lu2, Zhongmin Tian*, 1 and Zhe Yang*, 1

1

The Key Laboratory of Biomedical Information Engineering of Ministry of

Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China 2

Instrument Analysis Centre, Xi'an Jiaotong University, Xi’an 710049, China.

† These authors contribute equally to this work. * Corresponding author * E-mail: [email protected]; [email protected] * Tel:

0086-29-82667331

ACS Paragon Plus Environment

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

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Abstract Due to their abilities of transforming into bulk cancer cells and resistance to radiotherapy and chemotherapy, cancer stem cells (CSCs) are currently considered as a major obstacle for cancer treatment. Application of multiple drugs using nano-carriers is a promising approach to simultaneously eliminate non-cancer stem cells (non-CSCs) and CSCs. Herein, to employ the advantages of nano-medicine while avoiding new excipients, pH-responsive pro-drug (PEG-CH=N-DOX) was employed as the surfactant to fabricate cargo-free nano-medicine for co-delivery of DOX conjugated prodrug with SN38 to synergistically eradicate breast cancer stem cells (bCSCs) and non-bCSCs. Through the intermolecular interaction between DOX and SN38, PEG-CH=N-DOX and SN38 were assembled together to form a stable nano-medicine.

This nano-medicine

not

only

dramatically

enhanced

drug

accumulation efficiency at the tumor site, but also effectively eliminated bCSCs and non-bCSCs, which resulted in achieving a superior in vivo tumor inhibition activity. Additionally, the biosafety of this nano-medicine was systematically studied through immunohistochemistry, blood bio-chemistry assay, blood routine examination and metabolomics. The results revealed that this nano-medicine significantly reduced the adverse effects of DOX and SN38. Therefore, this simple yet efficient nano-medicine provided a promising strategy for future clinical applications.

Keywords Carrier free nano-medicine, pH-responsive, breast cancer stem cells, synergistic therapy, biosafety


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1

Introduction

Cancer stem cells (CSCs), a type of cancer cells exist in a very small proportion and possess the capacities of proliferation and self-renewal to form new tumors, have been widely

studied

recently.1-3

Additionally,

CSCs’

resistance

to

standard

chemotherapeutics is the major cause of tumor metastasis or recurrence after traditional therapies, since traditional treatments only shrink the tumor volume by primarily killing the non-CSCs which only have limited proliferative potential.2, 4, 5 To achieve a superior cancer therapeutic efficiency, there are many promising approaches that have been developed to simultaneously eliminate CSCs and non-CSCs, such as the combined treatment of multiple chemotherapeutic drugs.6, 7 Doxorubicin (DOX), a conventional chemotherapy drug, can be used to block the replication processes of a wide range of cancer cells through interacting with DNA and

inhibiting

topoisomerase

II

(TOP II).8,

9

SN38

(7-ethyl-10-hydroxyl

camptothecin), an active metabolite of irinotecan (CPT-11), displays 100-1000 folds enhancement in inhibiting topoisomerase I (TOP I) in comparison to CPT-11, causing poor DNA replication and transcription.10, 11 Topoisomerases in CSCs are crucial for facilitating the rates of transcription, repairing the DNA and improving the expression of anti-apoptotic proteins. Thus, suppressing both TOP I and TOP II with DOX and SN38 can effectively kill CSCs.12-14 Unfortunately, the conventional chemotherapy using DOX and SN38 still suffers from several limitations, including poor bioavailability, rapid in vivo clearance, low accumulation efficiency at tumor site and adverse effects for normal tissues.10, 15 To address these limitations, great efforts have been made to encapsulate these insoluble drugs into nano-carriers, such as polymeric nanoparticles, liposomes and inorganic materials.16 With assistance of nano-vehicles, hydrophobic drugs can be delivered to the action sites with the enhanced therapeutic efficiency and reduced side effects in comparison to free drugs.17 However, the fabrication of these nano-carriers has been proved to be extremely complicated in most cases. The challenges include difficulties



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in materials synthesis and purification, preventing manufacturing at large scale.18-20 Moreover, almost all currently used carriers and their degradations have no therapeutic effect, but instead induce obvious side effects, including cardiovascular effects, mitochondrial damage, inflammation and so on.20, 21 Thus, to take advantages of nano-formulation while excluding any unnecessary materials, we believe that cargo-free nano-medicine will achieve the combination therapy by using multiple chemotherapeutics against cancers.22-24 For example, Shen et al. utilized irinotecan hydrochloride (CPT11) as surfactant to prepare the carrier-free CPT-11/SN38 nano-medicine for cancer therapy, which not only improved the bioavailability of these two drugs but also enhanced their antitumor efficacy.19 However, application of cargo-free nano-medicines in synergistic eradication of breast cancer stem cells (bCSCs) has seldom been reported. Herein, we employed a simple method to fabricate the cargo-free and pH-responsive nano-medicine for co-delivery of DOX and SN38, in order to simultaneously eliminate bCSCs and non-bCSCs (Figure 1). This cargo-free nano-medicine was composed of prodrug (PEG-CH=N-DOX) and SN38, which could self-assemble into nanoparticles (PEG-DOX/SN38 NPs) through hydrophobic interactions between DOX and SN38. Due to the pH-sensitive imine linker between PEG and DOX,25, 26 a rapid drug release in acidic condition could be achieved. Additionally, other features of PEG-DOX/SN38 NPs involve the effective cellular uptake capacity, high tumor penetrating ability, and enhanced passive targeting ability through the enhanced permeability and retention (EPR) effect. These effects could empower this cargo-free nano-medicine to eliminate bCSCs and non-bCSCs at the same time, improving both in vitro and in vivo therapeutic efficiency. More importantly, through systematical characterizations

on

bio-safety

(including

immunohistochemistry,

blood

bio-chemistry assay, blood routine examination and metabolomics studies), it is demonstrated that this cargo-free and pH-responsive nano-medicine presents the preferable biosafety for further medical application.


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Figure 1. Schematic illustration of cargo-free and pH responsive nano-medicne with enhanced cross-membrane transportation and controlled drug release.

2

Experimental Section

2.1 Materials mPEG-CHO (Mw: 850 Da) was obtained from Yare Biotechnology Co. Ltd. (Shanghai, China). Doxorubicin hydrochloride (DOX·HCl) and SN38 were purchased from Mesochem technology Co., Ltd. (Beijing, China). MTT was purchased from Amresco (USA). MCF7 cells (human breast carcinoma cell line) were obtained from Shanghai cell bank of Chinese Academy of Science, and cultured in DMEM (Gibco) with 10% FBS at 37 °C in a humidified 5% CO2 incubator. MCF7 mammospheres were cultured according to our previously reported method.27, 28

2.2 Synthesis of PEG-CH=N-DOX The Schiff ’s base reaction was employed to synthesize PEG-CH=N-DOX.25,

26

Briefly, DOX·HCl (69.5 mg) in DMSO (2.0 mL) was first deprotonated through reacting with triethylamine (TEA) under magnetically stirring for 2 h. Afterwards, mPEG-CHO (100 mg) dissolved in DMSO (2.0 mL) was added into the above



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solution and reacted at room temperature for 24 h. After reaction, the crude product was dialyzed against DMSO (dialysis bag: MWCO 1000 Da) to remove the unreacted DOX. Then the resultant solution was mixed with cold water, followed by dialyzing against deionized water (dialysis bag: MWCO 1000 Da) to remove the organic solution for lyophilization and further studies. The structure of PEG-DOX was confirmed by proton NMR spectroscopy (1H NMR) and Fourier transform infrared spectrometry (FTIR). Additionally, the critical micelle concentration (CMC) value of PEG-DOX was measured using our previously reported method.29, 30

2.3 Modelling self-assembly of SN38 and PEG-DOX Materials Studio was used to set up the 3D model of SN38 and PEG-DOX. Force filed parameters was gotten from COMPASS II force field. These two molecules were mixed at a molar ratio of 3:1 (PEG-DOX:SN38) and initially distributed randomly in space.

Drug

molecules

were

merged

in periodic

TIP3P water box

of

36.215*36.215*36.215 Å3. Dynamic process was calculated by “Forcite tools” model in NPT ensemble with a temperature of 300 K and a pressure of 1 atm. Berendsen method was applied to control temperature and pressure.31 Ewald method was performed to calculate long-range electrostatic and VDW interaction.32 Cutting off value for VDW interaction was 12 Å. Time step was 2.0 fs. Total simulating time was 1.5 ns.

2.4 Fabrication and characterization of PEG-DOX/SN38 NPs PEG-DOX/SN38 NPs were prepared using dialysis method. Briefly, PEG-DOX (5 mg) and SN38（0.4 mg）in DMSO (400 µL) was injected into PBS (pH 7.4, 0.01M) under sonication on ice for 2 min at 14 W (Sonics & Materials, VCX500). Afterwards, the resultant emulsion was dialyzed against PBS overnight. The size distribution and zeta potential of the obtained nano-medicine were characterized using dynamic light scattering analyzer (Malvern, ZS90). The morphology of PEG-DOX/SN38 NPs were observed using a transmission electron microscope (TEM, JEM-200CX).


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2.5 Encapsulation efficiency and drug loading content of nano-medicine The content of DOX and SN38 in nano-medicine were determined using HPLC (Agilent Technologies 1100 system) equipped with a C18 reverse-phase column (5 µm, 250 mm×4.6 mm). UV detection at wavelength of 380 nm and 480 nm was performed to measure the signals of SN38 and DOX, respectively. HPLC analyses were performed under the following conditions: the mobile phase (acetonitrile/water, 60/40) was pumped at a flow rate of 1.0 mL/min and maintained at 25 °C. Moreover, the loading efficiency and encapsulation efficiency were determined using the following equations: Loading efficiency (%)=Drug weight in NPs/NPs weight×100% Encapsulation efficiency (%)=Drug weight in NPs/Total drug weight×100%

(1) (2)

2.6 pH-sensitive release of DOX and SN38 The in vitro drug release of DOX and SN38 from PEG-DOX/SN38 NPs was measured at 37 ℃ under varying pH conditions (pH 7.4 and 5.0). Briefly, 3 mL of PEG-DOX/SN38 NPs suspension was added to a dialysis bag (MWCO 1000) against 10 mL of PBS (pH = 7.4 or 5.0) containing 0.2% Tween 80 (v/v) at 37 °C on a rotary shaker set at 300 rpm. 3 mL of dialysate was withdrawn and replenished with the same volume of fresh buffer at pre-determined time intervals. The amounts of DOX and SN38 released from nano-medicine were measured using HPLC according to the method described above.

2.7 In vitro cellular uptake studies As demonstrated in our previous studies, the proportion of bCSCs in MCF7 mammosphere cells was higher than that in MCF7 adherent cells.27, 28 Thus, in this study, MCF7 mammosphere cells or MCF7 mammospheres were employed to investigate the cross-membrane transportation ability and therapeutic efficiency of PEG-DOX/SN38 NPs.



In cellular uptake studies, MCF7 mammosphere cells and MCF7 mammosphers were incubated with free PEG-DOX+SN38 or PEG-DOX/SN38 NPs (DOX (2 µg/mL) and SN38 (0.96 µg/mL)) for 2 h. Subsequently, the medium was removed, and the cells were washed using PBS for three times. Then the intracellular distribution and mammosphere penetration capacity of PEG-DOX/SN38 NPs were evaluated using confocal laser scanning microscope (CLSM, Carl Zeiss, LSM700). The excitation wavelength for detecting DOX and SN38 were 540 nm and 360 nm, respectively. The cellular uptake efficiency of free PEG-DOX+SN38 or PEG-DOX/SN38 NPs was also quantified using flow cytometry. After the treatment on MCF7 mammosphere cells and MCF7 mammosphers for 2 h, the cells were harvested and rinsed with PBS (pH 7.4, 0.01 M) for three times. Subsequently, the cells were re-suspended in 0.5 mL of PBS (pH 7.4, 0.01 M) and analyzed using the flow cytometer (FACS Canto, BD).

2.8 MTT assay for cell viability The capacity of free PEG-DOX+SN38 or PEG-DOX/SN38 NPs killing MCF7 mammosphere cells was measured using MTT assay. Briefly, MCF-7 mammosphere cells were cultured in 96-well plates at density of 5000 cells per well and incubated overnight at 37 ℃. Subsequently, mammosphere cells were treated with free PEG-DOX, free SN38, free PEG-DOX+SN38 and PEG-DOX/SN38 NPs at different concentrations for 48 h. After incubation, the percentage of alive cell was estimated using MTT assay and the absorbance of medium was measured via a microplate reader at a wavelength of 570 nm (Tecan infinite 2000).

2.9 Cell apoptosis measurement MCF7 mammosphere cells were seeded into ultralow attachment 6-well culture plate at density of 2×104 cells per well. Mammosphere cells were collected and stained with Annexin V-FITC/PI Apoptosis Detection Kit (Bestbio, China) after the treatment of free PEG-DOX+SN38 or PEG-DOX/SN38 NPs for 24 h. Subsequently, the apoptotic cells were measured using flow cytometry.


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2.10 Proportion of CD44+/CD24- cells assay To estimate the killing ability of PEG-DOX/SN38 NPs against bCSCs, the percentage of CD44+/CD24- cells were measured after treating MCF7 mammosphere cells with free PEG-DOX+SN38 or PEG-DOX/SN38 NPs. In brief, MCF7 mammosphere cells were added into ultralow attachment 6-well culture plate at density of 2×104 cells/well and incubated with different drug formulations for 10 days. Then, the cells were harvested, washed and re-suspended at density of 1×105 cells/mL in PBS containing 1% BSA, followed by staining with CD44-PE and CD24-FITC antibodies (BD Biosciences) for 30 min at 4 ℃. Finally, the stained cells were analyzed using flow cytometry.

2.11 Western blotting analysis of ALDH1 In order to further evaluate PEG-DOX/SN38 NPs’ capacity of eliminating bCSCs, another hallmarker of bCSCs, ALDH1,33 was measured using western blot after treating MCF7 mammospheres with free PEG-DOX+SN38 or PEG-DOX/SN38 NPs. Briefly, after the same treatment described above, the protein was extracted from mammosphere in each group, separated by 10% SDS–PAGE, transferred to PVDF membranes (Millipore, USA), and blocked with 5% fat-free milk in TBST buffer (0.1 M, pH 7.4). Each sample was treated with anti-ALDH1 (1:700, Abgent) or anti-β-actin (1:1000, Cwbiotech) primary antibodies at 4 °C overnight. Then, the membrane was incubated with secondary antibody for 1 h at room temperature. Protein bands were detected using the enhanced chemiluminescence imaging system (Clinx Science Instruments, China).

2.12 Mammosphere inhibition assay The capability of PEG-DOX/SN38 NPs against MCF7 mammospheres was estimated by measuring the mammosphere’s volume after treatment. In brief, the mammospheres with a diameter of 100 µm

were incubated with free



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PEG-DOX+SN38 or PEG-DOX/SN38 NPs. Then, the volume of mammosphere was recorded under an inverted microscope (IX51, Olympus) during the 8-day incubation. The major (dmax) and minor (dmin) diameters of mammospheres in each group were measured using ImageJ software. The volume of mammosphere was calculated by the following equation:27, 28 V = 0.5×dmin2×dmax

(3)

At the end of the treatment, the relative volume of mammosphere was estimated using the following equation: N = (Vn/V0)×100%

(4)

where Vn represents the volume of mammosphere at day n after treatment, and V0 represents the volume of mammospheres at day 0.27, 28

2.15 Mammosphere formation assay MCF7 mammosphere cells (10,000 cells/well) were cultured in ultralow attachment 6-well culture plate and treated with free PEG-DOX+SN38 or PEG-DOX/SN38 NPs for 7 days. Following the above treatment, the mammospheres were photographed using the inverted microscope. Besides, the number of mammospheres was also counted to assess their efficacy of anti-mammosphere formation.

2.16 Passive targeting study Animal experiments were conducted through the protocols approved by the Animal Experimentation Ethics Committee of Xi’an Jiaotong University. To obtain the mice bearing xenograft MCF7 tumor, approximately 1×107 of MCF7 cells in a mixture of PBS and Matrigel (1:1, v/v) were subcutaneously inoculated in right flank region of balb/c mice (six-week old, female). The tumor volume was calculated using the equation: Volume=0.5×length×(width)2

(5)

To assess the in vivo passive targeting capacity, eight mice bearing tumor with volume of 200~300 mm3 were randomly divided into two groups. Then, the mice were


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injected with free PEG-DOX+SN38 or PEG-DOX/SN38 NPs at DOX and SN38 dosages of 5 mg/kg and 2.5 mg/kg via tail vein administration. Moreover, MCF7 tumors were harvested at 24 h post-injection. The contents of DOX and SN38 in tumor tissues were detected using UPLC-MS (Vion IMS QTOF system, Waters).

2.17 In vivo antitumor efficacy When the tumor volume reached approximately 50-100 mm3, tumor-bearing mice were randomly divided into 5 groups (n=5) and injected (i.v) with different drug formulations at doses of 5 mg/kg (DOX) and 2.5 mg/kg (SN38) on day 0, 2, 4, 6 and 8, respectively. And the tumor dimension was tracked for 26 days. During this period, tumor growth inhibition (TGI) was also quantified using the equation: TGI = (1-Vt/Vn)×100%

(6)

where Vn is the tumor volume in 0.9% NaCl group, and Vt is the tumor volume in treatment group, both of which were recorded on the same day.27, 28 Besides, the body weight of mice was also tracked during the whole experiment. At the end of experiment, the tumor tissues were harvested and stained using hematoxylin and eosin (H&E) and Tunel assay to further confirm the antitumor efficacy.

2.18 Proportion of bCSCs in tumor after therapy After the therapy as described above, the tumors were sheared into small pieces and washed with cold PBS (pH 7.4, 0.01 M) containing 1% penicillin/streptomycin for 3 times. Afterwards, these fragments were immersed in digestion solution with 1 mg/mL of collagenase type I and incubated at 37 ℃ for 3 h. The dispersed cells were filtered through a sterile nylon 200-mesh sieve and washed with cold PBS (pH 7.4, 0.01 M). Then, the cells were cultured in ultralow attachment 6-well culture plate using CSC culture medium for 10 days. Following the above treatment, the formed mammospheres were stained with CD44-PE and CD24-FITC antibodies, with the proportion of CD44+/CD24- cells being measured using the flow cytometry. Moreover, ALDH1 expression level of tumor was measured using western blot.



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2.19 Immunohistochemistry and blood bio-chemistry assay To estimate biosafety of PEG-DOX/SN38 NPs, the major organs, such as heart, liver, spleen, lung and kidney, were harvested and stained with H&E following the above treatment. Besides, the blood samples were collected for blood routine examination and blood bio-chemistry.

2.20 Metabolomics analysis To

further

assess

in

vivo

bio-safety

of

PEG-DOX/SN38

NPs,

gas

chromatography-mass spectrometry (GC-MS) was employed to analyze the metabolomics after therapy. In brief, 100 mg of tissues were extracted with 1 mL of mixture solution (methanol/water/chloroform=2.5/1/1, myristic acid d-27 (3 mg/ml) as an internal standard, 1:5 (m/v)), and 30 µL of blood samples were added into 1 mL of extracted solution (isopropanol/acetonitrile/water=3/3/2, v/v) and vortexed for 30 s. After storing at -20 °C overnight, samples were thawed on ice and centrifuged at 12,000 g for 10 min at 4 °C. A portion of supernatant was transferred to a 2 mL of vial for vacuum freeze-drying. Then, the samples were reacted with 80 µL of methoxyamine hydrochloride at 37 °C for 90 min. Afterwards, 80 µL of N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) were added to the above mixture and incubated at 60 °C for 60 min. 1 µL aliquot of the resultant solution was injected into the Agilent 7890A gas chromatograph equipped with a DB-5 MS column (Agilent 119, USA) and a quadrupole mass spectrometer (5975C). The column temperature was kept at 80 °C for 2 min and then increased from 80 °C to 300 °C at a rate of 5 °C/min. The column temperature was kept at 300 °C for 2 min.

2.21 Statistical analysis The data was presented as the means ± standard deviations (SD) from at least three repeated experiments. Statistical analyses were performed using a two-sided Student's t-test. P-value of 0.05 or less was considered to be statistically significant.


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3

Results and Discussion

3.1 Synthesis of PEG-CH=N-DOX Figure S1 exhibited that the Schiff’s base reaction was employed to synthesize the pH-responsive pro-drug (PEG-CH=N-DOX). Its chemical structure was first determined using 1H NMR spectrum (400 MHz, d6-DMSO). As shown in Figure 2A, the disappearance of aldehyde proton peak at 10.53 ppm (-CHO), the appearance of ethylene protons signals of PEG at 3.51 ppm (-CH2CH2O-), and phenyl protons of DOX at 7.65~7.9 ppm in 1H NMR spectrum of PEG-CH=N-DOX demonstrated that PEG-CHO was successfully conjugated to the amino group of DOX. The successful synthesis of PEG-CH=N-DOX was also confirmed using FTIR spectroscopy. In FTIR spectrum of PEG-CH=N-DOX (Figure 2B), the absorption band at 1718 cm-1 (-CHO) almost disappeared and the strong absorption bands at 1100 cm-1 (CH2-O-CH2) and 1623 cm-1 (-CO-) were still present, further confirming that PEG-CH=N-DOX was successfully synthesized. Besides, the CMC value of PEG-CH=N-DOX was also measured, which is 2.3 µg/mL (Figure S2). Thus, we believe that such sufficient low CMC value is beneficial for in vivo applications. To simplify the description, PEG-CH=N-DOX was named as PEG-DOX in following text.

3.2 Modelling self-assembly of SN38 and PEG-DOX As shown in Figure S3 A-C, the interaction distance between SN38 and PEG-DOX (3.8 Å) was similar to that between two SN38 molecules (3.4 Å) and closer than that between two PEG-DOX molecules (4.4 Å), indicating that there existed a strong interaction between SN38 and PEG-DOX molecules. Moreover, due to the amphipathicity and sufficiently low CMC value of PEG-DOX, we speculate that the structure of this cargo-free nano-medicine is similar to the micellar system consisted of amphipathic polymers. Thus, we believed that PEG layer would be exposed on the surface of nano-medicine to improve NPs’ stability, and SN38 would be encapsulated into NPs through the strong interaction between SN38 and PEG-DOX molecules. This



hypothesis was also demonstrated by modelling the self-assembly of SN38 and PEG-DOX, which was shown in Figure S3D and further confirmed that PEG-DOX and SN38 could self-assemble together and form a particle.

Figure 2. (A) 1H NMR spectra and (B) FTIR spectrum of mPEG-CHO, DOX and PEG-DOX; (C) size, zeta potential, drug loading and entrapment efficiency of PEG-DOX/SN38 NPs; (D) TEM image of PEG-DOX/SN38 NPs; (E) in vitro cumulative drug release of PEG-DOX/SN38 NPs in PBS with di℃erent pH values (pH = 5.0 or 7.4) at 37 ℃. Data were presented as the mean ± SD (n = 3).

3.3 Preparation and characterization of PEG-DOX/SN38 NPs As shown in Figure 2C, the average hydrodynamic size of PEG-DOX/SN38 NPs was 185.3 nm with the PDI of 0.354. Additionally, they showed the negatively charged surface. The drug loading of DOX and SN38 in PEG-DOX/SN38 NPs were 215.9 µg per mg NPs (DOX) and 111.0 µg per mg NPs (SN38), respectively. And their


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encapsulation efficiencies of DOX and SN38 were 22.7% and 33.1%, respectively. The morphology of PEG-DOX/SN38 NPs was observed using TEM, which exhibited a spherical shape (Figure 2D). Besides, the serum stability of PEG-DOX/SN38 NPs was studied by measuring their size change after the incubation in a mimic physiological environment (PBS with 10% FBS, pH=7.4). As shown in Figure S4, PEG-DOX/SN38 NPs only showed a slight diameter increase in PBS containing 10% FBS (v/v) at pH 7.4 for 24 h, indicating the better stability of PEG-DOX/SN38 NPs. However, due to the pH-sensitive imine linker in PEG-DOX, an obvious change of NPs’ size distribution under acidic environment (PBS with 10% FBS, pH=5.0) was observed with the results shown in Figure S4 C. It is obvious that PEG-DOX/SN38 NPs quickly assembled into large agglomerates, owing to the PEG detachment from the surface of NPs at pH 5.0. Additionally, these results were confirmed by the TEM images presented in Figure S4 D-F. Therefore, it is further demonstrated that PEG-DOX and SN38 can form into a stable and pH-responsive nano-medicine.

3.4 In vitro drug release The in vitro drug release profiles of PEG-DOX/SN38 NPs were investigated in PBS with different pH values (pH 7.4 and 5.0) at 37 °C to imitate physiological and intracellular conditions, respectively. As shown in Figure 2E, both DOX and SN38 could be rapidly released at acidic condition (pH 5.0). Especially after 24 h incubation, there were 86 % of DOX and 72 % of SN38 released from NPs at pH 5.0, which were 3.3 and 1.6 folds higher than those at pH 7.4, respectively. As we expected, the imine bonds between PEG and DOX would be cleaved under acid environments, resulting in the detachment of hydrophilic PEG layer and the disassembly of nano-medicine.23, 34

Thus, both the DOX and SN38 release from PEG-DOX/SN38 NPs is a

pH-dependent behavior.

3.5 Intracellular drug distribution and cellular uptake To

investigate

the

cellular

uptake

efficiency

of


PEG-DOX/SN38

NPs,


PEG-DOX/SN38 NPs were incubated with MCF7 mammosphere cells for 2 h. Moreover, the physical mixture of free PEG-DOX and SN38 was employed as control. As shown in Figure 3A, both PEG-DOX/SN38 NPs and free PEG-DOX+SN38 could be taken up by MCF7 mammosphere cells, and the results of flow cytometry analysis indicated that there was just a slight increase of MFI (mean fluorescence intensity) in PEG-DOX/SN38 NPs group compared to that in free PEG-DOX+SN38 group (Figure 3C). To further detect the potential tumor penetration property of PEG-DOX/SN38 NPs, MCF7 mammospheres were cultured to mimic tumor tissues which treated with PEG-DOX/SN38 NPs and free PEG-DOX+SN38 subsequently. As shown in Figure 3B and Figure S5, the stronger fluorescence intensities of DOX and SN38 appeared throughout the whole mammosphere, while only weak fluorescence signals were present in the core of the mammosphere treated with free PEG-DOX+SN38. This result was also consistent with that measured using the flow cytometry. In Figure 3D, MFI values of DOX and SN38 in PEG-DOX/SN38 NPs were 1.68 folds and 1.40 folds than those in free PEG-DOX+SN38 group, respectively. More importantly, the percentage of the cells simultaneously exhibiting the fluorescence signals of DOX and SN38 in both MCF7 mammospheres and MCF7 mammosphere cells (Q2 region of Figure 3E) showed a remarkable difference between PEG-DOX/SN38 NPs and free PEG-DOX+SN38 group (Figure 3E). Specifically, the percentage of cells in Q2 region in PEG-DOX/SN38 NPs group were 50.9% and 54.1% in MCF7 mammosphere cells and MCF7 mammosphere, respectively, which were significantly higher than those in free PEG-DOX+SN38 group (10.3% and 20.8%). Based on these results, PEG-DOX/SN38 NPs showed excellent properties of co-delivering DOX and SN38 into the same tumor cells and the deep site of mimic tumor tissues, which are the prerequisites to realize in vitro and in vivo synergistic therapy effect.


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Figure 3. Intracellular cellular uptake and drug distribution. CLSM images of (A) MCF7 mammosphere cells and (B) MCF-7 mammospheres incubated with free PEG-DOX+SN38 or PEG-DOX/SN38 NPs for 2 h; (C) flow cytometry statistic results of MCF7 mammosphere cells or (D) MCF-7 mammospheres incubated with free PEG-DOX+SN38 or PEG-DOX/SN38 NPs for 2 h; (E) flow cytometry statistic results of co-localization percentage of DOX and SN38 in MCF7 mammosphere cells or MCF-7 mammospheres after incubated with free PEG-DOX+SN38 or PEG-DOX/SN38 NPs for 2 h. Data were presented as the mean ± SD (n = 3). * represents p < 0.05, ** represents p < 0.01.

3.6 In vitro cytotoxicity studies To evaluate the anticancer capacity of PEG-DOX/SN38 NPs, MCF-7 mammosphere cells were treated with free PEG-DOX, free SN38, free PEG-DOX+SN38 as well as PEG-DOX/SN38 NPs at various concentrations for 48 h. The MTT assay results suggested that the cell proliferation of MCF7 mammosphere cells was restricted after the different treatments for 48 h (Figure 4A). However, because there was no synergistic therapy effect, the single drug formulations (free PEG-DOX or SN38) showed weaker capacity of suppressing MCF7 mammosphere cells growth compared to the dual drug formulations (free PEG-DOX+SN38 and PEG-DOX/SN38 NPs). Especially for PEG-DOX/SN38 NPs, they exhibited a much higher cytotoxicity on MCF7 mammosphere cells than other groups. To further confirm this result, the IC50 values of different drug formulations were calculated. The IC50 values of



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PEG-DOX/SN38 NPs were 0.2 mg/mL (DOX) and 0.08 mg/mL (SN38), respectively, which were also lower than other groups (Figure 4B). Besides, to estimate the synergistic therapy efficacy of PEG-DOX/SN38 NPs, the combination indexes (CI) of DOX and SN38 were calculated using the following equation: CI=C1/Cm1+C2/Cm2

(7)

where C1 and C2 are the IC50 values of PEG-DOX and SN38 in dual drug formulations, and Cm1 and Cm2 are IC50 values of PEG-DOX and SN38 in single drug formulations, respectively.17, 27, 28 The CI value lower than, equal to, and higher than 1 represent the synergism, additivity and antagonism, respectively. As shown in Figure 4B, the CI value of PEG-DOX/SN38 NPs (0.13) was lower than that of free PEG-DOX+SN38 (0.22), indicating that the synergistic therapy effect of PEG-DOX and SN38 was further improved when they formed into NPs. Moreover, we studied the cell apoptosis induced by different drug formulations. MCF7 mammosphere cells were incubated with different drug formulations at concentration of 0.5 µg/mL (DOX) and 0.24 µg/mL (SN38) for 24 h. As shown in Figure 4C, PEG-DOX/SN38 NPs showed higher percentage of apoptotic MCF7 mammosphere cells (29.6%) compared to other groups. These results demonstrated that PEG-DOX/SN38 NPs could induce apoptosis in more MCF7 mammosphere cells, which was consistent with the results determined using MTT assays.


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Figure 4. Inhibitory capacity of different drug formulations against MCF7 mammosphere cells. (A) Cytotoxicity of MCF7 mammosphere cells incubated with different drug formulations at varying concentrations for 48 h; (B) IC50 (µg/mL) and CI values of different drug formulations; (C) cell apoptosis after treatment of different drug formulations for 24 h. Data were presented as mean ± SD (n = 5), * represents p < 0.05, ** represents p < 0.01.

3.7 The inhibition of breast cancer stem cells 3.7.1 The measurement of hallmark of bCSC As CD44+/CD24- have been used as the specific markers for bCSCs, we estimated the bCSCs-killing capacity of PEG-DOX/SN38 NPs through detecting the proportion of CD44+/CD24- cells after different treatments.27,

28

As shown in Figure 5A,



PEG-DOX/SN38 NPs significantly decreased the percentage of CD44+/CD24- cells in comparison to other drug formulations after MCF7 mammosphere cells obtained a 10-day treatment. Additionally, in order to further confirm the inhibitory ability of PEG-DOX/SN38 NPs against bCSCs, the expression level of ALDH1 in mammospheres was measured following the different treatments. As shown in Figure 5B and Figure S6, a remarkably reduced expression of ALDH1 was measured in PEG-DOX/SN38 NPs group, indicating that PEG-DOX/SN38 NPs displayed the strongest synergistic capacity of inhibiting bCSCs proliferation.

Figure 5. Inhibitory effect of different drug formulations against bCSCs. (A) The percentage of CD44+/CD24- cells measured by flow cytometry and (B) ALDH1 protein levels of MCF7 mammospheres treated with different drug formulations for 10 days; (C) the images and (D) re-formation number of MCF-7 mammosphere cells


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after different treatments for 10 days; (E) the size of mammosphere treated with various drug formulations for 8 days and (F) volume change of mammospheres at the end of treatment (volume normalized to the original volume). Data were presented as mean ± SD (n = 3), ** represents p < 0.01.

3.7.2 Mammosphere formation assay The self-renewal capacity of CSCs is responsible for tumor growth and proliferation.35 According to our assumption, through inhibiting the proliferation of bCSCs using PEG-DOX/SN38 NPs, the tumor regrowth could be finally limited. To demonstrate that, we used MCF7 mammospheres as the mimic tumor models in vitro. Through determining the number and size of mammospheres after different treatments, their inhibitory effects on mammospheres’ regeneration were evaluated. As presented in Figure 5C, the mammosphere cells in untreated group still maintained their tumorigenic capacity of re-forming MCF7 mammosphere. In contrast, both the single drug formulations and the physical mixture of PEG-DOX and SN38 were able to inhibit the self-renewal ability of MCF7 mammosphere cells to some extent. The treatment of PEG-DOX/SN38 NPs significantly suppressing the reformation of MCF7 mammosphere, and the number of the reformed mammospheres was less 10, which was significantly less than other groups (Figure 5D).

3.7.3 MCF7 mammospheres destruction assay Next, the destruction level of MCF7 mammospheres was quantified following the different treatments. As shown in Figure 5E and F, the treatment of PEG-DOX slightly inhibited the mammosphere growth, and the mammosphere volume was 2-fold larger than the original volume of mammasphere after an 8-day treatment. Besides, the inhibitor efficiency of free PEG-DOX+SN38 on mammosphere growth was slightly higher in comparison to that under free SN38 treatment, indicating that there was no synergism or additivity effect for the physical mixture of these two drugs. By contrast, when these two drugs were formed into NPs, the most obvious inhibitory ability against MCF7 mammospheres growth was observed. We believed that both the synergetic inhibitory effect of DOX and SN38 and enhanced penetration ability of



PEG-DOX/SN38 NPs acted the essential roles in killing bCSC, because this cargo-free nano-medicine could deliver more DOX and SN38 into the center region of mammospheres, which was beneficial to maximize the synergism of these two chemotherapeutics.

3.8 In vivo antitumor effects 3.8.1 Passive targeting study In aim to evaluate in vivo antitumor capacity of PEG-DOX/SN38 NPs, we also investigated the passive targeting ability of PEG-DOX/SN38 NPs. As depicted in Figure 6A, there were only 1.0 µg of DOX and 1.9 µg of SN38 accumulated in per mg tumor tissue for free PEG-DOX+SN38 following the administration (i.v.) of different drug formulations. However, for PEG-DOX/SN38 NPs, they exhibited a better drug accumulation in tumors, and the drug content showed 12-fold (DOX) and 4-fold (SN38) enhancement over the groups treated with free drugs, respectively. The better tumor targeting effect for NPs, mainly ascribed to EPR effect, revealed that PEG-DOX/SN38 NPs has satisfied the precondition to exert the effective in vivo therapy effect.

3.8.2 In vivo antitumor efficiency To study the synergistic anticancer efficacy of PEG-DOX/SN38 NPs, MCF7 tumor-bearing mice were injected with different drug formulations through tail vein. As illustrated in Figure 6B, PEG-DOX/SN38 NPs were more effective in comparison to other drug formulations on suppressing tumor growth, and no remarkable recurrence was observed during the whole experiment. On the contrary, a remarkable regrowth of tumor appeared among free PEG-DOX, free SN38 and free PEG-DOX+SN38 groups after ceasing these treatments. At the end of therapy, the tumor volume in PEG-DOX/SN38 NPs group was 7.8, 7.1, 2.8 and 2.3-fold smaller than that in 0.9% NaCl, free PEG-DOX, free SN38 and free PEG-DOX+SN38 groups, respectively (Figure 6B and 6D). And the tumor weight in PEG-DOX/SN38 NPs


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group was the lightest compared to other groups on day 26 post-injection (Figure 6C). Moreover, the TGI of PEG-DOX/SN38 NPs (87.2%) was higher than that of the sum of PEG-DOX (8.6%) and SN38 (64.5%) as well as that of physical mixture of PEG-DOX and SN38 (69.8%), suggesting a significantly synergistic effect for PEG-DOX/SN38 NPs on suppressing tumor growth (Table S1). In addition, the immunohistochemical analysis was performed to confirm the antitumor activity of PEG-DOX/SN38 NPs. As shown in Figure 6E, the H&E images in PEG-DOX/SN38 NPs group depicted more tumor necrosis, indicating PEG-DOX/SN38 NPs treatment possessed a better antitumor efficiency. This finding was consistent with the TUNEL staining assay, which further confirmed the excellent in vivo synergism of PEG-DOX/SN38 NPs.

3.8.3 In vivo anti-bCSCs efficiency To demonstrate whether PEG-DOX/SN38 NPs can eliminate bCSCs in vivo, we also measured the percentage of bCSCs in tumor. Figure 6F shows that the proportion of bCSCs marked with CD44+/CD24- is extremely lower in the tumors treated with PEG-DOX/SN38 NPs, which was only 1.2%. Moreover, ALDH1 level in tumors treated with PEG-DOX/SN38 NPs was significantly down-regulated compared to other groups (Figure 6G). Thus, it is concluded that this cargo-free nano-medicine can effectively eliminate bCSC in vivo, which would be beneficial for achieving the prolonged tumor suppression. Based on these in vivo anti-tumor results, it was found that PEG-DOX/SN38 NPs presented the strongest antitumor capacity in comparison to other groups. As we expected, when PEG-DOX and SN38 self-assemble into NPs, this nano-medicine can effectively accumulate at tumor site with the enhanced cellular uptake and tumor penetration ability, which not only improve the retention of these drugs in tumor, but also is beneficial for these two drugs to enter the center region of tumor. Thus, these advantages will maximize these two drugs’ synergistic therapy efficiency and facilitate them to simultaneously kill non-bCSCs and bCSC, resulting in this novel



nano-medicine displaying an improved in vivo therapeutic efficiency.

Figure 6. PEG-DOX/SN38 NPs exhibit an excellent in vivo anti-tumor capacity. (A) Drug content in tumor tissue of the nude balb/c mice treated with free PEG-DOX+SN38 or PEG-DOX/SN38 NPs; (B) the profiles of tumor growth during the whole experiment; (C) the weight and (D) images of the excised tumor at the end of the treatment; (E) the histological sections of the tumors stained by H&E and TUNEL assay; (F) the proportion of CD44+/CD24- cells and (G) the ALDH1 expression in tumor at the end of the treatment; (H) the body weight of the mice during the experimental period. Data are presented as the mean±SD (n = 5), * represents p < 0.05, ** represents p < 0.01.

3.9 Biosafety 3.9.1 Immunohistochemistry and blood bio-chemistry assay As emphasized above, the biosafety of nano-medicine is a necessary prerequisite for their clinic application.36,

37

Hence, the biosafety of PEG-DOX/SN38 NPs was

systematically evaluated from different aspects. In addition to the negligible body weight loss (Figure 6H), there was no obvious histological damage or inflammation


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appearing in H&E stained images of heart, liver, spleen, lung and kidney after the treatment of PEG-DOX/SN38 NPs (Figure 7A). However, the H&E images in free PEG-DOX+SN38 group showed that there were some inflammatory injuries in heart, liver and kidney.

Figure 7. Biosafety analysis. (A) Histological analysis of normal tissues treated with different drug formulations; (B-E) the blood level of ALT, AST, CRE and BUN and (F-I) blood routine analysis in the mice after different treatments; significantly altered metabolic pathways in plasma (J) and liver (L) (PEG-DOX+SN38 vs. 0.9% NaCl group); significantly altered metabolic pathways in plasma (K) and liver (M) (PEG-DOX/SN38 NPs vs. 0.9% NaCl group). Data are presented as the mean±SD (n = 5), * represents p < 0.05, ** represents p < 0.01.



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Then, the blood analyses including blood chemistry and blood routine examination were performed at the end of in vivo anti-tumor experiment. As presented in Figure 7B and C, the liver function markers including alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were estimated to be normal in each group. However, the kidney function markers, creatinine (CRE) and blood urea nitrogen (BUN), notably decreased in mice treated with free PEG-DOX+SN38 while exhibiting no statistical differences between normal group and PEG-DOX/SN38 NPs group, indicating the obvious advantage of PEG-DOX/SN38 NPs on reducing kidney disturbance (Figure 7D and E). In blood routine examination (Figure 7F-I and Figure S7), we found that all parameters in PEG-DOX/SN38 NPs group displayed normal in comparison to those in 0.9% NaCl group. In contrast, the results of lymphocyte, neutrophil number, mean corpuscular

volume

and

mean

corpuscular

hemoglobin

content

in

free

PEG-DOX+SN38 group showed a statistical difference compared to those in normal group.

3.9.2 Metabolomics profiles of different drug formulations’ toxicity The omics technologies are particularly suitable to estimate the toxicity in both in vitro and in vivo systems.38 Especially for metabolomics, it can rapidly screen for biomarkers related to predefined pathways or processes in bio-fluids and tissues, which can provide mechanistic insight into nano-toxicity.39,

40

In this study, the

GC-MS based metabolomics approach was employed to assess the biochemical changes after chemotherapy with different drug formulations, the low molecular weight compounds in different tissues (69 metabolites for plasma, 112 metabolites for liver, 79 metabolites for kidney) were successfully identified. Then, to search the metabolic pathways and functions related to the in vivo toxicity, the altered metabolites were comprehensively analyzed with Metabo Analyst 3.5 online database. As shown in Table S2, in free PEG-DOX+SN38 group, there were 20 metabolites


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significantly altered in plasma compared to normal group, including the decrease in pyruvic acid, lactic acid, 3-phosphoglycerate, malic acid and amino acids and the increase in palmitoleic acid, which indicated an abnormality in energy metabolism and amino acids metabolism.40,

41

In contrast, only 7 significantly altered plasma

metabolites in PEG-DOX/SN38 NPs vs. normal group were distinguished (Table S3). Moreover, as shown in Table S4, 21 significantly altered liver metabolites in free PEG-DOX+SN38 vs. normal group were identified, such as an increase in malic acid, fumaric acid and so on, while there were only 4 significantly altered liver metabolites in PEG-DOX/SN38 NPs vs. normal group (Table S5). Since malic acid and fumaric acid participate in the TCA cycle, their accumulation in liver would hinder the energy metabolism of liver mitochondria.42,

43

These results were also confirmed by the

metabolism pathway analysis. In Figure 7J and L, after the treatment of free PEG-DOX+SN38, there were more than 20 pathways (including valine, leucine and isoleucine biosynthesis, methane metabolism, phenylalanine metabolism and so on) being over-activated through the analysis of plasma sample and liver tissue, whereas only few pathways were activated by the PEG-DOX/SN38 NPs treatment (Figure 7K and M). Based on the results of immunohistochemistry analysis, blood bio-chemistry assay and metabolomics, it is demonstrated that this cargo-free and pH-responsive nano-medicine possesses the preferable biosafety for further medical application, because they have no additional toxicity induced by carrier materials or chemical modifications as well as they showed the enhanced passive targeting ability.

4

Conclusions

In summary, we have developed a cargo-free and pH-responsive nano-medicine for co-delivery of PEG-DOX and SN38 to simultaneously eliminate bCSCs and non-bCSCs. As a result of pH-sensitive cleavage between PEG and DOX, PEG-DOX/SN38 NPs could release DOX and SN38 more efficiently in acidic environment (pH 5.0) than that in physiological condition (pH 7.4). Due to the



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synergistic anticancer efficacy of DOX and SN38, PEG-DOX/SN38 NPs exhibited the most cytotoxicity against both bCSCs and non-bCSCs. In addition, since their improved passive tumor targeting abilities with an enhanced cellular uptake, PEG-DOX/SN38 NPs also showed a significantly improved in vivo therapeutic efficacy with fewer side effects. Overall, this cargo-free nano-medicine may provide a promising approach for the combinational therapy in breast cancer treatment along with the properties of easy scale-up and clinical translation.

Associated Content: The supporting information of this paper is available free of charge on the ACS Publications website (http://pubs.acs.org), and the contents of the material are listed as following: Synthetic route of PEG-DOX; CMC value measurement of PEG-DOX; modelling the self-assembly of SN38 and PEG-DOX; stability of mPEG-DOX/SN38 NPs; 3D images of MCF7 mammospheres obtained by CLSM; ALDH1 expression levels of MCF7 mammospheres; blood routine analysis; tumor growth inhibition (TGI) during the treatment; significantly altered metabolites in plasma and liver after the treatment of 0.9% NaCl, PEG-DOX+SN38 and PEG-DOX/SN38 NPs.

Acknowledgements This work was supported by the National Natural Science Foundation of China (51703178, 81770728, 81570655), the Fundamental Research Funds for the Central Universities

(xjj2016084),

the

China

Postdoctoral

Science

Foundation

(2015M580855), and the Postdoctoral Science Foundation of Shaanxi Province (2016BSHEDZZ100). Additionally, we thank Axin Lu at instrument analysis center of Xi'an Jiaotong University for her assistance with the UPLC-MS analysis.

References


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


1.

Huang, R.; Rofstad, E. K. Cancer stem cells (CSCs), cervical CSCs and targeted

therapies. Oncotarget 2017, 8 (21), 35351-35367. 2.

Reya, T.; Morrison, S. J.; Clarke, M. F.; Weissman, I. L. Stem cells, cancer, and

cancer stem cells. Nature 2001, 414 (6859), 105-111. 3.

Meacham, C. E.; Morrison, S. J. Tumour heterogeneity and cancer cell plasticity.

Nature 2013, 501 (7467), 328-337. 4.

Clarke, M. F.; Dick, J. E.; Dirks, P. B.; Eaves, C. J.; Jamieson, C. H. M.; Jones, D.

L.; Visvader, J.; Weissman, I. L.; Wahl, G. M. Cancer stem cells--perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res. 2006, 66 (19), 9339-44. 5.

Agliano, A.; Calvo, A.; Box, C. The challenge of targeting cancer stem cells to

halt metastasis. Semin. Cancer Biol. 2017, 44, 25-42. 6.

Li, S.-Y.; Sun, R.; Wang, H.-X.; Shen, S.; Liu, Y.; Du, X.-J.; Zhu, Y.-H.; Wang, J.

Combination therapy with epigenetic-targeted and chemotherapeutic drugs delivered by nanoparticles to enhance the chemotherapy response and overcome resistance by breast cancer stem cells. J. Control. Release 2015, 205, 7-14. 7.

Al Faraj, A.; Shaik, A. S.; Ratemi, E.; Halwani, R. Combination of

drug-conjugated SWCNT nanocarriers for efficient therapy of cancer stem cells in a breast cancer animal model. J. Control. Release 2016, 225, 240-251. 8.

Gill, M. R.; Harun, S. N.; Halder, S.; Boghozian, R. A.; Ramadan, K.; Ahmad, H.;

Vallis, K. A. A ruthenium polypyridyl intercalator stalls DNA replication forks, radiosensitizes human cancer cells and is enhanced by Chk1 inhibition. Sci. Rep. 2016, 6. 9.

Yang, Z.; Zhang, X.; Luo, X.; Jiang, Q.; Liu, J.; Jiang, Z. Enzymatic Synthesis of

Poly(butylene-co-sebacate-co-glycolate) Copolyesters and Evaluation of the Copolymer Nanoparticles as Biodegradable Carriers for Doxorubicin Delivery. Macromolecules 2013, 46 (5), 1743-1753. 10. Zhang, H.; Wang, J.; Mao, W.; Huang, J.; Wu, X.; Shen, Y.; Sui, M. Novel SN38 conjugate-forming nanoparticles as anticancer prodrug: In vitro and in vivo studies. J.



Control. Release 2013, 166 (2), 147-158. 11. Yang, Z.; Luo, H.; Cao, Z.; Chen, Y.; Gao, J.; Li, Y.; Jiang, Q.; Xu, R.; Liu, J. Dual-targeting hybrid nanoparticles for the delivery of SN38 to Her2 and CD44 overexpressed human gastric cancer. Nanoscale 2016, 8 (22), 11543-11558. 12. Wang, H.; Agarvval, P.; Zhao, S.; Xu, R. X.; Yu, J.; Lu, X.; He, X. Hyaluronic acid-decorated dual responsive nanoparticles of Pluronic F127, PLGA, and chitosan for targeted co-delivery of doxorubicin and irinotecan to eliminate cancer stem-like cells. Biomaterials 2015, 72, 74-89. 13. Hirose, S.; Suzuki, Y. Invitro transcription of eukaryotic genes is affected differently by the degree of DNA supercoiling. Proc. Natl. Acad. Sci. U. S. A. 1988, 85 (3), 718-722. 14. Liu, L. F.; Wang, J. C. Supercoiling of the DNA-template during transcription. Proc. Natl. Acad. Sci. U. S. A. 1987, 84 (20), 7024-7027. 15. Glasgow, M. D. K.; Chougule, M. B. Recent developments in active tumor targeted multifunctional nanoparticles for combination chemotherapy in cancer treatment and imaging. J. Biomed. Nanotechnol. 2015, 11 (11), 1859-1898. 16. Shi, J.; Kantoff, P. W.; Wooster, R.; Farokhzad, O. C. Cancer nanomedicine: progress, challenges and opportunities. Nat. Rev. Cancer 2017, 17 (1), 20-37. 17. Chen, F.; Zhao, Y.; Pan, Y.; Xue, X.; Zhang, X.; Kumar, A.; Liang, X.-J. Synergistically enhanced therapeutic effect of a carrier-free HCPT/DOX nanodrug on breast cancer cells through improved cellular drug accumulation. Mol. Pharmaceut. 2015, 12 (7), 2237-2244. 18. Sun, Q.; Radosz, M.; Shen, Y. Challenges in design of translational nanocarriers. J. Control. Release 2012, 164 (2), 156-169. 19. Hu, S.; Lee, E.; Wang, C.; Wang, J.; Zhou, Z.; Li, Y.; Li, X.; Tang, J.; Lee, D. H.; Liu, X.; Shen, Y. Amphiphilic drugs as surfactants to fabricate excipient-free stable nanodispersions of hydrophobic drugs for cancer chemotherapy. J. Control. Release 2015, 220, 175-179. 20. Mou, Q.; Ma, Y.; Zhu, X.; Yan, D. A small molecule nanodrug consisting of


Page 30 of 41

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


amphiphilic targeting ligand-chemotherapy drug conjugate for targeted cancer therapy. J. Control. Release 2016, 230, 34-44. 21. Moghimi, S. M.; Hunter, A. C.; Murray, J. C. Nanomedicine: current status and future prospects. Faseb J. 2005, 19 (3), 311-330. 22. Hu, M.; Huang, P.; Wang, Y.; Su, Y.; Zhou, L.; Zhu, X.; Yan, D. Synergistic combination chemotherapy of camptothecin and floxuridine through self-assembly of amphiphilic drug-drug conjugate. Bioconjugate Chem. 2015, 26 (12), 2497-2506. 23. Zhang, Y.; Yang, C.; Wang, W.; Liu, J.; Liu, Q.; Huang, F.; Chu, L.; Gao, H.; Li, C.; Kong, D.; Liu, Q.; Liu, J. Co-delivery of doxorubicin and curcumin by pH-sensitive prodrug nanoparticle for combination therapy of cancer. Sci. Rep. 2016, 6. 24. Zhang, T.; Huang, P.; Shi, L.; Su, Y.; Zhou, L.; Zhu, X.; Yan, D. Self-assembled nanoparticles of amphiphilic twin drug from floxuridine and bendamustine for cancer therapy. Mol. Pharmaceut. 2015, 12 (7), 2328-2336. 25. Ohya, Y.; Kuroda, H.; Hirai, K.; Ouchi, T. Synthesis and cytotoxic activity of conjugates of monomethoxy poly(ethylene-glycol) end-capped with doxorubicin via ester, amide, or Schiff-base bond. Journal of Bioactive and Compatible Polymers 1995, 10 (1), 51-66. 26. Saito, H.; Hoffman, A. S.; Ogawa, H. I. Delivery of doxorubicin from biodegradable PEG hydrogels having Schiff base linkages. Journal of Bioactive and Compatible Polymers 2007, 22 (6), 589-601. 27. Yang, Z.; Sun, N.; Cheng, R.; Zhao, C.; Liu, J.; Tian, Z. Hybrid nanoparticles coated with hyaluronic acid lipoid for targeted co-delivery of paclitaxel and curcumin to synergistically eliminate breast cancer stem cells. J. Mater. Chem. B 2017, 5 (33), 6762-6775. 28. Yang, Z.; Sun, N.; Cheng, R.; Zhao, C.; Liu, Z.; Li, X.; Liu, J.; Tian, Z. pH multistage responsive micellar system with charge-switch and PEG layer detachment for co-delivery of paclitaxel and curcumin to synergistically eliminate breast cancer stem cells. Biomaterials 2017, 147, 53-67.



29. Zhang, X. F.; Liu, B.; Yang, Z.; Zhang, C.; Li, H.; Luo, X.; Luo, H. Y.; Gao, D.; Jiang, Q.; Liu, J.; Jiang, Z. Z. Micelles of enzymatically synthesized PEG-poly(amine-co-ester) block copolymers as pH-responsive nanocarriers for docetaxel delivery. Colloids and Surfaces B-Biointerfaces 2014, 115, 349-358. 30. Liu, B.; Zhang, X. F.; Chen, Y.; Yao, Z. C.; Yang, Z.; Gao, D.; Jiang, Q.; Liu, J.; Jiang, Z. Z. Enzymatic synthesis of poly(omega-pentadecalactone-co-butylene-co-3,3 '-dithiodipropionate) copolyesters and self-assembly of the PEGylated copolymer micelles as redox-responsive nanocarriers for doxorubicin delivery. Polymer Chemistry 2015, 6 (11), 1997-2010. 31. Berendsen, H. J. C.; Postma, J. P. M.; Vangunsteren, W. F.; Dinola, A.; Haak, J. R. Molecular-dynamics with coupling to an external bath. J. Chem. Phys. 1984, 81 (8), 3684-3690. 32. Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual molecular dynamics. J. Mol. Graph. Model. 1996, 14 (1), 33-38. 33. Li, W.; Ma, H.; Zhang, J.; Zhu, L.; Wang, C.; Yang, Y. Unraveling the roles of CD44/CD24 and ALDH1 as cancer stem cell markers in tumorigenesis and metastasis. Sci. Rep. 2017, 7. 34. Yu, J.; Ha, W.; Chen, J.; Shi, Y.-p. pH-Responsive supramolecular hydrogels for codelivery of hydrophobic and hydrophilic anticancer drugs. RSC Adv. 2014, 4 (103), 58982-58989. 35. O'Brien, C. A.; Kreso, A.; Jamieson, C. H. M. Cancer Stem Cells and Self-renewal. Clin. Cancer Res. 2010, 16 (12), 3113-3120. 36. Su, H.; Wang, Y.; Gu, Y.; Bowman, L.; Zhao, J.; Ding, M. Potential applications and human biosafety of nanomaterials used in nanomedicine. J. Appl. Toxicol. 2018, 38 (1), 3-24. 37. Schuetz, C. A.; Juillerat-Jeanneret, L.; Mueller, H.; Lynch, I.; Riediker, M.; NanoImpactNet, C. Therapeutic nanoparticles in clinics and under clinical evaluation. Nanomedicine 2013, 8 (3), 449-467. 38. Schnackenberg, L. K.; Sun, J.; Beger, R. D. Metabolomics techniques in


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nanotoxicology studies. Methods in molecular biology (Clifton, N.J.) 2012, 926, 141-56. 39. Wishart, D. S. Emerging applications of metabolomics in drug discovery and precision medicine. Nat. Rev. Drug Discov. 2016, 15 (7), 473-484. 40. Zhou, X.; Meng, X.; Cheng, L.; Su, C.; Sun, Y.; Sun, L.; Tang, Z.; Fawcett, J. P.; Yang, Y.; Gu, J. Development and application of an MSALL-based approach for the quantitative analysis of linear polyethylene glycols in rat plasma by liquid chromatography triple-quadrupole/time-of-flight mass spectrometry. Anal. Chem. 2017, 89 (10), 5193-5200. 41. Cambien, F.; Warnet, J. M.; Vernier, V.; Ducimetiere, P.; Jacqueson, A.; Flament, C.; Orssaud, G.; Richard, J. L.; Claude, J. R. An epidemiologic appraisal of the associations between the fatty-acids esterifying serum-cholesterol and some cardiovascular risk-factors in middle-aged men. Am. J. Epidemiol. 1988, 127 (1), 75-86. 42. Patel, D. P.; Krausz, K. W.; Xie, C.; Beyoglu, D.; Gonzalez, F. J.; Idle, J. R. Metabolic profiling by gas chromatography-mass spectrometry of energy metabolism in high-fat diet-fed obese mice. Plos One 2017, 12 (5). 43. Tretter, L.; Patocs, A.; Chinopoulos, C. Succinate, an intermediate in metabolism, signal transduction, ROS, hypoxia, and tumorigenesis. BBA-Bioenergetics 2016, 1857 (8), 1086-1101.



Figure 1. Schematic illustration of cargo-free and pH responsive nano-medicne with enhanced crossmembrane transportation and controlled drug release. 171x101mm (300 x 300 DPI)


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Figure 2. (A) 1H NMR spectra and (B) FTIR spectrum of mPEG-CHO, DOX and PEG-DOX; (C) size, zeta potential, drug loading and entrapment efficiency of PEG-DOX/SN38 NPs; (D) TEM image of PEG-DOX/SN38 NPs; (E) in vitro cumulative drug release of PEG-DOX/SN38 NPs in PBS with different pH values (pH = 5.0 or 7.4) at 37 ℃. Data were presented as the mean ± SD (n = 3). 171x161mm (300 x 300 DPI)



Figure 3. Intracellular cellular uptake and drug distribution. CLSM images of (A) MCF7 mammosphere cells and (B) MCF-7 mammospheres incubated with free PEG-DOX+SN38 or PEG-DOX/SN38 NPs for 2 h; (C) flow cytometry statistic results of MCF7 mammosphere cells or (D) MCF-7 mammospheres incubated with free PEG-DOX+SN38 or PEG-DOX/SN38 NPs for 2 h; (E) flow cytometry statistic results of co-localization percentage of DOX and SN38 in MCF7 mammosphere cells or MCF-7 mammospheres after incubated with free PEG-DOX+SN38 or PEG-DOX/SN38 NPs for 2 h. Data were presented as the mean ± SD (n = 3). * represents p < 0.05, ** represents p < 0.01. 171x90mm (300 x 300 DPI)


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Figure 4. Inhibitory capacity of different drug formulations against MCF7 mammosphere cells. (A) Cytotoxicity of MCF7 mammosphere cells incubated with different drug formulations at varying concentrations for 48 h; (B) IC50 (µg/mL) and CI values of different drug formulations; (C) cell apoptosis after treatment of different drug formulations for 24 h. Data were presented as mean ± SD (n = 5), * represents p < 0.05, ** represents p < 0.01. 82x157mm (300 x 300 DPI)



Figure 5. Inhibitory effect of different drug formulations against bCSCs. (A) The percentage of CD44+/CD24cells measured by flow cytometry and (B) ALDH1 protein levels of MCF7 mammospheres treated with different drug formulations for 10 days; (C) the images and (D) re-formation number of MCF-7 mammosphere cells after different treatments for 10 days; (E) the size of mammosphere treated with various drug formulations for 8 days and (F) volume change of mammospheres at the end of treatment (volume normalized to the original volume). Data were presented as mean ± SD (n = 3), ** represents p < 0.01. 170x178mm (300 x 300 DPI)


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Figure 6. PEG-DOX/SN38 NPs exhibit an excellent in vivo anti-tumor capacity. (A) Drug content in tumor tissue of the nude balb/c mice treated with free PEG-DOX+SN38 or PEG-DOX/SN38 NPs; (B) the profiles of tumor growth during the whole experiment; (C) the weight and (D) images of the excised tumor at the end of the treatment; (E) the histological sections of the tumors stained by H&E and TUNEL assay; (F) the proportion of CD44+/CD24- cells and (G) the ALDH1 expression in tumor at the end of the treatment; (H) the body weight of the mice during the experimental period. Data are presented as the mean±SD (n = 5), * represents p < 0.05, ** represents p < 0.01. 171x132mm (300 x 300 DPI)



Figure 7. Biosafety analysis. (A) Histological analysis of normal tissues treated with different drug formulations; (B-E) the blood level of ALT, AST, CRE and BUN and (F-I) blood routine analysis in the mice after different treatments; significantly altered metabolic pathways in plasma (J) and liver (L) (PEGDOX+SN38 vs. 0.9% NaCl group); significantly altered metabolic pathways in plasma (K) and liver (M) (PEG-DOX/SN38 NPs vs. 0.9% NaCl group). Data are presented as the mean±SD (n = 5), * represents p < 0.05, ** represents p < 0.01. 171x189mm (300 x 300 DPI)


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Table of contents graphic 66x34mm (300 x 300 DPI)


Cargo-Free Nanomedicine with pH Sensitivity for Codelivery of DOX

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