Highly Uniform Synthesis of Selenium Nanoparticles with EGFR

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Biological and Medical Applications of Materials and Interfaces

Highly Uniform Synthesis of Selenium Nanoparticles with EGFR Targeting and Tumor Microenvironment Responsive Ability for Simultaneous Diagnosis and Therapy of Nasopharyngeal Carcinoma Jing Huang, Wei Huang, Zehang Zhang, Xueran Lin, Hao Lin, Lijiao Peng, and Tianfeng Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b22678 • Publication Date (Web): 01 Mar 2019 Downloaded from http://pubs.acs.org on March 2, 2019

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ACS Applied Materials & Interfaces

Highly Uniform Synthesis of Selenium Nanoparticles with EGFR Targeting and Tumor Microenvironment Responsive Ability for Simultaneous Diagnosis and Therapy of Nasopharyngeal Carcinoma

Jing Huang 1#, Wei Huang 2#, Zehang Zhang2, Xueran Lin2, Hao Lin 3, Lijiao Peng 1*, Tianfeng Chen 2*

1. Oncology Center, Affiliated Hospital of Guangdong Medical University, Zhanjiang 524001, China. 2. Department of Chemistry, Jinan University, Guangzhou 510632, China. 3. Department of Spinal Surgery, Affiliated Hospital of Guangdong Medical University, Zhanjiang 524001, China. # Authors contribute equally to this study * To whom correspondence may be addressed. E-mail: [email protected] (L. P.) E-mail: [email protected]. Tel: +86 20-85225962 (T. C.) Keywords: EGFR targeting, nanomedicine, nasopharyngeal carcinoma, theranosis, tumor microenvironment

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Abstract Rational design of multifunctional and smart drug-delivered nanoplatforms is promising strategy to achieve simultaneous diagnosis, real-time monitoring and therapy of cancers. Herein, highly uniform and stable selenium nanoparticles with EGFR targeting and tumor microenvironment responsive ability (Se-5Fu-Gd-P(Cet/YI-12)) were designed and synthesized by using epidermal growth factor receptor (EGFR) as targeting molecule, gadolinium chelate as magnetic resonance imaging contrast agent, 5-fluorouracil (5Fu) and cetuximab as drug payload, polyamidoamine (PAMAM) and 3,3'-dithiobis (sulfosuccinimidyl propionate) (DTSSP) as response agents of intratumoral glutathione and pH, for treatment and diagnosis of nasopharyngeal carcinoma (NPC). This Se nanoplatform showed excellent magnetic resonance imaging capability, and possess the potential clinical application as a diagnostic agent for tumors tissue specimen. Additionally, in vitro cellular experiments showed that by means of introducing of clinical targeted drugs and peptide not only validly increased intracellular uptake of Se nanoplatform in NPC cells, but also enhanced its penetration ability toward CNE tumor spheroids, resulting in simultaneous inhibition of CNE cells growth, invasion and migration. In addition, the sequentially-triggered bioresponsive property of the nanoplaform in tumor microenvironment effectively improved the targeting delivery and anticancer efficiency of payloads. Overall, this study not only provides a strategy for facile synthesis of highly-uniform and stable nanomedicines and tailing of the bioresponsive property, but also sheds light on its application in targeting theranosis of NPC.


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1. Introduction Nasopharyngeal carcinoma (NPC), a malignancy that rises from nasopharyngeal epithelia, is highly prevalent in South China and Southeast Asia.1-3 Characterized by its concealed location and strong invasiveness, more than 70% patients with locoregionally advanced NPC at their initial diagnosis and possess a poor 5-year survival.4 Despite the standard concurrent chemoradiotherapy yielding great improvement of survival outcome, 20-30% of patients absolutely at higher risk failed because of local-regional relapse and/or distant metastasis after treatment.5-7 Therefore, there is an urgent need for better treatment strategies in order to improve the efficacy of advanced NPC patients. Epidermal growth factor receptor (EGFR), a transmembrane protein, is highly expressed in more than 80% of NPC patients and is associated with poor prognosis.8 Cetuximab (Cet), a human-mouse chimeric antibody blocking EGFR approved by FDA, has been proved to inhibit tumor cell growth/survival and metastasis, angiogenesis. In recent years, anti-EGFR targeted therapy has been frequently applied in combination treatment of NPC, and shown promising advantages for NPC treatment.9-11 However, anti-EGFR therapy related toxicities (mainly skin reaction and mucositis), as well as its unwelcome cost are important factors restricting its wild application.12 Besides, as reported, only 13% of the head and neck tumors benefitted from the use of Cet.13 And drug resistance is a common problem in the treatment of molecular-targeted drugs.14-16 Thus it’s an urgent problem in NPC management to clarify the specificity and efficacy of targeted therapy and to find out how to combine targeted drugs with other cytotoxic drugs or radiotherapy so as to achieve better therapeutic effect. Cancer nanotechnology, as an integrated platform, displays vast applications in cancer diagnosis, imaging and therapy, due to their enhanced permeability and retention effect and versatile chemical modification.17-20 Among many organic and inorganic nanoparticles, selenium (Se), a necessary and trace element for human, exhibited welldocumented anticancer activity and antioxidant activity in early researches, and then garnered unprecedented concerns.21,

22

And target connection of SeNPs not only

improve the therapeutic effect, but also decrease toxic and side effects induced by the ACS Paragon Plus Environment


precise transmission of drugs.23-25 Meanwhile, our groups have synthesized and reported anticancer drugs-delivered SeNPs such as SeNPs-doxorubicin (DOX)26, SeNPs-5-fluorouracil (5Fu)27 which not only show synergistic enhanced chemotherapy effect, but also reduced the possibility of drug resistance. Notability, although previous studies showed that impactful grafting of cationic polymers and/or polysaccharide (such as polyethylenimine-polyethylene glycol (PEIPEG),28 chitosan (Cs),29 polysaccharides–protein complexes (PSP)30 to the surface of SeNPs enhances their stability in aqueous solution and promotes their intracellular uptake in a certain range. However, similar with other kinds of nanomedicine, the synthesis of highly uniform and stable SeNPs is still the bottleneck for its future clinical application. Tween-80 (TW-80), a synthetic nonionic surfactant, is prepared by the copolymerization of sorbitol and its dehydrated mono oleic ester with ethylene oxide under alkali catalysis. According to previous reports,31 TW-80 was used as a protective agent to mediate the rapid synthesis of stable conjugates of DNA/Gold nanoparticles. Moreover, TW-80 is also applied to be an excellent stabilizer which could significantly enhanced the stability and dispersibility of the modified silver NPs.32 Briefly, TW-80 has been widely used in cosmetics, food and pharmaceutical preparations, especially in the aspect of modification of nanoparticles.33 Besides, “smart” nanosystems have been widely designed to realize precise release of drugs at tumor sites, resulting in excellent drugs activity.34-36 3,3'-dithiobis (sulfosuccinimidyl propionate) (DTSSP) has the same active groups at both ends, which can covalently combine with amino groups, mercapto groups and hydroxyl groups. Meanwhile, because the disulfide bond existed in DTSSP was easily destructed under high concentration of glutathione (GSH), hence DTSSP was commonly used to crosslinking agent in the field of nanomedicine, especially in these nanoparticles responding to GSH. For example, Huang et al. have reported SPIO@DOX-ICG nanoparticles by the crosslinking of DTSSP to continuously release drugs in response of intratumoral GSH, laser and X-ray irradiation.37 Polyamidoamine (PAMAM) possess a unique three-dimensional dendritic symmetric structure. Because of the protonation effect of PAMAM in the acid environment, the surface decoration of ACS Paragon Plus Environment

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nanomaterials by PAMAM maybe endows the nanosystem pH-responsiveness ability. Furthermore, multifunctional nanosystems are integrated diagnoses and treatment, which have been acquired intense attention currently.38, 39 In clinical, tumor diagnoses mainly divided to biopsy of living tissue and molecular imaging technologies such as magnetic resonance imaging (MRI), photoacoustic imaging (PA) and X-ray computed tomography et.al.40-42 Among them, owing to the reason of safety, non-invasive and high sensitivity, MRI has becoming a commonly used diagnostic tool in clinic, especially in evaluation of clinical staging of NPC according to the International Union against Cancer/American Joint Committee on Cancer (UICC/AJCC) staging system. At present,

based

on

its

acceleration

of

T1

relaxation,

gadolinium

diethylenetriaminepentaacetic acid (Gd-DTPA) is most diffusely applied T1-weighted contrast agent. Meanwhile, nanocomposites loaded with gadolinium chelates (Gd) have been developed as an effective means for the diagnosis of cancer.43, 44 For example, Zhao et al. groups prepared Gd-hybridized Au-nanosystem to enhance the permeability of DOX in MRI-guided therapy.45 Based on these facts in mind, we first prepared highly uniform SeNPs by using TW-80 as surfactant and utilized it to further construct a multifunctional and smart drug-delivered Se nanoplatform (Se-5Fu-Gd-P(Cet/YI-12)) with EGFR as targeting molecule, Gd as MRI contrast agent, 5Fu (a most widely used anti-metabolites currently in clinical practice

46-48)

and Cet as drug payloads, PAMAM and DTSSP as response

reagents of intratumoral GSH and acid for the treatment and diagnosis of NPC. The results of transmission electron microscope (TEM) and atomic force microscope (AFM) showed that this Se nanoplatform possess highly uniform size and morphology. Meanwhile, Se-5Fu-Gd-P(Cet/YI-12) exhibited low hemolysis rate and high stability under physiological condition. Most importantly, Se-5Fu-Gd-P(Cet/YI-12) not only exhibited excellent MR imaging capability in vitro and in vivo, but also possess the same potential clinical diagnostic capabilities for tumor tissue specimens as other conventional reagents. Furthermore, in vitro cellular experiments showed that the connection of clinical targeted drugs and peptide not only valid increased intracellular uptake of Se nanoplatform in NPC cells by receptor-mediated endocytosis, but ACS Paragon Plus Environment


enhanced its penetration toward CNE tumor spheroids we cultured in vitro, resulting to great anticancer efficacy by inducing the reduction of intracellular ROS. As expected, Se-5Fu-Gd-P(Cet/YI-12) simultaneously inhibits the growth, invasion and migration of the CNE cells. Besides, the morphology and size of the nanoplaform enabled to sequentially change in the tumor microenvironment, which may improve the efficiency of 5Fu. Taken together, this study provides an available strategy for synthesis of multifunctional Se nanoplatform to integrate treatment and diagnosis against NPC.

2. Results and Discussion 2.1 Synthesis and Characterization of highly uniform and stable SeNPs Similar with other kinds of nanoplatforms, the synthesis of highly uniform and stable SeNPs is still one of the bottlenecks affecting its future clinical application. Here, we first used lentinan (Let) and Cs as surfactants to acquire Let/Cs-SeNPs according to previous literatures. 25 The results of size distributions, digital photographs and TEM images which exhibited in Figure 1A showed that despite the prepared Let-SeNPs and Cs-SeNPs showed sphere morphology with an average size of about 110 nm and 126.1 nm, but the uniformity and dispersivity of Let/Cs-SeNPs need to be improved. TW-80, a common nonionic surfactant, is considered to be an effective method to modify NPs and then improve stability of NPs due to its inherent steric effects and hydrogen bond effect from ether bond, thus achieving the purpose of dispersion of NPs possibility. As a result, we employed TW-80 as decorator to prepare TW-80-SeNPs with ideal size and regularity by using traditional oxidation-reduction method. As showed in Figure 1A, the size of TW-80-SeNPs was 37.1 nm with a 0.036 of polydispersity index (PDI), which is the smallest of the three SeNPs. Meanwhile, the dispersion of TW-80-SeNPs was better than that of Let/Cs-SeNPs obviously. The synthesis mechanism of highly uniform selenium NPs by using TW-80 was proposed in Figure 1 B. Hence, we selected TW-80-SeNPs as drug delivery carriers of anticancer drugs and contrast agents. PAMAM with a large of amino groups in the surface, a special three-dimensional dendritic structure, endows the nanosystem ability to responsive pH. DTSSP, a common crosslinking agent, not only simultaneity realize the connection ACS Paragon Plus Environment

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1E-G) of final NPs under different scales appear as highly regular sphericity in shape with the rough size of 71.4 nm and good dispersion uniformity. Meanwhile, the results of the elemental mapping based on high-resolution TEM showed that Se, F, Gd, S and N were evenly distributed in SeNPs, which confirmed the successful loading of 5Fu and contrast agents in NPs (Figure 1H). Additionally, the average height of final NPs was consistency as exhibited by the 3D view of the AFM images (Figure 1I-K). Next, to test the T1 contrast capabilities of Se-5Fu-Gd-P(Cet-YI-12) NPs in MR images, we introduced the 3.0 T MR scanner used in clinical to measure the T1weighted signal of Gd. As shown in Figure 2A-2C, the white signal was gradually increasing with the increase of Gd concentration ranging between 0-2.59 mM and the relaxation rate (r1 value) of Se-5Fu-Gd-P(Cet-YI-12) was higher than of Gd alone, suggesting this resultant Se nanoplatform exhibited enhanced relaxation behavior, maybe due to the large number of Gd gathered in its inside. Subsequently, some of the commonly used methods of characterization including HPLC, fourier transform infrared spectroscopy (FT-IR), UV-vis and zeta potential were employed to prove the successful delivery of 5Fu, Cet, YI-12 in Se-5Fu-Gd-P(Cet-YI12) NPs. First, it could be observed form Figure 2D a weak absorbance at 266 nm which was belonged to 5Fu. Second, the 5Fu concentration and encapsulation efficiency of NPs were determine by using high performance liquid chromatography (HPLC) and the value of EE was 17.74%.


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Besides, in order to understand the chemical connection between drugs, targeted peptides and TW-80-SeNPs, FI-IR was investigated. For example, in Figure 2H and 2I, the spectrum of 5Fu showed the peaks of -C-F band, -C=O and C-N bond at 1430 cm-1, 1663 cm-1 and 1248 cm-1, respectively. In the spectrums of Se-5Fu-Gd NPs and Se-5Fu-Gd-P(Cet-YI-12) NPs, the characteristic peaks of 5Fu mentioned above were be observed obviously, which indicated the 5Fu was connected to SeNPs by Se-N and Se-O bond possibility. Meanwhile, the presence of disulfide bond (543 cm-1) in the IR spectra of Se-5Fu-Gd-P(Cet-YI-12) NPs indicates DTSSP were existed in final NPs. And the characteristic peaks primary amine and secondary amine at 3420 cm-1 and 3284 cm-1 were existed in the spectrums of PAMAM and Se-5Fu-Gd-P(Cet-YI-12) NPs which showed the PAMAM was successfully decorated on the surface of NPs. Moreover, the successful connection of Cet and Se-5Fu-Gd-P NPs was characterized by using BCA assay (Figure 2F and 2G). Finally, as we observe from Figure 2J, with the addition of 5Fu, Gd, Cet and YI-12, the surface potential of NPs was slightly changed. It should be noted that after the modification of PAMAM, reverses of surface potential form negative to positive could be observed, which may be come from large of amino groups of PAMAM. Therefore, based on above results, an targeted Se-5FuGd-P(Cet/YI-12) nanoplatform which programmed PAMAM-SeNPs-loaded Gd and 5Fu covered with Cet and YI-12 peptide by DTSSP as bridging agent was established.

2.2 Safety and stability of Se-5Fu-Gd-P(Cet-YI-12)NPs Drug stability is an important factor in the investigation of nanomedicine. Thus, all samples were incubated with water, fetal bovine serum (FBS) and dulbecco's Modified Eagle Medium (DMEM) environments in equal proportion, which was an unavoidable environment for nanodrug. Stability of different Se-containing nanosystems was measured through monitoring its sizes change in DMEM and FBS solution within 84 h. The results showed that Se-5Fu-Gd-P(Cet-YI-12) NPs remained stable either in FBS or DMEM within 84 h, which may be attributed to the important role played by TW-80 (Figure 3A-C).


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A

Gd Concentration (mM) 0

0.1618

0.32375

0.6475

1.295

H

2.59

DTSSP PAMAM YI-12 Cet PAMAM-DTSSP-CET-YI-12"

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Se-5Fu-Gd-P(Cet-YI-12)

3425

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3420

3284

Se-5Fu-Gd

Gd

0.012

546 3284 3421

0.02 -1

1/T1 (mS )

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1/T1 (mS )

3423

0.008

0.004

Y=0.0045x+0.0007

3421

3600

0.01

r1 = 0.0045 mM-1mS-1

0.000 0.5

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TW-SeNPs Gd Se(5Fu)-Gd Nps 5Fu

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BCA

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Cet Se(5-FU)-Gd-Cet BCA

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10 Zeta potetial (mv)

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r1 = 0.0091 mM-1mS-1

0.00 0.0

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Y=0.0091x+0.0003

Control

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


0

-10

-20 480

560

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720

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Wavenumber (nm)

Figure 2 Characterization of Se-5Fu-Gd-P(Cet-YI-12) NPs. (A) MR images in vitro were acquired to evaluate the magnetic property of Gd and Se-5Fu-Gd-P(Cet-YI-12) NPs. T1 relaxation rate (r1) of Gd (B) and Se-5Fu-Gd-P(Cet-YI-12) NPs (C) under clinical 3-T MRI scanner. (D) UV-vis spectra of Se, Gd, 5Fu and Se-5Fu-Gd-P(CetYI-12) NPs. (E) HPLC spectra of 5Fu and Se-5Fu-Gd-P(Cet-YI-12) NPs to determine the load of NPs for 5Fu. (F) UV–vis spectra exhibiting the changes of absorbance at 560 nm to determine the connection between Cet and Se-5Fu-Gd-P NPs by using BCA assay. (G) Photograph of BCA solution after addition of Cet, BSA and Se-5FuGd-P(Cet) NPs.(H) (I) FT-IR spectra of various raw materials and products in the synthesis (J) Zeta potential of these NPs.



120

Size (nm)

Size (nm)

80 60

FBS

TW-80-Se Se-5Fu-Gd Se-5Fu-Gd-P(Cet) Se-5Fu-Gd-P(Cet-YI-12)

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1.6 Hemolytic rate (%)

200

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Water


100

Size (nm)

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1.2

TW-80-Se Se-5Fu-Gd Se-5Fu-Gd-P(Cet) Se-5Fu-Gd-P(cet/YI-12)

15.6 AM

Se-5Fu-Gd-P(Cet)

0.8

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25 Am

0.0

30

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Figure 3 Hemolysis and stability analyses of different NPs. The changes of size of NPs after incubation with water (A), FBS (B) and DMEM (C) solution. (D, E, F) Analysis of hemolytic rates of different NPs with different concentrations. (G) Agglutination of human erythrocytes in different NPs treated groups. Each value are expressed as mean ±SD (n = 3)

Besides, biocompatibility has always been an important problem which needs to be solved before NPs enter clinical application. Thus, hemolysis analysis was performed to evaluate the hemocompatibility of Se-5Fu-Gd-P(Cet-YI-12)NPs. red blood cells (RBCs) were incubated with different concentrations NPs including TWSe, Se-5Fu-Gd, Se-5Fu-Gd-P(Cet) and Se-5Fu-Gd-P(Cet-YI-12) NPs for 30, 120, 240 and 360 min. The hemoglobin which released from RBCs was used to assess its damages. As shown in Figure 3D-3F, all NPs exerted very low hemolysis rates (less


EGFR antibody

EGFR-targeted nanoparticles

EGFR antibody


NPC tissues

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


Non-tumor tissues

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EGFR antibody


Figure 4 Cancer diagnoses in clinical NPC specimens using CSe-5Fu-Gd-P(Cet-YI12) NPs. Clinical NPC tissues representing different clinical stages and corresponding normal tissues, were stained by anti-EGFR antibody (up row) and CSe-5Fu-GdP(Cet-YI-12) NPs (down row). Compared to the normal tissues, NPC tissues exhibited stronger positive staining for anti-EGFR antibody (brown) and CSe-5FuGd-P(Cet-YI-12) nanoparticles (green fluorescence). The tumors were staged using the American Joint Committee on Cancer (AJCC) Cancer Staging Manual, 8th Edition. Red scale bar was 100 R%S white scale bar, 400 R%

than 5.0%), even in the high concentration nanodrugs environment (15.6 RM). Consistency, the morphology of the erythrocytes treated with TW-80-Se and Se-5FuGd-P(Cet-YI-12) NPs basically remained round and surface smooth. As contrast, the Se-5Fu-Gd-P(Cet) and Se-5Fu-Gd NPs with high concentration treatments caused slight destruction for erythrocytes based on the feature of surface rough (Figure 3G). Taken together, this functionalized NPs exhibited high safety and stability when applied in physiological condition.



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Table 1 Histological analysis of CSe-5Fu-Gd-P(Cet-YI-12) NPs staining of clinical NPC and normal nasopharyngeal mucosa tissue specimens. a) Histological analysis of CSe-5Fu-Gd-P(Cet-YI-12) NPs staining. b) Immunohistochemical staining for EGFR antibody Tissues

Positive/casesa)

Positive/casesb)

(sensitivity) Normal tissues

0/7 (0%)

0/7

NPC tissues

28/30(93.3%)

28/30

5/5(100%)

5/5

6/6(100%)

6/6

9/10(90%)

9/10

8/9(88.9%)

8/9

2.3 Targeting efficacy (Immunofluorescence analysis) Since EGFR has been used as a targeting marker for tumor diagnosis and therapy, the specificity of CSe-5Fu-Gd-P(Cet-YI-12) NPs targeting to EGFR-expressed NPC was investigated using a set of human NPC tissue samples and corresponding normal tissue samples. As shown in Figure 4 and Table 1, except for 2 NPC tissue samples with EGFR weak or negative expressed, Coum-6 labeled Se-5Fu-Gd-P(Cet-YI-12) NPs showed different intensity of fluorescence staining in 28 out 30 NPC tissue samples, whereas no obvious fluorescence staining were detected in the whole 7 normal tissue samples, which confirming the specific tumor-binding reactivity of the final NPs. Compared with conventional antibody-based histological methods for cancer detection in clinics, our novel CSe-5Fu-Gd-P(Cet-YI-12) NPs-based method has the equivalent sensitivity and specificity, and the advantage of a rapid examination time with 1-2 h incubation of nanoparticles rather than the 4 h or even overnight required for immunohistochemistry. Moreover, our method does not require the secondary or enzyme-labeled third antibody, which directly avoided multistep incubation and reduce


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activity, but its toxic and side effects cannot be ignored. One of the aims of this study is to use EGFR-targeted SeNPs as a carrier of 5Fu to overcome drawbacks of low selectivity of SeNPs and 5Fu between cancer and normal cells, thus enhancing its antitumor efficacy. Therefore, the PAMAM-modified non-targeted and EGFR-targeted drug loaded nanoparticles were tested for their cytotoxicity in representative NPC cell lines (CNE, CNE2) and normal liver cell line L02 by using 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay.

The cytotoxicity of

raw TW-80-Se, Gd and 5Fu were performed for completely comparison. First, Figure S1A-1C demonstrated that free Gd chelates we prepared have no significant cytotoxicity toward CNE, CNE2 and normal L02 cells. Meanwhile, as calculated by 5Fu, it was found that the viability of 5Fu was significantly higher than that of Se-5FuGd-P(Cet-YI-12) NPs in CNE and CNE2 cells, and lower than that of Se-5Fu-GdP(Cet-YI-12) NPs in L02 cells, which revealed that the EGFR-targeted design maybe improved the selectivity toward normal and cancer cells, thus enhancing the antitumor efficacy (Figure S1D-F). Moreover, Figure 5A provides the IC50 values of different nanodrugs obtained after 72 h of incubation according to the calculation of Se concentration, and shows TW-80-Se, Se-5Fu-Gd, Se-5Fu-Gd-P(Cet) and Se-5Fu-GdP(Cet-YI-12) NPs repress the proliferation of CNE and CNE2 cells. We also noticed that individual Cet-conjugated NPs shows higher IC50 value compared to that in Cet and EGFR-targeted peptide co-conjugated group, which could be attributed to the lower binding affinity of Cet than EGFR-targeted peptide to SeNPs. Notability, Se-5Fu-GdP(Cet-YI-12) NPs not only exhibited great cytotoxicity against CNE with the lowest IC50 value 4.51 R

but also indicated decided selectivity between normal cell line L02

and cancer cell line CNE. Therefore, CNE cells were selected models for all subsequent cellular experiments. Second, Figure 5B-D exhibited the cells viability decreased with the increase of intracellular NPs concentration. The safety index (SI) of drugs was calculated by the formula that is IC50 (normal cells)/IC50 (cancer cells)), which is an important index to evaluate safety and selectivity of drugs. Form Figure 5A, SI value of Se-5Fu-Gd-P(Cet-YI-12) NPs showed significantly higher than that of the other three groups. Furthermore, we captured CNE and L02 cells images after treatments ACS Paragon Plus Environment

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functionalized NPs for 72 h. From Figure 5E, the morphology and number of CNE cells have changed and obviously decreased with dose-dependent manner. In contrast, no significant difference in treated L02 cells could be observed compared with that of control group. These results revealed that Se-5Fu-Gd-P(Cet-YI-12) NPs possessed great selectivity between cancer cells and normal cells than Se-5Fu-Gd-P(Cet) NPs and have prominent antitumor activity.

2.5 Se-5Fu-Gd-P(Cet-YI-12) NPs inhibit CNE cell proliferation, migration and invasion Malignant tumors are often characterized by abnormal proliferation, tissue invasion and metastasis. Then, we performed colony formation to evaluate whether Se-5Fu-GdP(Cet-YI-12) effect on CNE cell proliferation. As shown in Figure 6A-B, both Se-5FuGd and Se-5Fu-Gd-P(Cet-YI-12) NPs exhibited dose-independent proliferation inhibition. For example, compared to the same concentration of Se-5Fu-Gd NPs (15.6 R 8 Se-5Fu-Gd-P(Cet-YI-12) NPs showed a more significant proliferation inhibition, up to two-fold of Se-5Fu-Gd NPs. It well known that both the migration of tumor cells to adjacent organs and distant metastasis are major causes leading treatment failure. Subsequently, we focused on detecting whether Se-5Fu-Gd-P(Cet-YI-12) NPs affect cell migration and invasion ability of NPC cells in vitro. Wound-healing assay shown in Figure 6C indicated that Se-5Fu-Gd-P(Cet-YI-12) NPs effectively inhibited CNE cell migration with the increase of slight toxic concentration, compared to the control group. Moreover, the inhibitory action of Se-5Fu-Gd-P(Cet-YI-12) NPs was superior to that of Se-5Fu-Gd NPs at the same concentrations. Similarly, Matrigel invasion assay demonstrated that the permeability through Matrigel of Se-5Fu-Gd NPs were inferior to than that of EGFR-targeted NPs. It could be noticed that from the images of Figure 6D

that

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Figure 6 Effect of Se-5Fu-Gd-P(Cet-YI-12) NPs on CNE cell proliferation, migration and invasion. (A) Colony formation assay were performed in 6-well plates with the application of Se-5Fu-Gd NPs and Se-5Fu-Gd-P(Cet-YI-12) NPs for 10 days. (B) The colony formation abilities in each group were statistically analyzed. (C) Wound healing assay showed the different effects of Se-5Fu-Gd NPs and Se-5Fu-Gd-P(Cet-YI-12) NPs on cell migration, compared to the control. Scale bar was 1000 R% (D) Metrigel invasion assay were performed to explore the cell invasion ability exposed to Se-5FuGd NPs and Se-5Fu-Gd-P(Cet-YI-12) NPs for 24 h. Original magnification, 100×. All experiments were repeated three times.

CNE cells penetrated in 7.8 R

Se-5Fu-Gd treated group, whereas no cells were

observed in the parallel Se-5Fu-Gd-P(Cet-YI-12) treated group. Taken together, these findings suggested that EGFR-targeted delivery nanosystem Se-5Fu-Gd-P(Cet-YI-12)


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NPs effectively suppress proliferation, migration and invasion ability of NPC cells.

2.6 Selective cellular uptake, intra-cellular translocation and its uptake mechanism of CSe-5Fu-Gd-P(Cet-YI-12) NPs It well known that the cellular uptake efficacy is a vital factor affecting the anticancer ability of drugs. Thus, to further investigate the reasons for different antitumor activities of these nanoparticles, on the one hand, we quantitatively analyzed the selective cellular uptake of Coum-6 loaded Se-5Fu-Gd, Se-5Fu-Gd-P(Cet) and Se-5Fu-Gd-P(Cet-YI-12) NPs in CNE and L02 cells, respectively within 12 h by fluorescence quantitative. We observed from Figure 7A that the intracellular accumulation of these NPs in CNE cells presents time-effect manner and reached highest uptake in 8 h. Meanwhile, the accumulation of CSe-5Fu-Gd-P(Cet-YI-12) NPs in CNE cells was higher than that of other two nanoparticles. More strikingly, the overall cellular uptake efficiency of these NPs in L02 cells was relatively lower than that in CNE cells (Figure 7B). On the other hand, we preformed YI-12 peptide competition assay to further demonstrate the selective cellular uptake of CSe-5Fu-Gd-P(Cet/YI-12) NPs by flow cytometric analysis. As shown in Figure 7C, quantitative analysis confirmed that the intracellular fluorescence intensity of Se-5Fu-Gd-P(Cet/YI-12) NPs decreased in a dose-dependent manner on adding YI-12 (0-0.5 mg mLU ), which indicates that YI-12 blocked the EGFR receptor and then prevented the drug from entering the cell. Besides, we introduced Alexa Fluor 488 phalloidin and Hoechst 33342 fluorescent probe to marker the cytoskeleton and nucleus of CNE cells to intuitively observe the cellular fluorescent intensity of NPs, which further reflects the cellular uptake of NPs. As shown in Figure 7D, in CSe-5Fu-Gd-P(Cet-YI-12) NPs-treated CNE cells, the green fluorescence signal inside cells was stronger, whereas the fluorescence of CSe5Fu-Gd and CSe-5Fu-Gd-P(Cet) NPs mostly remained on the side of the cell (mag be cell membrane), which consisted with above results of drug cellular uptake. Overall, these results indicated that EGFR receptor mediated tumor targeted therapy is an effective means to improve the drug accumulation in tumor




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cells, thus producing significant cytotoxicity to tumor cells. Endocytosis is one of the most pivotal pathways for drugs absorption especially for macromolecular drugs such as NPs. Herein, in order to understand the subcellular localization of NPs, we used lyso-tracker (red) and Hoechst 33342 (blue) to label lysosome and nucleus respectively. As shown in Figure 8A and S1, after 2 h incubation with CSe-5Fu-Gd-P(Cet-YI-12) NPs, green fluorescence has overlapped with lysosomes fluorescence. And the fluorescence signal became stronger and stronger, and gradually diffused into the entire cytoplasm after 8 h incubation, which indicated a higher accumulation of CSe-5Fu-Gd-P (Cet-YI-12). Meanwhile, no obvious green fluorescence was detected in the nucleus during the whole observation, suggesting that lysosomes rather than nucleus were the main cellular target of CSe-5Fu-Gd-P(Cet-YI12) NPs. Afterwards, we used the different endocytosis inhibitors including sodium azide in combination of 2-deoxy-D-glucose (NaN3+DOG), Sucrose, Nystain and Dynasore which represents energy-related, clathrin-mediated, nystatin-related and dynaminrelated, respectively to further validate the mechanisms of final NPs endocytosis. The results illustrated in Figure 8B demonstrated that cellular uptake of CSe-5Fu-GdP(Cet-YI-12) NPs were affected differently by these endocytosis inhibitor. For example, the internalization of CSe-5Fu-Gd-P(Cet-YI-12) NPs were significantly inhibited by Dynasore with a nearly 58.7% inhibition compared with the control. The addition of NaN3+DOG, Sucrose and Nystain reduced CSe-5Fu-Gd-P(Cet-YI-12) NPs uptake by 43.9%, 23.4% and 30.9% compared with the control. These results indicate that dynamin-related lipid raft-mediated endocytosis was preferably the main pathway of the internalization of CSe-5Fu-Gd-P(Cet-YI-12)NPs.

2.7 Bioresponsive Property of Se-5Fu-Gd-P(Cet-YI-12) NPs Microenvironment of most solid tumors is often characterized with hypoxia, low pH and high levels of GSH, which is considered to be a key factor affecting therapeutic effect. Therefore, based on these features of solid tumor, researchers have designed




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different pH values ( pH 5.3 and 7.4) and high concentration of GSH (10 mM) or both mixed solution.

and synthesized a series of tumor-microenvironment responsive drugs-delivered nanoplaforms for reduction the drugs toxicity. As previously reported, the crosslinking of DTSSP and the decoration of PAMAM may endow nanosystems the abilities in response of intratumoral GSH, acid environment, because a disulfide bond existed in DTSSP was easily to destroyed and a plenty of amino groups form PAMAM can protonated under acidic environment.49-51 Therefore, DTSSP was used to achieve the connection between the targeted peptides and PAMAM-modified SeNPs and then was effectively broken in GSH environment, which may be conducive to responsive drug controlled release. Herein, to prove the speculation, we simulated the acidic and high reducing environment of tumor tissue by using phosphate buffered saline (PBS) (pH= 5.3), GSH (10 mM) and GSH+PBS medium (10 mM, pH= 5.3) to investigated the in vitro release of 5Fu from various NPs. First, the samples were incubated in different media for 12 h, and centrifuged to obtain supernatant solution. Then, the absorbance intensity of supernatants was examined by UV-vis spectroscopy. The results (Figure 8C and D) showed that the 5Fu releases behaviors of Se-5Fu-Gd-P(Cet-YI-12) NPs at GSH+PBS solution was likely faster than that of at pH 5.3 and GSH circumstances. As visualized by size distribution and morphology of NPs (Figure 8E and 8F), the morphology and size of NPs in pH 7.4 PBS solution remain approximately the same as those of in aqueous solution which suggesting NPs has excellent stability in PBS solution. Meanwhile, it could be observed that the size of NPs at GSH circumstances decreased from 80.1 nm to 53.6 nm, which may be caused by the fracture of disulfide bond from NPs. Additionally, compared with slow size-increase features in pH 5.4 PBS solution, the size-increase speeds of NPs was found to be much faster in GSH+PBS solution. Eventually, the size of NPs in GSH+PBS solution has enhanced to micron, which have exhibited same phenomenon in previous study. 28 The breakage of disulfide bonds in DTSSP on the surface of NPs under GSH environment results in an increase the number of naked leakage amino groups in Se-5Fu-Gd-P NPs, which may be the ACS Paragon Plus Environment


fundamental reason for the differences of size variation of NPs in GSH+PBS and PBS environments. More importantly, we could be noticed from the TEM images that the edges of NPs occurred obvious reduced and same small particles were appeared, which probably means the Cet or 5Fu releases form NPs. Therefore, these results demonstrated that Se-5Fu-Gd-P(Cet/YI-12) NPs have the possibility of redox and pH dual-sensitive, which has the potential to achieve precise treatment in the future and reduce the side effects of drugs.

2.8 Decline of ROS production by Se-5Fu-Gd-P(Cet-YI-12) NPs The maladjustment of the redox system in organisms was recognized as major action mechanism for drugs to induce the death of cancer cells. Hence, ROS variation was examined in CNE cells for 2 h during the treatment of drugs by using dihydroethidium (DHE) as a fluorescence dye. As shown in Figure 9A-B, ROS levels showed a linear decrease in the first 30 min after exposing to different NPs with various concentrations, following with a steady decline. To prove the results intuitively, we reported the cells photographs by using fluorescence confocal microscopy to observe the ROS intensity. As reflected in Figure 9C, a weaker fluorescence intensity was showed in Se-5Fu-GdP(Cet-YI-12) treated group than its controls, demonstrating ROS scavenging stimulated by Se-5Fu-Gd-P(Cet-YI-12) NPs. Of course, to deep-seated verify the phenomenon, ABTS•+ free radical scavenging assay was performed to evaluate the antioxidant potential of Se-5Fu-Gd-P(Cet-YI-12) NPs. The reduction of absorbance at 734 nm was observed in Figure 9D-F that free radicals were scavenged by the Se-5Fu-Gd-P(Cet-YI-12) in a concentration dependent manner. Meanwhile, the final NPs exhibited higher scavenging capability than that of Se-5FuGd and Se-5Fu-Gd-P(Cet) NPs under the same conditions. Accordingly, the percentage of inhibition enhanced as the concentration of final NPs improved (Figure 9F). For example, the clearance ABTS•+ rate of the Se-5Fu-Gd-P(Cet-YI-12) NPs at 7.8 RM concentrations was about 49.4% and the scavenging ABTS•+ radical of this NPs with a concentration of 0.975 RM was high as high 90.1%. Furthermore, we have


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Figure 9 Decline of ROS stimulated by Se-5Fu-Gd-P(Cet-YI-12) NPs (A) The intracellular ROS level in CNE cells exposed to same concentrations of NPs (15.6 RM) for 2 h. (B) ROS scavenging stimulated by different NPs (7.8 RM) were calculated by measuring the fluorescence intensity. (C) Representative DHE staining images of ROS reduction in CNE cells after the incubation of 15.6 R

NPs for

various times. Original magnification: 20×. (D) Different NPs with the same concentration scavenged ABTS•+ free radicals. (E) Different concentrations of Se5Fu-Gd-P (Cet-YI-12) NPs scavenged ABTS•+ free radicals. (F) Relationship between the clearance ABTS•+ rate and concentration of final NPs. (G) Optical picture of ABTS•+ solution after addition of different concentrations of Se-5Fu-Gd, Se-5Fu-GdP(Cet) and Se-5Fu-Gd-P (Cet-YI-12) NPs for 120 min.




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A

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Figure 11 Biodistribution of Se-5Fu-Gd and Se-5Fu-Gd-P(Cet-YI-12) NPs in vivo. (A) T1-weighted MR imaging of CNE-bearing xenograft nude mice before and 4 h, 24 h after i.v. injected with Se-5Fu-Gd and Se-5Fu-Gd-P(Cet-YI-12) NPs. Changes of T1 MR signal intensity in tumor sites before and after the injection of Se-5Fu-Gd (B) and Se-5Fu-Gd-P(Cet-YI-12) NPs (C) at various time intervals (0, 4 and 24 h). (D) The concentration of Se-5Fu-Gd and Se-5Fu-Gd-P(Cet-YI-12) NPs in major organs and tumor from tumor-bearing nude mice at the 24 h post-injection (n=3, as calculated by Se).

understand the intratumoral behaviors of nanoparticle penetration in real time with a confocal microscopy. Figure 10A showed the process of progressive formation of tumor spheroids. 3D tumor spheroids were successfully cultivated approximately 200



R% in 7th day to mimic the extracellular microenvironment and diffusion processes in actual solid tumors. These tumor spheroids were incubated with different nanoparticles at equal concentration, and continuously monitored the growth volume for 6 days. Figure 10B showed that the volume of CNE tumor spheroids gradually increased to 1.2 fold than that of the original during the 6 days. In the contrary, the volume of tumor spheroids in treated groups reduced gradually, especially for final group treatment, which revealed that tumor spheroids growth were repressed by the nanoparticles. For example, the spheroids volume ratio of Se-5Fu-Gd-P(Cet-YI-12) NPs treated group was reduced to 67.1%, much less than that of Se-5Fu-Gd (92.3%) and Se-5Fu-Gd-P(Cet) (78.4%), which may be induced by the difference in cellular uptake of nanoparticles in tumor spheroids, resulting in various permeability to nanosystem. Hence, we then evaluated the permeability of functionalized CSe-5Fu-Gd-P(Cet-YI-12) on spheroids by a confocal laser scanning microscope. Figure 10C showed the fluorescence intensity of different NPs in different scan sections of tumor spheroids. Results revealed that CSe-5Fu-Gd-P(Cet-YI-12) penetrated deeper (40-50 R%8 than CSe-5Fu-Gd and CSe5Fu-Gd-P(Cet-YI-12), with more extensive and stronger green fluorescence intensity when compared with the same scan level. These results indicated that CSe-5Fu-GdP(Cet-YI-12) NPs had great advantages in penetrating into NPC, which would play an important role in cancer therapy.

2.10 MR images and biodistribution of Se-5Fu-Gd-P(Cet-YI-12) NPs in vivo CNE-bearing xenograft nude mice model were built to further demonstrate the targeting ability of Se-5Fu-Gd-P(Cet-YI-12) NPs in vivo. First, equivalent concentrations of Se5Fu-Gd and Se-5Fu-Gd-P(Cet-YI-12) NPs (Se concentration, 4 mg kgU , 0.2 mL) were intravenously (i.v.) injected to mice. Next, dynamic MRI was performed by acquiring the T1-weighted signal of Gd within 24 h of injection under a clinical 3.0 T MRI scanner. As reflected in Figure 11A, after i.v. injection of Se-5Fu-Gd or Se-5Fu-GdP(Cet-YI-12) NPs for 4 h, a significant brightening phenomenon could be found in the tumor region, demonstrating that these Se nanoplatforms could be used as a promising contrast agents for MRI. Meanwhile, stronger accumulation of Se-5Fu-Gd-P(Cet-YIACS Paragon Plus Environment

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12) NPs in tumor region at 24 h than that of Se-5Fu-Gd treatment, as presented by the stronger T1-weighted signal, which suggesting the strengthened targeting ability of Se5Fu-Gd-P(Cet-YI-12) NPs in vivo (Figure 11A-11C). Furthermore, at the end of the experiment, the mice were sacrificed and the heart, liver, spleen, lungs, kidneys and tumor were exploited to measure the content of Se by ICP-MS. Tumoral amount of Se from Se-5Fu-Gd-P(Cet-YI-12) NPs was 3.2 folds higher than that of Se-5Fu-Gd, which further proved the above result (Figure 11D). Overall, based on above results in vivo, we can conclude that the as-synthesized Se nanosystems could recognize the tumor, accumulate in the tumor and improve the efficiency of treatment and diagnosis.

3. Conclusion SeNPs have drawn the wide attention and showed great antitumor potential, attributing to its unique features such as biocompatibility, intrinsic anticancer activities and drugloading capacity. Despite its tremendous advantages, nonuniformity and inaccurate release remains to be the major obstacles hampering the achievement of optimal therapeutic outcomes. Herein, we have engineered and synthesized a highly uniform and stable SeNPs by using TW-80 as a modification agent due to its inherent steric effects and hydrogen bond effect from ether bond. And then we employed TW-80-SeNPs to further construct a multifunctional and smart drug-delivered Se nanoplatform (Se-5Fu-Gd-P(Cet/YI-12)) which programmed PAMAM-SeNPs-loaded Gd and 5Fu covered with Cet and YI-12 peptide by DTSSP as bridging agent against NPC. This Se nanoplatform exhibited uniform size, morphology and high stability under physiological conditions. It was noteworthy that Se-5Fu-GdP(Cet/YI-12) showed excellent MR imaging capability and possess the potential clinical application as a diagnostic agent for tumors tissue specimens. In vitro cellular experiments showed that by surface connecting of EGFR targeted drugs and YI-12 peptide not only effective increased intracellular accumulation of Se nanoplatform in



NPC cells by receptor-mediated endocytosis, but also enhanced its penetration toward CNE tumor spheroids we cultured in vitro, resulting to simultaneous CNE cells inhibition of growth, the invasion and migration. Besides, the morphology and size of the nanoplaform enabled to sequentially change in the tumor microenvironment, which may improve the efficiency of 5Fu. Overall, this study not only provides a strategy for facile synthesis of highly-uniform and stable nanomedicines and tailing of the bioresponsive property, but also sheds light on its application in targeting theranosis of NPC.

4. Experimental Section Chemicals and reagents: G5 PAMAM with the core of ethylenediamine, TW-80, Na2SeO3 and 5Fu were purchased from Sigma-Aldrich. L-cysteine (L-cys) was purchased from Guangzhou chemical reagent factory. Cet was acquired from BristolMyers Squib. The anti-EGFR antibody for IHC analysis was obtained from Cell Signaling Technology (#4267). The YI-12 peptide was synthesized by GL Biochem (Shanghai) Ltd. Besides, experimental water was obtained from Milli-Q system when it value reached 18.2 X which was conform to the experimental demand.

Synthesis of Gd chelates stock solution: Gd chelates stock solution was synthesized according to the previous reports.37 In brief, 50 R6 GdCl3•6H2O (1 M) was added to sodium citrate solution with the drastic stirring. Next, ammonia (NH4OH) was added to the mixed solution and reacted a few minutes.

Preparation of TW-80/Cs/Let-Se, Se-5Fu-Gd, Se-5Fu-Gd-P nanoparticles: First, we prepared the TW-80/Cs/Let-SeNPs by using L-cys as reducing agents, Na2SeO3 as Se source and TW-80 (1 mg mLU )/Cs (0.8 mg mLU )/PSR (0.3 mg mLU ) as modification agents. Next, 5Fu and Gd chelates loaded-SeNPs (Se-5Fu-Gd NPs) were synthesized by the electrostatic interaction and formation of Se-O and Se-N bond


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according the description of the existing literature 27. In brief, 1.5 mM 5Fu (1 mL) and 0.17 M Gd (1 mL) were mixed with Na2SeO3 solution (1 mL) containing TW-80 (1 mg mLU ) under mid agitation, and then slowly injected to L-cys. The mixture solution was stirred in -4 °C environments for 12 h to acquire the sample (Se-5Fu-Gd NPs). Se-5FuGd NPs (1 mM, 10 mL) we prepared were mixed with PAMAM (50 R68 to acquire amino groups functioned Se-5Fu-Gd NPs (Se-5Fu-Gd-P NPs) and then were used to connect Cet and YI-12 peptides. All of the NPs were dialyzed against Milli-Q water for one day to remove reactant.

Fabrication of Se-5Fu-Gd-P(Cet/YI-12) nanoparticles: DTSSP can covalently combine with amino groups, mercapto groups and hydroxyl groups by substituting sulfonic acid in alkaline environment.52 Hence, Se-5Fu-Gd-P(Cet/YI-12) NPs were synthesized by the following methods. In short, DTSSP, Cet and YI-12 were added to 10 mL of Se-5Fu-Gd-P NPs solution, following by the addition of NaOH (1%) to adjust the pH value of the reacted solution to 10. After reacted for 12 h in alkaline environment, Se-5Fu-Gd-P(Cet/YI-12) NPs were acquired by further purification.

MR images in vitro: The clinical 3.0 T MRI scanner (Bruker Biospin Corporation, Billerica, MA, USA) was introduced in order to compare the magnetic properties of Gd alone and Se-5Fu-Gd-P(Cet/YI-12) NPs at the same concentration in vitro. In short, Gd chelates solution and Se-5Fu-Gd-P(Cet/YI-12) NPs solution with different concentrations including 0.1618, 0.32375, 0.6475, 1.295 and 2.59 RM was placed in tube and then scanned by 3.0 T MRI scanner.

Characterization of Se-5Fu-Gd-P(Cet/YI-12) nanoparticles: The morphology of Se-5Fu-Gd-P(Cet/YI-12) NPs was characterized by using TEM and AFM. We employed Zetasizer Nano ZS particle analyzer to measure the size distribution and Zeta potential of Se-5Fu-Gd-P(Cet/YI-12) NPs. FT-IR and UV-vis (Carry 5000 spectrophotometer) were introduced to testify the connection Cet and YI-12 by using the KBr-disk method. Meanwhile, we conducted bicinchoninic acid (BCA) kit (Pierce) ACS Paragon Plus Environment


for Cet and YI-12 determination. In addition, the 5Fu concentration of as-prepared samples was quantitatively measured using an HPLC system (Agilent 1100) with a model UV-1000 UV finder, and the wavelength was set at 266 nm. The column used was R2G

D C18 (4×300 mm, Grom, Germany) followed by previous studies.53

Gd and Se concentration of NPs were analyzed by inductively coupled plasma mass spectrometry (ICP-MS).

Labeling Se-5Fu-Gd-P(Cet-YI-12) NPs with coumarin-6: coumarin-6 (Coum-6) is a common green fluorescent dye, which have widely labeled in various nanosystems.54-56 In dark environment, Coum-6 (2 mg LU ) was mixed with Se-5FuGd-P(Cet-YI-12) NPs (10 mL) and stirring for 12 h. Finally, the hybrid solution (CSe5Fu-Gd-P(Cet-YI-12) NPs) was dialyzed against Milli-Q water for 24 h to remove excess Coum-6.

Stability analysis of Se-5Fu-Gd-P(Cet/YI-12) nanoparticles: The stability of NPs is one of the most basic supporting data for future application. In order to evaluate the stability of NPs, we measured the size of Se-5Fu-Gd-P(Cet/YI-12) NPs in FBS solution, DMEM and aqueous solution for 15 days by using nano-ZS instrument.

Hemolysis analysis: Whole blood extracted from healthy volunteers was centrifuged at 1500 rpm for 10 min to obtain RBCs. Subsequently, RBCs (0.5 mL) incubated with TW-80-Se, Se-5Fu-Gd, Se-5Fu-Gd-P(Cet) and Se-5Fu-Gd-P(Cet-YI-12) NPs at various concentrations at 37 °C for different time. Additionally, PBS solution and Triton X-100 (10 g LU ) incubated with RBCs were introduced as negative control (NC) and positive control (PC). On the one hand, the treated RBCs were centrifuged, while the clear supernatant was collected for calculating the rate of hemolysis by measuring the absorbance at 542 nm. The rate of hemolysis is calculated based on the formulas. Hemolytic rate (%) = (A sample-ANC)/APC –ANC) ×100%. On the other hand, after incubation for 6 h, we observed the morphology of RBCs ACS Paragon Plus Environment

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treated with different concentrations of NPs and different NPs by using microscope.

Staining of clinical specimens: The human tissues samples were collected from Affiliated hospital of Guangdong Medical University from January 2016 to December 2017. Our study was carried out with participants who had given their consent previously and the ethical approval of the Institutional Ethics Committee. All tissue sections, 4 R% in thickness, were baked for 1 h at 60 °C, and then deparaffinized with xylene and rehydrated with gradient ethanol. After antigen retrieval by microwave and EDTA buffer solution (pH 8.0), sections were blocked with non-antigen goat serum for 30 min in room temperature and then incubated with Coum-6-conjugated CSe-5Fu-GdP(Cet-YI-12) nanoparticles (31.2 R 8 for 1.5 h at 37 °C. The stained sections were analyzed under a fluorescent microscope (Life Technologies). Immunohistochemical staining of tissue sections by anti-EGFR antibody (1:50 dilution) was performed to compare tumor binding specificity and staining quality with CSe-5Fu-Gd-P(Cet-YI-12) NPs.

Cell culture and cytotoxicity evaluation: The human NPC cell lines including CNE and CNE2 were obtained from the Cancer Institute, Southern Medical University (Guangzhou, China). These cell lines were maintained in RPMI-1640 medium, while human normal liver cell L02, which were purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China), were maintained in high glucose DMEM. All the media were supplemented with heat-inactivated fetal bovine serum (10%, GIBCO 10270-106) and antibiotics (GIBCO 15070-063). Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. The cytotoxicity of the Gd, 5Fu, TW-80Se, Se-5Fu-Gd NPs, Se-5Fu-Gd-P(Cet) NPs, Se-5Fu-Gd-P(Cet-YI-12) NPs to different cells was detected by MTT assay. In brief, cells at a density of 20000 / per well were seeded on 96-well plates and allow to stick. Next, various concentrations of drugs were added to each well and cultured for 72 h, followed by the supplement of MTT for 4 h. Finally, microplate reader with wavenumber set at 570 nm was used to detect the viability of drugs. ACS Paragon Plus Environment


Cellular uptake and intracellular localization: CNE cells and L02 cells were digested into single cells suspension at a density of 1.5×105 cells mlU , which was inoculated into 6-well plate (2 ml per well). After incubation for 24 h, a certain concentration of CSe-5Fu-Gd NPs, CSe-5Fu-Gd-P(Cet) NPs and CSe-5Fu-Gd-P(CetYI-12)NPs were added and incubated continuously. At different time points, the cells were harvested and cellular uptake of the nanosystem was quantitatively analyzed by measuring the fluorescence intensity. YI-12 peptide competition assay was performed to prove the important of EGFR receptor effects cellular uptake. Briefly, CNE cells were pre-treated with different concentrations of YI-12 peptide (0–0.5 mg mLU ). And then, the cells were wash three times by PBS and incubated with CSe-5Fu-Gd-P(Cet/YI-12) NPs for 4 h. Finally, the internalized CSe-5Fu-Gd-P(Cet/YI-12) NPs was examined through monitoring its fluorescence intensity change by flow cytometric analysis. Intracellular localization of nanoparticles was tracked by immunofluorescence staining. After treatment with CSe-5Fu-Gd-P(Cet-YI-12) NPs, the cells were labeled by Hoechst 33342 (blue) and Lyso-tracker DND-99 (Red). And the culture medium was removed and washed by PBS for 3 times to ensure the clear environment. Finally, the cells were monitored by fluorescence microscope (IX51, Olympus) to record the intracellular trafficking.

Mechanism of cellular uptake: CNE cells were seeded at a density of 8×103 cells/well into 96-well plates and incubated for 24 h. After cell adherence, inhibit agents about endocytosis including Sucrose (0.45 mM), NaN3 (10 mM), Dynasore (80 R 8 and Nystatin (10 R mLU ) were added into each well and incubated for 30 min or 60 min, respectively. And the medium was removed and then a certain concentration of CSe-5Fu-Gd-P(Cet-YI-12)NPs were added to wells and incubated for 4 h. Meanwhile, in the control group, some concentration of CSe-5Fu-Gd-P(Cet-YI-12) NPs was added to well without the addition of inhibit agents. Subsequently, the cells were washed with PBS for three times and lysed with 1% Triton X-100 in 0.1 N NaOH solutions. Finally, ACS Paragon Plus Environment

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we introduced the microplate reader to measure the fluorescence intensity of NPs with excitation and emission wavelengths set at 430 and 485 nm.

Cytoskeleton staining: For cytoskeleton staining, a total of 1.0×105 cells were seeded in confocal culture dish and incubated for at least 12 h to allow cell attachment. Then, 2 mL fresh medium containing CSe-5Fu-Gd NPs, CSe-5Fu-Gd-P-Cet NPs or CSe-5Fu-Gd-P(Cet-YI-12)NPs were added to each dishes. After incubating for 4 h, the seeded cells were fixed in 4% formaldehyde solution and stained subsequently with Alexa Fluor 555-conjugated phalloidin (Cell Signaling Technology, CST) and Hoechst 33342 (CST). Images were acquired using an Eclipse fluorescent microscope (Life Technologies) with a ×100 oil-immersion objective.

Colony formation assay: CNE cells (2500 cells/well) were seeded and allowed to attach for 24 h in 6-well plates. The cells were incubated with Se-5Fu-Gd NPs and Se5Fu-Gd-P(Cet-YI-12) NPs at different concentrations for 10 days after the attachment in the 6-well plates. After that the cells were fixed and stained with 0.5% crystal violet solution. The positive colonies (colonies containing Z # cells) was counted under a microscope. Plate colony formation efficiency=number of colonies/ number of cells inoculated ×100%.

Wound healing assay: Cells were cultured in 24-well plates to 80~90% confluence under standard conditions. Scratches were constructed with the 10 R6 micropipette, followed by washing for 3 times and the addition of fresh serumcontaining medium plus with different concentrations of Se-5Fu-Gd-P(Cet-YI-12) NPs. Wild CNE cells treated with Se-5Fu-Gd NPs were used as controls. Cells were stained with Hoechst 33342 for 30 min. After incubation for 24 h fluorescence microscope was utilized to capture the images of cells.

Examination of Cell invasion: Cell invasion ability was assessed in 24-well transwell inserts (8.0 R% pore size, Corning Inc.). Firstly, 200 R6 of serum-free medium ACS Paragon Plus Environment


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containing 2.5×105 CNE cells was added to the top chambers, while the lower chambers were filled with 500 R6 complete RPMI 1640 media containing 10% FBS. Different concentrations of Se-5Fu-Gd-P(Cet-YI-12) NPs and Se-5Fu-Gd NPs were supplied in the upper media. After 18-24 h of incubation, the invaded cells

under the surface of

the membrane were stained with 0.1% crystal violet after fixation. Cells in random fields were visualized and counted under a microscope.

ROS generation: The effects of Se-5Fu-Gd-P(Cet-YI-12) NPs on intracellular superoxide generation in CNE cells were measured by using DHE, a fluorescent marker for ROS which permeates live cells and is deacetylated by intracellular esterase. Briefly, CNE cells were seeded into 96-well plate (3.0×104 cells per well) for cell attachment, and incubated with 10 R

DHE at 37 °C for 30 min. After that, the cells were treated

with different concentration of Se-5Fu-Gd NPs, Se-5Fu-Gd-P(Cet) NPs and Se-5FuGd-P(Cet-YI-12) NPs. At specific time intervals, the intracellular ROS level was recorded by a microplate reader. Meanwhile, to observe the fluorescence level of ROS vividly, CNE cells were seeded to 2 cm dish for attachment and stained with DHE for 30 min. Next, the medium was displaced by phosphate buffer solution containing nanodrugs. Finally, fluorescence microscope was used to capture the fluorescence intensity of treated CNE cells in different times.

ABTS radical scavenging activity: Based on the requirement of the Total Antioxidant Capacity Assay Kit, ABTS method was used to evaluate the antioxidant activity of Se-5Fu-Gd-P(Cet-YI-12) NPs. ABTS solution was reacted with excess manganese dioxide (MnO2) to gain ABTS•+ stock solution and then the stock solution was diluted with PBS to acquire the working solution that the absorbance value was about 0.40 ± 0.02. Then, in dark environment, the mixed solution including different concentrations of NPs and ABTS•+ working solution was detected by a microplate reader with the wavenumber set at 734 nm. The absorbance value at 734 nm of mixture represented the content of ABTS radical.


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Penetrating and inhibitory effects to CNE multicellular tumor spheroids: 3-dimensional (3D) multicellular tumor spheroids which have applied in the research of cellular proliferation and differentiation, and the response of tumors to chemotherapy have been widely used in biology due to the simple simulation of tumor environment in vivo. As described in previous study,57 the CNE cells (6.0×105 cells in 2 mL of complete media) were inoculated to ultra-low attachment multiple well plates (6-well) to form tumor spheroids. To evaluate the tumor-penetrating ability of nanoparticles, the spheroids were exposed to CSe-5Fu-Gd NPs, CSe-5Fu-Gd-P(Cet) NPs and CSe-5FuGd-P(Cet-YI-12) NPs with the same concentration, respectively. After incubation for 12 h, these treated CNE spheroids were observed in sequential thin-layer using confocal laser scanning microscope (Zeiss LSM, Germany). In order to investigate the inhibitory effect of NPs on spheroids growth, CNE spheroids were exposed to the same concentration of nanodrugs. Meanwhile, few of spheroids incubated with drug-free completed media were used as control. The growth of spheroids was monitored with an EVOS fluorescence microscope (Life Technologies) for 5 days.

MR images in vivo and biodistribution of Se-5Fu-Gd-P(Cet/YI-12) NPs: First, Female BALB/c nude mice were acquired from animal center of Guangdong province and the operation were conducted in accordance with the scheme approved by the Laboratory Animal Center of Jinan University. CNE xenograft mice were scanned by MRI before and after i.v. injection of Se-5Fu-Gd and Se-5Fu-Gd-P(Cet-YI-12) NPs (4 mg kgU , 0.2 mL). Finally, after 24 h, these mice were executed, and main organs and tumor tissues were collected for Se concentration detection by ICP-MS.

Statistical analysis: All experiments were carried out at least in triplicate and results were calculated as means ± SD.

Supporting Information



Intracellular localization of CSe-5Fu-Gd-P(Cet-YI-12) NPs in CNE cells (Figure S1). The Supporting Information is available free of charge on the ACS Publication website.

Conflict of Interest The authors declare no conflict of interest. J. Huang and W. Huang contributed equally to this work.

Acknowledgements This work was supported by Natural Science Foundation of China (21877049), National Program for Support of Top-notch Young Professionals (W02070191), YangFan Innovative & Entepreneurial Research Team Project (201312H05) and Fundamental Research Funds for the Central Universities.

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Highly Uniform Synthesis of Selenium Nanoparticles with EGFR

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