Smart Cancer Cell Targeting Imaging and Drug Delivery System by

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A Smart Cancer Cell Targeting Imaging and Drug Delivery System by Systematically Engineering Periodic Mesoporous Organosilica Nanoparticles Nan Lu, Ying Tian, Wei Tian, Peng Huang, Ying Liu, Yuxia Tang, Chunyan Wang, Shouju Wang, Yunyan Su, Yunlei Zhang, Jing Pan, Zhaogang Teng, and Guangming Lu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b09585 • Publication Date (Web): 15 Jan 2016 Downloaded from http://pubs.acs.org on January 19, 2016

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A Smart Cancer Cell Targeting Imaging and Drug Delivery System by Systematically

Engineering

Periodic

Mesoporous

Organosilica

Nanoparticles

Nan Lu,†,⊥ Ying Tian,† Wei Tian,† Peng Huang,⊥,§ Ying Liu,† Yuxia Tang,† Chunyan Wang,† Shouju Wang,† Yunyan Su,† Yunlei Zhang,† Jing Pan,† Zhaogang Teng,*,†,‡ and Guangming Lu*,†,‡ †

Department of Medical Imaging, Jinling Hospital, Medical School of Nanjing University, Nanjing,

210002 Jiangsu, P.R. China ‡

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical

Engineering, Nanjing University, Nanjing, 210093 Jiangsu, P.R. China ⊥

Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical

Imaging and Bioengineering (NIBIB), National Institutes of Health, Bethesda, Maryland 20892, United States §

Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging, Department of

Biomedical Engineering, School of Medicine, Shenzhen University, Shenzhen, 518060 Guangdong, P.R. China

ABSTRACT: The integration of diagnosis and therapy into one nanoplatform, known as theranostics, has attracted increasing attention in the biomedical areas. Herein, we firstly present a cancer cell targeting imaging and drug delivery system based on engineered thioether-bridged periodic mesoporous organosilica nanoparticles (PMOs). The PMOs are stably and selectively conjugated with near-infrared fluorescence (NIRF) dye Cyanine 5.5 (Cy5.5) and anti-Her2 affibody on the outer surfaces to endow them with excellent NIRF imaging and cancer targeting properties. Also, taking the advantage of the thioether-group-incorporated mesopores, the release of ACS Paragon Plus Environment

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chemotherapy drug doxorubicin (DOX) loaded in the PMOs is responsive to the tumor-related molecule glutathione (GSH). The drug release percentage reaches 84.8 % in 10 mM of GSH solution within 24 h, which is more than two-fold higher than that without GSH. In addition, the drug release also exhibits pH-responsive, which reaches 53.6 % at pH 5 and 31.7 % at pH 7.4 within 24 h. Confocal laser scanning microscopy and flow cytometry analysis demonstrate that the PMOs-based theranostic platforms can efficiently target to and enter Her2 positive tumor cells. Thus, the smart imaging and drug delivery nanoplatforms induce high tumor cell growth inhibition. Meanwhile, the Cy5.5 conjugated PMOs perform great NIRF imaging ability, which could monitor the intracellular distribution, delivery and release of the chemotherapy drug. In addition, cell viability and histological assessments show the engineered PMOs have good biocompatibility, further encouraging the following biomedical applications. Over all, the systemically engineered PMOs can serve as a novel cancer cell targeting imaging and drug delivery platform with NIRF imaging, GSH and pH dual-responsive drug release, and high tumor cell targeting ability.

KEYWORDS: HER2 positive cancer, periodic mesoporous organosilica, cancer cell targeting, near-infrared fluorescence imaging, drug delivery

1. INTRODUCTION Cancer is a main cause of death worldwide, leading an estimated 8.2 million cancer deaths in 2012.1 It is urgent to develop new method to diagnose and treat cancers. The emergence of nanoplatforms with potentially transformative advances in biomedicine provides considerable promise for cancer diagnosis and treatment.2-5 Compared with traditional drugs and methods, the nanoplatforms hold several advantages for diagnostics and therapeutics, including harnessed various imaging modalities of near-infrared fluorescence (NIRF) imaging,6 magnetic resonance imaging (MRI),7 computed ACS Paragon Plus Environment


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tomography (CT),8 positron emission tomography (PET),9 ultrasound (US) imaging,10 and photoacoustic imaging (PAI),11 with selectable therapeutic functionalities, such as chemotherapy,12 photodynamic therapy (PDT),13 and photothermal therapy (PTT).14 What’s more, conveniently modified surface of nanoparticles makes it possible for accumulation at tumor via extension of circulating half-life and active or passive targeting effects.15-16 More recently, integration of imaging and therapeutic capabilities into single nanoplatform, resulting in an approach termed "theranostics", has been realized and drawn increasing interest for drug delivery tracking, therapy response monitoring, and image-guided therapy.17-21 At present, various theranostic agents based on various inorganic or organic materials, such as silica,22 gold,23 iron,24 palladium,5 and polymers25 have been constructed. The integration of organic and inorganic fragments can endow the hybrid materials with colorful functional groups and good biocompatibility for biomedical applications.26 As a new type of inorganic–organic hybrid materials, periodic mesoporous organosilica nanoparticles (PMOs) have received increased attention because of their well-defined mesopores, large surface areas, and organic group-incorporated frameworks.27-29 Simultaneously, It is reported that the PMOs are biodegradable and have better hemocompatibility than pure mesoporous silica nanoparticles (MSNs).26,30 Therefore, the PMOs have great promise for different biomedical applications. However, to the best of our knowledge, PMOs have not been used to construct theranostic platforms. For an ideal PMO-based cancer theranostic nanoplatform, several issues should be considered and resolved. Firstly, both imaging and targeting molecules must be stably conjugated on the PMOs. Secondly, the modified imaging and targeting molecules should not block the mesochannels so that treatment molecules, such as chemotherapy drug, could be loaded and released. Thirdly, to reduce side effects, the loaded treatment molecules should be stimuli-responsive to tumor cells.

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Herein, we firstly reported PMO based cancer cell targeting imaging and drug delivery agents. NIRF dye Cyanine 5.5 (Cy5.5) and anti-Her2 affibody molecules are stably and selectively modified on the PMOs outer surfaces by click chemistry. Chemotherapy drug DOX is further loaded in the mesochannels of the PMOs. Owing to the specific recognition of affibody and Her2 expressed on tumor cells, the theranostic particles exhibit excellent targeting ability towards Her2 positive cells. After enter the tumor cells, DOX could be released from theranostic platforms via both the cleavage of disulfide in PMOs by glutathione (GSH) and acidic environment, inducing obvious toxicity in tumor cells. At the same time, NIRF imaging could efficiently monitor the drug distribution, delivery and release in cells.

2. MATERIALS AND METHODS 2.1 Materials. Analytical reagents of tetraethoxysilane (TEOS), cetyltrimethylammonium bromide (CTAB), anhydrous ethanol, concentrated ammonia aqueous solution (25 wt%), dioxane, triphenylphosphine, N,N-dimethylformamide (DMF), concentrated HCl (37%), and GSH were obtained

from

Sinopharm

Chemical

Reagent

Co.,

Ltd.

(Shanghai,

China).

1,4-Bis(triethoxysily)propane tetrasulfide (TESPTS), 2-carboxyethl phosphine hydrochloride (TCEP), and doxorubicin (DOX) in the form of hydrochloride salt were purchased from Sigma−Aldrich (St. Louis, MO, USA). Maleimide derivative cyanine dye (Cy5.5-maleimide) and polyethylene glycol (PEG) polymer with two distal maleimide reaction sites (Mal-PEG-Mal) (MW = 3.4 kDa) were bought from Seebio Biotechnology Co., Ltd. (Shanghai, China). Anti-Her2 affibody (ZHER2:342) was bought from Affibody (Solna, Sweden). Deionized water (Millipore) with a resistivity of 18 MΩ cm was used in all experiments. Dulbecco's Modified Eagle's Medium (DMEM), heat-inactivated fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), 0.05% trypsin–EDTA, and penicillin–streptomycin solution were obtained from Gibco Laboratories (NY, ACS Paragon Plus Environment


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USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Nanjing Keygen Biotech. Co., Ltd. (Nanjing, China). The human cancer MCF-7 cell line and MDA-MB-435 cell line were bought from American Type Culture Collection (ATCC). 2.2 Synthesis of Thioether-Bridged PMOs. Thioether-bridged PMOs with a unique yolk–shell structure were prepared via a surfactant-directed sol–gel process in a solution containing water, ethanol, ammonia, CTAB, TEOS, and TESPTS according to our previously reported method.31 Typically, 0.12 g of CTAB was dissolved in a mixture of concentrated ammonia aqueous solution (1 mL, 25 wt %), ethanol (30 mL), and water (75 mL). Afterward, the mixed solution was heated to 35 °C, and TESPTS (0.1 mL) and TEOS (0.25 mL) was added under vigorous stirring. The molar ratio of the reaction mixture was 1.00 TEOS: 0.179 TESPTS: 0.294 CTAB: 11.82 NH3: 3720 H2O: 460 C2H5OH. After reacting at 35 °C for 24 h, the white product was collected by centrifugation at 11000 rpm for 10 min and washed three times with ethanol. Then, the as-made mesostructured nanoparticles were dispersed in water (30 mL) and transferred to a 50 mL Teflon-lined stainless-steel autoclave. After heated in an air flow electric oven at 150 °C for 24 h, the yolk–shell structured thioether-bridged PMOs were obtained. 2.3 Modification of Cy5.5 and Anti-Her2 Affibody on the Thioether-Bridged PMOs. Firstly, the disulfide bonds on the outer surfaces of PMOs were transformed into thiol groups according to the previously reported method.32 In a typical procedure, the thioether-bridged PMOs (0.065 g) was dispersed in dioxane (1.1 mL) and water (0.3 mL). Then, triphenylphosphine (0.10 g) was added to the suspension. The mixture was heated to 40 °C, and two drops of concentrated HCl (37%) were added. After reacting at 40 °C under N2 for 2 h, the PMOs with thiol groups on their surfaces were washed with ethanol three times, and dispersed in 2 mL of ethanol. To link near-infrared dye Cy5.5, Cy5.5-maleimide (0.3 mg) was added to the above obtained suspension in a mixture of water (12.0 mL) and DMF (1.2 mL). Afterward, the suspension was shaken at room temperature for 12 h. ACS Paragon Plus Environment

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Finally, the Cy5.5 grafted yolk–shell PMOs (denoted as PMO-Cy5.5) were obtained and thoroughly washed with water. The obtained blue product was then dispersed in water. To evaluate the efficiency of Cy5.5-maleimide conjugated to the nanoparticles, the supernatant and washed solutions were collected and the free dye content was measured by using an UV-vis spectrometer at a wavelength of 673 nm. To link anti-Her2 affibody (ZHER2:342), Cysteine (Cys) derivative ZHER2:342 (ZHER2:342-Cys) was first subjected to reduction with 5 mM TCEP and then linked Mal-PEG-Mal. The molar ratio of affibody and Mal-PEG-Mal was 1 : 1. The Mal-PEG-Mal connected anti-Her2 affibody was then conjugated to PMO-Cy5.5. Typically, ZHER2:342-Cys (4.5 µg) was added to 5 mM TCEP in 15 mL of water and shaken at room temperature for 30 min. Then a mixture of Mal-PEG-Mal (2.2 µg) in ethanol (2.5 mL) and DMF (1.5 mL) was added to the above obtained suspension, and the resulting mixture was shaken at room temperature for 3 h. Afterward, PMO-Cy5.5 (0.045 g) was dispersed in the above mixture and shaken at room temperature for 3 h. Finally, the Cy5.5 and affibody grafted yolk–shell PMOs (denoted as PMO-Cy5.5-affibody) were obtained and thoroughly washed with water. To remove the surfactant (CTAB), the as-synthesized materials PMO-Cy5.5 or PMO-Cy5.5-affibody were transferred to a solution containing ethanol (30 mL) and concentrated HCl (60 µL, 37%) and stirred at 60 °C for 3 h. The solvent-extraction step was repeated three times to ensure complete removal of CTAB. Finally, the product was washed with ethanol three times. 2.4 Loading Anti-cancer Drug. Typically, PMO-Cy5.5 (10 mg) or PMO-Cy5.5-affibody (10 mg) and DOX (5 mg) were mixed with 10 mL 1 x phosphate-buffered saline (PBS) solution. After stirring for 24 h under dark conditions, DOX loaded PMO-Cy5.5 (denoted as [email protected]) or PMO-Cy5.5-affibody (denoted as [email protected]) were centrifuged at 11000 rpm for 10 min. To remove free DOX, [email protected] or [email protected] were further washed with PBS three times. The resultant [email protected] or [email protected] ACS Paragon Plus Environment


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were dried under high vacuum for further use. To evaluate the DOX-loading capacity, the supernatant and washed solutions were collected and the residual DOX content was measured by using a UV-vis spectrometer at a wavelength of 490 nm. 2.5 In Vitro GSH and pH-Induced Drug Release. In vitro reducing-responsive DOX releasing was conducted in PBS at the GSH concentration of 0, 5, and 10 mM. And in vitro pH-responsive DOX releasing was executed in PBS solution of pH 5 and pH 7.4. In brief, the above-prepared [email protected] (1 mg) were dissolved in GSH solution (1.5 mL) at different concentrations (0, 5 and 10 mM) or PBS solution (1.5 mL) at pH 5 and pH 7.4, which was further put into a shaking table with a speed of 100 rpm at 37 °C. At certain time intervals, the supernatant was collected by centrifugation and replaced with 1.5 mL of fresh media for each sample. The obtained supernatant was tested by UV-vis spectroscopy at 490 nm. 2.6 Western Blot Analysis of Her2 Protein Expression. MDA-MB-435 and MCF-7 cells were separately seeded in a 6-well plate at a density of 1 × 105 cells/mL and cultured in 2.5 mL of DMEM containing 10% FBS for 24 h under a humidified 5% CO2 atmosphere. Subsequently, cells were washed with cold PBS buffer thoroughly and lysed with cell lysis buffer. Cells were scraped, under ultrasound for three times and the protein extracts were harvested by centrifugation at 4 °C for 15 min. The concentration of protein extracts was determined by using the Bio-Rad protein assay (Hercules, CA, USA). The obtained protein extracts were then incubated in a sample buffer at 95 °C for 10 min. Thereafter, the proteins were separated on a 10% polyacrylamide gel containing sodium dodecyl sulfate (SDS) (Bio-Rad, Hercules, CA, USA) by gel electrophoresis. The proteins were then transferred from SDS gel surface to a polyvinylidene difluoride (PVDF) membrane (Millipore) by a semi-dry transfer cell (Bio-Rad). Subsequently, the membranes were blocked with 5% milk in Tris-buffered saline-Tween 20 (TBST) for 2 h, followed by incubating with primary anti-Her2 antibody (1 : 400) and GAPDH (1 : 1000) at 4 °C overnight. After being washed with TBST three ACS Paragon Plus Environment

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times, the membranes were incubated with a secondary horseradish peroxidase conjugated antibody for 2 h at room temperature. After the membranes were washed with TBST again, the signal of Her2 protein was detected on a Bio-Rad ChemiDoc Touch imaging system (Hercules, CA, USA). The expression of GAPDH protein was detected as control. 2.7 Targeted Uptake Evaluation by Inductively Coupled Plasma (ICP) Analysis. First, 2.5 mL of DMEM medium containing approximately 2.5 × 105 MDA-MB-435 and MCF-7 cells were respectively added into six-well plates. After 24 h incubation under a humidified 5% CO2 at 37 °C, the medium was removed and 2.5 mL of DMEM containing 100 µg/mL PMOs equivalent of PMO-Cy5.5-affibody was respectively added. After culturing for 2 h, the medium was removed and the cells were washed with PBS three times. MDA-MB-435 and MCF-7 cells were harvested with centrifugation after digested with 0.05% trypsin–EDTA. Afterward, the cells were separately washed with PBS two times and dispersed in 1 mL of PBS. 200 µL of the cells suspension was centrifuged and lysed with cell lysis buffer, followed by protein extracts determined by using the Bio-Rad protein assay (Hercules, CA, USA). The rest 800 µL of the cells suspension was centrifuged and then lysed with 50 µL of 95% HNO3 at 95 °C. After the solution turned achromic, 100 µL of 14 M NaOH was added to dissolve the PMOs. Finally, 5 mL of water was added to 11 µL of above obtained solution. The intracellular concentration of silicon in MDA-MB-435 and MCF-7 cells was quantitatively measured by using an inductively coupled plasma spectrometer (Perkin-Elmer, Inc., Waltham MA, USA). 2.8 Targeted Uptake Evaluation by Confocal Laser Scanning Microscopy. MDA-MB-435 cells and MCF-7 cells (1×105 cells per mL per well) were separately planted into a Lab-Tek Chamber Slide system (Thermo Fisher Scientific, Rochester, NY, USA) in 300 µL of DMEM containing 10% FBS under a humidified 5% CO2 atmosphere at 37 °C. After incubation for 24 h, the medium was carefully removed and replaced with 200 µL of medium containing 50 µg/mL PMOs ACS Paragon Plus Environment


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equivalent of [email protected] or [email protected]. After 4 h, the cells were washed three times with PBS and stained with UltraCruz Mounting Medium (Santa Cruz Biotechnology, TX, USA). The confocal laser scanning microscopy (CLSM) images of the cells were performed using a confocal laser scanning microscope (LSM 710, Carl Zeiss, Germany) by blue fluorescing (λex = 405 nm), green fluorescing (λex = 673 nm), and red fluorescing (λex = 480 nm). The fluorescence intensity of DOX fluorescence images was measured by ImageJ (NIH, Bethesda, MD). 2.9 Targeted Uptake Evaluation by Flow Cytometry. MDA-MB-435 cells (1×105 cells per mL per well) were seeded in a twelve-well plate and maintained in 2 mL of DMEM with 10% FBS for 24 h under a humidified 5% CO2 atmosphere at 37 °C. Afterward, the medium was removed and 2 mL

of

DMEM

containing

50

µg/mL

PMOs

equivalent

[email protected]

and

[email protected] were added separately. After another 0.5, 2, and 6 h, the DMEM was respectively removed and washed with PBS three times. The cells were digested with 0.05% trypsin–EDTA and harvested by centrifugation. The flow cytometry was performed on a BD Accuri C6 (BD, Franklin Lakes, NJ, USA). The DMEM without adding nanoparticles was used as control. 2.10 In Vitro Cytotoxicity. MDA-MB-435 cells were grown in DMEM medium supplemented with 10% FBS at 37 °C in a humidified atmosphere with 5% CO2. The cytotoxicity of PMO-Cy5.5-affibody, [email protected], [email protected] or free DOX against MDA-MB-435 cells was measured by MTT assay. The cells were seeded in 96-well plates at a density of 1 × 104 cells/well and allowed to grow for 24 h. Then the culture medium was replaced with 100 µL of fresh medium containing different concentrations of PMO-Cy5.5-affibody, [email protected], [email protected] or free DOX at pH 7.4 and the cells were incubated for 2 h. Then the cells were washed with PBS thoroughly, and incubated in fresh medium for another 24 h. Afterward, 10 µL of MTT reagent (5 mg/mL in culture medium) was added. Following incubation for 4 h, the culture medium was removed and DMSO (150 µL) was added to ACS Paragon Plus Environment

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each well for dissolving the formazan crystals. Finally, the absorbance of the solution was determined at 570 nm using a microplate reader (BioTek Instruments, Winooski, VT, USA). Statistical analyses were examined using independent-samples T test by SPSS version 16.0 (SPSS, Chicago, IL, USA). It was considered significant when P < 0.05. 2.11 In Vivo Toxicity. Animal procedures were in agreement with the guidelines of the Institutional Animal Care and Use Committee. 1.5 mg/mL PMO-Cy5.5-affibody (100 µL) was injected into 4 week-old male ICR mice (n = 3) via the tail vein every two days for three times. To examine in vivo toxicity, the heart, liver, spleen, lung, and kidney were removed after injection and fixed in 10% formalin solution. Then, the tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The histological sections were observed using an optical microscope (IX71; Olympus, Tokyo, Japan). 2.12 Characterization. Transmission electron microscopy (TEM) images were captured by using a HT7700 microscope (Hitachi, Tokyo, Japan) at 100 kV. The zeta potential and hydrodynamic size were measured by using a Brookhaven analyzer (Brookhaven Instruments Co., Holtsville, NY, USA). UV-vis spectra were obtained by using a Lambda 35 UV-vis spectrophotometer (PerkinElmer, Inc., Waltham, MA, USA). Fourier transform infrared (FT-IR) spectra were recorded on a Nicolet Nexus 870 spectrometer (Nicolet Instruments Inc. Madison, WI, USA). NIRF imaging was performed by IVIS Lumina XR system (Xenogen Corporation-Caliper, Alameda, CA, USA) under the Cy5.5 filter (λex = 673 nm, λem = 707 nm). Nitrogen sorption isotherms were measured by a Micromeritics Tristar 3000 analyzer (Micromeritics Instruments Corporation, Atlanta, GA, USA) at -196 °C. The Brunauer-Emmett-Teller (BET) and nonlocal density functional theory (NLDFT) methods were used to calculate the specific surface areas and the pore sizes, respectively. The adsorbed amount at p/p0 = 0.995 was used to estimate the total pore volume.



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3. RESULTS AND DISCUSSION As shown in scheme 1, yolk–shell structured thioether-bridged PMOs were first synthesized by hydrothermal treatment of mesosturcured organosilica nanospheres (step 1). The disulfide bonds on the surface of PMOs were then selectively reduced to thiol groups (step 2). Cy5.5-maleimide and anti-Her2 affibody molecules were selectively conjugated on the outer surfaces of PMOs by click chemistry of thiol and maleimide groups (step 3). After the extraction of CTAB (step 4), chemotherapy drug DOX was loaded in the mesochannels of PMOs (step 5), resulting in the cancer targeting theranostic platforms. The imaging and drug delivery system can be internalized by Her2 positive tumor cells with the help of anti-Her2 affibody. The anti-cancer drug DOX in nanoparticles would be released on responsive to tumor-related molecule GSH and acidic environment. Meanwhile, Cy5.5 functionalized particles could monitor the distribution, delivery and release of drug in cells by NIRF imaging.

Scheme1. Illustration of the construction of the PMO-based smart cancer targeting imaging and drug delivery system and the GSH and pH dual-responsive drug release in tumor cell. Step 1: preparation of yolk–shell structured thioether-bridged PMOs; Spet 2: breaking the disulfide bonds on the surface of PMOs; Step 3: conjugation of Cy5.5-maleimide and anti-Her2 affibody molecules on the PMOs via click chemistry; Step 4: extraction of CTAB; Step 5: loading anti-cancer drug DOX in the ACS Paragon Plus Environment

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mesopores.

The TEM image showed that the prepared PMOs have a well-defined yolk–shell structure with a diameter of approximately 245 nm (Figure 1a). FT-IR of the PMOs show C-H bands at 2930, 1450, and 1410 cm-1 and C-S band at 694 cm−1, indicating the thioether-bridged frameworks33 (Figure 1b). Dynamic light scattering (DLS) and size distribution showed that the sizes of PMOs, PMO-Cy5.5, [email protected], [email protected] are 333, 336, 300, 301 nm, respectively (Figure 1c and Figure S1). The size difference of the PMOs measured by TEM and DLS was mainly attributed to DLS presenting wet samples.34 After modified with Cy5.5, the hydrodynamic size of PMO-Cy5.5 was almost consistent with that of PMOs, owing to the molecule Cy5.5 is small. After loaded with anticancer drug DOX, the hydrodynamic sizes of [email protected] and [email protected] were decreased to about 300 nm, which is mostly likely attributed to the compress of the electric double layer on the particles by the DOX. The zeta potential of PMOs was -19.0 mV, which turned to -32 mV when coupled with Cy5.5, owing to negatively charged Cy5.5.35 When affibody was introduced, the zeta potential became less negative, from -32.0 mV to -19.5 mV, which is in accordance to a previous affibody modified surface study (Figure 1d).36 FT-IR spectrum of [email protected] showed a band of amide I at around 1650 cm-1 (Figure S2, black arrow), confirming the peptide groups in affibody.37 The above results indicated that Cy5.5 and affibody were successfully conjugated on the PMOs. The PMO-Cy5.5 showed strong fluorescence signal, while the supernatant after centrifugation of PMO-Cy5.5 presented no signal (Figure 1e), indicating the NIRF dye are effectively connected on the PMOs by the click chemistry of thiol and maleimide groups. Simultaneously, owing to the thiol groups are incorporated in the PMO frameworks, no free dye was detected form the supernatant of PMO-Cy5.5 after 7 days, indicating the Cy5.5 is stably modified on the PMOs. In addition, PMO-Cy5.5 also exhibited the ACS Paragon Plus Environment


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typical UV-vis absorbance peak of Cy5.538 (Figure 1f), further confirming Cy5.5 has been linked on the PMOs. Owing to the NIRF dye are stable linked on the PMOs, it can be used as a reliable optical imaging agent to monitor the location of the particles in tumor cells. Because the Cy5.5 and anti-Her2 affibody molecules are selectively conjugated on the outer surfaces of PMOs, the positively charged DOX can be loaded into the mesochannels of negatively charged PMO-Cy5.5 or PMO-Cy5.5-affibody by electrostatic interaction.39 The zeta potential values of PMO-Cy5.5 and PMO-Cy5.5-affibody changed from -32 mV and -19.5 mV to 17.6 mV and 12.4 mV, respectively, after loading with DOX (Figure 1d). Nitrogen sorption isotherms of the PMO-Cy5.5 showed a typical-IV curve, with a surface area of as high as 728 m2/g. In contrast, the curve of [email protected] showed a lower adsorption volume and a decreased surface area of 235 m2/g (Figure S3a). The peaks of the pore size distribution curves of [email protected] were significantly decreased compared to PMO-Cy5.5. Simultaneously, the pore volume of PMO-Cy5.5 and [email protected] were measured to be 0.77 and 0.34 cm3/g (Figure S3b). These results clearly indicated the anti-cancer drug is successfully loaded in the mesopores of PMOs. The DOX-loading capacity of PMO-Cy5.5 and PMO-Cy5.5-affibody reached 140 and 112 mg DOX per gram of PMO-Cy5.5 and PMO-Cy5.5-affibody particles, respectively. Consistently, the resulted [email protected] showed characteristic excitation peak of DOX at 490 nm40 (Figure 1f), and an absorption band at around 1700 cm-1 of C=O carbonyl stretching contained in DOX in FT-IR spectrum41 (Figure S2, red arrow), further indicating successful DOX loading in the PMOs.


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Figure 1. (a) TEM images of yolk–shell structured thioether-bridged PMOs. (b) FT-IR spectrum of the

thioether-bridged

PMOs.

(c)

Particle

size

distributions

of

PMOs,

PMO-Cy5.5,

[email protected], and [email protected]. (d) Zeta potentials of PMOs, PMO-Cy5.5, PMO-Cy5.5-affibody, [email protected], and [email protected]. (e) Near-infrared fluorescence signal of water, supernatant of PMO-Cy5.5, and PMO-Cy5.5. (f) UV-vis spectra of PMO-Cy5.5, [email protected], and Cy5.5 dispersed in PBS.

Drug release profile is very important for drug delivery. It is well known that the disulfide bonds are physiologically responsive and can be broken up by GSH via the redox reaction.42 Considering the fact that the level of intracellular GSH concentration can reach 1-11 mM, which is much higher than that of extracellular (10 µM),43 and GSH concentration of tumor cells is significantly higher than that of normal cells,44 tumor intracellular GSH will break up the disulfide bonds within the thioether groups in the PMOs framework and trigger the release of loaded DOX. The in vitro drug release profile of [email protected] was measured at GSH concentrations of 0, 5, and 10 mM, which simulated the GSH situation in body fluid (Figure 2a). The release rate and percentage of DOX from [email protected] was obviously GSH dependent and in proportion to the GSH concentration. In the absence of GSH, the release rate was relatively slow with a 31.7 % releasing ACS Paragon Plus Environment


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percentage in 24 h. As a comparison, with the addition of GSH, the release of DOX was sharp in the first 2 h and became slower in the following releasing, mainly owing to the delaying cleavage of disulfide bonds. Finally, the release rates increased to 71.1 and 84.8 % at the GSH concentrations of 5 and 10 mM respectively within 24 h. It has been demonstrated that PMOs exhibit time-dependent biodegradable in GSH solution of 10 mM,42 and we examined PMOs treated with 10 mM GSH solution for 24 h by TEM. As shown in Figure S4, the shape of PMOs has changed, and the edges became fuzzy and merged each other, indicating the dissolution of the PMOs. In addition, after the breakage of disulfide bonds in framework, the hydrophobic interaction between PMOs and DOX would be broken up,42 triggering the DOX release from [email protected]. Considering that the electrostatic interaction of DOX with the PMOs, we further examined the drug release profile of DOX from [email protected] at pH values of 7.4 and 5.0 for 24 h (Figure 2b). As shown in Figure 2b, DOX release could also be accelerated in acidic environment. The release rate was 53.6 % at pH 5.0 and 31.7 % at pH 7.4 within 24 h, indicating a pH-responsive drug release property. Considering the stimuli-responsive drug release ability of the nanoplatforms, [email protected] can selectively deliver the drug into the cells, avoiding much premature extracellular release. Taking into account that tumor cells exhibit much higher GSH concentration, and are more acidic than normal cells,41 the GSH and pH-dual responsive [email protected] are expected to use in on demand cancer treatment in cancer.


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Figure 2. DOX release profiles from [email protected] at (a) different concentrations of GSH and (b) different pH values.

Her2 over expressed cancer cell line of MDA-MB-435 was chosen to evaluate the targeting ability and intracellular distribution of the theranostic platforms, and cancer cell line of MCF-7 without Her2 receptor was used as control. Firstly, Her2 expression of MDA-MB-435 and MCF-7 was determined by Western blot. As shown in Figure 3a, Her2 was highly expressed in MDA-MB-435 cells, while negative in MCF-7 cells. The targeting ability and quantitative cellular uptake of PMO-Cy5.5-affiboy was evaluated by ICP analysis. The results showed internalized PMOs (mg) per milligram protein in the corresponding cells (%). Because of the specific recognition of Her2 expressed on MDA-MB-435 cells and anti-Her2 affibody on the surface of PMOs, after incubation for 2 h, the uptake of PMO-Cy5.5-affibody by MDA-MB-435 was significantly higher than that by MCF-7 (Figure 3b). Thanks to the NIRF property of the theranostics, the detailed cellular uptake, intracellular distribution and drug release of [email protected] in MDA-MB-435 and MCF-7 cells was monitored by confocal laser scanning microscopy (Figure 4). For each cell line, the DOX concentration (10 µg/mL) and image exposure times were kept constant for comparison purposes. Both cell lines could internalize [email protected] and [email protected], showing by Cy5.5 (green) and DOX (red) fluorescence. For MDA-MB-435, owing to the ACS Paragon Plus Environment


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combination of Her2 and anti-Her2 affibody, the internalization of [email protected] was significantly higher than that of [email protected], corresponding to the strong green and red fluorescence (Figure 4a). In comparison, [email protected] and [email protected] exhibited similar uptake in MCF-7 cells, owing to the lack of specificity of anti-Her2 affibody to Her2 (Figure 4b). Furthermore, according to the quantified intensity of red fluorescence (Figure S5), DOX entered cells was significantly high in MDA-MB-435 treated by [email protected], owing to the high uptake of the particles. In addition, the NIRF showed that the [email protected] and [email protected] were located in the cytoplasm of MDA-MB-435 and MCF-7 cells (Figure 4a, b). Due to the cleavage of disulfide bonds in PMOs by GSH, DOX released from the nanoparticles and entered the nucleus, showing by the red fluorescence. Further investigation of [email protected] internalization in MDA-MB-435 was performed by flow cytometry. As shown in Figure 5, the uptake of nanoplatforms was time dependent, simultaneously monitored by both Cy5.5 (Figure 5a) and DOX (Figure 5b) fluorescence. The corresponding mean fluorescence intensity (MFI) was inserted separately, showing the fluorescence intensity increased with incubation time. In addition, the target cell uptake was evaluated by flow cytometry for 6 h incubation. Both increased fluorescence of Cy5.5 (Figure S6a) and DOX (Figure S6b) indicated the internalization of target particles was higher than that of non-target group. The above results demonstrated that [email protected] exhibited targeted sustained drug release with Cy5.5 and DOX fluorescence monitoring, which could be a valuable nanoplatform for targeted cancer imaging and therapy.


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Figure 3. (a) Expression of Her2 in MCF-7 and MDA-MB-435 cells determined by Western blot, and (b) uptake of PMO-Cy5.5-affibody by MDA-MB-435 and MCF-7 cells after incubation for 2 h.

Figure 4. Confocal laser scanning microscopy of MDA-MB-435 (a) and MCF-7 cells (b) treated with [email protected] and [email protected] for 4 h (relative DOX = 5.6 µg/mL). ACS Paragon Plus Environment


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(Blue fluorescence is associated with DAPI; green fluorescence stands for Cy5.5, red fluorescence is expressed by free DOX, released DOX, and DOX in PMOs.) Scale bar: 50 µm.

Figure

5.

Flow

cytometry

analysis

of

MDA-MB-435

cells

incubated

with

[email protected] nanoparticles for 0.5, 2, and 6 h. The uptake of nanoparticles was monitored by the fluorescence of (a) Cy5.5 and (b) DOX.

We further investigated the toxicity of the promising targeted theranostic platforms by MTT assay. Figure 6a showed good biocompatibility of blank PMO-Cy5.5-affibody in MDA-MB-435 cell lines, with a wide range of PMO concentrations up to 200 µg/mL. After loaded with DOX, [email protected] presented statistically cell toxicity than [email protected] in MDA-MB-435 cells at DOX concentrations of 1, 2, 4, and 8 µg/mL using independent-samples T ACS Paragon Plus Environment

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test (P < 0.05). It is observed that less than 40 % of MDA-MB-435 cells were alive in the targeting group at the DOX concentration of 8 µg/mL, while at the same DOX concentration nearly 60 % of cells were still alive in non-targeting group, suggesting the targeting nanoplatforms presented higher toxicity in MDA-MB-435 cells because of their much more cellular internalization. In contrast, free DOX showed very low toxicity to the cells line at various concentrations, mainly owing to the short incubation time. Therefore, [email protected] exhibited potential for biocompatible targeted drug delivery to Her2 over expressed cell lines, which was important for cancer targeting therapy and clinical applications.

Figure 6. Cell viability of MDA-MB-435 cells incubated with (a) blank PMO-Cy5.5-affibody, and (b)

free

DOX,

[email protected],

and

[email protected]

(b)

for

2

h.

Independent-samples T test “*” p < 0.05.

The in vivo toxicity of PMO-Cy5.5-affibody was investigated by histological assessment. Sections were obtained from the organs harvested as described above (heart, liver, spleen, lung and kidney). As shown in Figure 7, cardiac samples exhibited no hydropic degeneration. Liver samples were normal without inflammatory infiltrates and steatosis. In the lung samples, there was no pulmonary fibrosis or inflammation. And in the other tissues, no apparent inflammation, injury, or necrosis was observed. These results further indicated the good biocompatibility of blank PMO-Cy5.5-affibody, which promoted biological applications of the theranostic platforms. ACS Paragon Plus Environment


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Figure 7. Representative tissue sections of mice stained with hematoxylin and eosin. All images shown are of 100× magnification.

4. CONCLUSIONS We presented Her2 positive cancer cell-targeted imaging and drug delivery system by conjugating Cy5.5 and anti-Her2 affibody on the surfaces of thioether-bridged PMOs and loading DOX in the mesopores. The Cy5.5 is stably modified on the PMOs, which make the platforms suitable for NIRF imaging, monitoring the cell distribution, drug delivery and release. The loaded DOX shows a significant dual stimuli-responsive release via the cleavage of disulfide bonds in PMOs by GSH and acidic environment. Moreover, affibody modified [email protected] can be more efficiently internalized by Her2 positive tumor cells via the specific recognition of Her2 and affibody. In addition, the imaging and drug delivery system shows an excellent in vitro and in vivo biocompatibility. Overall, this study reports for the first time a smart cancer cell targeting imaging and drug delivery system with the NIRF imaging, GSH and pH dual-responsive drug release, and cancer targeting properties by engineering PMOs. ASSOCIATED CONTENT Supporting Information Size distribution of PMOs, PMO-Cy5.5, [email protected], and [email protected]; FT-IR spectra of [email protected]; Nitrogen sorption isotherms and pore size distribution curves of PMO-Cy5.5 and [email protected]; TEM images of PMOs treated with 10 mM GSH solution for 24 h; quantified intensity of DOX fluorescence images of MDA-MB-435 and MCF-7 incubated with [email protected] and [email protected] for 4 h; flow cytometry of MDA-MB-435 incubated with [email protected] and [email protected] ACS Paragon Plus Environment

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for 6 h. This information is available free of charge via the Internet at http://pubs.acs.org/. AUTHOR INFORMATION Corresponding Author

*Fax: +86 25 8480 4659. [email protected].

Tel:

+86

25

8086

0185.

E-mail:

[email protected];

Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing ﬁnancial interest. ACKNOWLEDGMENTS We greatly appreciate financial support from the National Key Basic Research Program of the PRC (2014CB744504 and 2011CB707700), the Major International (Regional) Joint Research Program of China (81120108013), the National Natural Science Foundation of China (81201175), the Natural Science Foundation of Jiangsu Province (BK20130863), the National Science Foundation for Post-doctoral Scientists of China (2013T60939 and 2012M521934), and the Science Foundation of Nanjing University of Posts and Telecommunications (NY213045 and 214096).

REFERENCES (1) Torre, L. A.; Bray, F.; Siegel, R. L.; Ferlay, J.; Lortet-Tieulent, J.; Jemal, A. Global Cancer Statistics, 2012. CA-Cancer J. Clin. 2015, 65, 87-108. (2) Liang, X. L.; Li, Y. Y.; Li, X. D.; Jing, L. J.; Deng, Z. J.; Yue, X. L.; Li, C. H.; Dai, Z. F. PEGylated Polypyrrole Nanoparticles Conjugating Gadolinium Chelates for Dual-Modal MRI/Photoacoustic Imaging Guided Photothermal Therapy of Cancer. Adv. Funct. Mater. 2015, 25, 1451-1462. (3) Zhang, L. E.; Zeng, L. Y.; Pan, Y. W.; Luo, S.; Ren, W. Z.; Gong, A.; Ma, X. H.; Liang, H. Z.; Lu, G. M.; Wu, A. G. Inorganic Photosensitizer Coupled Gd-Based Upconversion Luminescent Nanocomposites for In Vivo Magnetic Resonance Imaging and Near-Infrared-Responsive Photodynamic Therapy in Cancers. Biomaterials. 2015, 44, 82-90. (4) Wang, S. G.; Li, X.; Chen, Y.; Cai, X. J.; Yao, H. L.; Gao, W.; Zheng, Y. Y.; An, X.; Shi, J. L.; Chen, H. R. A Facile One-Pot Synthesis of a Two-Dimensional MoS2/Bi2S3 Composite Theranostic Nanosystem for Multi-Modality Tumor Imaging and Therapy. Adv. Mater. 2015, 27, 2775-2782. (5) Zhu, X. L.; Chi, X. Q.; Chen, J. H.; Wang, L. R.; Wang, X. M.; Chen, Z.; Gao, J. H. Real-Time Monitoringin In Vivo Behaviors of Theranostic Nanoparticles by Contrast-Enhanced T1 Imaging. Anal. Chem. 2015, 87, 8941-8948. (6) Wu, J. B.; Shi, C. H.; Chu, G. C.-Y.; Xu, Q. J.; Zhang, Y.; Li, Q. L.; Yu, J. S.; Zhau, H. E.; Chung, L. W. K. Near-Infrared Fluorescence Heptamethine Carbocyanine Dyes Mediate Imaging and Targeted Drug Delivery for Human Brain Tumor. Biomaterials 2015, 67, 1-10. (7) Yang, L. J.; Zhou, Z. J.; Liu, H. Y.; Wu, C. Q.; Zhang, H.; Huang, G. M.; Ai, H.; Gao, J. H. Europium-Engineered Iron Oxide Nanocubes with High T-1 and T-2 Contrast Abilities for MRI in Living Subjects. Nanoscale 2015, 7, 6843-6850. (8) Dong, W. J.; Li, Y. S.; Niu, D. C.; Ma, Z.; Liu, X. H.; Gu, J. L.; Zhao, W. R.; Zheng, Y. Y.; Shi, J. L. A Simple Route to Prepare Monodisperse Au NP-Decorated, Dye-Doped, Superparamagnetic Nanocomposites for Optical, MR, and CT Trimodal Imaging. Small 2013, 9, 2500-2508. ACS Paragon Plus Environment


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

(9) Zhou, M.; Li, J. J.; Liang, S.; Sood, A. K.; Liang, D.; Li, C. CuS Nanodots with Ultrahigh Efficient Renal Clearance for Positron Emission Tomography Imaging and Image-Guided Photothermal Therapy. ACS Nano 2015, 9, 7085-7096. (10) Zhao, Y. J.; Song, W. X.; Wang, D.; Ran, H. T.; Wang, R. H.; Yao, Y. Z.; Wang, Z. G.; Zheng, Y. Y.; Li, P. Phase-Shifted PFH@PLGA/Fe3O4 Nanocapsules for MRI/US Imaging and Photothermal Therapy with Near-Infrared Irradiation. ACS Appl. Mater. Interfaces 2015, 7, 14231-14242. (11) Zha, Z. B.; Deng, Z. J.; Li, Y. Y.; Li, C. H.; Wang, J. R.; Wang, S. M.; Qu, E. Z.; Dai, Z. F. Biocompatible Polypyrrole Nanoparticles as a Novel Organic Photoacoustic Contrast Agent for Deep Tissue Imaging. Nanoscale 2013, 5, 4462-4467. (12) Parambadath, S.; Rana, V. K.; Zhao, D. Y.; Ha, C.-S. N,N′-Diureylenepiperazine-Bridged Periodic Mesoporous Organosilica for Controlled Drug Delivery. Microporous Mesoporous Mater. 2011, 141, 94-101. (13) Zeng, L. Y.; Ren, W. Z.; Xiang, L. C.; Zheng, J. J.; Chen, B.; Wu, A. G. Multifunctional Fe3O4-TiO2 Nanocomposites for Magnetic Resonance Imaging and Potential Photodynamic Therapy. Nanoscale 2013, 5, 2107-2113. (14) Zha, Z. B.; Yue, X. L.; Ren, Q. S.; Dai, Z. F. Uniform Polypyrrole Nanoparticles with High Photothermal Conversion Efficiency for Photothermal Ablation of Cancer Cells. Adv. Mater. 2013, 25, 777-782. (15) Ma, X. H.; Gong, A.; Xiang, L. C.; Chen, T. X.; Gao, Y. X.; Liang, X. J.; Shen, Z. Y.; Wu, A. G. Biocompatible Composite Nanoparticles with Large Longitudinal Relaxivity for Targeted Imaging and Early Diagnosis of Cancer. J. Mater. Chem. B 2013, 1, 3419-3428. (16) Yu, M. X; Zheng, J. Clearance Pathways and Tumor Targeting of Imaging Nanoparticles. ACS Nano 2015, 9, 6655-6674. (17) Xie, J.; Lee, S.; Chen, X. Y. Nanoparticle-Based Theranostic Agents. Adv. Drug Delivery Rev. 2010, 62, 1064-1079. (18) Choi, K. Y.; Liu, G.; Lee, S.; Chen, X. Y. Theranostic Nanoplatforms for Simultaneous Cancer Imaging and Therapy: Current Approaches and Future Perspectives. Nanoscale 2012, 4, 330-342. (19) Sun, Y.; Zheng, Y. Y.; Li, P.; Wang, D.; Niu, C. C.; Gong, Y. P.; Huang, R. Z.; Wang, Z. B.; Wang, Z. G.; Ran, H. T. Evaluation of Superparamagnetic Iron Oxide-Polymer Composite Microcapsules for Magnetic Resonance-Guided High-Intensity Focused Ultrasound Cancer Surgery. BMC Cancer 2014, 14, 800-810. (20) Kunjachan, S.; Ehling, J.; Storm, G.; Kiessling, F.; Lammers, T. Noninvasive Imaging of Nanomedicines and Nanotheranostics: Principles, Progress, and Prospects. Chem. Rev. 2015, DOI: 10.1021/cr500314d. (21) Zhao, Z. H.; Wang, X. M.; Zhang, Z. J.; Zhang, H.; Liu, H. Y.; Zhu, X. L.; Li, H.; Chi, X. Q.; Yin, Z. Y.; Gao, J. H. Real-Time Monitoring of Arsenic Trioxide Release and Delivery by Activatable T-1 Imaging. ACS Nano 2015, 9, 2749-2759. (22) Lee, J. E.; Lee, N.; Kim, T.; Kim, J.; Hyeon, T. Multifunctional Mesoporous Silica Nanocomposite Nanoparticles for Theranostic Applications. Acc. Chem. Res. 2011, 44, 893-902. (23) Xia, Y. N.; Li, W. Y.; Cobley, C. M.; Chen, J. Y.; Xia, X. H.; Zhang, Q.; Yang, M. X.; Cho, E. C.; Brown, P. K. Gold Nanocages: From Synthesis to Theranostic Applications. Acc. Chem. Res. 2011, 44, 914-924. (24) Liao, M. Y.; Lai, P. S.; Yu, H. P.; Lin, H. P.; Huang, C. C. Innovative Ligand-Assisted Synthesis of NIR-Activated Iron Oxide for Cancer Theranostics. Chem. Commun. 2012, 48, 5319-5321. (25) Liu, F. Y.; He, X. X.; Chen, H. D.; Zhang, J. P.; Zhang, H. M.; Wang, Z. X. Gram-Scale Synthesis of Coordination Polymer Nanodots with Renal Clearance Properties for Cancer Theranostic Applications. Nat. Commun. 2015, 6, 8003-8011. (26) Teng, Z. G.; Wang, S. J.; Su, X. D.; Chen, G. T.; Liu, Y.; Luo, Z. M.; Luo, W.; Tang, Y. X.; Ju, H. ACS Paragon Plus Environment

Page 24 of 26

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X.; Zhao, D. Y.; Lu, G. M. Facile Synthesis of Yolk-Shell Structured Inorganic-Organic Hybrid Spheres with Ordered Radial Mesochannels. Adv. Mater. 2014, 26, 3741-3747. (27) Asefa, T.; MacLachlan, M. J.; Coombs, N.; Ozin, G. A. Periodic Mesoporous Organosilicas with Organic Groups Inside the Channel Walls. Nature 1999, 402, 867-871. (28) Chen, Y.; Xu, P. F.; Chen, H. R.; Li, Y. S.; Bu, W. B.; Shu, Z.; Li, Y. P.; Zhang, J. M.; Zhang, L. X.; Pan, L. M.; Cui, X. Z.; Hua, Z. L.; Wang, J.; Zhang, L. L.; Shi, J. L. Colloidal HPMO Nanoparticles: Silica-Etching Chemistry Tailoring, Topological Transformation, and Nano-Biomedical Applications. Adv. Mater. 2013, 25, 3100-3105. (29) Teng, Z. G.; Su, X. D.; Zheng, Y. Y.; Zhang, J. J.; Liu, Y.; Wang, S. J.; Wu, J.; Chen, G. T.; Wang, J. D.; Zhao, D. Y.; Lu, G. M. A Facile Multi-Interface Transformation Approach to Monodisperse Multiple-Shelled Periodic Mesoporous Organosilica Hollow Spheres. J. Am. Chem. Soc. 2015, 137, 7935-7944. (30) Croissant, J.; Cattoën, X.; Man, M. W. C.; Gallud, A.; Raehm, L.; Trens, P.; Maynadier, M.; Durand, J.-O. Biodegradable Ethylene-Bis(Propyl)Disulfide-Based Periodic Mesoporous Organosilica Nanorods and Nanospheres for Efficient In-Vitro Drug Delivery. Adv. Mater. 2014, 26, 6174-6180. (31) Teng, Z. G.; Su, X. D.; Lee, B. H.; Huang, C. G.; Liu, Y.; Wang, S. J.; Wu, J.; Xu, P.; Sun, J.; Shen, D. K.; Li, W.; Lu, G. M. Yolk-Shell Structured Mesoporous Nanoparticles with Thioether-Bridged Organosilica Frameworks. Chem. Mater. 2014, 26, 5980−5987. (32) Besson, E.; Mehdi, A.; Reyé, C.; Corriu, R. J. P. Soft Route for Monodisperse Gold Nanoparticles Confined within SH-Functionalized Walls of Mesoporous Silica. J. Mater. Chem. 2009, 19, 4746-4752. (33) Kim, J. H.; Fang, B. Z.; Song, M. Y.; Yu, J. S. Topological Transformation of Thioether-Bridged Organosilicas into Nanostructured Functional Materials. Chem. Mater. 2012, 24, 2256-2264. (34) Goel, S.; Chen, F.; Hong, H.; Valdovinos, H. F.; Hernandez, R.; Shi, S. X.; Barnhart, T. E.; Cai, W. B. VEGF121-Conjugated Mesoporous Silica Nanoparticle: A Tumor Targeted Drug Delivery System. ACS Appl. Mater. Interfaces 2014, 6, 21677-21685. (35) Hue, J. J.; Lee, H. J.; Jon, S.; Nam, S. Y.; Yun, Y. W.; Kim, J. S.; Lee, B. J. Distribution and Accumulation of Cy5.5-Labeled Thermally Cross-Linked Superparamagnetic Iron Oxide Nanoparticles in the Tissues of ICR Mice. J. Vet. Sci. 2013, 14, 473-479. (36) Yang, M.; Cheng, K.; Qi, S.; Liu, H.; Jiang, Y.; Jiang, H.; Li, J.; Chen, K.; Zhang, H.; Cheng, Z. Affibody Modified and Radiolabeled Gold-Iron Oxide Hetero-Nanostructures for Tumor PET, Optical and MR Imaging. Biomaterials 2013, 34, 2796-2806. (37) Habicht, G.; Haupt, C.; Friedrich, R. P.; Hortschansky, P.; Sachse, C.; Meinhardt, J.; Wieligmann, K.; Gellermann, G. P.; Brodhun, M.; Gotz, J.; Halbhuber, K. J.; Rocken, C.; Horn, U.; Fandrich, M. Directed Selection of a Conformational Antibody Domain that Prevents Mature Amyloid Fibril Formation by Stabilizing Abeta Protofibrils. Proc. Natl. Acad. Sci. U S A 2007, 104, 19232-19237. (38) Lin, J. Y.; Li, Y.; Li, Y. X.; Wu, H. J.; Yu, F.; Zhou, S. F.; Xie, L. Y.; Luo, F. H.; Lin, C. J.; Hou, Z. Q. Drug/Dye-Loaded, Multifunctional PEG-Chitosan-Iron Oxide Nanocomposites for Methotraxate Synergistically Self-Targeted Cancer Therapy and Dual Model Imaging. ACS Appl. Mater. Interfaces 2015, 7, 11908-11920. (39) Slowing, I. I.; Vivero-Escoto, J. L.; Wu, C. W.; Lin, V. S. Y. Mesoporous Silica Nanoparticles as Controlled Release Drug Delivery and Gene Transfection Carriers. Adv. Drug Delivery Rev. 2008, 60, 1278-1288. (40) Yang, G. B.; Gong, H.; Liu, T.; Sun, X. Q.; Cheng, L.; Liu, Z. Two-Dimensional Magnetic WS2@Fe3O4 Nanocomposite with Mesoporous Silica Coating for Drug Delivery and Imaging-Guided Therapy of Cancer. Biomaterials 2015, 60, 62-71. ACS Paragon Plus Environment


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

(41) Tang, Y. X.; Teng, Z. G.; Liu, Y.; Tian, Y.; Sun, J.; Wang, S. J.; Wang, C. Y.; Wang, J. D.; Lu, G. M. Cytochrome C Capped Mesoporous Silica Nanocarriers for pH-Sensitive and Sustained Drug Release. J. Mater. Chem. B 2014, 2, 4356-4362. (42) Chen, Y.; Meng, Q. S.; Wu, M. Y.; Wang, S. G.; Xu, P. F.; Chen, H. R.; Li, Y. P.; Zhang, L. X.; Wang, L. Z.; Shi, J. L. Hollow Mesoporous Organosilica Nanoparticles: A Generic Intelligent Framework-Hybridization Approach for Biomedicine. J. Am. Chem. Soc. 2014, 136, 16326-16334. (43) Bauhuber, S.; Hozsa, C.; Breunig, M.; Göpferich, A. Delivery of Nucleic Acids via Disulfide-Based Carrier Systems. Adv. Mater. 2009, 21, 3286-3306. (44) Lee, M. H.; Yang, Z.; Lim, C. W.; Lee, Y. H.; Dongbang, S.; Kang, C.; Kim, J. S. Disulfide-Cleavage-Triggered Chemosensors and Their Biological Applications. Chem. Rev. 2013, 113, 5071-5109.


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Smart Cancer Cell Targeting Imaging and Drug Delivery System by

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