Targeted delivery of Auristatin PE to Hep G2 cells using folate - conjugated boron nitride nanotubes

Journal Pre-proof Targeted delivery of Auristatin PE to Hep G2 cells using folate conjugated boron nitride nanotubes

Wei Li, Xi Xie, Tiantian Wu, Huan Yang, Yanan Peng, Lijie Luo, Yongjun Chen PII:

S0928-4931(19)31677-7

DOI:

https://doi.org/10.1016/j.msec.2019.110509

Reference:

MSC 110509

To appear in:

Materials Science & Engineering C

Received date:

6 May 2019

Revised date:

10 October 2019

Accepted date:

28 November 2019

Please cite this article as: W. Li, X. Xie, T. Wu, et al., Targeted delivery of Auristatin PE to Hep G2 cells using folate - conjugated boron nitride nanotubes, Materials Science & Engineering C (2018), https://doi.org/10.1016/j.msec.2019.110509

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© 2018 Published by Elsevier.

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Targeted delivery of Auristatin PE to Hep G2 cells using folate conjugated boron nitride nanotubes Wei Li,a Xi Xie,b Tiantian Wu,a Huan Yang,a Yanan Peng,a Lijie Luo,a and Yongjun Chena* a

State Key Laboratory of Marine Resource Utilization in South China Sea, School of Materials

Hainan Provincial Key Laboratory for Tropical Hydrobiology and Biotechnology, College of

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b

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Science and Engineering, Hainan University, Haikou 570228, China.

Marine Science, Hainan University, Haikou 570228, China *

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Corresponding author at: State Key Laboratory of Marine Resource Utilization in South China

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Sea, School of Material Science and Engineering, Hainan University, Haikou 570228, China

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E-mail: [email protected]

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Abstract Auristatin PE (PE) as an anti-microtubule agent possesses good anticancer activity. However, the poor target effect and strong side effect limit its clinical

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applications. Targeted delivery of PE may overcome the disadvantages associated with PE, being very conducive to continuing clinical trials of PE. Boron nitride nanotubes (BNNTs) with unique physical and chemical properties have attracted considerable attention in drug delivery. Herein, a targeted drug delivery strategy based on folate-conjugated boron nitride nanotubes (BNNTs-FA) was used to improve the efficacy of PE. It was found that PE was successfully loaded onto BNNTs-FA via π-π stacking and hydrogen bonding interactions. BNNTs-FA@PE exhibited stronger cytotoxicity to Hep G2 cells than free PE and BNNTs@PE complexes due to the increased cellular uptake of PE mediated by the FA receptor.

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BNNTs-FA@PE showed excellent antiproliferative activities in a dose- and time-dependent manner. Furthermore, BNNTs-FA@PE induced apoptosis of Hep G2 cells via an intrinsic mitochondria–mediated pathway by reducing the mitochondrial membrane potential, activating Caspase-9 and Caspase-3. The construction of BNNTs-FA@PE system successfully improves the target effect of PE and may be

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very promising for the treatment of liver cancer in the future.

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Keywords: Auristatin PE; Boron nitride nanotubes; Targeted drug delivery;

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Cytotoxicity

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1. Introduction

Liver cancer has a very high prevalence among cancers in general and remains to be

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one of the leading causes of death across the world.[1] More and more attentions have

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been focusing on marine bioactive peptides due to their novel chemical and diverse

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biological properties against cancer cells.[2] Auristatin PE (PE)，a derivative of dolastatin 10, [3] is a microtubular targeting and depolymerizing agent. PE exhibits excellent antitumor activity both in vitro and in vivo.[4-6] The anticancer activities of PE are stronger than other drugs including paclitaxel, vincristine and adriamycin.[7] PE has ever entered Phase II clinical trials, however, as many other small molecule drugs, PE also has shortcomings such as short circulation time, poor target effect, strong side effect, and inefficiency as a single agent.[8] Thus, its clinical development was no longer pursued. In order to overcome the disadvantages assisted with PE, analogues of PE have been designed and synthesized but only one of them showed a better safety potential than PE.[9] Efforts should be took to search for more ways to

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solve the problem assisted with PE. Nanoscale materials such as liposomes[10], protein nanoparticles[11], micelle[12] and carbon nanotubes(CNTs)[13] could deliver small molecule drugs selectively to the therapeutic sites, reducing their side effects and enhancing their therapeutic efficacy.[14, 15] Nanotechnology as an emerging technology paves new way for targeted PE delivery and clinical trials of PE.

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Boron nitride nanotubes (BNNTs) and carbon nanotubes (CNTs) are analogous in structure but different in properties. BNNTs are wide band-gap semiconductors with

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optical transitions in the UV range above 5.5 eV independent of tube diameter and

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chirality[16] which makes BNNTs very different from CNTs and attractive for

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applications as fluorescence materials.[17] Furthermore, BNNTs possess excellent

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biocompatibility with lower cytotoxicity as compared to CNTs when they are

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incubated with various cells.[18] BN nanomaterials are now regarded as very promising materials in biotechnology. For example, BN nanospheres can be used as a

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carrier for the delivery of doxorubicin (DOX).[19,20] hBN nanosheets have been investigated for various sensor applications, e.g. serotonin detection,[21] anticancer drug detection,[22] β-agonists determination,[23] cypermethrin (CYP) detection,[24] and diethylstilbestrol analysis.[25] BN quantum dots were developed and applied for sensor applications, e.g. organophosphate pesticides[26] and cTnI detection.[27] BNNTs are regarded as one of the most fascinating nanomaterials proposed for cancer therapies, including boron neutron capture therapy (BNCT)[28], β- emission source[29], gene and drug delivery[30-32]. In vivo studies revealed that BNNTs could accumulate in liver and be ultimately eliminated via renal excretion with no

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impairments in blood, liver and kidney functionality, and no significant adverse effects were observed after BNNTs were injected in rabbits continuously for 7 days,[33, 34] hinting that BNNTs could be a good carrier for the drugs in treating liver disease and could be eliminated after drug release. Furthermore, functionalization of BNNTs with targeting moieties folic acid (FA) whose receptors are over-expressed on

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the surfaces of human tumors[35] can improve the targeted drug delivery efficiency of BNNTs. However, the application of BNNTs in biomedicine remains largely

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unexplored.

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In the present work, a novel targeted PE delivery system against liver cancer cells

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based on FA conjugated BNNTs (BNNTs-FA) was constructed. The cytotoxicity of

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BNNTs-FA@PE on liver cancer cells (Hep G2 cells) was evaluated. Furthermore, the

2. Experimental

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possible mechanisms that BNNTs-FA@PE kills cancer cells were studied.

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2.1 Preparation BNNTs-FA@PE

BNNTs were synthesized using a solid-state reaction.[36] Then, these synthesized BNNTs were hydroxylated and FA was grafted onto them according to the work of Ferreira et al.[35] 2.5 mg BNNTs-FA and 20 µL of 5 mgmL-1 PE (MedChemExpress, USA) were added into deionized water (5 mL). The mixture was ultrasonically treated at room temperature in the dark for 6 h, followed by dialysis to remove unloaded PE. The standard curve and regression equation of PE (y=2.362 x+0.0605, R2 =0.9988) were established to calculate the content of PE. The loading capacity of BNNTs for PE was calculated using the following equation:

Journal Pre-proof Loading capacity (gmg-1 )=

Mass of initial PE-Mass of free PE Mass of BNNTs-FA

The remaining BNNTs-FA@PE in the dialysis bag was collected and freeze dried. Transmission electron microscopy (TEM, JEM 2100, JOEL), fourier transform infrared (FTIR, PerkinElmer) spectroscopy, ultraviolet-visible (UV-Vis) absorption spectroscopy

(Mapada

3000

spectrophotometer)

and

X-ray

photoelectron

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spectroscopy (XPS, Kratos AXIS-SUPRA) were used to analyze BNNTs-FA and BNNTs-FA@PE. Size distribution and Zeta potential were measured using a Zeta

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sizer Nano ZS system (NanoPlus-3/NanoPlus-AT, Micromeritics, America).

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BNNTs@PE, which is regarded as the control in some in vitro test, were prepared

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via the same method except the conjunction of FA, and will be reported elsewhere.

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2.2 In vitro cytotoxicity of BNNTs-FA complexes

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In vitro cytotoxicity was analyzed using Cell Counting Kit-8 assay (CCK-8, Beyotime, China). Hep G2 cells and L02 cells were incubated with different

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concentrations of BNNTs-FA (0-100 gmL-1). The obtained cultures were then treated with 100 L culture medium supplemented with 10 L CCK-8 solution for 2 h. Absorbance at 450 nm was measured using a microplate reader (Multiskan FC, Thermo Fisher Scientific, USA). 2.3 Cellular uptake Internalization of BNNTs-FA was assessed by transmission electron microscopy (TEM). Hep G2 cells were incubated with 50 gmL-1 BNNTs-FA for 4 h. After that, the cells were fixed with 2.5% glutaraldehyde and 1% OsO4, and then embedded in epoxy resin (EMBed 812) and baked at 60 C for 48 h. Ultra-thin sections (70 nm

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thick) were cut with an ultramicrotome (Leica UC7). Finally, the cells were examined with a Bio-TEM (HITACHI, HT7700). Interactions between BNNTs-FA@PE and Hep G2 cells were further investigated by scanning electron microscope (SEM, Hitachi S-4800). Hep G2 cells were incubated with BNNTs-FA and BNNTs-FA@PE at an equivalent concentration of

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BNNTs-FA (13.78 gmL-1) for 24 h, and sequentially fixed and dehydrated. All samples were sputtered with gold prior to SEM observation. The elemental mappings

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of B and N were conducted by energy-dispersive X-ray spectroscopy (EDS) under

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SEM.

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2.4 Actin staining

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Hep G2 cells were treated with BNNTs-FA or BNNTs-FA@PE (at an equivalent

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BNNTs-FA concentration of 12.37 gmL-1) for 24 h. Actin staining of the cells was performed with a 100 M FITC-phalloidin solution (Shanghai Yeasen Biotechnology

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Co., Ltd, China) following the manufacturer’s recommendations. Mounted samples were imaged using a confocal laser scanning microscope (CLSM, Leica TCS SP8). The fluorescence emission of BNNTs-FA was tested from 603 nm to 731 nm at an excitation wavelength of 561 nm. 2.5 In vitro anticancer effects of BNNTs-FA@PE on Hep G2 cells The cell viability of the cultures after 24 h incubation with BNNTs-FA@PE was assessed with the ReadyProbes™ Cell Viability Imaging Kit (Blue/Green) (Invitrogen™). Stained cells were imaged using fluorescence microscopy (BX71,

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Olympus, Japan). The cell cytotoxicity of PE, BNNTs@PE and BNNTs-FA@PE was further analyzed using CCK-8 assay. 2.6 Flow cytometry analysis of apoptosis Hep G2 cells were exposed to BNNTs-FA@PE for 24 h. The apoptosis was measured through Annexin V-FITC/PI apoptosis kit (Shanghai Yeasen Biotechnology

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Co., Ltd, China) following the manufacturer’s recommendations and analyzed by flow cytometry on guava easyCyte (Merck Millipore, USA).

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2.7 Determination of Mitochondrial Membrane Potential (ΔΨ)

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Hep G2 cells were incubated with PE, BNNTs@PE and BNNTs-FA@PE (at an

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equivalent PE concentration of 3.2 ngmL-1) for 12 h. The JC-1 mitochondrial

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Apoptosis Detection Kit (Beyotime, Shanghai, China) was used to detect ΔΨ

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disruption following the manufacturer’s protocols. The fluorescence images were recorded by fluorescence microscope.

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2.8 Western Blot Analysis

Hep G2 cells were treated with PE, BNNTs@PE and BNNTs-FA@PE (at an equivalent PE concentration of 3.2 ngmL-1) for 12 h. The expression of Bax, Bcl-2, procaspase 9, procaspase 3 and ɑ-tubulin in Hep G2 cells was analyzed by western blot analysis. GAPDH was used as internal control. 2.9 Detection of Caspase-3/7 activity After treatment with control culture, PE and BNNTs@PE for 24 h, respectively, Hep G2 cells were incubated with 100 μL of Caspase-Glo3/7 Luminescence reagent (Promega, Corp., Madison, WI, USA) at room temperature for 1 h. The activities of

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Caspase 3/7 were determined by quantifying the luminescence property (Promega, USA). 2.10 Statistical analysis Statistical analyses were performed using the one-way ANOVA method. Data were presented as mean ± standard deviation (SD). A P value of < 0.05 was determined to

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be significant. Error bars in all figures represented the standard error of the mean.

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3 Results and Discussion

3.1 Characterization and in vitro cytotoxicity of BNNTs-FA complexes

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The SEM and TEM images (Figure S1a-c) show that large quantities

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bamboo-shaped BNNTs with an average diameter of about 90 nm are synthesized.

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These BNNTs exhibit three main photoluminescence (PL) emission bands centered at

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419, 489 and 594 nm, indicating the excellent PL property of BNNTs in visible-light range that favors the localization and tracing of BNNTs in cells (Figure S1d). These

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BNNTs were treated with nitric acid to introduce -OH group, then, FA is grafted onto BNNTs-OH via the esterification reaction between the -COOH groups on FA and -OH groups on the BNNTs-OH (Figure S2a-c).[37] Cytotoxicity of vehicles is a crucial issue that needs to be addressed before their utilization as drug carrier. Although BNNTs and FA were reported to be biocompatible, it is necessary to validate the cytotoxicity of BNNTs-FA complexes against L02 cells and Hep G2 cells because of no relevant report before. As shown in Figure S3, the viability of Hep G2 and L02 cells is slightly decreased with the increase of BNNTs-FA concentrations up to 100 gmL-1 after 72 h incubation, but

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they are still higher than 90%, indicating no adverse effects of BNNTs-FA on L02 cells and Hep G2 cells in terms of metabolic activity. 3.2 Characterization of BNNTs-FA@PE complexes The morphologies/structures of the BNNT-FA@PE nanohybrids were also analyzed by TEM (Figure 1 a-b). The images show the presence of amorphous-like

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layers on the sidewalls of BNNTs (indicated by red arrows in Figure 1b), which are not present on the starting BNNTs-OH (Figure 1a) or BNNTs (Figure S1c), indicating

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the wrapping of BNNTs-OH with FA and PE layer. The size distribution was

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measured in deionized water. As shown in Figure 1c-d, the average hydrodynamic

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diameter of BNNTs-OH and BNNTs-FA@PE is 1367.6 nm and 1422.6 nm,

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respectively. The zeta potential was measured in deionized water to evaluate the

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surface charge of BNNT-FA@PE, and the results show that zeta potential values of BNNTs-OH (Figure 1e) changed from -44.31 mV to -38.34 mV in BNNTs-FA@PE

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(Figure 1f). These differences between BNNTs-OH and BNNTs-FA@PE indicate FA and PE are successfully loaded onto BNNTs-OH and BNNTs-FA@PE is relative stable.

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Figure 1. TEM images of (a) BNNTs-OH (the insert is the enlarged TEM images) and (b) BNNTs-FA@PE (the insert is the enlarged TEM images). Zeta potentials of (c) BNNTs-OH and (d) BNNTs-FA@PE, and the size distribution of (e) BNNTs-OH and (f) BNNTs-FA@PE.

The FTIR spectra of PE, BNNTs-FA and BNNTs-FA@PE were shown in Figure 2. Compared with BNNTs-FA, BNNTs-FA@PE has additional absorption bands at 2937-2829 cm-1 and 1250-950 cm-1 (Figure 2a), which are attributed to the symmetric stretching and asymmetric stretching of C-H bonds in the methylene group (CH2) of PE molecules, respectively. The wave form of correlation peaks is fitted based on gaussian model (Figure 2b). It can be found that the peaks of hydroxyl group (-OH) at 3431 cm-1 and ester bonds (C-O-B) at 1149 cm-1 and 1035 cm-1 in BNNTs-FA sample

Journal Pre-proof shift to 3425 cm-1, 1172 cm-1 and 1055 cm-1 in BNNTs-FA@PE sample, respectively. And the peak of amide II band (N-H) at 1633 cm-1 in PE sample[38] shifts to 1618 cm-1 in BNNTs-FA@PE sample. These shifts reveal the existence of intermolecular hydrogen bonds between the non-binding electron pair of N or O atoms (amide group) in the PE and O atoms (-OH groups or ester bonds) in the BNNTs-FA. In BNNTs-FA, the peak centered at 1276 cm-1 can be assigned to N-B-N variable-angle vibration, and

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the peaks at 1392 cm-1, 1470 cm-1 and 1530 cm-1 can be ascribed to the typical B-N

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stretching vibration.[39-41] In BNNTs-FA@PE, however, these peaks shift to 1265,

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1385, 1462, and 1528 cm-1, respectively. It is believed that these shifts are caused by

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π-π stacking interactions existed between the aromatic groups of PE and

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BNNTs-FA.[42]

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Figure 2. (a) The original FTIR spectra of PE, BNNTs-FA and BNNTs-FA@PE. (b) The fitting spectra of BNNTs-FA and BNNTs-FA@PE based on Gaussian model.

The UV-Vis spectrum of BNNTs-FA@PE in Figure 3 illustrates an increased intensity at 207 nm caused by the benzene ring of PE, also hinting the successful loading of PE onto BNNTs-FA. A weak peak at 195.5 nm in BNNTs-FA sample

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which may be assigned to the band gap transition shifts to 191 nm in BNNTs-FA@PE

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sample, evidencing that the electronic structure of BNNTs-FA is affected by the

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charge transfer derived from the π-π interactions between BNNTs-FA and PE.[42]

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According to the standard curve and regression equation of PE established by UV-Vis

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191 207

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spectrum, 17.47 ± 0.29 µg PE are carried on 1 mg BNNTs-FA.

BNNTs-FA@PE

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Abs (a.u.)

207

195.5

PE

BNNTs-FA

200

250

300

350

400

Wave Length (nm)

Figure 3. UV-Vis spectra of BNNTs-FA@PE, PE and BNNTs-FA.

The surface chemical composition of BNNTs-FA@PE was investigated by XPS. Wide XPS spectra show that B, N, C, and O elements are present in both BNNTs-FA

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and BNNTs-FA@PE samples (Figure 4). And there is no Fe element in both BNNTs-FA and BNNTs-FA@PE samples according to the Wide XPS spectra, indicating the remnant catalyst particles were removed. The C peak (285.0 eV) and O peak (532.3 eV) in BNNTs-FA sample are due to the graft of FA on the surface of BNNTs.[28] After loading with PE, the ratios of C/B and O/B increase slightly (Table

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1), and the O peak shifts to 531.3 eV. In BNNTs-FA, the O 1s peak is split into three peaks at 533.52 eV, 532.36 eV and 531.76 eV, corresponding to O=C-O, B-O[43] and

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O=C-NH bonds[44], respectively. In BNNTs-FA@PE, the O 1s peak can be split into

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four peaks at 533.65 eV, 533.15 eV, 532.21 eV and 531.52 eV, corresponding to

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C-OCH3, O=C-O, B-O[43] and O=C-NH bonds[44], respectively. Garcia-Gil et al

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stated that the shift of O 1s peak is caused by hydrogen bond.[45] Therefore, we

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believe that intermolecular hydrogen bonds between PE and BNNTs-FA are formed in the current study, which is also in agreement with the former FTIR results. The

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schematic diagram presenting the fabrication mechanism is shown in Scheme 1. The BNNT walls are oxidized under acid treatment introducing -OH groups on B site which will react with the carboxyl groups of FA, thus grafting FA onto the BNNT wall (Scheme 1a). After that, PE is loaded onto BNNTs-FA via the π-π interactions assisted with hydrogen bond (Scheme 1b). As mentioned above, the aromatic group of PE can interact with BNNTs-FA via π-π stacking, and the non-binding electron pairs of the N and O atoms in amide group of PE can form intermolecular hydrogen bond with -OH groups or ester bonds on the BNNTs-FA. Additionally, Van der Waals’ forces may also exist.

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Figure 4. Wide XPS spectra of BNNTs-FA and BNNTs-FA@PE, and narrow-scan high-resolution

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XPS spectra of O1.

Table 1. The atomic percentage calculations according to XPS results of BNNTs-FA and

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BNNTs-FA@PE

Atomic percentage (mol %)

Sample

C1s

O1s

N1s

B1s

C/B

O/B

BNNTs-FA

21.94

8.96

21.94

37.95

0.57

0.24

BNNTs-FA@PE

28.20

11.54

27.20

33.06

0.85

0.35

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Scheme 1. The conjugation of FA and PE on the surface of a BNNT

3.3 Cellular uptake of BNNTs-FA@PE

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Figure 5. TEM images of Hep G2 cells incubated with BNNTs-FA for 4 h. (a) Low magnification and (b) high-magnification image of the boxed region, showing a bamboo-shaped BNNTs inside cytoplasm vesicles.

TEM analysis was carried out to investigate internalization of BNNTs-FA, which reveals that large aggregates and individual nanotubes are accumulated inside

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cytoplasm vesicles or transfer into cytoplasm after incubating with BNNTs-FA for 4 h (Figure 5a). The enlarged image (Figure 5b) clearly illustrates the presence of

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noncellular electron-dense material, verifying the existence of BNNTs-FA in

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cytoplasmic vacuoles.

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Hep G2 cells were incubated with BNNTs-FA and BNNTs-FA@PE for 24 h at an

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equivalent BNNTs-FA concentration of 6.8 gmL-1, then the cells were observed by

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SEM equipped with EDS to study the interactions. The results in Figure 6 demonstrate that both BNNTs-FA and BNNTs-FA@PE can insert into cells

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membrane. However, the treatment with BNNTs-FA@PE induces the morphological changes of Hep G2 cells including cell shrinkage, size decrease and rounding, which are related to the apoptosis of Hep G2 cells (Figures 6e-f).

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Figure 6. SEM images of Hep G2 cells after 24 h incubation with (a) BNNTs-FA, showing the presence of nanotubes agglomerates over the cell membrane. (b) High magnification image of the boxed region, showing the crossing of BNNTs-FA with cell membrane. (c-d) The elemental mappings of boron (B) and nitride (N) in the crossing region between the BNNTs-FA and the membrane. (e-f) SEM images of Hep G2 cells after incubation with BNNTs-FA@PE for 24 h.

Qualitative examination of F-actin was carried out to investigate internalization of BNNTs-FA@PE and the effect of BNNTs-FA@PE on cytoskeleton organization (Figure 7a). The internalization of BNNTs-FA, BNNTs@PE and BNNTs-FA@PE can be found clearly and more BNNTs are internalized after modified with FA,

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indicating that the modification of BNNTs with FA can improve the transmission efficiency of PE. The BNNTs-FA@PE have an average hydrodynamic diameter of about 1422.6 nm. Studies pointed out that the size of BNNTs can strongly affect the efficiency of cellular uptake.[46, 47] In general, the carriers with diamters of 200~1000 nm prefer to enter cells in a manner of caveolae-mediated endocytosis, and those with diameters

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less than 200 nm prefer to enter cells in a manner of clathrin mediated endocytosis,

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while those with diameters larger than 1 μm can hardly enter into the cells. However,

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elongated particles including BNNTs can be internalized via clatherin-dependent

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endocytosis as demonstrated in a previous study by Ciofani et al.[48] Previous studies

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verified that the functionalization of BNNTs with targeting moieties FA whose

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receptors are over-expressed on the surfaces of Hela cells permits BNNTs to enter into the cells by a FA receptor-mediated endocytosis pathway.[35] Even if Hep G2

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cells do not have the high level expression of folate receptors as other cancer cells, their increased expression relative to normal cells is well documented [49,50], and several studies have been reported on the folate ligand-based targeted drug delivery for Hep G2 cells.[51, 52] As far as we know, the binding between the receptors and their ligands has the characteristics of specificity, selectivity, strong affinity, and obvious biological effect.[53] Therefore, it is speculated that PE can be transported into Hep G2 cells by BNNTs-FA via the endocytosis pathway assisted with FA receptor-mediated endocytosis pathway instead of a passive diffusion pathway, avoiding the drug efflux pump (e.g. P-glycoprotein).[27] However, the internalization

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mechanism of BNNTs-FA@PE needs further investigation with the usage of specific

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inhibitors of various endocytic pathways.

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Figure 7. (a) Confocal laser scanning microscopy images (F-actin in green, nuclei in blue, BNNTs in red) of Hep G2 cells after 24 h incubation with control culture, BNNTs-FA, BNNTs@PE and

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BNNTs-FA@PE, respectively. (b) Western blot analysis of ɑ-tubulin expression level. GAPDH is used as internal control. (c) Quantification of ɑ-tubulin expression level. (n = 3, *P< 0.05).

PE is an antitubulin chemotherapeutic agent that inhibits the microtubule polymerization and perturbs microtubule dynamics. F-actin in the BNNTs-FA treated cells is the same as that in the control sample, howerer, the F-actin disappears in the BNNTs@PE and BNNTs-FA@PE treated cells. ɑ-Tubulin expression was also measured by western blotting. It is clear from Figure 7b, ɑ-Tubulin is down-regulated after exposure to BNNTs-FA@PE. As shown in Figure 7c, the expression of

Journal Pre-proof ɑ-Tubulin is as low as 0.72 fold in the BNNTs-FA@PE group, and 0.95 and 0.89 fold in the PE and BNNTs@PE group, respectively. 3.4 In vitro anticancer effect of BNNTs-FA/ PE complexes The cytotoxicity of BNNTs-FA@PE complexes was evaluated and compared with PE and BNNTs@PE (at equivalent PE concentration). It is noteworthy that

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BNNTs-FA@PE complexes exhibit stronger cytotoxicity than BNNTs@PE and free PE (Figure 8a). The cytotoxicity of BNNTs-FA@PE against Hep G2 cells was further

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studied using the Ready Probes® Cell Viability (Blue/Green) assay and CCK-8 assay.

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As shown in Figure 8b, the optical cell viability decreases when the BNNTs-FA@PE

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concentration rises after 24 h incubation. Furthermore, CCK-8 results show that the

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toxic action of BNNTs-FA@PE is increased with the prolonged incubation time and

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the increased BNNTs-FA@PE dose (Figure 8c). The growth of Hep G2 cells for 72 h is also investigated and the alterations in the cell density are shown in Figure 8d.

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Compared with the control cultures, proliferation of the cells treated with BNNTs-FA@PE decreases significantly, demonstrating strong anticancer activity of BNNTs-FA@PE.

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Figure 8. (a) Effects of PE, BNNTs@PE and BNNTs-FA-PE (at equivalent PE concentration) on the viability of Hep G2 cells after 24 h incubation. (n=3, P < 0.05). (b) Cell viability determined by ReadyProbes® Cell Viability Imaging Kit after incubation with different concentrations (0-179.78 ng·mL-1) of BNNTs-FA@PE for 24 h. (c-d) Effects of BNNTs-FA@PE on the viability and proliferation of Hep G2 cells. (n=4, P < 0.05).

Double-label flow cytometry analysis was performed to confirm BNNTs-FA@PE could induce the apoptosis of Hep G2 cells. The early apoptotic/late apoptotic ratios of the Hep G2 cells after treatment with 89.89 ngmL-1, 179.78 ngmL-1 and 359.56 ngmL-1 BNNTs-FA@PE are 61.6%/19.0%, 50.2%/31.7% and 54.1%/41.7%,

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respectively, indicating a concentration-dependent manner of cell apoptosis (Figure 9). Apoptosis is a form of cells death which is genetically programmed and morphologically distinct, and the pathways of inducing apoptosis may be via the intrinsic

mitochondria-mediated

pathway

(MMP)

and

extrinsic

death

receptor-dependent pathway (DRP). MMP is triggered from the cell interior and

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mediated by mitochondrial outer membrane permeabilization, resulting in activation of Caspase-9.[54, 55] While DRP is triggered through ligand activation of the

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abnormal cell surface death receptors, leading to the activation of Caspase-8.[56]

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Watanabe et al reported that PE could induce apoptosis via Caspase-3-dependent

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pathway,[57] which is important in both MMP and DRP.

Figure 9. Double-label flow cytometry analysis of cell apoptosis.

The collapse of ΔΨ is a hallmark for apoptosis which is closely related to the early apoptotic cascade in MMP. JC-1 probe is used to explore the influence of

Journal Pre-proof BNNTs-FA@PE on the ΔΨ. Polarized mitochondria are marked by punctate orange-red fluorescent staining. Upon depolarization, the punctate orange-red staining will change into green monomer fluorescence. As shown in Figure 10a, after treatment with PE, BNNTs@PE and BNNTs-FA@PE, the orange-red fluorescence transfers to green monomer fluorescence gradually, indicating an obvious collapse of

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ΔΨ. Therefore, it can be concluded that MMP is triggered by BNNTs-FA@PE. Proteins of the Bcl-2 family including antiapoptotic proteins and pro-apoptotic

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proteins play a major role in the MMP apoptosis pathway. The ratio of antiapoptotic

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protein Bcl-2 to pro-apoptotic protein Bax (Bcl-2/Bax) affects the fate of cells. When

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the apoptosis signal is received, Bax relocates to the mitochondrial surface and forms

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a hole across the mitochondrial membrane on the mitochondrial surface, resulting in a

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decrease of ΔΨ.[58] At the same time, the rise of Bax expression promotes the release of pro-apoptotic proteins (e.g. cytochrome c) from the inter-membrane space to the

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cytosol. Cytochrome c can then bind Apaf-1 to form apoptosome and activate Caspase-9 and Caspase-3, initiating the caspase cascade, and eventually leading to the apoptosis of cells.[56]

To further examine the possible mechanism underlying of apoptosis, the expression of related proteins was examined. As shown in Figure 10b, exposing to BNNTs-FA@PE up-regulates Bax and down-regulates Bcl-2, procaspase9 and procaspase 3 expression levels. Further analysis shows that the ratio of Bax/Bcl-2 is higher in the BNNTs-FA@PE group than that in the PE and BNNTs@PE group (Figure 10c), indicating BNNTs-FA@PE promote Hep G2 cells apoptosis. The

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expression of procaspase 9 is 0.95, 0.92 and 0.88 fold in the PE, BNNTs@PE and BNNTs-FA@PE group (Figure 10d), respectively. The expression of procaspase 3 is 0.84, 0.72 and 0.69 fold in the PE, BNNTs@PE and BNNTs-FA@PE group (Figure 10e), respectively. Meanwhile, the activity of Caspase-3/7 improves with 1.13, 1.27 and 1.35 fold in the PE, BNNTs@PE and BNNTs-FA@PE group (Figure 10f),

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respectively. The changed expression of apoptosis-related proteins and Caspase-3/7 enzymatic activity comfirm that BNNTs-FA is efficient drug carrier which can

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transport higher amounts of PE into the cancer cells and cause cells apoptosis via

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MMP. However, the detailed apoptosis mechanism needs to be studied in the future,

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and additional in vitro and in vivo experiments are needed to be performed to

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understand the selectivity of nanocarrier against Hep G2 cells.

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Figure 10. (a) The mitochondrial membrane potential (ΔΨ) of control, PE, BNNTs@PE,

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BNNTs-FA@PE and positive control group. Positive control is conducted using carbonyl cyanide m-chlorophenylhydrazone. (b) Western blot analysis of Bax, Bcl-2, procaspase 9 and procaspase 3 expression level. GAPDH is used as internal control. (c) The ratio of Bax/Bcl-2. (n = 3, *P<0.05). (d-e) Quantification of procaspase 9, procaspase 3 and ɑ-tubulin expression level. (n = 3, *P< 0.05). (f) Caspase 3/7 activities in Hep G2 cells. (n = 3, *P< 0.05).

4. Conclusions The BNNTs-FA@PE drug-delivery system is successfully constructed and its in vitro anticancer effects against Hep G2 cells is investigated. As a vehicle of PE, BNNTs-FA shows no toxicity on both liver cells (L02) and liver cancer cells (Hep G2) in terms of metabolic activity and quick cellular uptake, indicating that

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BNNTs-FA can be a good carrier in liver-targeted drug delivery system. PE is loaded onto the BNNTs-FA via π-π stacking and hydrogen bonding interactions. PE can be transported into the cells via the endocytosis pathway assisted with FA receptor-mediated endocytosis pathway instead of a passive diffusion pathway, avoiding to be flush out by the drug efflux pump (e.g. P-glycoprotein). Excess

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accumulation of PE in Hep G2 cells results in the depolymerization of microtubule bundles, which further inhibits normal proliferation, followed by apoptosis. The

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decreased ΔΨ, changed apoptosis-related proteins expression (Bcl-2, Bax,

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procaspasse 9 and procaspase 3) and Caspase-3/7 enzymatic activity all confirm that

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BNNTs-FA@PE can induce cells apoptosis via MMP. We anticipate that this

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BNNTs-FA@PE drug-delivery system can overcome the disadvantages associated

Competing interests

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with PE and facilitate the clinical trials of PE.

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The authors have declared that no competing interests exist. Acknowledgements

The work is supported by the Natural Science Foundation of Hainan Province (No. 518MS021), National Natural Science Foundation of China (No. 51702072) and the Graduate Student Innovation Project of Hainan Province (No. Hyb2017-01). Appendix A. Supplementary data Supplementary

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doi.org/10.1016/xxxx.xxxx.xx.xxx. References

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Highlights The targeted Auristatin PE-delivery system is designed and fabricated based on folate-conjugated boron nitride nanotubes. Auristatin PE is successfully loaded onto BNNTs-FA via π-π stacking and hydrogen bonding interactions.

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Auristatin PE can be transported into the cells via the endocytosis pathway assisted with folate receptor-mediated endocytosis pathway instead of a passive diffusion

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pathway.

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This complex possesses preferable anticancer activity in contrast to free Auristatin

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PE and excellent antiproliferative activities. Furthermore, this complex can induce

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cells apoptosis via a mitochondria–mediated pathway.