AuNP flares-capped mesoporous silica nanoplatform for MTH1 detection and inhibition

Accepted Manuscript AuNP flares-capped mesoporous silica nanoplatform for MTH1 detection and inhibition Wen Gao, Wenhua Cao, Yuhui Sun, XuePing Wei, Kehua Xu, Huaibin Zhang, Bo Tang PII:

S0142-9612(15)00679-1

DOI:

10.1016/j.biomaterials.2015.08.021

Reference:

JBMT 17016

To appear in:

Biomaterials

Received Date: 24 April 2015 Revised Date:

7 August 2015

Accepted Date: 8 August 2015

Please cite this article as: Gao W, Cao W, Sun Y, Wei X, Xu K, Zhang H, Tang B, AuNP flares-capped mesoporous silica nanoplatform for MTH1 detection and inhibition, Biomaterials (2015), doi: 10.1016/ j.biomaterials.2015.08.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

AuNP flares-capped mesoporous silica nanoplatform for MTH1 detection and inhibition

RI PT

Wen Gao, Wenhua Cao, Yuhui, Sun, XuePing Wei, Kehua Xu, Huaibin Zhang, Bo Tang*

College of Chemistry, Chemical Engineering and Materials Science, Collaborative

SC

Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education,

M AN U

Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals,

EP

TE D

Shandong Normal University, Jinan 250014, P.R. China.

AC C

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 61 62 63 64 65

*

Corresponding authors. Tel.: +86 531 86180010; Fax: +86 531 86180017. E-mail addresses: [email protected] (Bo Tang). 1

ACCEPTED MANUSCRIPT ABSTRACT The human mutT homologue MTH1, a nucleotide pool sanitizing enzyme, represents a vulnerability factor and an attractive target for anticancer therapy. However, there is

RI PT

currently a lack of selective and effective platforms for the detection and inhibition of MTH1 in cells. Here, we demonstrate for the first time a gold nanoparticle (AuNP) flares-capped mesoporous silica nanoparticle (MSN) nanoplatform that is capable of detecting MTH1 mRNA and simultaneously suppressing MTH1 activity. The AuNP

SC

flares are made from AuNPs that are functionalized with a dense shell of MTH1 recognition sequences hybridized to short cyanine (Cy5)-labeled reporter sequences

M AN U

and employed to seal the pores of MSN to prevent the premature MTH1 inhibitors (S-crizotinib) release. Just like the pyrotechnic flares that produce brilliant light when activated, the resulting AuNP flares@MSN (S-crizotinib) undergo a significant burst of red fluorescence enhancement upon MTH1 mRNA binding. This hybridization event subsequently induces the opening of the pores and the release of S-crizotinib in

TE D

an mRNA-dependent manner, leading to significant cytotoxicity in cancer cells and improved therapeutic response in mouse xenograft models. We anticipate that this nanoplatform may be an important step toward the development of MTH1-targeting

EP

theranostics and also be a useful tool for cancer phenotypic lethal anticancer therapy. Keywords: AuNP flares; Mesoporous silica nanoparticle; MTH1; Detection;

AC C

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 61 62 63 64 65

Inhibition; Cancer theranostic

2

ACCEPTED MANUSCRIPT 1. Introduction

Redox dysregulation in cancer cells results in reactive oxygen species (ROS) overproduction, damaging DNA and free bases in deoxyribonucleotide triphosphates

RI PT

(dNTPs) pools [1-3]. The MutT Homolog1 protein, MTH1, can effectively degrade oxidized nucleotides to prevent their incorporation into DNA, and thereby is

SC

important for minimizing cancer-associated damage in the dNTP pool as required for cancer cell survival. Experimental evidence has shown that inhibition of MTH1

M AN U

activity selectively and effectively kills cancer cell lines, but is considerably less toxic to normal cells, which validates MTH1 as an “Achilles heel” for cancer cells and a new target for cancer treatment [4, 5]. However, the localization and relative

TE D

abundance of MTH1 mRNA in different cell types, as well as the regulation of intracellular release of MTH1 inhibitors, are poorly characterized. Northern blots [6] and reverse transcription polymerase chain reaction (RT-PCR) [4, 5] are commonly

EP

used to detect MTH1 mRNA transcripts. However, these methods require millions of cells and are not applicable to real-time studies of live single cells. Although in situ

AC C

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 61 62 63 64 65

staining [6, 7], green fluorescent protein (GFP) [8], and fluorescence resonance energy transfer (FRET) sensors [8] have been used to detect MTH1 mRNA in living cells, these probes are often difficult to transfect, require additional agents for cellular internalization, and are unstable in cellular environments. This leads to a high background signal and an inability to detect targets. Small molecules such as TH287, TH588, SCH51344 and S-crizotinib inhibit the MTH1 catalytic activity [4, 5], but the 3

ACCEPTED MANUSCRIPT therapeutic effects may be limited by their insolubility and instability in cellular environments as well as their poor penetration into tumor sites. Moreover, these inhibitors lack selectivity and distribute indiscriminately into all cells, requiring a high

RI PT

dose to significantly affect tumor volume in mouse xenografts. The uncontrolled release mechanism makes it difficult for MTH1 inhibitors to reach their full therapeutic efficacy. Therefore, the detection and visualization of MTH1 mRNA in

SC

living cells and controlled release of the inhibitors according to MTH1 mRNA levels

M AN U

remains a challenge.

Nanomaterials have showed their powerful ability to construct multifunctional platforms for intracellular research [9, 10]. Gold nanoparticles (AuNPs) conjugated to short, fluorophore-labeled oligonucleotide duplexes can be intracellular “nanoflares”

TE D

and are of significant interest because of their strong fluorescence signals, low background and probe stability [11, 12], which enables the visualization and

EP

quantification of MTH1 mRNA in living cells, without adding external transfection agents. Another motivation for employing AuNP flares as an MTH1 probe is that they have the potential of becoming a switch to control the release of MTH1 inhibitors in

AC C

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 61 62 63 64 65

response to intracellular MTH1 mRNA levels. Surface-functionalized, end-capped mesoporous silica nanoparticles (MSNs) are promising scaffolds to construct mRNA-responsive controlled release systems, because of their unique mesoporous structures, large surface areas, tunable pore sizes, and good biocompatibility, both in vitro and in vivo [13-15]. These end-capped MSNs with “controlled release” 4

ACCEPTED MANUSCRIPT properties have been synthesized with different pore-blocking caps such as organic molecules [16, 17], supramolecular assemblies [18, 19], nucleotides [14, 20] and nanoparticles (NPs) [21, 22]. AuNPs in particular have successfully controlled the

RI PT

opening and closing of the pore entrance of MSN through either a reversible pH-dependent boronate ester bond [23], an acetal linker [24] or photo-controlled electrostatic interactions [25]. We hypothesized that the AuNP flares could be

SC

employed as caps to encapsulate MTH1 inhibitors within the porous channels of the

M AN U

MSNs, which will be selectively opened in the presence of MTH1 mRNA targets. Herein, we present a novel and effective strategy for combined MTH1 detection and inhibition based on AuNP flares-capped MSN nanoplatform. As shown in Fig. 1, the AuNP flares consisted of AuNP functionalized with a dense shell of 20-base

TE D

MTH1 recognition sequences hybridized to short cyanine (Cy5) dye-labeled reporter sequences via a gold-thiol bond. After entrapping MTH1 inhibitors (S-crizotinib) in

EP

the mesopores of MSN, the AuNP flares were immobilized on the exterior surfaces of the MSN through the linkage of reporter sequences. In the bound state, the Cy5 fluorescence was quenched, and the AuNP blocked the pores. In the presence of

AC C

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 61 62 63 64 65

MTH1 mRNA, the recognition sequences hybridized with this complementary target sequences by forming the longer and more stable duplexes, causing the liberation of AuNP from the reporter sequences and the opening of the pores, which can then produce fluorescent signals correlated with the relative amount of the MTH1 mRNA and release of S-crizotinib in an mRNA-dependent manner. These AuNP 5

ACCEPTED MANUSCRIPT flares-capped MSNs were successfully used to detect and quantify MTH1 mRNA and inhibit MTH1 activity both in vitro and in vivo. To the best of our knowledge, this is the first time that AuNP flares have been employed as pore-blocking caps to construct

RI PT

mRNA-responsive controlled release system based on MSNs, which allows a single nanoplatform capable of both detecting and regulating MTH1. By targeting MTH1, a

EP

TE D

M AN U

useful tool for cancer diagnosis and therapy.

SC

novel cancer phenotypic lethal, AuNP flares-capped MSNs have the potential to be a

AC C

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 61 62 63 64 65

Fig. 1. Schematic illustration of AuNP flares-capped MSN nanoplatform for detection and inhibition of MTH1.

2. Materials and methods

2.1. Materials

6

ACCEPTED MANUSCRIPT DNA oligonucleotides were synthesized and purified by Sangon Biotechnology Co., Ltd (Shanghai, China). The sequences of these oligonucleotides are shown in Table S1. Cetyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (3-aminopropyl)

1-ethyl-3-(3-dimethylaminopropyl)

triethoxysilane carbodiimide

(APTES),

RI PT

(TEOS),

hydrochloride

(EDC),

3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), S-crizotinib

(III)

(HAuCl4·4H2O,

99.99%),

Trisodium

citrate

M AN U

tetrachloroaurate

SC

and Rhodamine B (RhB) were purchased from Sigma-Aldrich. Hydrogen

(C6H5Na3O7·2H2O), Sodium dodecylsulfate (SDS), mercaptoethanol (ME), MgCl2, CaCl2, KCl were obtained from Sinopharm Chemical Reagent. Co. Ltd. (Shanghai, China). Deoxyribonuclease I (DNase I) was purchased from Solarbio Science and

TE D

Technology Co., Ltd. (Beijing, China). Cell culture products, unless mentioned otherwise, were purchased from GIBCO. DAPI were bought from Beyotime Inst.

EP

Biotech, (Haimen, China). All chemicals and solvents used were of analytical grade. Water was purified with a Sartorius Arium 611 VF system (Sartorius AG, Germany) to a resistivity of 18.2 MΩ·cm.

AC C

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 61 62 63 64 65

2.2. Characterization

High resolution transmission electron microscopy (HRTEM) was carried out on a JEM-2100 electron microscope. N2 adsorption-desorption isotherms were recorded on a Micromeritics ASAP2020 surface area and porosity analyzer. The samples were 7

ACCEPTED MANUSCRIPT degassed at 150°C for 5 h. The specific surface areas were calculated from the adsorption data in the low pressure range using the BET model and pore size was determined using the Barrett-Joyner-Halenda (BJH) method. X-ray diffraction (XRD)

RI PT

analysis was carried out on a D/Max 2500 V/PC X-ray diffractometer using Cu (40 kV, 30 mA) radiation. Absorption spectra were measured on a pharmaspec UV-1700 UV-visible spectrophotometer (Shimadzu, Japan). Dynamic light scattering (DLS)

SC

measurements were performed on a Malvern zeta sizer Nano-ZS90. FTIR spectra

M AN U

were collected on a Nicolet Impact 410 FTIR spectrometer in the range of 400-4000 cm−1. X-ray photoelectron spectroscopy (XPS) spectra were performed with a Phobios 100 electron analyzer (SPECS GmbH) equipped 5 channeltrons, using an unmonochromated Mg Kα X-ray source (1253.6 eV). The deconvolution of the XPS

TE D

peaks was done by a XPS Peak fitting program (version 4.1). Fluorescence spectra were obtained with FLS-920 Edinburgh Fluorescence Spectrometer with a Xenon

EP

lamp and 1.0 cm quartz cells at the slits of 3.0/3.0 nm. All pH measurements were performed with a pH-3c digital pH-meter (Shanghai LeiCi Device Works, Shanghai, China) with a combined glasscalomel electrode. Absorbance was measured in a

AC C

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 61 62 63 64 65

microplate reader (RT 6000, Rayto, USA) in the MTT assay. Confocal fluorescence imaging studies were performed with a TCS SP5 confocal laser scanning microscopy (Leica Co., Ltd. Germany) with an objective lens (×40). In vivo imaging were performed with Caliper IVIS Lumina III (Caliper Co., USA).

2.3. Preparation of the AuNP flares 8

ACCEPTED MANUSCRIPT First, the 5 nm AuNP were prepared by the modified citrate reduction method [26]. 5.9 mg of HAuCl4 was dissolved in 1 mL of ultrapure water and added into 47 mL of ultrapure water to the final concentration of 0.3 mM. The solution was

RI PT

incubated with constant stirring at 25 °C for 10 min. 0.5 mL of 50 mM sodium citrate solution was added to the reaction mixture to a final concentration of 0.5 mM, and the solution was incubated under constant stirring for another 10 min. 0.67 mL of cooled

SC

0.1 M NaBH4 was added into the stirred mixture; the solution developed an

M AN U

orange-red color. The resulting nanoparticles were screened for their size and uniformity by HRTEM. The prepared AuNP were stored at 4 °C. Second, the thiolated recognition strand were mixed with reporter strand (1:1.2) in phosphate buffered saline (PBS: 137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl,

TE D

pH 7.4), heated to 75 °C and maintained 10 min, then slowly cooled to room temperature, and stored in the dark for at least 12 hours to allow complete

EP

hybridization and flare duplex formation. Then, the flare duplexes added to a solution of AuNP (3 nM) with a final concentration of 150 nM and shaken overnight. After 12 hours, SDS solution (10%) was added to the mixture to achieve 0.1 % SDS

AC C

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 61 62 63 64 65

concentration. Phosphate buffer (0.1 M, pH 7.4) was added to the mixture to achieve 0.01 M phosphate concentration and the NaCl concentration of the mixture was slowly increased to 0.7 M over an eight-hour period. The resulting AuNP flares were purified from unreacted materials by three successive rounds of centrifugation (14000 rpm, 20 min, 4 °C), supernatant removal, and resuspension in PBS with a 9

ACCEPTED MANUSCRIPT concentration of 15 nM as stock solution at 4 °C. The AuNP flares was diluted to certain concentration for use in all subsequent experiments. The concentration of AuNP flares was determined by measuring their extinction at 528 nm (ε = 2.7×108 L

2.4. Quantitation of DNA duplexes loaded on the AuNP flares

RI PT

mol-1 cm-1).

SC

The flare duplexes loaded on AuNP were quantitated according to the previous

M AN U

protocol [27]. Briefly, mercaptoethanol (ME) was added (final concentration 20 mM) to the AuNP flares solution (3 nM). After it was incubated overnight with shaking at room temperature, the flares were released. Then the released flares were separated via centrifugation and the fluorescence was measured with a fluorescence

TE D

spectrometer. The fluorescence of Cy5 labelled flare was excited at 648 nm and measured at 688 nm. The fluorescence was converted to molar concentrations of

EP

flares by interpolation from a standard linear calibration curve that was prepared with known concentrations of flares with identical buffer pH, ionic strength and ME concentrations. By dividing molar concentrations of the flares by the original AuNP

AC C

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 61 62 63 64 65

flares concentration, we calculated that there was 18 ± 1 flares per AuNP.

2.5. Preparation of AuNP flares@MSN nanoplatform

First, amino modified mesoporous silica was synthesized according to the typical co-condensation method reported previously with some modifications [28]. 10

ACCEPTED MANUSCRIPT Cetyltrimethylammonium bromide (CTAB, 0.25 g, 0.69×10-3 mol) was first dissolved in 120 mL sartorius ultrapure water. NaOH (2 M, 0.75 mL) was then added to the above solution and the mixture was stirred for 5 min, followed by adjusting the

RI PT

solution temperature to 80 °C. After that, TEOS (1.25 mL, 5.6×10-3 mol) was first introduced dropwise to the surfactant solution, followed by the dropwise addition of APTES (0.25 mL, 1.07×10-3 mol). The mixture was allowed to stir for 2 h to give rise

SC

to white precipitates (as synthesized NH2-MSN). The solid product was filtered,

M AN U

washed with ultrapure water and methanol, and then dried in air. To remove the surfactant template (CTAB), 0.5 g as-synthesized amino modified mesoporous silica was refluxed for 24 h in a solution of 3 mL of HCl (37.4%) and 50 mL of methanol followed by extensive washes with ultrapure water and methanol. Finally, the

TE D

precipitates were dried for 24 h in vacuum.

Then, AuNP flares@MSN was obtained by coupling the carboxyl groups of the

EP

reporter strands and the amino groups on the surface of NH2-MSN to form the CONH amide bonds. 3 μL EDC solution (2.8 mM) was added to 500 μL of AuNP flares (3 nM) solution and the solution was mixed and reacted for 30 min at room temperature

AC C

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 61 62 63 64 65

to activate carboxyl groups. Then the mixture was added to 0.5 mL of a previously prepared NH2-MSN solution (2 mg/mL; MES buffer: 10 mM, pH 6.0) with gentle stirring in darkness. The solution was reacted for 1 h to result in the formation of the amide bond. After that, the precipitates were centrifuged (14000 rpm, 25 min, 4 °C) and washed with PBS buffer (10 mM, pH 7.4) for three times. 11

ACCEPTED MANUSCRIPT 2.6. Binding experiment

50 μg of AuNP flares@MSN was redispersed in 1 mL of hybridization buffer

RI PT

(10 mM PBS, pH 7.4, 100 mM NaCl, and 1 mM MgCl2) and incubated with different concentrations of complementary DNA targets (0, 2, 5, 10, 25, 50, 100 and 200 nM). After incubation for 1 h at 37 °C, the fluorescence of Cy5 was excited at 648 nm and

SC

measured at 688 nm. All experiments were repeated at least three times.

M AN U

2.7. Specificity experiment

The complementary DNA target for AuNP flares@MSN and other targets (MTH1 single-base mismatched target, survivin target, K-ras target, Galnac-T target,

TE D

c-myc target, HER-2/neu target) were spiked in 1 mL hybridization buffer containing 50 μg/mL nanoplatform, while the DNA targets were 200 nM. All experiments were

EP

repeated at least three times.

2.8. Loading experiment

AC C

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 61 62 63 64 65

For RhB loading, 1 mg NH2-MSN was added and dispersed in 1 mL RhB

solution (0.5 mg/mL). The mixture was stirred for 24 h in darkness to reach the maximum loading. The other procedures for capping the RhB loaded NH2-MSN were the same as the methods mentioned above. The prepared AuNP flares@MSN (RhB) was centrifuged, washed several times with PBS buffer (at least 6) to get rid of any RhB physisorbed, and dried under high vacuum. The supernatant and all the washings 12

ACCEPTED MANUSCRIPT solutions were collected and the amount of RhB washed was measured using fluorescent spectroscopy (λex=532 nm, λem=575 nm). The loading of RhB 0.036 mg/mg AuNP flares@MSN) was calculated with the difference between the original

RI PT

and the washed amount of RhB. MTH1 inhibitor (S-crizotinib, 0.5 mg/mL, DMSO solution) was loaded and capped by following the same procedure. The loading

to be 0.051 mg/mg AuNP flares@MSN.

M AN U

2.9. Capping efficiency of AuNP flares@MSN (RhB)

SC

content of S-crizotinib was monitored by UV/Vis spectroscopy at 270 nm calculated

To evaluate the capping efficiency, 1 mL as-prepared AuNP flares@MSN (RhB) (50 μg/mL) or 1 mL MSN (RhB) (50 μg/mL) was placed at room temperature in

TE D

darkness and the fluorescence intensity of the sample was measure at 0, 6, 12, 24, 36, 48, 60, and 72 h, respectively. The percentage of dye leaking from the nanoplatform

EP

was calculated as follows: (fluorescence intensity of each sample) / (fluorescence intensity of the loaded RhB).

AC C

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 61 62 63 64 65

2.10. Controlled release of AuNP Flares@MSN (RhB)

50 µg of AuNP flares@MSN (RhB) was incubated with 200 nM perfectly

matched target in 1.0 mL of hybridization buffer (10 mM PBS, pH 7.4, 100 mM NaCl, and 1 mM MgCl2) for 24 h at 37 °C. Single-base mismatched DNA target (200 nM) was added in the control reaction. The release of RhB from AuNP flares@MSN 13

ACCEPTED MANUSCRIPT was also studied as a function of the amount of the DNA targets (0, 2, 5, 10, 25, 50, 100, 200 nM; 2 h at 37 °C). RhB release was monitored by fluorescent spectroscopy

RI PT

at appropriate time intervals and calculated as the same formula above.

2.11. Nuclease assay

Two groups of 50 μg/mL AuNP flares@MSN (RhB) in PBS buffer (10 mM, pH

SC

7.4, 2.5 mM MgCl2, and 0.5 mM CaCl2) were placed in a 96-well fluorescence

M AN U

microplate at 37 ºC. After allowing the samples to equilibrate (10 min), 1.3 μL of DNase I in assay buffer (2 U/L) was added to one group. The fluorescence of these samples was monitored for 12 h and was collected at 2 h intervals during this period. Then 100 nM DNA targets were paralleled added into the two samples with

TE D

incubation for 1 h at 37 ºC, the fluorescence of RhB and Cy5 was measured at appropriate excitation wavelength, respectively.

EP

2.12. Cell culture

Human cervical carcinoma cell line HeLa, Human hepatocellular liver carcinoma

AC C

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 61 62 63 64 65

cell line HepG2 and human hepatocyte cell line HL-7702 were obtained from the Committee on Type Culture Collection of Chinese Academy of Sciences. Cells were grown in cell culture media and incubated at 37 °C in a 5% CO2/ 95% air humidified incubator (MCO-15AC, SANYO). The cell culture medium was RPMI-1640 (2000 mg/L D-Glucose, 300 mg/L L-Glutamine, Hyclone, USA) supplemented with 10% 14

ACCEPTED MANUSCRIPT heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA).

RI PT

2.13. TEM imaging of cells

HeLa, HepG2 and HL7702 cells were treated with 50 μg/mL AuNP flares@MSN (S-crizotinib) for 1 h and 3 h, respectively. Then, cells were trypsizined,

SC

centrifuged, and fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH

M AN U

7.4) for 1 h in room temperature and rinsed. Cells were then post fixed 1 h in 2% osmium tetroxide with 3% potassium ferrocyanide and rinsed, next enbloc staining with a 2% aqueous uranyl acetate solution and dehydration through a graded series of alcohol, two changes of propylene oxide, a series of propylene oxide/Epon dilutions,

TE D

and embedded in 100% Epon. The thin (70 nm) sections were cut on a Leica UC6 ultramicrotome, and images were taken on a JEOL 1200 EX (JEOL, Ltd. Tokyo,

EP

Japan) using an AMT 2k digital Camera.

2.14. Confocal fluorescence imaging

AC C

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 61 62 63 64 65

HeLa, HepG2 and HL7702 cells in the logarithmic growth phase were seeded at

an initial density of 5 × 104 cells/dish in 20-mm glass bottom dishes (SPL Life Sciences, Seoul, Korea) and incubated for 24 h before adding the test substance. Then these cells were respectively treated with AuNP flares@MSN (S-crizotinib) (50 μg/mL) for 3 h and 12 h. After the nanoplatform incubation, the medium was 15

ACCEPTED MANUSCRIPT removed and washed three times with PBS. The cell nuclei were then stained with DAPI (10 μg/mL) for 30 min in darkness at 37 °C. Cells were finally washed three times with PBS and examined by confocal laser scanning microscopy (CLSM) with

RI PT

405 nm and 633 nm excitation.

2.15. RT-PCR

SC

Total RNA from sorted cells was extracted with the RNeasy Mini Kit (Qiagen,

M AN U

Valencia, CA). cDNA synthesis was performed using an iScript kit (Bio-Rad). RT-PCR was carried out with SYBR Green I (Qiagen) on an ABI PRISM 7000 sequence detection system. Relative level of tumor mRNA was calculated from the quantity of tumor mRNA PCR products and the quantity of GAPDH PCR products.

EP

2.16. MTT assay

TE D

The primers used in this experiment were shown in Table S1.

Cytotoxicity was measured by using the MTT assay in the logarithmic phase of cell growth. HeLa, HepG2 and HL7702 cells were seeded at a density of 5×104

AC C

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 61 62 63 64 65

cells/well in a 96 well-plate and incubated for 24 h before adding the test substance. Then fresh medium containing increasing concentrations of AuNP flares@MSN, AuNP flares@MSN (S-crizotinib) and free S-crizotinib was added to each well, respectively. After 12 h incubation, medium was removed and replaced with medium containing MTT (0.5 mg/mL). Cells were incubated at 37 °C for another 4 h after 16

ACCEPTED MANUSCRIPT which medium was removed. DMSO (100 μL) was added to lyse the cells and dissolve the formazan produced. The absorbance at 570 nm of each well was monitored using a RT 6000 microplate reader. Viability was calculated based on the

RI PT

recorded data. In the co-incubation experiment, HepG2 cells (5000 cells/well) and equivalent HL7702 cells were cultured in 96 a well-plate and incubated for 24 h before

SC

treatment. Then fresh medium containing 50 μg/mL of AuNP flares@MSN

M AN U

(S-crizotinib) and free S-crizotinib was added to each well, respectively. After 12 h incubation, the cells were treated as mentioned above.

2.17. In vivo imaging of MTH1

TE D

All animal experiments were carried out according to the Principles of Laboratory Animal Care (People's Republic of China) and the Guidelines of the

EP

Animal Investigation Committee, Biology Institute of Shandong Academy of Science, China. Male nude mice (6-8 week old, ~20 g) were housed under normal conditions with 12 h light and dark cycles and given access to food and water ad libitum.

AC C

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 61 62 63 64 65

Right flank of the mice were injected a suspension of 1 × 106 HepG2 cells in

PBS (150 μL). When tumor grew up to an average volume of 300-500 mm3, 100 μL of AuNP flares@MSN (S-crizotinib) was intravenously injected. The NIR fluorescent signals were observed using Caliper IVIS Lumina III at 0, 3, 6, 12 and 24 h time points. At 24 h postinjection, tumors and normal organs including heart, liver, spleen, 17

ACCEPTED MANUSCRIPT lung, and kidney from HepG2 tumor-bearing nude mice were collected and visualized with Caliper IVIS Lumina III. Tumor-to-muscle ratios (T/M) were determined by image processing on Living Image Software. Briefly, fluorescence images were

RI PT

background subtracted using a rolling-ball radius of 150. Region-of-interests (ROIs) were defined by drawing a contour around the tumor (Tumor ROI) and around the entire animal except the tumor (Muscle ROI). T/M was determined by dividing the

SC

average pixel intensity of the Tumor ROI by the average pixel intensity in the Muscle

2.18. In vivo antitumor efficacy

M AN U

ROI. Values are presented as means ± S.D. calculated for groups of five animals.

HepG2 cells (1×106 cells per 150 μL of PBS) were injected in the right flank of

TE D

nude mice. When the tumor volume reached to about 80-100 mm3 (two weeks), 100 μL PBS, free S-crizotinib (25mg/kg), AuNP flares@MSN (25 mg/kg), and AuNP

EP

flares@MSN (S-crizotinib) (25 mg/kg containing S-crizotinib 1.25mg) was administered intratumorly once daily for 30 days. The mouse body weight and tumor size was recorded. The tumor volume (V) was determined by measuring length (L)

AC C

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 61 62 63 64 65

and width (W), and calculated as V=L×W×W×0.52. The relative tumor volumes were calculated for each mouse as V/V0 (V0 was the tumor volume when the treatment was initiated). At termination, tumors and normal organs from PBS- and AuNP flares@MSN (S-crizotinib)-treated mice were collected and applied for analyzing the nanoplatform toxicity. 18

ACCEPTED MANUSCRIPT 3. Results and discussion

3.1. Synthesis and characterization of AuNP flares-capped MSN nanoplatform

RI PT

The AuNP flares were designed using 5 nm AuNP because this size is an efficient quencher [29] and it can be suitable for capping the MSN pores. The thiol-modified

single-stranded

DNA

SC

(5’-GAAATTCCACGGGTACTTCAAAAAA-(CH2)6-SH-3’) was designed as the

M AN U

recognition sequence to target intracellular MTH1 mRNA, and the carboxyl-modified single-stranded DNA (5’-COOH-(Cy5)TGAAGTACCCG-3’) also labeled with Cy5 dye was designed as the reporter sequence. After hybridization, the resulting flare duplexs were immobilized on the surface of AuNP via the thiol groups of recognition

TE D

sequence. High-resolution transmission electron microscopy (HRTEM) images of AuNP and AuNP flares are shown in the Supporting Information, Fig. S1. The results

EP

showed that the border of the AuNP was clear and that of the AuNP flares was ambiguous. This is indicative of the assembly of flare duplexes on the surface of the AuNP. The UV/Vis absorption spectra indicated that the maximum absorption of the

AC C

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 61 62 63 64 65

AuNP was at 513 nm and that it was red-shifted to 528 nm for the AuNP flares, which further confirmed that the AuNP was successfully functionalized with flare duplexes (Fig. S2). Based on the previously established method [27], each AuNP was calculated to carry 18 ± 1 Cy5-labeled flares targeting MTH1. Details of the characterization are provided in Supporting Information and Fig. S3. 19

ACCEPTED MANUSCRIPT MSNs were then synthesized according to a previously reported method [28]. The honeycomb-like mesoporous structure was confirmed by HRTEM, scanning electron microscopy (SEM) and N2 sorption analysis. As shown in Fig. 2a and S4a,

RI PT

the MSN particle is spherical with an average diameter of 80 nm. The N2 adsorption-desorption isotherm of MSNs also showed a typical Type IV curve with a total surface area of 405 m2/g and average pore diameter of 2.3 nm with a narrow

SC

pore-size distribution (Fig. S5). The surface of the MSN was functionalized with

M AN U

APTES to result in NH2-MSN, which was then reacted with COOH groups on the reporter sequences to cap the AuNP flares onto the pores (AuNP flares@MSN). The TEM and SEM images verified the successful capping with AuNP flares, as distinctive dark, crystalline spots with diameters of 5-6 nm on the surfaces of the

TE D

MSN (Fig. 2b, S4b). These AuNP flares (5-6 nm diameter) were large enough to block the 2.3 nm pores of the MSNs and thus inhibit the release of the cargo. Powder

EP

X-ray diffraction (XRD) in 30° < 2θ < 70° range (Fig. S6) showed similar (111), (200), and (220) diffraction peaks to those observed for AuNP flares, supporting the presence of AuNP flares on MSN. The successful fabrication of AuNP flares@MSN

AC C

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 61 62 63 64 65

was further confirmed by dynamic light scattering, Fourier transform infrared and X-ray photoelectron spectroscopy (see the Supporting Information Table S2, Fig. S7 and S8).

20

SC

is a high-resolution TEM image.

M AN U

Fig. 2. HRTEM micrographs of (a) MSN and (b) AuNP flares@MSN. The inset in (b)

To evaluate the feasibility of AuNP flares@MSN for the detection of MTH1 mRNA, we first examined the response of this nanoplatform to synthetic DNA

TE D

targets. The addition of the perfectly matched DNA target resulted in a 7.7-fold increase in fluorescence signal upon target recognition and binding. In contrast, the

EP

signal did not obviously change in the presence of a single-base mismatched target and was of comparable magnitude to background fluorescence (Fig. 3a). These results

AC C

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 61 62 63 64 65

RI PT

ACCEPTED MANUSCRIPT

indicated that the nanoplatform was efficient at signaling the presence of specific targets. Fig. 3b showed that the fluorescence intensity of the nanoplatform increases with increasing concentration of the DNA targets from 0 to 200 nM, thus suggesting that the hybridization of the AuNP flares in the nanoplatform and DNA targets led to fluorescence recovery and that the fluorescence intensity is associated with concentration of the DNA targets. The selectivity of the AuNP flares for the 21

ACCEPTED MANUSCRIPT nanoplatform was also performed and is shown in Fig. S9. The results revealed that the AuNP flares were specifically bound to the DNA target and generated 6- to 7-fold

M AN U

SC

RI PT

higher fluorescent signal compared with other targets.

Fig. 3. The nanoplatform responds to synthetic target DNA. (a) Fluorescence spectra of the 50 μg/mL nanoplatform alone (black), with 200 nM target (red), and with 200 nM single-base mismatched target (blue). (b) Fluorescence spectra of the

TE D

nanoplatform (50 μg/mL) in the presence of various concentrations of DNA target (0, 2, 5, 10, 25, 50, 100 and 200 nM). Inset: calibration curves for the fluorescence

EP

intensity versus corresponding target concentrations. To investigate the mRNA-induced controlled release property of the AuNP

AC C

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 61 62 63 64 65

flares@MSN nanoplatform in an extracellular environment, rhodamine B (RhB) was used as a model cargo molecule. The RhB-loaded AuNP flares@MSN (RhB) sample was prepared, and the loading of RhB was determined to be 0.036 mg/mg of AuNP flares@MSN as described in Supporting Information (Fig. S10). To examine the capping efficiency, the AuNP flares@MSN (RhB) was first placed for 72 h in darkness and the fluorescence intensity in aqueous solution was measured at 22

ACCEPTED MANUSCRIPT appropriate time intervals. As shown in Fig. S11, about 89% RhB was leaked after 72 h for the control MSN without capping, while less than 8% RhB was leaked after 72 h for AuNP flares@MSN, indicating that the capping strategy was successful with good

RI PT

efficiency. Then release experiments were performed and the release profile of RhB was displayed in Fig. 4a. As can be seen, without target or with a single-base mismatch,

SC

the AuNP flares@MSN was tightly capped and showed a negligible release of RhB

M AN U

(curve i and curve ii). In contrast, the presence of the complementary DNA target induced the hybridization between recognition sequences on the AuNP flares@MSN and DNA target, the opening of the pores, and a remarkable release of the dye (curve iii). Curve iii also showed that AuNP flares@MSN responded rapidly to the target and

TE D

within 2 h more than 52% of the RhB was released. This target-triggered pore uncapping could also clearly be seen in the HRTEM (Fig. 4b).

EP

The release of RhB from AuNP flares@MSN was also studied as a function of the amount of the DNA targets (see inset in Fig. 4a). Clearly, the release of the RhB was proportional to the target concentration. The maximum release was observed at a

AC C

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 61 62 63 64 65

target concentration of 100 nM; at higher concentrations the release was partially inhibited, most likely because excess DNA target was adsorbed onto the surface of the MSN, inducing partial pore capping.

23

SC

M AN U

Fig. 4. (a) Release of RhB from AuNP flares@MSN in the absence of the target (i), and in the presence of single-mismatched target (ii) and perfectly matched target (iii) at concentrations of 100 nM. Inset: Percentage of released RhB as a function of the concentration of DNA target after 2 hour of reaction. Data are shown as mean ± S.D.

TE D

of three independent experiments. (b) HRTEM micrographs of the sample obtained after hybridization with DNA target showing the pores opening.

EP

Because nuclease resistance is critical for the AuNP flares@MSN when used in living cells or in vivo, its nuclease stability was evaluated with enzyme

AC C

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 61 62 63 64 65

RI PT

ACCEPTED MANUSCRIPT

deoxyribonuclease I (DNase I) [30] under physiological conditions via analysis of Cy5 or RhB fluorescence. Fig. S12 showed that both Cy5 and RhB fluorescence intensity of the nanoplatform treated with DNase I was not obviously changed compared with the case without DNase I. However, the fluorescence intensity of the two solutions was all markedly enhanced after hybridization with the DNA targets, indicating that AuNP flares@MSN possessed high resistance to nucleases and the 24

ACCEPTED MANUSCRIPT release of RhB was indeed due to the hybridization with the targets instead of nuclease degradation. The above results confirmed the mRNA-responsive controlled-release mechanism and the feasibility of AuNP flares@MSN specific

3.2. In vitro imaging and inhibition of MTH1

RI PT

detection and targeted inhibition of MTH1.

SC

For intracellular applications of AuNP flares@MSN nanoplatform, a MTH1

M AN U

inhibitor (S-crizotinib) was chosen as the cargo molecule for a controlled release study in human cancer cells (HepG2 and HeLa, MTH1-positive) and normal liver cells (HL7702, MTH1-negative). The AuNP flares@MSN (S-crizotinib) was endocytosed rapidly by these three cell types and is localized mainly in the cytoplasm

TE D

and subcellular vesicles as determined by TEM (Fig. 5a). After the positive cancer cells were incubated with AuNP flares@MSN (S-crizotinib) for 3 h, strong red

EP

fluorescence was observed under confocal laser scanning microscopy (CLSM). Additionally, DAPI nuclear staining and bright-field imaging showed no DNA damage or apoptotic cells within the initial 3 h. Inhibition of MTH1 by S-crizotinib

AC C

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 61 62 63 64 65

could selectively induce an increase in DNA single-strand breaks leading to DNA damage and cytotoxicity in human cancer cells [5]. After 12 h, we observed both HepG2 and HeLa cells undergoing apoptosis with clear nuclear DNA condensation and cell shrinkage. Red fluorescent signals were also observed in two cancer cells similar to the case at 3 h. When the HL7702 cells were incubated under the same 25

ACCEPTED MANUSCRIPT condition, the fluorescent signals became faint and these cells were viable throughout the imaging experiments (Fig. 5b). Our experiments indicated that the relative expression levels of MTH1 mRNA in HepG2 and HeLa were higher than that in

RI PT

HL7702, which is consistent with real-time reverse transcription-PCR (RT-PCR) results (Fig. S13). The binding of MTH1 mRNA triggered the release of S-crizotinib, which could effectively induce DNA damage and cell death in cancer cells. Thus, the

SC

AuNP flares@MSN (S-crizotinib) nanoplatform can be used to distinguish cancer

M AN U

cells from normal cells as well as selectively release S-crizotinib on the basis of

EP

TE D

MTH1 mRNA levels.

AC C

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 61 62 63 64 65

Fig. 5. (a) Time-course changes in subcellular localization of AuNP flares@MSN (S-crizotinib) within three types of cells (see arrows). (b) The time course of confocal images of the three cells after incubated with AuNP flares@MSN (S-crizotinib).

26

ACCEPTED MANUSCRIPT Arrows indicate DNA condensation and cell shrinkage of apoptotic cells. Scale bar = 25 µm. To further evaluate the therapeutic efficacy of this nanoplatform, an MTT assay

RI PT

was used for quantitative testing of the viability of the three model cells in the presence of AuNP flares@MSN, AuNP flares@MSN (S-crizotinib), and free

SC

S-crizotinib. As shown in Fig. 6, the AuNP flares@MSN had no obvious effect on HepG2, HeLa and HL7702 cell viability at concentrations up to 100 μg/mL, whereas

M AN U

AuNP flares@MSN (S-crizotinib) exhibited a statistically significant cytotoxic effect on the two cancer cells but not on the normal cells. Notably, the half-maximal inhibitory concentration (IC50) of AuNP flares@MSN (S-crizotinib) against the two

TE D

cancer cells was similar and calculated to be ∼36 μg/mL (the loaded S-crizotinib concentrations are ∼1.8 μg/mL). These values were 4.4% that of free S-crizotinib (IC50 of 41 μg/mL), suggesting that the nanoplatform could reduce the dosage and

EP

greatly improve the therapeutic effect of S-crizotinib. This is mainly because the encapsulation of S-crizotinib within AuNP flares@MSN overcomes many severe

AC C

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 61 62 63 64 65

problems including solubility and poor cellular uptake when administered as free S-crizotinib. Moreover, AuNP flares@MSN (S-crizotinib) could prevent unexpected release in normal cells and therefore cause a marked dose escalation within cancer cells. This additional benefit was validated by the co-incubation experiment of HepG2 and HL7702. As shown in Fig. S14, the mixed cells (HepG2: 0.1 mL, 5 × 104/mL, HL7702: 0.1 mL, 5 × 104/mL) still maintained about 74.5% of the cell viability after 27

ACCEPTED MANUSCRIPT incubation with free S-crizotinib (50 μg/mL), which was higher than when an equal amount of AuNP flares@MSN (S-crizotinib) was added (cell viability, 50.2%). AuNP flares@MSN (S-crizotinib) reduced the retention of S-crizotinib in normal cells and

RI PT

allowed the localized release of highly concentrated S-crizotinib within cancer cells. These results, plus intracellular imaging experiment, further confirmed that this nanoplatform for the detection and inhibition of MTH1 is effective at the in vitro

TE D

M AN U

SC

level, therefore could be further extended to in vivo application.

Fig. 6. In vitro viability of (a) HeLa, (b) HepG2, and (c) HL7702 cells incubated with

h.

EP

AuNP flares@MSN, AuNP flares@MSN (S-crizotinib), and free S-crizotinib for 12

3.3. In vivo MTH1 imaging

AC C

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 61 62 63 64 65

We next studied the in vivo MTH1 imaging ability and tumor selectivity of

AuNP flares@MSN (S-crizotinib) in a subcutaneous HepG2 tumor model. The AuNP flares@MSN (S-crizotinib) (25 mg/kg) was intravenously administered, and fluorescence images were acquired at different time points post-injection. Excitation and emission wavelengths were set as 640/670 nm for Cy5. After injection, MTH1 28

ACCEPTED MANUSCRIPT mRNA was bound to AuNP flares in the tumor area and the quenching effect between AuNP and Cy5 was lessened, resulting in fluorescence signal recovery over time. The tumor-to-muscle tissue (T/M) ratios were 1.55 ± 0.32, 2.42 ± 0.21, 3.15 ± 0.44, and

RI PT

2.11 ± 0.23 at 3, 6, 12, and 24 h post-injection, showing high levels of MTH1 in tumor regions (Fig. 7a and 7b). Decrease at the 24 h time point was probably due to the clearance of AuNP flares@MSN in vivo. To confirm our hypothesis, ex vivo

SC

imaging was performed as shown in Fig. 7c. In addition to accumulation in the

M AN U

HepG2 tumors, we found a high level of fluorescence activity in the livers and kidneys at 24 h, indicating the hepatic and renal clearances of AuNP flares@MSN. The uptake in other organs was much lower as determined by ex vivo imaging. These imaging results suggested that AuNP flares@MSN (S-crizotinib) could specifically

TE D

accumulate at the tumor site via a passive mechanism and enabled clear visualization of MTH1 mRNA levels in tumors. Moreover, the ability of AuNP flares@MSN to be

EP

eliminated from the body would decrease the biological persistence of this nanoplatform injected into the body and eliminate its risks of long-term toxicity.

AC C

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 61 62 63 64 65

29

SC M AN U

Fig. 7. (a) NIR fluorescent imaging of MTH1 mRNA in HepG2 tumor-bearing mice

TE D

intravenously received AuNP flares@MSN (S-crizotinib). Images were acquired at indicated time points, and fluorescent signals were normalized by the maximum

EP

average value. The color bar indicates radiant efficiency (low, 0; high, 3.33 × 108). White circles were used to indicate tumors location. (b) Tumor/muscle (T/M) ratio of HepG2 tumor-bearing mouse model. Data are shown as means ± S.D., n = 5 per

AC C

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 61 62 63 64 65

RI PT

ACCEPTED MANUSCRIPT

group. (c) Ex vivo fluorescence images of major organs and tumors excised at 24 h post-injection of AuNP flares@MSN (S-crizotinib).

3.4. In vivo antitumor efficacy

30

ACCEPTED MANUSCRIPT To investigate the in vivo potential of AuNP flares@MSN (S-crizotinib) to suppress tumor growth, we established HepG2 tumors in the flank of 20 mice. Two weeks after tumor engraftment the mice were randomized into four treatment groups

RI PT

(n = 5/group). Each group received intra-tumoral injections of either phosphate buffered saline (PBS), free S-crizotinib (25mg/kg) [5], AuNP flares@MSN (25 mg/kg) or AuNP flares@MSN (S-crizotinib) (25 mg/kg, loaded S-crizotinib was

SC

1.25mg/kg). To study the therapeutic effects, we continued to monitor and record

M AN U

HepG2 tumor growth rates (Fig. 8a). Following sacrifice of the mice after 30 days, the tumors on mice received AuNP flares@MSN (S-crizotinib) treatment showed a greater decrease in tumor volume (78%) compared to free S-crizotinib (42%). In marked contrast, other control groups with PBS only or AuNP flares@MSN without

TE D

loading S-crizotinib did not show significant tumor suppression (Fig. 8b). We also examined the major organs from HepG2 tumor-bearing nude mice including heart,

EP

liver, spleen, lung and kidney. We did not observe any noticeable organ damage compared to the control group (Fig. 8c) suggesting that AuNP flares@MSN (S-crizotinib) did not have toxic side effects in HepG2 tumor-bearing nude mice. The

AC C

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 61 62 63 64 65

body weight data further confirmed this (Fig. 8d). Collectively, our results verified that the ability to package S-crizotinib with controlled release of this inhibitor according to MTH1 mRNA levels allowed AuNP flares@MSN (S-crizotinib) to effectively suppress tumor growth in mice and greatly improve the therapeutic

31

ACCEPTED MANUSCRIPT efficacy of S-crizotinib. The AuNP flares@MSN (S-crizotinib) can both detect and

TE D

M AN U

SC

RI PT

inhibit MTH1 and is an ideal platform for image-guided cancer therapy.

Fig. 8. In vivo anticancer activity of AuNP flares@MSN (S-crizotinib). (a) HepG2

EP

tumor growth rate in each group after treatment (control groups: 50 μL PBS; therapeutic groups: 25 mg/kg; intratumorally daily). Tumor volumes were normalized to their initial size. (b) The effect on tumor growth following 30-day treatment.

AC C

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 61 62 63 64 65

Images depict representative tumors for each treatment group. (c) Photographs of the major organs excised after 30-day treatment with AuNP flares@MSN (S-crizotinib). No noticeable abnormality was found in the heart, liver, spleen, lung, or kidney. (d) Body weight curves of HepG2 tumor-bearing mice for each group. All data are shown as mean ± S.D.; n = 5 per group.

32

ACCEPTED MANUSCRIPT 4. Conclusion

In summary, we have successfully fabricated a multifunctional nanoplatform

RI PT

based on AuNP flares-capped MSNs that can detect MTH1 mRNA and simultaneously suppressed MTH1 catalytic activity both in vitro and in vivo. This nanoplatform signaled the presence of MTH1 mRNA with the liberation of AuNP

SC

flares, which resulted in the release of S-crizotinib from the pores of the MSNs into the cytosol. These MTH1 inhibitors specifically induced DNA damage and cell death

M AN U

in cancer cells. Furthermore, the high specificity, good nuclease stability, enhanced cellular uptake and MTH1 mRNA-dependent release properties enabled the nanoplatform to clearly visualize the MTH1 in tumors as well as efficiently reduce

TE D

tumor volume by more than 70%. Therefore, the AuNP flares@MSN (S-crizotinib) integrating MTH1 detection and regulation in a single nanoplatform is a promising first step toward the development of MTH1 directed theranostics and is expected to be

EP

a novel cancer phenotypic lethal anticancer strategy.

AC C

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 61 62 63 64 65

Acknowledgements.

This work was supported by 973 Program (2013CB933800), National Natural

Science Foundation of China (21227005, 21390411, 21305081).

33

ACCEPTED MANUSCRIPT References

[1] JS D, VA P. Understanding redox homeostasis and its role in cancer. J Clin Diagn

RI PT

Res 2012;6:1796-1802. [2] Oka S, Ohno M, Tsuchimoto D, Sakumi K, Furuichi M, Nakabeppu Y. Two distinct pathways of cell death triggered by oxidative damage to nuclear and

SC

mitochondrial DNAs. EMBO J 2008;27:421-432.

M AN U

[3] Jorgenson TC, Zhong W, Oberley TD. Redox imbalance and biochemical changes in cancer. Cancer Res 2013;73:6118-6123.

[4] Gad H, Koolmeister T, Jemth AS, Eshtad S, Jacques SA, Ström CE, et al. MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool. Nature

TE D

2014;508:215-221.

[5] Huber KVM, Salah E, Radic B, Gridling M, Elkins JM, Stukalov A, et al.

EP

Stereospecific targeting of MTH1 by (S)-crizotinib as an anticancer strategy. Nature 2014;508:222-227.

[6] Oda H, Nakabeppu Y, Furuichi M, Sekiguchi M. Regulation of expression of the

AC C

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 61 62 63 64 65

human MTH1 gene encoding 8-oxo-dGTPase. Alternative splicing of transcription products. J Biol Chem 1997;272:17843-17850.

[7] Yoshimura D, Sakumi K, Ohno M, Sakai Y, Furuichi M, Iwai S, et al. An oxidized purine nucleoside triphosphatase, MTH1, suppresses cell death caused by oxidative stress. J Biol Chem 2003;278:37965-37973. [8] Schmidt MC, Mccartney RR, Zhang X, Tillman TS, Solimeo H, Lfl SW, et al. 34

ACCEPTED MANUSCRIPT Std1 and Mth1 proteins interact with the glucose sensors to control glucose-regulated gene expression in saccharomyces cerevisiae. Mol Cell Biol 1999;19:4561-4571.

RI PT

[9] Gao W, Ji L, Li L, Cui G, Xu K, Li P, et al. Bifunctional combined Au-Fe2O3 nanoparticles for induction of cancer cell-specific apoptosis and real-time

SC

imaging. Biomaterials 2012;33:3710-3718.

[10] Lu H-Y, Chang Y-J, Fan N-C, Wang L-S, Lai N-C, Yang C-M, et al. Synergism

M AN U

through combination of chemotherapy and oxidative stress-induced autophagy in A549 lung cancer cells using redox-responsive nanohybrids: A new strategy for cancer therapy. Biomaterials 2015;42:30-41.

[11] Seferos DS, Giljohann DA, Hill HD, Prigodich AE, Mirkin CA. Nano-flares:

TE D

probes for transfection and mRNA detection in living cells. J Am Chem Soc 2007;129:15477-15479.

EP

[12] Li N, Chang C, Pan W, Tang B. A multicolor nanoprobe for detection and imaging of tumor-related mRNAs in living cells. Angew Chem Int Ed 2012;51

AC C

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 61 62 63 64 65

7426-7430.

[13] Meng H, Mai WX, Zhang H, Xue M, Xia T, Lin S, et al. Codelivery of an optimal drug/siRNA combination using mesoporous silica nanoparticles to overcome drug resistance in breast cancer in vitro and in vivo. ACS Nano 2013;7:994-1005. [14] Climent E, Martínez-Máñez R, Sancenón F, Marcos MD, Soto J, Maquieira A, et 35

ACCEPTED MANUSCRIPT al. Controlled delivery using oligonucleotide-capped mesoporous silica nanoparticles. Angew Chem Int Ed 2010;49:7281-7283. [15] Calvo A, Yameen B, Williams FJ, Soler-Illia GJAA, Azzaroni O. Mesoporous

RI PT

films and polymer brushes helping each other to modulate ionic transport in nanoconfined environments. An interesting example of synergism in functional

SC

hybrid assemblies. J Am Chem Soc 2009;131:10866-10868.

[16] Mal NK, Fujiwara M, Tanaka Y. Photocontrolled reversible release of guest from

coumarin-modified

2003;421:350-353.

mesoporous

silica.

Nature

M AN U

molecules

[17] Casasús R, Climent E, Marcos MD, Martínez-Máñez R, Sancenón F, Soto J, et al. Dual aperture control on pH- and anion-driven supramolecular nanoscopic

TE D

hybrid gate-like ensembles. J Am Chem Soc 2008;130:1903-1907. [18] Saha S, Leung KCF, Nguyen TD, Stoddart JF, Zink JI. Nanovalves. Adv Funct

EP

Mater 2007;17:685-693.

[19] He D, He X, Wang K, Chen M, Zhao Y, Zou Z. Intracellular acid-triggered drug delivery system using mesoporous silica nanoparticles capped with T-Hg2+-T

AC C

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 61 62 63 64 65

base pairs mediated duplex DNA. J Mater Chem B 2013;1:1552-1560.

[20] Li N, Yu Z, Pan W, Han Y, Zhang T, Tang B. A near-infrared light-triggered nanocarrier with reversible DNA valves for intracellular controlled release. Adv Funct Mater 2013;23:2255-2262. [21] Ma M, Chen H, Chen Y, Wang X, Chen F, Cui X, et al. Au capped magnetic 36

ACCEPTED MANUSCRIPT core/mesoporous

silica

photothermo-/chemo-therapy

shell and

nanoparticles multimodal

for

imaging.

combined Biomaterials

2012;33:989-998.

RI PT

[22] Muhammad F, Guo M, Qi W, Sun F, Wang A, Guo Y, et al. pH-triggered controlled drug release from mesoporous silica nanoparticles via intracelluar

SC

dissolution of ZnO nanolids. J Am Chem Soc 2011;133:8778-8781.

[23] Aznar E, Marcos MD, Martínez-Máñez R, Sancenón F, Soto J, Amorós P, et al.

M AN U

pH- and photo-switched release of guest molecules from mesoporous silica supports. J Am Chem Soc 2009;131:6833-6843.

[24] Liu R, Zhang Y, Zhao X, Agarwal A, Mueller LJ, Feng P. pH-responsive nanogated ensemble based on gold-capped mesoporous silica through an

TE D

acid-labile acetal linker. J Am Chem Soc 2010;132:1500-1501. [25] Vivero-Escoto JL, Slowing II, Wu CW, Lin VS. Photoinduced intracellular

EP

controlled release drug delivery in human cells by gold-capped mesoporous silica nanosphere. J Am Chem Soc 2009;131:3462-3463.

AC C

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 61 62 63 64 65

[26] Zikich D, Borovok N, Molotsky T, Kotlyar A. Synthesis and AFM characterization

of

poly(dG)-poly(dC)-gold

nanoparticle

conjugates.

Bioconjugate Chem 2010;21:544-547.

[27] Demers LM, Mirkin CA, Mucic RC, Reynolds RA, Letsinger RL, Elghanian R, et al. A fluorescence-based method for determining the surface coverage and hybridization efficiency of thiol-capped oligonucleotides bound to gold thin 37

ACCEPTED MANUSCRIPT films and nanoparticles. Anal Chem 2000;72:5535-5541. [28] Kim J, Kim HS, Lee N, Kim T, Kim H, Yu T, et al. Multifunctional uniform nanoparticles composed of a magnetite nanocrystal core and a mesoporous silica

Angew Chem Int Ed 2008;47:8438-8441.

RI PT

shell for magnetic resonance and fluorescence imaging and for drug delivery.

SC

[29] Fan C, Wang S, Hong JW, Bazan GC, Plaxco KW, Heeger AJ. Beyond superquenching: Hyper-efficient energy transfer from conjugated polymers to

M AN U

gold nanoparticles. Proc Natl Acad Sci U S A 2003;100:6297-6310. [30] Seferos DS, Prigodich AE, Giljohann DA, Patel PC, Mirkin CA. Polyvalent

EP

TE D

DNA nanoparticle conjugates stabilize nucleic acids. Nano Lett 2009; 9:308-311.

AC C

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 61 62 63 64 65

38

Figure captions Click here to download Captions: Fig caption.docx

ACCEPTED MANUSCRIPT Fig. 1. Schematic illustration of AuNP flares-capped MSN nanoplatform for detection and inhibition of MTH1. Fig. 2. HRTEM micrographs of (a) MSN and (b) AuNP flares@MSN. The inset in (b)

RI PT

is a high-resolution TEM image.

Fig. 3. The nanoplatform responds to synthetic target DNA. (a) Fluorescence spectra

SC

of the 50 μg/mL nanoplatform alone (black), with 200 nM target (red), and with 200 nM single-base mismatched target (blue). (b) Fluorescence spectra of the

M AN U

nanoplatform (50 μg/mL) in the presence of various concentrations of DNA target (0, 2, 5, 10, 25, 50, 100 and 200 nM). Inset: calibration curves for the fluorescence intensity versus corresponding target concentrations.

TE D

Fig. 4. (a) Release of RhB from AuNP flares@MSN in the absence of the target (i), and in the presence of single-mismatched target (ii) and perfectly matched target (iii) at concentrations of 100 nM. Inset: Percentage of released RhB as a function of the

EP

concentration of DNA target after 2 hour of reaction. Data are shown as mean ± S.D.

AC C

of three independent experiments. (b) HRTEM micrographs of the sample obtained after hybridization with DNA target showing the pores opening. Fig. 5. (a) Time-course changes in subcellular localization of AuNP flares@MSN (S-crizotinib) within three types of cells (see arrows). (b) The time course of confocal images of the three cells after incubated with AuNP flares@MSN (S-crizotinib). Arrows indicate DNA condensation and cell shrinkage of apoptotic cells. Scale bar = 25 µm.

ACCEPTED MANUSCRIPT Fig. 6. In vitro viability of (a) HeLa, (b) HepG2, and (c) HL7702 cells incubated with AuNP flares@MSN, AuNP flares@MSN (S-crizotinib), and free S-crizotinib for 12 h.

RI PT

Fig. 7. (a) NIR fluorescent imaging of MTH1 mRNA in HepG2 tumor-bearing mice intravenously received AuNP flares@MSN (S-crizotinib). Images were acquired at indicated time points, and fluorescent signals were normalized by the maximum

SC

average value. The color bar indicates radiant efficiency (low, 0; high, 3.33 × 108).

M AN U

White circles were used to indicate tumors location. (b) Tumor/muscle (T/M) ratio of HepG2 tumor-bearing mouse model. Data are shown as means ± SD, n = 5 per group. (c) Ex vivo fluorescence images of major organs and tumors excised at 24 h post-injection of AuNP flares@MSN (S-crizotinib).

TE D

Fig. 8. In vivo anticancer activity of AuNP flares@MSN (S-crizotinib). (a) HepG2 tumor growth rate in each group after treatment (control groups: 50 μL PBS;

EP

therapeutic groups: 25 mg/kg; intratumorally daily). Tumor volumes were normalized to their initial size. (b) The effect on tumor growth following 30-day treatment.

AC C

Images depict representative tumors for each treatment group. (c) Photographs of the major organs excised after 30-day treatment with AuNP flares@MSN (S-crizotinib). No noticeable abnormality was found in the heart, liver, spleen, lung, or kidney. (d) Body weight curves of HepG2 tumor-bearing mice for each group. All data are shown as mean ± S.D.; n = 5 per group.

ACCEPTED MANUSCRIPT

SUPPORTING INFORMATION

RI PT

AuNP flares-capped mesoporous silica nanoparticles for MTH1 detection and inhibition

SC

Wen Gao, Wenhua Cao, Yuhui, Sun, XuePing Wei, Kehua Xu, Huaibin Zhang, Bo Tang*

M AN U

College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong

AC C

EP

TE D

Normal University, Jinan 250014, P.R. China.

*

Corresponding authors. Tel.: +86 531 86180010; Fax: +86 531 86180017. E-mail addresses: [email protected] (Bo Tang).

S1

ACCEPTED MANUSCRIPT

1. Supporting Table: Table S1. DNA sequences employed in this work. Oligonucleotide

Sequence 5’-GAAATTCCACGGGTACTTC(A)6 -(CH2)6-SH-3’

Flare reporter sequence

5’- COOH-(Cy5)TGAAGTACCCG -3’

MTH1 perfectly matched target

5’-GAAGTACCCGTGGAATTTC-3’

MTH1 single-base mismatched target

5’-GAAGTAGCCGTGGAATTTC-3’

MTH1 forward

5’-GTGCAGAACCCAGGGACCAT-3’,

MTH1 reverse

5’-GCCCACGAACTCAAACACGA-3’

GAPDH forward

5’-AAGGTCGGAGTCAACGGATT-3’,

GAPDH reverse

5’-CTCCTGGAAGATGGTGATGG-3’

M AN U

SC

RI PT

MTH1 recognition sequence

Table S2. Zeta potentials and size distributions of different nanoparticles in DI water. Zeta potential (mV)

Size distribution (nm)

AuNP

-21.8 ± 0.9

5.8 ± 1.1

AuNP flares NH2-MSN

-77.5 ± 5.1

8.9 ± 0.8

33.1 ± 1.4

87.7 ± 4.2

-1.7 ± 0.4

102.3 ± 10.9

EP

AuNP flares@MSN

TE D

Sample

AC C

The zeta potential of AuNP were -21.8 mV, owing to the citrate groups. The decreased zeta potential (-77.5 mV) of AuNP flares suggested that the negatively charged DNA duplexs were successfully introduced onto the surface of AuNP. Zeta potential of NH2-MSN was 33.1 mV, while that of the AuNP flares@MSN was -1.7 mV due to the capping of negatively charged AuNP flares onto MSN. Moreover, the AuNP flares capping of MSN induced a slight increase in the dynamic particle size, but the particle size distribution was still narrow, suggesting that AuNP flares@MSN can be uniformly dispersed in aqueous solution.

S2

ACCEPTED MANUSCRIPT

M AN U

SC

RI PT

2. Supporting Figures:

AC C

EP

TE D

Fig. S1. HRTEM images of (a) AuNP and (b) AuNP flares. Scale bar = 50 nm.

Fig. S2. UV/Vis spectra for unmodified AuNP and the AuNP flares. The maximum optical absorption was shifted from 513 nm to 528 nm after the DNA assembly on the surface of AuNP.

S3

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Fig. S3. Standard linear calibration curves of Cy5.

Fig. S4. SEM images of (a) MSN and (b) AuNP flares@MSN. Arrows indicate AuNP flares. Scale bar = 50 nm.

S4

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

Fig. S5. N2 sorption isotherms of MSN (black) and AuNP flares@MSN (red). (a) BET nitrogen sorption isotherms, and (b) BJH pore size distribution. The blocking of the MSN pores by AuNP flares was confirmed by the N2 adsorption/desorption isotherms. A change in the shape of the isotherm from type IV to type I after capping and the disappearing of the maximum peak in the BJH pore size distribution were observed.

S5

SC

RI PT

ACCEPTED MANUSCRIPT

M AN U

Fig. S6. XRD of AuNP flares and AuNP flares@MSN. The 30° < 2θ < 70° range showed similar (111), (200), and (220) diffraction peaks to those observed for AuNP flares,

AC C

EP

TE D

supporting the presence of AuNP flares on AuNP flares@MSN.

S6

ACCEPTED MANUSCRIPT

Fig. S7. FITR spectra of AuNP flares, NH2-MSN and AuNP flares@MSN. AuNP flares displayed feature absorption band at 761 cm-1, which were attributed to Au-S groups [1]. The intensity of the peak at 3400 cm-1 and 1722 cm-1 due to the introduction of COOH

RI PT

groups derived from reporter strands [2]. It provides the direct evidence that flare duplexs were coupled to AuNP. NH2-MSN displayed strong absorption signals at 1091 cm-1, which were assigned to asymmetric stretching of Si-O-Si bridges. Peaks at 1638 cm-1 and 1556 cm-1 were attributed to the stretching bands of N-H groups. After covalently

SC

coupled with COOH groups on the report strands of AuNP flares, a strong peak at 1650 cm-1 was observed, due to the stretching vibration of amide [3]. These results suggest that

AC C

EP

TE D

M AN U

AuNP flares@MSN was successfully synthesized.

Fig. S8. XPS spectra of AuNP flares@MSN. The fully scanned spectra demonstrated that Au, Si, P, N, C and O elements existed in the as-prepared nanoplatform. The binding energy for the C 1s peak at 284.6 eV was used as the reference for calibration. The peak S7

ACCEPTED MANUSCRIPT

located at 84.0, 101.6, 133.9, 154.4 and 400.5 eV were assigned to Au 4f, Si 2p, P 2p, Si 2S, and N 1S [4, 5], respectively. An unsymmetrical O 1s signal peak at 531.2 eV can be deconvoluted to three peaks at 528.4 eV, 531.6 eV and 534.3 eV, which were ascribed to

TE D

M AN U

SC

RI PT

P-O* and HN-C=O* and Si-O* groups [6, 7], respectively.

EP

Fig. S9. Specificity of the nanoplatform over several DNA targets (1: without target, 2: MTH1 perfectly matched target, 3: MTH1 single-base mismatched target, 4: survivin

AC C

target, 5: K-ras target, 6: Galnac-T target, 7: c-myc target, 8: HER-2/neu target). Data are shown as mean ± S.D. of three independent experiments.

S8

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Fig. S10. Standard linear calibration curves of RhB.

Fig. S11. The leakage of the cargo from (black) MSN (RhB) and (red) AuNP flares@MSN (RhB) over a time profile at 0, 6, 12, 24, 36, 48, 60, and 72 h, respectively. Data are shown as mean ± S.D. of three independent experiments.

S9

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. S12. Nuclease stability of AuNP flares@MSN (RhB) in the presence or absence of DNase I. Fluorescence curves of the nanoplatform (50 µg/mL) in PBS(10 mM) without DNase I (-●-), in the presence of DNase I (-■-). Insets: fluorescence spectra after hybridization of the nanoplatform with DNA target in the presence (red) and absence (black) of DNase I. (a) Cy5 measured with excitation at 648 nm. (b) RhB measured with excitation at 550 nm. Data are shown as mean ± S.D. of three independent experiments.

S10

SC

RI PT

ACCEPTED MANUSCRIPT

M AN U

Fig. S13. Detection of the levels of MTH1 mRNA by RT-PCR in HeLa, HepG2 and HL7702 cells. The relative level of tumor mRNA was calculated from the quantity of tumor mRNA PCR products and the quantity of GAPDH PCR products and normalized to the expression level in HL7702 cells. Data are shown as mean ± S.D. of three

AC C

EP

TE D

independent experiments.

Fig. S14. In vitro viability of the mixed cells incubated with AuNP flares@MSN (S-crizotinib) and free S-crizotinib for 12 h. The mixed cells contain equal amounts of HepG2 and HL7702 cells (0.1 mL, 5×104/mL). Data are shown as mean ± S.D. of three independent experiments. S11

ACCEPTED MANUSCRIPT

References

[1] Švorčík V, Kolská Z, Kvítek O, Siegel J, Řezníčková A, Řezanka P, et al. “Soft and dithiols

and

Au

nanoparticles

grafting

on

plasma-treated

RI PT

rigid”

polyethyleneterephthalate. Nanoscale Res Lett 2011;6:607-614.

[2] Muhammad F, Guo M, Qi W, Sun F, Wang A, Guo Y, et al. pH-triggered controlled drug release from mesoporous silica nanoparticles via intracelluar dissolution of

SC

ZnO nanolids. J Am Chem Soc 2011;133:8778-8781.

[3] Luo Z, Cai K, Hu Y, Zhao L, Liu P, Duan L, et al. Mesoporous silica nanoparticles

M AN U

end-capped with collagen: redox-responsive nanoreservoirs for targeted drug delivery. Angew Chem Int Ed 2011;50:640-643.

[4] Lee CY, Gong P, Harbers GM, Grainger DW, Castner DG, Gamble LJ. Surface coverage

and

structure

of

mixed

DNA/alkylthiol

monolayers

on

gold:

characterization by XPS, NEXAFS, and fluorescence intensity measurements. Anal

TE D

Chem 2006;78:3316-3325.

[5] Sugimoto K, Kishi K, Ikeda S, Sawada Y. X-ray photoelectron spectra of passivated 18Cr-8Ni stainless steels. Chem Soc Jpn 1974;38:54-62. [6] Liu J, Zhang J, Cheng S, Liu Z, Han B. DNA-mediated synthesis of microporous

EP

single crystal-like NaTi2(PO4)3 nanospheres. Small 2008;4:1976-1979. [7] Peeling J, Hruska FE, McIntyre NS. ESCA spectra and molecular charge for

AC C

distributions

some

pyrimidine

1978;56:1555-1561.

S12

and

purine

bases.

Can

J

Chem