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A multifunctional lymph-targeted platform based on Mn@mSiO2 nanocomposites: Combining PFOB for dual-mode imaging and DOX for cancer diagnose and treatment

Tian Liu1,2, Guangyu Wu3, Jiejun Cheng3, Qing Lu3, Yanjie Yao1,2, Zhenjing Liu1, Dongchen Zhu1, Juan Zhou2,Jianrong Xu3 (*), Jun Zhu2 (*), and Dannong He1 (*)

Nano Res., Just Accepted Manuscript • DOI: 10.1007/s12274-015-0929-1

http://www.thenanoresearch.com on October. 28, 2015

© Tsinghua University Press 2015

Just Accepted

This is a “Just Accepted” manuscript, which has been examined by the peer-review process and has been accepted for publication. A “Just Accepted” manuscript is published online shortly after its acceptance, which is prior to technical editing and formatting and author proofing. Tsinghua University Press (TUP) provides “Just Accepted” as an optional and free service which allows authors to make their results available to the research community as soon as possible after acceptance. After a manuscript has been technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Please note that technical editing may introduce minor changes to the manuscript text and/or graphics which may affect the content, and all legal disclaimers that apply to the journal pertain. In no event shall TUP be held responsible for errors or consequences arising from the use of any information contained in these “Just Accepted” manuscripts. To cite this manuscript please use its Digital Object Identifier (DOI®), which is identical for all formats of publication.

Nano Research DOI 10.1007/s12274-015-0929-1

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A Multifunctional Lymph-Targeted Platform

Based on Mn@mSiO2 Nanocomposites:

Combining PFOB for Dual-Mode Imaging and

DOX for Cancer Diagnose and Treatment

Tian Liu1,2,§, Guangyu Wu3,§, Jiejun Cheng3, Qing

Lu3, Yanjie Yao1,2, Zhenjing Liu1, Dongchen

Zhu1, Juan Zhou2, Jianrong Xu3*, Jun Zhu2*, and

Dannong He1*

1 Shanghai Jiao Tong University, China 2 National Engineering Research Center for

Nanotechnology, China 3 Shanghai Renji Hospital, Shanghai Jiao Tong

University School of Medicine, China § These authors contributed equally to this work.

A universal platform, with Mn doping and HA modification based on

mSiO2, is utilized for further dual-mode imaging and cancer diagnose and

treatment through absorbing hydrophobic PFOB and hydrophilic DOX.

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A Multifunctional Lymph-Targeted Platform Based on Mn@mSiO2 Nanocomposites: Combining PFOB for Dual-Mode Imaging and DOX for Cancer Diagnose and Treatment

Tian Liu1,2,§, Guangyu Wu3,§, Jiejun Cheng3, Qing Lu3, Yanjie Yao1,2, Zhenjing Liu1, Dongchen Zhu1, Juan Zhou2, Jianrong Xu3 (*), Jun Zhu2 (*), and Dannong He1 (*) 1 Country School of Materials Science and Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P. R.

China. 2 National Engineering Research Center for Nanotechnology, 28 East Jiang Chuan Road, Shanghai 200241, P. R. China. 3 Department of Radiology, Shanghai Renji Hospital, Shanghai Jiao Tong University School of Medicine, 1630 Dong Fang Rd,

Shanghai 200127, P. R. China. § These authors contributed equally to this work.

Received: day month year Revised: day month year Accepted: day month year (automatically inserted by the publisher)

© Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2014

KEYWORDS multifunctional platform, dual-mode imaging, MRI, ultrasound imaging, cancer diagnose

ABSTRACT A universal platform with Mn doping and HA modification based on mSiO2 is designed and prepared, which is used as a basic multifunctional material with MR imaging. Furthermore, we add some functions flexibly through endowing some functional molecules. Specially, two typical compounds, hydrophobic PFOB (perfluorocty bromide) and hydrophilic DOX, are loaded into channels to obtain PFOB@Mn@mSiO2@HA (PMMH) or DOX@Mn@mSiO2@HA (DMMH) nanoparticles for dual-mode imaging, and imaging and therapy, respectively. We present evidence that the PMMH and DMMH nanoparticles are highly targeted to lymph system in vitro and in vivo, and highlight the MR and ultrasound imaging of PMMH nanoparticles in lymph system, and MR imaging and chemotherapy of DMMH nanoparticles in cancer. The results show that both PMMH and DMMH nanoparticles are potential in high lymph targeting efficiency, PMMH nanoparticles are a dual-mode contrast agent about both ultrasound and MR imaging for lymph system and DMMH nanoparticles arepowerful agents for combined diagnosis and therapy of cancer in vivo.

Nano Research DOI (automatically inserted by the publisher) Research Article

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1 Introduction

The rapid development in nanotechnology has allowed the emerging of various functional nanomaterials with multiple discrete function-related components integrated in one nanoparticle for applications in biomedical imaging and therapy to break through the limitations of each single function [1, 2]. These multifunctional nanomaterials are highly sought in biomedical sciences because of their potentials in areas such as diagnosis, drug delivery, hyperthermia, stem cell therapy, and tissue engineering. On the one hand, it is interesting to design a multifunctional nanoprobe for united imaging because each imaging modality has its own limitations and advantages. For instance, a lot of nanoprobes with luminescent imaging [3-5], computed tomography (CT) and magnetic resonance (MR) imaging [6-8], and so on, have been reported for cancer diagnosis to improve the sensitivity and accuracy [3-8]. On the other hand, it is also meaningful to develop a multifunctional nanoarchitecture for united therapy or diagnosis and therapy through encapsulating functional materials within a designed nanoparticle system. For example, the combing of chemotherapy and imaging or photothermal- and chemotherapies was designed in many reports [3, 9-12]. However, despite the extensive applications provided by these multifunctional nanoarchitectures, their functions will always be restricted once these multifunctional nanoarchitectures are prepared. In other words, it is difficult to add or change the functions of these multifunctional nanoarchitectures flexibly. Therefore, the development of a more universal platform is necessary, which can be conveniently endowed with varies properties by different post processing methods after their synthesis according to diagnostic and therapeutic demands.

To make this design feasible, a basic material with a sophisticated structure and fundamental function is needed to carry the above multifunctional entities. Among them, mesoporous silica (mSiO2) have attracted substantial attentions in recent years due to their advantageous structural properties, such as high internal surface area, pore volume, tunable pore sizes, colloidal stability and the possibility to specifically functionalize the inner

pore system or the external particle surface. These highly attractive features enable mSiO2 to be a promising and widely applicable platform for diverse biomedical applications including bioimaging, biosensing, biocatalysis, bonerepair and scaffold engineering, and drug delivery. Many efforts are made in recent years to create multifunctional nanoparticles consisting of mSiO2 as host materials, such as the multistage delivery system and multimodal imaging system. Specially, some metal ions for magnetic resonance imaging, such as Mn2+ and Gd3+, have been doped into the framework of mSiO2, which contributes to a platform with MR imaging. The platform could further absorb the drug molecules to fabricate a multifunctional nanoarchitecture with the drug delivery and MR imaging. Thus, on our opinions, a modified mSiO2 can be used as a basic material with a sophisticated structure and fundamental function, which can produce and change their multifunctional properties conveniently through absorbing different molecules.

As we well known, hyaluronic acid (HA) is a natural polysaccharide widely distributed in the extracellular matrix. Due to its biocompatibility and diverse biological functions as a targeting ligand and signaling molecule, HA has served as a superior biomaterial for the formulation of nanospheres, hydrogels, and scaffolds for drug delivery and tissue engineering [13]. In addition, because of the interaction between HA and the CD44 receptor, HA is widely used as a tumor vector for the targeting procedure because CD44 is expressed in a variety of tumors such as breast, colon, intestinal, and brain [14, 15]. Furthermore, LYVE-1, as a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for HA, which can bind both soluble and immobilized HA [16-18]. However, unlike CD44, the LYVE-1 molecule can colocalize with HA on the luminal face of the lymph vessel wall and is completely absent from blood vessels. Therefore, hyaluronic acid has showed a highly promising lymphatic targeting carrier. In our preliminary report [19], pure HA molecule is used as a targeted matrix to lymphatic system. The results show that HA molecule is capable in defining the structure of the lymphatic system and highly targeted to lymphatic system in vitro and in vivo.

The paper aims to design a universal and multifunctional platform, which comes from some

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popular materials including mSiO2 for a media, HA for a targeted matrix and Mn for a MRI element. The platform is cheap, practical and flexible. Based on the platform, we further develop a multifunctional probe with MR and ultrasound imaging to improve sensitivity and resolution for lymph imaging, and a multifunctional vehicle with accurate positioning and chemotherapy for target lymph tumor. According to the previous reports, some nano-/micro-carriers with PFOB (perfluorocty bromide) have been utilized as ultrasound contrast agents [20]. Therefore, in present experiment, PFOB is loaded into Mn@mSiO2@HA (MMH) nanoparticles to obtain PFOB@Mn@mSiO2@HA (PMMH) nanoparticles, in which PFOB can enhance sound reflection function and further improve ultrasound contrast signal. DOX (a general chemotherapeutic drug) is also loaded into MMH nanoparticles to obtain DOX@Mn@mSiO2@HA (DMMH) nanoparticles. Thus, two typical compounds, hydrophobic PFOB and hydrophilic DOX, are used as models to obtain PMMH or DMMH nanoparticles for dual-mode imaging, as well as imaging and therapy, respectively. We present evidence that the PMMH and DMMH nanoparticles are highly targeted to lymph system in vitro and in vivo, and highlight the MR and ultrasound imaging of PMMH nanoparticles in lymph system, and MR imaging and chemotherapy of DMMH nanoparticles in cancer. What is the most innovative development is that we provide a demonstration about how to put the new universal platform to further exploitations, which has great potential in biological area.

2 Experimental

2.1 Preparation of Mn@mSiO2 nanoparticles

Manganese doped mesoporous silica (Mn@mSiO2, MM) nanoparticles were synthesized as following: 1.2 mmol of cetyltrimethyl ammonium bromide (CTAB) was dissolved in 180 mL of double-distilled water at room temperature. And then 6 mmol of NaOH were added to the solution followed by 10 mmol of triethoxysilane (TEOS) under rigorous stirring. Subsequently, 0, 0.1, 0.5, 1 and 5 mmol MnCl2·4H2O were added rapidly. The obtained precipitates were recovered by filtration after 24 h rigorous stirring, washed and refluxed in 7.2 mg/mL ammonium nitrate ethanol solution for removing CTAB.

2.2 Preparation of Mn@mSiO2@HA nanoparticles

The preparation of Mn@mSiO2@HA (MMH) nanoparticles included the surface modification of MM nanoparticles with 3-aminopropyl triethoxysilane (APTES) and the conjugation of HA on them. In a typical experiment, 0.1 g of MM nanoparticles was reacted with 110 µL APTES in 10 mL toluene. The amino-modified MM nanoparticles were washed with methanol and water, centrifuged and dried at 60 °C for 12 h. Then, HA was activated using N-hydroxysuccinimide (NHS) and N-ethyl-N’-[3-(dimethylamino)propyl] carbodiimide hydrochloride (EDC). Specially, 1.25 g of HA and the obtained amino-modified MM nanoparticles were dissolved in 200 mL double-distilled water. Subsequently, 0.23 g of EDC and 0.14 g of NHS were added into the solution and stirred for 24 h at room temperature. Finally, the resulting products were collected by centrifuging, washing with ethanol and water three times, and drying at 60 °C in sequence.

2.3 Preparation of PFOB@Mn@mSiO2@HA and DOX@Mn@mSiO2@HA nanoparticles

0.1 g of MMH nanoparticles was dispersed into 30 mL double-distilled water, then 500 µL, 2 mg/mL of DOX or 100 µL of PFOB was injected. After stirring for 24 h, MMH nanoparticles with PFOB (PFOB@Mn@mSiO2@HA, PMMH) or DOX (DOX@Mn@mSiO2@HA, DMMH) were fabricated and separated. To remove the free PFOB and DOX in the solutions of nanoparticles, PMMH and DMMH were washed by double-distilled water about 5 times until the supernatant liquid was clear.

2.4 Characterization

Powder X-ray diffraction (XRD) patterns were recorded on a Rigaku D/MAX-2250V diffractometer using Cu-Karadiation (40 kV and 40 mA). The morphology of the samples were obtained using a high resolution transmission electron microscope (HRTEM, JEOL, JEM-2100F) and a field-emission scanning electron microscope (FESEM, Hitachi, S-4800), respectively. The Fourier transformation infrared (FT-IR) spectra were recorded on a Nicolet 6700 FT-IR spectrometer. The UV-vis spectra were investigated using a PerkinElmer Lambda 950 UV-vis spectrophotometer. The Brunauere-Emmette-Teller

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(BET) surface area and pore size were measured using a micromeritics ASAP 2020 instrument. X-ray photoelectron spectroscopy (XPS) was performed with a VG Scientic ESCLAB 220iXL X-ray photoelectron spectrometer. The average hydrodynamic sizes were measured by a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, Worcestershire, U.K.) based on dynamic light scattering. The contents of Mn were analyzed with inductively coupled plasma optic emission spectrometry (ICP-MS) using a PLASMA-SPEC-II instrument (Horiba JOBIN YVON). Fluorine content was determined by high performance ion chromatography (Dionex 500, USA) with conductivity detection through oxygen flask combustion (Separating column: IonPac AG14-AS14; Eluent: NaHCO3 0.0010M+Na2CO3 0.0035M). Based on the result, the loading content of PFOB in PMMH nanoparticles was calculated.

2.5 Cell viability experiments

HCT116 cells (5 × 103 cells per well) in log phase were incubated into 96-well flat-bottomed plates. The cells were incubated for 24 h at 37 °C under 5% CO2. The obtained PMMH nanoparticles at concentrations of 0, 25, 50, 75, 100 and 125 mg/mL in PBS were added to the wells of the treatment group, respectively. The cells were incubated for 4, 8 and12 h at 37 °C under 5% CO2. Then, all nanoparticles were removed from the medium and 10% CCK/ PBS solution was added to each well and incubated at 37 °C for 4 h. The amount of cell proliferation was measured by reading the optical density value at 450nm using a plate reader. The following formula was used to calculate the viability of cell growth: Viability (%) = (mean of absorbance value of treatment group/mean absorbance value of control) × 100%. The results were expressed as an average over five nominally identical measurement.

2.6 Confocal imaging of cells

Confocal imaging of cells was performed using a Leica laser scanning confocal microscope. HCT116 cells (1 ×106 cells/mL) were incubated with fluorescein isothiocyanate labelled nanoparticles (MM, MMH and PMMH) for 2 h for confocal imaging, fixed with 4% paraformaldehyde for 30 min and stained by DAPI for 8 min. All the cells were washed twice with PBS before confocal

imaging. Imaging of FITC (fluorescein isothiocyanate) labelled nanoparticles was carried out at 488 nm laser excitation, with its emission collected from 550 to 570 nm.

2.7 MRI in vitro

All MRI scans were carried out with a 3.0 T whole body system (3.0 T Intera Achieva, Philips Medical Systems, Best, The Netherlands) with brain Crossed Coil. Different mass concentrations of PMMH nanoparticles were placed in a series of PE tubes for T1-weighted MR imaging, using a standard spin echo sequence. The sequence parameters were TR/TE, 4.6/2.4 ms; flip angle, 12°; FOV, 26 cm; matrix, 320×256; effective slice thickness, 0.6 mm. The 3D MRIs were then reconstructed from the original data set at each time point using maximum-intensity projection (MIP).

2.8 Ultrasound imaging in vitro

Ultrasound imaging was performed using Acuson Sequoia 512 linear transducer (Siemens, Mountain View, CA). A dilute solution (1 mg/mL) of PMMH nanoparticles was placed in dialysis cassettes, examined by a Siemens Acuson Sequoia 512 with a 3.5 MHz linear transducer.

2.9 MRI in vivo

Animal experiments were approved by local governmental authorities. Experiments were carried out on New Zealand White Rabbits with the weight of 3~3.5 kg, which were anaesthetized by intraperitoneal injection of 3% pentobarbital solution. During the process, the rabbit body temperature was kept at physiological level. PMMH nanoparticles (400 µL, 0.10 mg/mL) and Gadopentetate (400 µL) were injected into the right and left knee popliteal fossa, respectively. The injection sites for the contrast agents were carefully disinfected. After the injection, the injection sites were gently massaged for approximately 30 second to improve lymph drainage. The interesting MR imaging area was from the upper thigh to the shank. Similarly，the nude mouse with the weight of 28.4 g was anaesthetized and injected in tumor region by 150 µL DMMH (0.10 mg/mL), and MR images were collected using small animal Crossed Coil.

All MR imaging was performed on a 3.0-T

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superconductive whole-body scanner (Signa HDXt GE Healthcare), using a dedicated six-channel phased array sensitivity encoding coil for optimized signal reception with the rabbits in the supine position. The conventional gradient system with an amplitude of 40 mT/m and a slew rate of 150 mT/m per millisecond. For interstitial MRI, a fast T1-weighted 3D gradient-echo sequence (LAVA) in the coronal plane was acquired after gadopentetate dimeglumine injection. To assess all phases of lymph node enhancement, we began acquiring images immediately after subcutaneous administration of two contrast agent every 10 mins. The sequence parameters were TR/TE, 4.6/2.4 ms; flip angle, 12°; FOV, 26 cm; matrix, 320×256; effective slice thickness, 0.6 mm. The 3D MRIs were then reconstructed from the original data set using maximum-intensity projection (MIP). During the acquisition, the examined rabbit remained in an unchanged position.

2.10 Ultrasound imaging in vivo

Animal experiments were approved by local governmental authorities. Experiments were carried out on New Zealand White Rabbits with the weight of 3~3.5 kg, which were anaesthetized by intraperitoneal injection of 3% pentobarbital solution. During the process, the rabbit body temperature was kept at physiological level. Furthermore, the skin of leg knee popliteal area was shed by NaS (6%). PMMH nanoparticles (400 µL, 0.10 mg/mL) were injected into the right knee popliteal fossa, before which a primary gray scale sonography was made to locate lymph nodes in the leg knee popliteal area. All ultrasound imaging was performed on High-Resolution In Vivo Ultrasound Micro-Imaging System with ECG 2500mv and Resp 2590mv.

2.11 Immunofluorescent antibody staining analysis

Rabbit anti-human LYVE-1 polyclonal primary antibodies were optimized and validated in formalin-fixed paraffin. Tissues in knee popliteal fossa region, which were imaged successfully by ultrasound and MR imaging, removed from rabbit, fixed in PBS, 4% paraformaldehyde, and embedded in paraffin wax. Prior to staining, the tissues were fixed, dehydrated, wax-impregnated, embedded and then sliced up. Primary antibody dilutions

with 1:50 were incubated with tissue slides for 90 min at room temperature. Lymph vessels from rabbit's leg knee popliteal fossa were used as positive control tissues for the validation of LYVE-1. The slides were viewed by a Zeiss Axioskop fluorescence microscope using fluorescent illumination.

2.12 DOX loading and in vitro drug release

50 mg MMH nanoparticles was added into 1 mg/mL, 50 mL DOX aqueous solution. After stirring for 24 h, the DMMH nanoparticles with loaded drug were fabricated and separated. 5 mg of DMMH nanoparticles was immersed in 10 mL phosphate buffer saline solution (PBS, pH = 5.5), which was introduced into dialysis cassettes (the width is 44 mm, the molecular weight cut-off is 8000-14000) as the drug donor. Furthermore, the bag was placed in 100 mL PBS as the release medium (receiver). The release medium (10 mL) was taken out for the determination of UV spectra around 490 nm at given time intervals and replaced with the same volume of fresh buffer solution. The curve of the release rate for DOX could be obtained from the relationship between the cumulative release amount and time t. Additionally, the standard curve of the DOX aqueous solution could be obtained from the relationship between the absorbance and the DOX concentration.

2.13 Growth inhibitory effect of DMMH nanoparticles on tumor cells

The growth inhibitory effect of DMMH nanoparticles on HCT116 tumor cells was evaluated by CCK assay according to the previously reported procedure [21-23]. The cells were seeded in a 96-well plate (5000 cells in 100 µL per well), and cultured for 24 h. The medium was then substituted with a series of concentrations of free DOX and DMMH nanoparticles. Two days later, the formulations were removed, and CCK solution was added and incubated for 4 h. Next, the CCK solution was removed, and PBS was added to the wells. The absorption representing cell viability was detected using a microplate reader (iMark/xMark Bio-Rad) to determine the cell viability at 450 nm.

2.14 Growth inhibition of DMMH nanoparticles on tumors in vivo

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Male Specific Pathogen Free nude mice, aging 4-5 weeks and weighing 18-20 g, were supplied by Department of Experimental Animals, Shanghai Jiaotong University, and maintained under standard housing conditions. All animal experiments were carried out in accordance with guidelines evaluated and approved by the ethics committee of Shanghai Jiaotong University.

YAC-1 (mouse lymphoma cells) tumor bearing mice were established by subcutaneous injection of 2×106 cells suspended in 200 µL PBS. Three groups of mice (n=6), eighteen totally, were subcutaneously injected with PBS , free DOX and DMMH nanoparticles (0.1 mg/mL) with the same dosage of free DOX inside, respectively, until the diameter of tumor was around 8-10 mm, after which all mice were returned to animal housing. The tumor volumes were tracked every 2 day using a vernier caliper, which was calculated by the formula V＝1/2 (L×W2), where L and W were length (longest dimension) and width (shortest dimension). At 23th day, all the animals were euthanized and the tumors were dissected and weighed.

2.15 Tissue distribution

The short-term distribution of MMH nanoparticles in mice was performed as following: eighteen mice were divided into six groups, three mice each group, besides three mice of a group were used as control group. Based on the preliminary experiment, the dosage in this experiment of MMH nanoparticles is 250 mg / kg, 0.1 mL. Every three mice of each group were sacrificed and anatomized at 4, 6, 8, 10 and 12 h after being injected by MMH nanoparticles in enterocoelia except the control group without any injection. About 100 µL of blood sample was obtained in each group firstly. Furthermore, next to blood, liver, lung, spleen, brain, heart and kidney were collected. Organs were weighed and homogenized. After subjecting the sample to a digestion process, the Mn content was determined by inductively coupled plasma mass spectrometry (ICP-MS).

The long-term distribution of MMH nanoparticles in mice was performed as following: nine mice were divided into three groups, three mice each group besides three mice of a group were used as control group. Based on the preliminary experiment, the dosage in this

experiment of MMH nanoparticles is 250 mg / kg, 0.1 mL. Every three mice of each group were sacrificed and anatomized at 1, 2 and 3 day, respectively after being injected by MMH nanoparticles in enterocoelia except the control group without any injection. From Day 1, every three mice of each group were intravenously injected once daily for 3 consecutive days. The injections were well tolerated and no adverse effects were observed during the entire experiment. At day 1, 2 and 3, about 100µL of blood sample was obtained in each group before liver, lung, spleen, brain, heart and kidney of the treatment-groups were collected at 12 h after injection. Organs were weighed and homogenized. After subjecting the sample to a digestion process, the Mn content was determined by ICP-MS.

2.16 Statistical analysis

Results are expressed as the mean ± standard deviation (SD). Statistical analysis was done using Student’s t-test. Differences were considered significant at a P value of < 0.05.

3 Results and discussion

A MM nanoparticles are obtained by mesoporous silica material doping Mn ions via a co-assemble process, which could contribute to basic multifunctional materials with MR imaging. Furthermore, the surface grafting of MM nanoparticles by HA results to a multifunctional platform for targeted delivery to special tumor cells and organism. In order to evaluate the flexiblity of the multifunctional platform, hydrophobic PFOB and hydrophilic DOX molecules are loaded to the platform for dual-mode imaging with MR and ultrasound imaging for lymph system, and combining diagnose and therapy with MR imaging and sustained release for a lymph tumor. The whole procedure is schematically illustrated in Fig. 1. Fig. S1 in the Electronic Supplementary Material shows that the multifunctional platform is chosen at 5 mmol Mn ions, which produces the enough surface area of 503 m2/g for further experiments. Moreover, the platform is used to absorb hydrophobic PFOB for the

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Figure 1 Schematic illustration of the formation of PMMH and DMMH nanoparticles. preparation of PMMH nanoparticles for dual-mode imaging, and PFOB loaded in MMH nanoparticles is determined to be 11.2 mg/g. Similar to Shi et al report [24], the driving force of the hydrophobic PFOB molecule in MMH nanoparticles is possibly the hydrophobic interaction between PFOB and HA molecule around HA molecule in mesoporous pores. The morphology and structure of PMMH nanoparticles were characterized. SEM and TEM images of the PMMH nanoparticles are presented in Fig. 2a and 2b, respectively. Fig. 2a shows that the PMMH nanoparticles are well-dispersed and uniform in size with an average diameter of about 150 nm, which is in accordance with the result of average hydrodynamic size in Fig. S2. Furthermore, it seems to find the regular hexagonal arrays of uniform pore opening in TEM image of as-synthesized PMMH nanoparticles (Fig. 2b). Additionally, the small angle XRD patterns also reveal the result. As shown in Fig. 2c, main (100) diffraction peak of typical mesoporous silica materials is displayed, which shows that mesoporous silica is maintained although the possible destroy of the channel frameworks by doping Mn ions. A typical type IV isotherm with a steep capillary condensation step also indicates the characteristic of an excellent quality mesoporous material with the surface area of 374 m2/g (Fig. 2d) and pore size of 3.0 nm (Fig. S3), although the mesoporous pores are filled by PFOB molecules. Moreover, as shown in Fig. S4, the decrease of the surface area could result from the HA modification and PFOB absorption, which is further characterized by FT-IR spectra of PFOB, HA and PMMH nanoparticles. As shown in Fig. 2e, the typical peaks of PFOB, such as 1245 and 1218 cm-1,

are monitored, which is attributed to characteristic asymmetric stretching vibrations of -CF2 group [12, 25, 26]. Furthermore, the typical peaks of HA, such as 1610 and 1040 cm-1 are found, which comes from the characteristic stretching vibrations of C=O and bending vibration of N-H, respectively. Compared to main characteristic peaks of PFOB and HA, all of peaks are found in FT-IR spectra of the PMMH nanoparticles besides 1150 cm-1, the characteristic stretching vibrations of C-N. The results indicate that PFOB and HA have been added in MM nanoparticles successfully, which is further approved by XPS, an analysis of the elemental composition and their chemical environment in PMMH nanoparticles. The desired elements, such as Si, C, N, O, Mn and F, are encapsulated in PMMH nanoparticles, and the Mn (2p) feature centered at 641.2 eV match with the typical binding energy of the metal-silicate (Mn-O-Si) [27-29]. Furthermore, EDX reveals that the Mn content is about 5.6% in PMMH nanoparticles. The above results show a universal platform with Mn doping and HA modification based on mSiO2 has successfully absorbed hydrophobic PFOB for a multifunctional platform.

Figure 2 (a) SEM, (b) TEM and (c) XRD pattern of PMMH nanoparticles, (d) N2 adsorption/desorption isotherm (1 - MMH nanoparticles and 2 - PMMH nanoparticles), (e) FT-IR spectra and (f) XPS spectra of PMMH nanoparticles.

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Figure 3 Laser scanning confocal microscopy images of HCT116 and HEK293 cells incubated with MM, MMH and PMMH nanoparticles. All images were taken under the identical instrumental conditions and presented at the same intensity scale.

To evaluate the targeting specificity of the PMMH nanoparticles to lymph system, cellular uptake efficiency of the PMMH nanoparticles was observed by confocal laser scanning microscopy. Based on previous studies [16, 30-32], receptor-mediated endocytosis, especially an interaction between HA and the CD44 receptor, was identified as the principal cellular uptake mechanism of HA-based nanoparticles. Furthermore, the amino acid sequence in the extracellular domain of lymphatic endothelial hyaluronan receptor-1 (LYVE-1), a lymphatic endothelial-specific marker in lymph system, and CD44 has 43% homology as homologues [33]. Therefore, some cells with wide expression of CD44 were always used to simulate the interaction of lymph system and HA-based nanoparticles. Thus, in present experiment, HCT116 cells were used as high CD44 receptor-expressing cells, while HEK293 cells were regarded as control groups because of the low expression of endogenous ligand receptor [34], and FITC (fluorescein isothiocyanate), an near-infrared fluorescence dye, was used for the detection of cellular uptake and distribution of the nanoparticles. As shown in Fig. 3, HCT116 and HEK293 cells with blue fluorescence were dyed with DAPI and green FITC-labeled nanoparticles. In HCT116 cells group, HA-based MMH nanoparticles embrace the cells

largely and distribute around them tightly (Fig. 3f), which is different to the result of MM nanoparticles (Fig. 3c), a general cellular uptake to nanoparticle. The result shows MMH has a strong affinity of HCT116 cells. Furthermore, the addition of PFOB in MMH nanoparticles has few influences on targeting specificity (Fig. 3i). However, In HEK293 cells group, all of MM, MMH and PMMH nanoparticles reveal poor ability of phagocytosis on cells (Fig. 3c’, 3f’ and 3i’). The above results imply that the CD44 receptor overexpressed lymph target ability of the developed HA-based nanoparticles is obtained, which is potential in high lymph targeting efficiency. The MR and ultrasound imaging of the PMMH

Figure 4 (a) T1-weighted maps of various Mn concentrations in vitro, (b) the linear relationship between relaxation rates (1/T1) and Mn concentrations and (c, d) ultrasound images in vitro of (c) pure H2O and (d) PMMH nanoparticles in a dialysis bag.

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nanoparticles were further investigated to evaluate their multifunctional diagnose. MR imaging signals of the PMMH nanoparticles were measured in vitro. Different mass concentrations of the PMMH nanoparticles in the centrifuge tubes, as well as pure water for the background signal, were measured for their T1 relaxation time by a 3T MR imaging scanner. T1-weighted maps in Fig. 4a show that the T1-weighted MR imaging signal intensity is continuously enhanced, resulting in brighter images with increasing the mass. Furthermore, as shown in Fig. 4b, the longitudinal proton relaxation rate as a function of PMMH nanoparticles concentration led to an r1 relaxivity of 13.6 mM-1s-1, which was much larger than that of Magnevist (r1=4.5 mM-1s-1) [35]. Additionally, ultrasound imaging experiments in vitro were performed. The results are shown in Fig.4c and 4d, which shows the ultrasound images of dialysis bag (labeled by the blue frame) with H2O and PMMH nanoparticles in cross-section. As shown in Fig. 4c, the weak signal is equal to that of H2O medium outside of the dialysis bag. However, PMMH nanoparticles show relatively strong ultrasound contrast to H2O medium outside of the dialysis bag because of large differences in acoustic impedances between perfluorocarbons and water (Fig. 4d). On the contrary, Fig. S5 shows the phenomenon of MMH nanoparticles, which is slightly different. The results reveal that PMMH nanoparticles are potential for biomedical engineering in molecular imaging including MR and ultrasound imaging.

Lymph ultrasound administration of the dual-mode contrast agent was performed in vivo in rabbit (Fig. 5). An attempt was made to locate lymph nodes in the knee popliteal fossa regions using primary gray scale sonography before PMMH nanoparticles being injected, which was helpful to observe the change of the lymph nodes after PMMH nanoparticles administration precisely. After the PMMH nanoparticles infusion, the increase of lymph nodes signal intensity is observed gradually (labeled by circle). When the enhancement time is 15 min (Fig. 5c), the lymph node is imaged clearly. With increasing the time to 35 min, the lymph node is brightest (Fig. 5e). Afterward, the signal decreased significantly at 55 and 65 min (Fig. 5g and 5h). Furthermore, closer observation reveals that lymph vessels around lymph nodes are also imaged (labeled by arrow). With the increase of enhancement time, more and more lymph vessels become clear gradually. Maximal signals for the PMMH nanoparticles are reached at 35 min, followed by decreased levels. The above results show that the PMMH nanoparticles can be returned to lymph nodes within 15 min through lymph vessels after hypodermic injection and retained on them at least 30 min (from 15 to 45 min). Therefore, the results reveal that the PMMH nanoparticles are potential in ultrasound imaging in vivo with a slower metabolism, which makes for longtime-carefully diagnosis without human body damage.

Figure 5 Ultrasound images in vivo of PMMH nanoparticles injection in rabbit knee popliteal fossa. Scanning time after hypodermic injection: (a) 0 min, (b) 5 min, (c) 15 min, (d) 25 min, (e) 35 min, (f) 45 min, (g) 55 min and (h) 65 min.

Furthermore, the MR imaging ability of the dual-mode contrast agent was evaluated. Based on

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the results of the ultrasound imaging, the injection site was chosen precisely and the MR images were collected from 15 to 45 min. As shown in Fig. 6a, the left lymph node (labeled by yellow arrow), imaged by commercial Gd-DTPA, appears obviously within 15 min whereas the right lymph node (labeled by red arrow), imaged by the PMMH nanoparticles, seems still blurry. Closer observation reveals that some vessels around lymph node in right leg can also be found (labeled by white arrow). With the increasing of enhancement time, the signal intensity of the left lymph node develops weaker but that of the right one is stronger (Fig. 6b and 6c). Additionally, those vessels are imaged gradually. At the enhancement time of 45 min, the lymph node of left leg is nearly not visible, which suggests that Gd-DPTA has begun to metabolize, but that of right one is still clear, which is similar to the result of ultrasound imaging. The above results illustrate that the enhancement caused by the PMMH nanoparticles is higher than that of the commercial Gd-DPTA. Thus, the dual-mode contrast agent is good at both ultrasound and MR imaging in vivo.

Figure 6 T1-weighted MR images in vivo of commercial Gd-DTPA (left leg) and PMMH nanoparticles (right leg) injection in rabbit knee popliteal fossa. Scanning time after hypodermic injection: (a) 15 min, (b) 25 min, (c) 35 min and (d) 45min.

Figure 7 Immunohistochemical characterization of lymphatic markers.

Immunofluorescence analysis of ultrasound or MR imaging tissue at 35 min post injection of the PMMH nanoparticles was performed to prove the ability of the PMMH nanoparticles penetrating into lymph system. As shown in Fig. 7, the yellow cells, colocalized with the anti-human LYVE-1 polyclonal primary antibodies, reveals that the tissue expresses LYVE-1 abundantly. Furthermore, the fluorescence image also indicates that some zone encircled by yellow cells should be lymph vessels or lymph capillaries (labeled by white arrows). The results suggest that the imaging tissue express obvious lymph characterization, where the PMMH nanoparticles could penetrate through so as to enhance their imaging effect. Thus, the dual-mode contrast agent is proved to have great lymph system-targeted ability in vivo.

The cytotoxicity was also investigated to examine the feasibility of the obtained PMMH nanoparticles for biomedical application. As shown in Fig. 8, the effect of different PMMH nanoparticles on cells

Figure 8 The CCK-8 assay results of the obtained PMMH nanoparticles.

Nano Res.

Figure 9 (a) SEM and (b) TEM of DMMH nanoparticles, (c) N2 adsorption/desorption isotherm (1 - MMH nanoparticles and 2 - DMMH nanoparticles)) and (d) DOX release curve from DMMH nanoparticles. proliferation was assessed with HCT116 cells by CCK-8 assay. Using the viability of untreated cells as control, the cellular viabilities decrease with an increasing concentrations of PMMH nanoparticles, and about 80% cell viabilities are maintained even up to a relatively high dose of 125 mg/mL after exposure for 12 h. In other words, upon incubating with the PMMH nanoparticles of different concentrations, less than 20% of cells died after 4, 8 and even 12 h. Therefore, the results demonstrate that the obtained nanoparticles have a low cytotoxicity.

Besides the preparation of the PMMH nanoparticles for dual-mode imaging of lymph system, the platform also could be loaded by hydrophilic DOX to obtain DMMH nanoparticles for combining diagnose and therapy with MR imaging and sustained release for lymph tumors. As shown in Fig. 9a, SEM image reveals that the DMMH nanoparticles are coated probably by organic nanoparticles compounds, with the average hydrodynamic size of 210 nm (Fig. S7). Compared to TEM image of MMH nanoparticles (Fig. S6), the surface of DMMH nanoparticles becomes blurrier (Fig. 9b). The N2 adsorption/desorption isotherm curve further confirms that the surface area is 284 m2/g ((Fig. 9c) and the average pore size of DMMH nanoparticles is 2.6 nm (Fig. S8), which implies the more DOX absorption in MMH nanoparticles than that of PFOB. Furthermore, TEM image proves the above speculation. Furthermore, the DOX loading in MMH nanoparticles is determined to be as high as 43.7 mg/g. The loading amount is much lower than

that of reported manganese oxide/MSNs composite (382 mg/g), but is similar to that of ZnO quantum dots modified mesoporous silica (40 mg/g) [36]. The difference could be that the mesoporous channels are pre-filled by HA molecules, which increases the difficulty of DOX absorption. As we seen, the loading content is much higher than that of PFOB in MMH nanoparticles, whose reason could come from the driving force among the hydrophilic and hydrophobic molecules penetrating into our mesoporous silica. As just discussed in Figure 2, the driving force of the hydrophobic PFOB molecule in MMH nanoparticles is the possible hydrophobic interaction between PFOB and HA molecule around HA molecule in mesoporous pores. However, similar to in previous report, the driving force of the hydrophilic DOX molecule in MMH nanoparticles is molecular interactions between DOX and HA molecule around HA molecule inside and/or outside mesoporous pores, such as hydrogen bonds, Van der Wals forces, and so on [37, 38]. The results are according to the decrease of the specific surface area, from 486 m2/g of MMH nanoparticles to 374 m2/g of PMMH nanoparticles and to 284 m2/g of DMMH nanoparticles (Fig.9c). Furthermore, we investigated the drug loading and release behavior of the DMMH nanoparticles. The UV-vis absorption spectra of DOX at 0.1 mg/mL concentration of the DMMH nanoparticles are shown in Fig. S9. One can see that the absorption spectra of DOX in the DMMH shows characteristic absorption peaks at 490 nm, which are similar to those reported in the literature [10, 39-42]. Fig. 9d further shows the release behavior of DOX from the DMMH nanoparticles in PBS (pH = 5.5), owing to the acid environment where tumors stay [56-58]. The final release is observed to be 31.8% at 80 h (Fig. 9d) and 49.1% at 10th day (Fig. S10), which demonstrates that the DOX inside channels of DMMH could be released out slowly. One can see that the drug release curve shows a sustained release behavior and the drug is progressively released by desorption and diffusion to the phosphate buffer saline solution. The result indicates that the DMMH nanoparticles have a favorable DOX release property.

To examine the feasibility of the obtained DMMH nanoparticles for in vitro growth inhibitory on tumor cells, their cell cytotoxicity and uptake on HCT116 cells were investigated. As shown in Fig.

Nano Res.

10a, growth

Figure 10 (a) Inhibitory effect of DMMH nanoparticles and free DOX on HCT116 cells as examined by CCK-8 assay and (b) Laser scanning confocal microscopy images of HCT116 cells incubated with DMMH nanoparticles. inhibition of cells is observed after incubation with free DOX and DMMH nanoparticles. The results indicate the dose-dependent cytotoxicity behavior, which reveals that the DMMH nanoparticles show remarkably higher anticancer efficiency than that of free DOX. Furthermore, the IC50 values are 7.9 mg/mL DOX in the DMMH nanoparticles and 10.6 mg/mL free DOX, also indicating that DMMH nanoparticles increased the in vitro growth inhibitory effect. The enhancement of anticancer efficiency could result from the high combining ability of HA and CD44 possibly enhancing their antitumor effect. Because of the excellent MR imaging in vitro and in vivo and targeting specificity of the universal platform, we further explored the potential to use the DMMH nanoparticles for diagnosis and treatment of a xenografted tumor model. As shown in photo picture

of Fig. 11a, the tumor-bearing nude mice with about 123.5 mm3 tumor volume were intratumorally injected with 0.2 mL, 0.10 mg/mL DMMH nanoparticles. Before and after 5, 10 min

post injection, the nude mice were imaged by MR (Fig. 11a). Compared to before injection, we can clearly see that the single of tumor region increases obviously in a typical T1-weighted MR image at 5 and 10 min after injection. Closer observation reveals that some subtle structures in tumor region are also found. The results suggest that the intratumoral injection of the DMMH nanoparticles leads to a quite uniform distribution of the particles within the tumor region, allowing for effective MR imaging of the whole tumor, which shows that the obtained DMMH nanoparticles have a great potential to be used as a contrast agent for in vivo tumor MR imaging.

To investigate therapeutic efficacy of the DMMH nanoparticles in vivo, comparative studies of inhibiting tumor effectiveness have been conducted. Eighteen tumor-bearing nude mice were randomly distributed into three groups, i.e., PBS, free DOX and DMMH nanoparticles group with the same dosage of free DOX. After two weeks feeding, the three groups of nude mice were intratumorally injected with DMMH nanoparticles (0.2 mL, 0.1 mg/mL), free DOX and PBS (0.2 mL), respectively. No mice died and their volume was measured during the course of therapy. As shown in Fig. 11b, the mean tumor volume of DMMH nanoparticles group increases most rapidly among the three groups, which shows that the DMMH nanoparticles have considerable growth inhibitory effect on tumors. Furthermore, on the twenty-third day, mice were sacrificed and tumors were excised and weighed. The tumor photograph and mean tumor weights in each group

Figure 11 (a) Photograph of the tumor-bearing nude mice for MR imaging and MR images at different scanning time, (b) Mean tumor volume of the mice in different groups after treatment, (c) Photograph of the tumors from different groups after treatment and (d) Mean body weights of the mice in different groups after treatment. Data represent the mean ± standard deviation of seven mice. P < 0.05 was considered to be statistically significant difference and shown by asterisks.

Nano Res.

Figure 12 Mean Mn concentrations (mg/kg) per gram organ over time after one single injection (a) and consecutive injection (b), error bars represent standard deviations. after the treatment are shown in Fig. 11c and 11d. The DMMH nanoparticles group also shows enhanced inhibition activity than PBS and free DOX groups. The mean tumor weight in the DMMH nanoparticles group (0.618 ± 0.137 g) is smaller than that of PBS group (1.270 ± 0.109 g) and free DOX group (0.978 ± 0.098 g, P<0.05). The results indicate that treatment with the DMMH nanoparticles showed significantly enhanced antitumor activity for a longtime, which attributes to DOX chemotherapy releasing from DMMH nanoparticles [10, 40, 41, 43, 44]. The above results reveal that the DMMH nanoparticles are a powerful agent for combined diagnosis and therapy of cancer in vivo.

The short-term and long-term distribution of the universe platform (MMH nanoparticles) in mice was performed to evaluate their safety. As shown in Fig. 12a of the single injection of MMH nanoparticles, Mn could be detected in any organ besides blood although they are not detected in any organ in control group, in which the maximum concentration is in liver. Furthermore, the concentration-time curves reveal a rapid decline in Mn concentration during 12 h after injection. At the time of 12 h, high Mn concentration is still measured in liver, spleen and kidneys, and other organ besides blood is in lower levels. The results show that MMH nanoparticles could be distributed mainly to liver followed by spleen and kidneys. Furthermore, repeated administration resulted in accumulation in liver, spleen and kidneys, indicating that these organs may be potential target organs for the excretion of MMH nanoparticles [45]. The above results indicate that the MMH nanoparticles could distribute in most of internal organs through blood flow and finally metabolized through liver, spleen and kidneys.

4 Conclusions

In summary, a new universal platform of MM nanoparticles with the MR imaging is designed through doping Mn2+ into the framework of mesoporous silica. HA-modified MM nanoparticles can be used for in vitro and in vivo targeted lymph system, to produce MMH nanoparticles. Then two typical compounds, hydrophobic PFOB and hydrophilic DOX, are loaded into channels to obtain PMMH or DMMH nanoparticles for dual-mode imaging, and imaging and therapy, respectively. It has been demonstrated that the PMMH and DMMH nanoparticles are highly targeted to lymph system in vitro and in vivo, and the MR/ultrasound imaging of PMMH nanoparticles in lymph system, and MR imaging and therapeutic ability of DMMH nanoparticles in cancer is highlighted. Furthermore, they are distributed and excreted mainly to liver followed by spleen and kidneys. The Mn@mSiO2-based nanoparticles developed herein are promising for many applications in biomedicine, including multimodality imaging, cell tracking, and cancer therapies. Our work shows a method about how to put the new universal platform to further exploitations, which has great potential in biological area.

Acknowledgements

The work is supported by Shanghai Rising-Star Program (13QB1402200), Shanghai Minhang district talent development special fund, National Key Technology Research and Development Program (No: 2014BAK05B02) and National Natural Science Foundation of China (No: 81271638 and 81371622).

References

[1] Jin, Y. D.; Gao, X. H. Plasmonic fluorescent

quantum dots. Nat Nanotechnol 2009, 4, 571-576.

[2] Hu, S. H.; Gao, X. H. Nanocomposites with

Spatially Separated Functionalities for Combined

Imaging and Magnetolytic Therapy. J. Am. Chem.

Soc. 2010, 132, 7234-7237.

[3] Fan, W. P.; Shen, B.; Bu, W. B.; Chen, F.; Zhao, K.

L.; Zhang, S. J.; Zhou, L. P.; Peng, W. J.; Xiao, Q.

F.; Xing, H. Y.; Liu, J. N.; Ni, D. L.; He, Q. J.; Shi,

J. L. Rattle-Structured Multifunctional

Nanotheranostics for Synergetic

Nano Res.

Chemo-/Radiotherapy and Simultaneous

Magnetic/Luminescent Dual-Mode Imaging. J. Am.

Chem. Soc. 2013, 135, 6494-6503.

[4]Passuello, T.; Pedroni, M.; Piccinelli, F.; Polizzi, S.;

Marzola, P.; Tambalo, S.; Conti, G.; Benati, D.;

Vetrone, F.; Bettinelli, M.; Speghini, A.

PEG-capped, lanthanide doped GdF3 nanoparticles:

luminescent and T-2 contrast agents for optical and

MRI multimodal imaging. Nanoscale 2012, 4,

7682-7689.

[5] Pellegatti, L.; Zhang, J.; Drahos, B.; Villette, S.;

Suzenet, F.; Guillaumet, G.; Petoud, S.; Toth, E.

Pyridine-based lanthanide complexes: towards

bimodal agents operating as near infrared

luminescent and MRI reporters. Chem. Commun.

2008, Doi 10.1039/B817343e, 6591-6593.

[6] Nishioka, T.; Shiga, T.; Shirato, H.; Tsukamoto, E.;

Tsuchiya, K.; Kato, T.; Ohmori, K.; Yamazaki, A.;

Aoyama, H.; Hashimoto, S.; Chang, T. C.;

Miyasaka, K. Image fusion between (18)FDG-PET

and MRI/CT for radiotherapy planning of

oropharyngeal and nasopharyngeal carcinomas. Int

J Radiat Oncol 2002, 53, 1051-1057.

[7] Wen, S. H.; Li, K. G.; Cai, H. D.; Chen, Q.; Shen,

M. W.; Huang, Y. P.; Peng, C.; Hou, W. X.; Zhu, M.

F.; Zhang, G. X.; Shi, X. Y. Multifunctional

dendrimer-entrapped gold nanoparticles for dual

mode CT/MR imaging applications. Biomaterials

2013, 34, 1570-1580.

[8] Kubota, K.; Yokoyama, J.; Yamaguchi, K.; Ono, S.;

Qureshy, A.; Itoh, M.; Fukuda, H. FDG-PET

delayed imaging for the detection of head and neck

cancer recurrence after radio-chemotherapy:

comparison with MRI/CT. Eur. J. Nucl. Med. Mol.

Imag. 2004, 31, 590-595.

[9] Cheng, S. H.; Lee, C. H.; Chen, M. C.; Souris, J. S.;

Tseng, F. G.; Yang, C. S.; Mou, C. Y.; Chen, C. T.;

Lo, L. W. Tri-functionalization of mesoporous

silica nanoparticles for comprehensive cancer

theranostics-the trio of imaging, targeting and

therapy. J. Mater. Chem. 2010, 20, 6149-6157.

[10] Cho, H. J.; Yoon, H. Y.; Koo, H.; Ko, S. H.; Shim,

J. S.; Cho, J. H.; Park, J. H.; Kim, K.; Kwon, I.

C.; Kim, D. D. Hyaluronic acid-ceramide-based

optical/MR dual imaging nanoprobe for cancer

diagnosis. J. Controlled Release 2012, 162,

111-118.

[11] Yang, K.; Hu, L. L.; Ma, X. X.; Ye, S. Q.; Cheng,

L.; Shi, X. Z.; Li, C. H.; Li, Y. G.; Liu, Z.

Multimodal Imaging Guided Photothermal

Therapy using Functionalized Graphene

Nanosheets Anchored with Magnetic

Nanoparticles. Adv. Mater. 2012, 24, 1868-1872.

[12] Zha, Z. B.; Wang, J. R.; Zhang, S. H.; Wang, S.

M.; Qu, E.; Zhang, Y. Y.; Dai, Z. F. Engineering

of perfluorooctylbromide polypyrrole

nano-/microcapsules for simultaneous contrast

enhanced ultrasound imaging and photothermal

treatment of cancer. Biomaterials 2014, 35,

287-293.

[13] Bhang, S. H.; Won, N.; Lee, T. J.; Jin, H.; Nam, J.;

Park, J.; Chung, H.; Park, H. S.; Sung, Y. E.;

Hahn, S. K.; Kim, B. S.; Kim, S. Hyaluronic

Acid-Quantum Dot Conjugates for In Vivo

Lymphatic Vessel Imaging. Acs Nano 2009, 3,

1389-1398.

[14] Jaggupilli, A.; Elkord, E. Significance of CD44

and CD24 as Cancer Stem Cell Markers: An

Enduring Ambiguity. Clinical & Developmental

Immunology 2012, Doi 10.1155/2012/708036.

[15] Kim, J. H.; Glant, T. T.; Lesley, J.; Hyman, R.;

Mikecz, K. Adhesion of lymphoid cells to

CD44-specific substrata: The consequences of

attachment depend on the ligand. Exp. Cell Res.

2000, 256, 445-453.

[16] Banerji, S.; Ni, J.; Wang, S. X.; Clasper, S.; Su, J.;

Tammi, R.; Jones, M.; Jackson, D. G. LYVE-1, a

new homologue of the CD44 glycoprotein, is a

lymph-specific receptor for hyaluronan. J. Cell

Biol. 1999, 144, 789-801.

[17] Jackson, D. G.; Prevo, R.; Clasper, S.; Banerji, S.

LYVE-1, the lymphatic system and tumor

lymphangiogenesis. Trends Immunol. 2001, 22,

317-321.

[18] Mizrahy, S.; Raz, S. R.; Hasgaard, M.; Liu, H.;

Soffer-Tsur, N.; Cohen, K.; Dvash, R.;

Landsman-Milo, D.; Bremer, M. G. E. G.;

Moghimi, S. M.; Peer, D. Hyaluronan-coated

Nano Res.

nanoparticles: The influence of the molecular

weight on CD44-hyaluronan interactions and on

the immune response. J. Controlled Release 2011,

156, 231-238.

[19] Wu, G. Y.; Zhang, H. J.; Zhan, Z. F.; Lu, Q.;

Cheng, J. J.; Xu, J. R.; Zhu, J. Hyaluronic

Acid-Gadolinium Complex Nanospheres as

Lymphatic System-Specific Contrast Agent for

Magnetic Resonance Imaging. Chinese J. Chem.

2015, Doi: 10.1002/cjoc.201500135.

[20] Ma, M.; Xu, H. X.; Chen, H. R.; Jia, X. Q.; Zhang,

K.; Wang, Q.; Zheng, S. G.; Wu, R.; Yao, M. H.;

Cai, X. J.; Li, F. Q.; Shi, J. L. A

Drug-Perfluorocarbon Nanoemulsion with an

Ultrathin Silica Coating for the Synergistic Effect

of Chemotherapy and Ablation by High-Intensity

Focused Ultrasound. Adv. Mater. 2014, 26,

7378-7385.

[21] Choi, K. Y.; Yoon, H. Y.; Kim, J. H.; Bae, S. M.;

Park, R. W.; Kang, Y. M.; Kim, I. S.; Kwon, I. C.;

Choi, K.; Jeong, S. Y.; Kim, K.; Park, J. H. Smart

Nanocarrier Based on PEGylated Hyaluronic

Acid for Cancer Therapy. Acs Nano 2011, 5,

8591-8599.

[22] Choi, K. Y.; Min, K. H.; Yoon, H. Y.; Kim, K.;

Park, J. H.; Kwon, I. C.; Choi, K.; Jeong, S. Y.

PEGylation of hyaluronic acid nanoparticles

improves tumor targetability in vivo.

Biomaterials 2011, 32, 1880-1889.

[23] Park, S. J.; Park, W.; Na, K. Photo-activatable

ternary complex based on a multifunctional

shielding material for targeted shRNA delivery in

cancer treatment. Biomaterials 2013, 34,

8991-8999.

[24] He, Q. J.; Shi, J. L. Mesoporous silica

nanoparticle based nano drug delivery systems:

synthesis, controlled drug release and delivery,

pharmacokinetics and biocompatibility. J. Mater.

Chem. 2011, 21, 5845-5855.

[25] Sim, L. N.; Majid, S. R.; Arof, A. K. FTIR studies

of PEMA/PVdF-HFP blend polymer electrolyte

system incorporated with LiCF3SO3 salt. Vib.

Spectrosc 2012, 58, 57-66.

[26] Sim, L. N.; Majid, S. R.; Arof, A. K. Effects of

1-butyl-3-methyl imidazolium

trifluoromethanesulfonate ionic liquid in

poly(ethyl

methacrylate)/poly(vinylidenefluoride-co-hexaflu

oropropylene) blend based polymer electrolyte

system. Electrochim. Acta 2014, 123, 190-197.

[27] Guillet-Nicolas, R.; Laprise-Pelletier, M.; Nair, M.

M.; Chevallier, P.; Lagueux, J.; Gossuin, Y.;

Laurent, S.; Kleitz, F.; Fortin, M. A.

Manganese-impregnated mesoporous silica

nanoparticles for signal enhancement in MRI cell

labelling studies. Nanoscale 2013, 5,

11499-11511.

[28] Bejar, A.; Ben Chaabene, S.; Jaber, M.; Lambert,

J. F.; Bergaoui, L. Mn-analcime: Synthesis,

characterization and application to cyclohexene

oxidation. Microporous Mesoporous Mater. 2014,

196, 158-164.

[29] Liu, Y.; Shen, J. M.; Chen, Z. L.; Liu, Y.

Degradation of p-chloronitrobenzene in drinking

water by manganese silicate catalyzed ozonation.

Desalination 2011, 279, 219-224.

[30] Pall, T.; Pink, A.; Kasak, L.; Turkina, M.;

Anderson, W.; Valkna, A.; Kogerman, P. Soluble

CD44 Interacts with Intermediate Filament

Protein Vimentin on Endothelial Cell Surface.

Plos One 2011. Doi:

10.1371/journal.pone.0029305

[31] Jones, M.; Tussey, L.; Athanasou, N.; Jackson, D.

G. Heparan sulfate proteoglycan isoforms of the

CD44 hyaluronan receptor induced in human

inflammatory macrophages can function as

paracrine regulators of fibroblast growth factor

action. J. Biol. Chem. 2000, 275, 7964-7974.

[32] Appaturi, J. N.; Adam, F. A facile and efficient

synthesis of styrene carbonate via cycloaddition

of CO2 to styrene oxide over ordered mesoporous

MCM-41-Imi/Br catalyst. Appl Catal B-Environ

2013, 136, 150-159.

[33] Banerji, S.; Hide, B. R. S.; James, J. R.; Noble, M.

E. M.; Jackson, D. G. Distinctive Properties of

the Hyaluronan-binding Domain in the

Lymphatic Endothelial Receptor Lyve-1 and

Their Implications for Receptor Function. J. Biol.

Nano Res.

Chem. 2010, 285, 10724-10735.

[34] Faul, C.; Donnelly, M.; Merscher-Gomez, S.;

Chang, Y. H.; Franz, S.; Delfgaauw, J.; Chang, J.

M.; Choi, H. Y.; Campbell, K. N.; Kim, K.;Reiser,

J.; Mundel, P. The actin cytoskeleton of kidney

podocytes is a direct target of the antiproteinuric

effect of cyclosporine A. Nat. Med. 2008, 14,

931-938.

[35] Stanisz, G. J.; Henkelman, R. M. Gd-DTPA

relaxivity depends on macromolecular content.

Magn. Reson. Med. 2000, 44, 665-667.

[36] Muharnmad, F.; Guo, M. Y.; Qi, W. X.; Sun, F. X.;

Wang, A. F.; Guo, Y. J.; Zhu, G. S. pH-Triggered

Controlled Drug Release from Mesoporous Silica

Nanoparticles via Intracelluar Dissolution of ZnO

Nanolids. J. Am. Chem. Soc. 2011, 133,

8778-8781.

[37] Zhao, W. W.; Cui, B.; Peng, H. X.; Qiu, H. J.;

Wang, Y. Y. Novel Method To Investigate the

Interaction Force between Etoposide and

APTES-Functionalized Fe3O4@nSiO(2)@mSiO(2)

Nanocarrier for Drug Loading and Release

Processes. J Phys Chem C 2015, 119, 4379-4386.

[38] Mathew, A.; Parambadath, S.; Park, S. S.; Ha, C.

S. Hydrophobically modified spherical MCM-41

as nanovalve system for controlled drug delivery.

Microporous Mesoporous Mater. 2014, 200,

124-131.

[39] de la Torre, C.; Casanova, I.; Acosta, G.; Coll, C.;

Moreno, M. J.; Albericio, F.; Aznar, E.; Mangues,

R.; Royo, M.; Sancenon, F.; Martinez-Manez, R.

Gated Mesoporous Silica Nanoparticles Using a

Double-Role Circular Peptide for the Controlled

and Target-Preferential Release of Doxorubicin

in CXCR4-Expresing Lymphoma Cells. Adv.

Funct. Mater. 2015, 25, 687-695.

[40] Niu, C. C.; Wang, Z. G.; Lu, G. M.; Krupka, T. M.;

Sun, Y.; You, Y. F.; Song, W. X.; Ran, H. T.; Li, P.;

Zheng, Y. Y. Doxorubicin loaded

superparamagnetic PLGA-iron oxide

multifunctional microbubbles for dual-mode

US/MR imaging and therapy of metastasis in

lymph nodes. Biomaterials 2013, 34, 2307-2317.

[41] Yang, X. Y.; Wang, Y. S.; Huang, X.; Ma, Y. F.;

Huang, Y.; Yang, R. C.; Duan, H. Q.; Chen, Y. S.

Multi-functionalized graphene oxide based

anticancer drug-carrier with dual-targeting

function and pH-sensitivity. J. Mater. Chem.

2011, 21, 3448-3454.

[42] Chen, Z. W.; Li, Z. H.; Lin, Y. H.; Yin, M. L.; Ren,

J. S.; Qu, X. G. Biomineralization inspired

surface engineering of nanocarriers for

pH-responsive, targeted drug delivery.

Biomaterials 2013, 34, 1364-1371.

[43] Sun, J. S.; Xianyu, Y. L.; Li, M. M.; Liu, W. W.;

Zhang, L.; Liu, D. B.; Liu, C.; Hu, G. Q.; Jiang,

X. Y. A microfluidic origami chip for synthesis of

functionalized polymeric nanoparticles.

Nanoscale 2013, 5, 5262-5265.

[44] Yu, L. L.; Bi, H. Facile synthesis and magnetic

property of iron oxide/MCM-41 mesoporous

silica nanospheres for targeted drug delivery. J.

Appl. Phys. 2012, Doi: 10.1063/1.3676203.

[45] Lankveld, D. P. K.; Oomen, A. G.; Krystek, P.;

Neigh, A.; Troost-de Jong, A.; Noorlander, C. W.;

Van Eijkeren, J. C. H.; Geertsma, R. E.; De Jong,

W. H. The kinetics of the tissue distribution of

silver nanoparticles of different sizes.

Biomaterials 2010, 31, 8350-8361.

Electronic Supplementary Material

17

A Multifunctional Lymph-Targeted Platform Based on Mn@mSiO2 Nanocomposites: Combining PFOB for Dual-Mode Imaging and DOX for Cancer Diagnose and Treatment

Tian Liu1,2, §, Guangyu Wu3, §, Jiejun Cheng3, Qing Lu3, Yanjie Yao1,2, Zhenjing Liu1, Dongchen Zhu1, Juan Zhou2, Jian-Rong Xu3 (*), Jun Zhu2 (*), and Dannong He1 (*) 1 Country School of Materials Science and Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P. R. China. 2 National Engineering Research Center for Nanotechnology, 28 East Jiang Chuan Road, Shanghai 200241, P. R. China. 3 Department of Radiology, Shanghai Renji Hospital, Shanghai Jiao Tong University School of Medicine, 1630 Dong Fang Rd, Shanghai 200127, P. R. China. § These authors contributed equally to this work.

18

Figure S1 N2 adsorption/desorption isotherm of MM nanoparticles at different molar Mn ions: (a) 0.1 mmol, (b) 0.5 mmol, (c) 1 mmol and (d) 5 mmol.

As shown in Fig. S1, the surface area of MM nanoparticles is decreased with the addition of Mn ions, which could result from the destroying of the channel frameworks by doping Mn ions. However, when 5 mmol Mn ions were doped, the surface area is still 503 m2/g, which could satisfy the further experiments and MR imaging.

19

Figure S2 DLS data of obtained PMMH nanoparticles.

As shown in Fig. S2, the average hydrodynamic size of the PMMH nanoparticles in deionized water is about 190 nm.

Figure S3 Pore size distribution curve of PMMH nanoparticles.

As shown in Fig.S3, the typical pore size distribution curve of further confirms that the average pore size of PMMH nanoparticles is 3.0 nm.

20

Figure S4 N2 adsorption/desorption isotherm of (a) MM (5 mmol Mn), (b) MMH and (c) PMMH nanoparticles.

As shown in Fig. S4, the surface areas of MM, MMH and PMMH nanoparticles are 503 m2/g, 486 m2/g and 374 m2/g, which implies that the decrease of the surface area could result from the HA modification and PFOB absorption.

Figure S5 Ultrasound image in vitro of MMH nanoparticles in a dialysis bag.

As shown in Fig. S5, the ultrasound signal of MMH nanoparticles is higher than that of H2O medium outside of the dialysis bag, which could be due to some bubbles absorbed in mesoporous pores. However, the signal of HA molecule is lower than that of MMH nanoparticles and higher than H2O medium.

21

Figure S6 TEM image of MMH nanoparticles.

Figure S7 DLS data of obtained DMMH nanoparticles.

As shown in Fig. S7, the average hydrodynamic size of the DMMH nanoparticles in deionized water is about 210 nm.

22

Figure S8 Pore size distribution curve of DMMH nanoparticles. As shown in Fig. S8, the typical pore size distribution curve of further confirms that the average pore size of DMMH nanoparticles is 2.6 nm.

Figure S9 UV-vis absorption spectra of DMMH nanoparticles

Figure S10 DOX release for 10 days curve from DMMH nanoparticles.

23

As shown in Fig. S10, there is 49.1% DOX released from DMMH, which demonstrates that the DOX inside channels of DMMH could be released out slowly.

Address correspondence to First A. Dannong He, hdn_nercn@163.com; Second B. Jun Zhu, yzjzhu@163.com; Third C. Jianrong Xu,renjixujr@163.com

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Silver Nanowires with Semiconducting Ligands for Low Temperature Transparent Conductors

Brion Bob,1 Ariella Machness,1 Tze-Bin Song,1 Huanping Zhou,1 Choong-Heui Chung,2 and Yang Yang1,*

1 Department of Materials Science and Engineering and California NanoSystems Institute,

University of California Los Angeles, Los Angeles, CA 90025 (USA)

2 Department of Materials Science and Engineering, Hanbat National University, Daejeon

305-719, Korea

Abstract

Metal nanowire networks represent a promising candidate for the rapid fabrication of transparent electrodes with high transmission and low sheet resistance values at very low deposition temperatures. A commonly encountered obstacle in the formation of conductive nanowire electrodes is establishing high quality electronic contact between nanowires in order to facilitate long range current transport through the network. A new system of nanowire ligand removal and replacement with a semiconducting sol-gel tin oxide matrix has enabled the fabrication of high performance transparent electrodes at dramatically reduced temperatures with minimal need for post-deposition treatments of any kind.

Keywords: Silver Nanowires, Sol-Gel, Transparent Electrodes, Nanocomposites

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1. Introduction. Silver nanowires (AgNWs) are long, thin, and possess conductivity values on the same order of magnitude as bulk silver

(Ag) [1]. Networks of overlapping nanowires allow light to easily pass through the many gaps and spaces between nanowires, while transporting current through the metallic conduction pathways offered by the wires themselves. The high aspect ratios achievable for solution-grown AgNWs has allowed for the fabrication of transparent conductors with very promising sheet resistance and transmission values, often approaching or even surpassing the performance of vacuum-processed materials such as indium tin oxide (ITO) [2-6].

Significant electrical resistance within the metallic nanowire network is encountered only when current is required to pass between nanowires, often forcing it to pass through layers of stabilizing ligands and insulating materials that are typically used to assist with the synthesis and suspension of the nanowires [7, 8]. The resistance introduced by the insulating junctions between nanowires can be reduced through various physical and chemical means, including burning off ligands and partially melting the wires via thermal annealing [9, 10], depositing additional materials on top of the nanowire network [11-14], applying mechanical forces to enhance network morphology [15-17], or using various other post-treatments to improve the contact between adjacent wires [18-21]. Any attempt to remove insulating materials the network must be weighed against the risk of damaging the wires or blocking transmitted light, and so many such treatments must be reined in from their full effectiveness to avoid endangering the performance of the completed electrode.

We report here a process for forming inks with dramatically enhanced electrical contact between AgNWs through the use of a semiconducting ligand system consisting of tin oxide (SnO2) nanoparticles. The polyvinylpyrrolidone (PVP) ligands introduced during AgNW synthesis in order to encourage one-dimensional growth are stripped from the wire surface using ammonium ions, and are replaced with substantially more conductive SnO2, which then fills the space between wires and enhances the contact geometry in the vicinity of wire/wire junctions. The resulting transparent electrodes are highly conductive immediately upon drying, and can be effectively processed in air at virtually any temperature below 300 °C. The capacity for producing high performance transparent electrodes at room temperature may be useful in the fabrication of devices that are damaged upon significant heating or upon the application of harsh chemical or mechanical post-treatments.

2. Results and Discussion

2.1. Ink Formulation and Characterization

Dispersed AgNWs synthesized using copper chloride seeds represent a particularly challenging material system for promoting wire/wire junction formation, and often require thermal annealing at temperatures near or above 200 °C to induce long range electrical conductivity within the deposited network [22, 23]. The difficulties that these wires present regarding junction formation is potentially due to their relatively large diameters compared to nanowires synthesized using other seeding materials, which has the capacity to enhance the thermal stability of individual wires according to the Gibbs-Thomson effect. We have chosen these wires as a demonstration of pre-deposition semiconducting ligand substitution in order to best illustrate the contrast between treated and untreated wires.

Completed nanocomposite inks are formed by mixing AgNWs with SnO2 nanoparticles in the presence of a compound capable of stripping the ligands from the AgNW surface. In this work, we have found that ammonia or ammonium salts act as effective stripping agents that are able to remove the PVP layer from the AgNW surface and allow for a new stabilizing matrix to take its place. Figure 1 shows a schematic of the process, starting from the precursors used in nanowire and nanoparticle synthesis and ending with the deposition of a completed film. The SnO2 nanoparticle solution naturally contains enough ammonium ions from its own synthesis to effectively peel the insulating ligands from the AgNWs and allow the nanoparticles to replace them as a stabilizing agent. If not enough SnO2 nanoparticles are used in the mixture, then the wires will rapidly agglomerate and settle to the bottom as large clusters. Large amounts of SnO2 in the mixture gradually begin to increase the sheet resistance of the nanowire network upon deposition, but greatly enhance the uniformity, durability, and wetting properties of the resulting films. We have found that AgNW:SnO2 weight ratios ranging between 2:1 and 1:1 produce well dispersed inks that are still highly conductive when deposited as films.

The nanowires were synthesized using a polyol method that has been adapted from the recipe described by Lee et al. [22, 23] Silver nitrate dissolved in ethylene glycol via ultrasonication was used as a precursor in the presence of copper chloride and PVP to provide seeds and produce anisotropic morphologies in the reaction products. Synthetic details can be found in the experimental section. Distinct from previous recipes, we have found that repeating the synthesis two times without cooling down the reaction mixture generally produces significantly longer nanowires than a single reaction step. The lengths of nanowires produced using this method fall over a wide range from 15 to 65 microns, with diameters between 125 and 250 nm. This range of diameters is common for wires grown using copper chloride seeds, although the double reaction produces a number of wires with roughly twice their usual diameter. The morphology of the as-deposited AgNWs as determined via SEM is shown in Figure 2(a), higher magnification images are also provided in Figures 2(c) and 2(d).

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The SnO2 nanoparticles were synthesized using a sol-gel method typical for multivalent metal oxide gelation reactions. A large excess of deionized water was added to SnCl4·5H2O dissolved in ethylene glycol along with tetramethylammonium chloride and ammonium acetate to act as surfactants. The reaction was then allowed to progress for at least one hour at near reflux conditions, after which the resulting nanoparticle dispersion can be collected, washed, and dispersed in a polar solvent of choice. The material properties of SnO2 nanoparticles formed using a similar synthesis method have been reported previously [24], although the present recipe uses excess water to ensure that the hydrolysis reaction proceeds nearly to completion.

After mixing with SnO2 nanoparticles, films deposited from AgNW/SnO2 composite inks show a largely continuous nanoparticle layer on the substrate surface with some nanowires partially buried and some sitting more or less on top of the film. Representative scanning electron microscopy (SEM) images of nanocomposite films are shown in Figure 2(b). Regardless of their position relative to the SnO2 film, all nanowires show a distinct shell on their outer surface that gives them a soft and slightly rough appearance, as is visible in the higher magnification images shown in Figure 2(e) and 2(f). The SnO2 nanoparticles do a particularly good job coating the regions near and around junctions between wires, and frequently appear in the SEM images as bulges wrapped around the wire/wire contact points.

The precise morphology of the SnO2 shell that effectively surrounded each AgNW was analyzed in more detail using transmission electron microscopy (TEM) imaging. Figures 3(a) to 3(c) show individual nanowires in the presence of different ligand systems: as-synthesized PVP in Figure 3(a), inactive SnO2 in Figure 3(b), and SnO2 activated with trace amounts of ammonium ions in Figure 3(c). The as-synthesized nanowires show sharp edges, and few surface features. In the presence of inactive SnO2, which is formed by repeatedly washing the SnO2 nanoparticles in ethanol until all traces of ammonium ions are removed, the nanowires coexist with somewhat randomly distributed nanoparticles that deposit all over the surface of the TEM grid. When AgNWs are mixed with activated SnO2, a thick and continuous SnO2 shell is formed along the nanowire surface. In when sufficiently dilute SnO2 solutions are used to form the nanocomposite ink, nearly all of the nanoparticles are consumed during shell formation and effectively no nanoparticles are left to randomly populate the rest of the image.

As the AgNWs acquire their metal oxide coatings in solution, the properties of the mixture change dramatically. Freshly synthesized AgNWs coated with residual PVP ligands slowly settle to the bottom of their vial or flask over a time period of several hours to one day, forming a dense layer at the bottom. The AgNWs with SnO2 shells do not settle to the bottom, but remain partially suspended even after many weeks at concentrations that are dependent on the amount of SnO2 present in the solution.

A comparison of the settling behavior of various AgNW and SnO2 mixtures after 24 hours is shown in Figures 3(d) and 3(e). The ratios 8:4, 8:16, and 8:8 indicate the concentrations of AgNWs and SnO2 (in mg/mL) present in each solution. The 8:8 uncoupled solution, in which the PVP is not removed from the AgNW surface with ammonia, produces a situation in which the nanowires and nanoparticles do not interact with one another, and instead the nanowires settle as in the isolated nanowire solution while the nanoparticles remain well-dispersed as in the solution of pure SnO2. The mixtures of nanowires and nanoparticles in which trace amounts of ammonia are present do not settle to the bottom, but instead concentrate themselves until repulsion between the semiconducting SnO2 clusters is able to prevent further settling.

Our current explanation for the settling behavior of the wire/particle mixtures is that the PVP coating on the surface of the as-synthesized wires is sufficient to prevent interaction with the nanoparticle solution. The addition of ammonia into the solution quickly strips off the PVP surface coating and allowing the nanoparticles to coordinate directly with the nanowire surface. This explanation is in agreement with the effects of ammonia has on a solution of pure AgNWs, which rapidly begin to agglomerate into clusters and sink to the bottom as soon as any significant quantity of ammonia is added to the ink.

We attribute the stripping ability of ammonia in these mixtures to the strong dative interactions that

occur via the lone pair on the nitrogen atom interacting with the partially filled d-orbitals of the Ag atoms

on the nanowire surface. These interactions are evidently strong enough to displace the existing

coordination of the five-membered rings and carbonyl groups contained in the original PVP ligands and

allow the ammonia to attach directly to the nanowire surface. Since ammonia is one of the original

surfactants used to stabilize the surface of the SnO2 nanoparticles, we consider it reasonable that ammonia

coordination on the nanowire surface would provide an appropriate environment for the nanoparticles to

adhere to the AgNWs.

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Scanning Energy Dispersive X-ray (EDX) Spectroscopy was also conducted on nanoparticle-coated AgNWs in order to image the presence of Sn and Ag in the nanowire and shell layer. The line scan results are shown in Figure 3(f), having been normalized to better compare the widths of the two signals. The visible broadening of the Sn lineshape compared to that of Ag is indicative of a Sn layer along the outside of the wire. The increasing strength of the Sn signal toward the center of the AgNW is likely due to the enhanced interaction between the TEM’s electron beam and the dense AgNW, which then improves the signal originating from the SnO2 shell as well. It is also possible that there is some intermixing between the Ag and Sn x-ray signals, but we consider this to be less likely as the distance between their characteristic peaks should be larger than the detection system’s energy resolution.

2.2. Network Deposition and Device Applications

For the deposition of transparent conducting films, a weight ratio of 2:1 of AgNWs to SnO2 nanoparticles was chosen in order to obtain a balance between the dispersibility of the nanowires, the uniformity of coated films, and the sheet resistance of the resulting conductive networks. Nanocomposite films were deposited on glass by blade coating from an ethanolic solution using a scotch tape spacer, with deposited networks then being allowed to dry naturally in air over several minutes.

The as-dried nanocomposite films are highly conductive, and require only minimal thermal treatment to dry and harden the film. Without the use of activated SnO2 ligands, deposited nanowire networks are highly insulating, and become conductive only after annealing at above 200 °C. The sheet resistance values of representative films are shown in Figure 4(a). The capability to form transparent conductive networks in a single deposition step that remain useful over a wide range of processing temperatures provides a high degree of versatility for designing thin film device fabrication procedures.

Figure 5(a) shows the sheet resistance and transmission of a number of nanocomposite films deposited from inks containing different nanowire concentrations. The deposited films show excellent conductivity at transmission values up to 85%, and then rapidly increase in sheet resistance as the network begins to reach its connectivity limit. The optimum performance of these networks at low to moderate transmission values is a consequence of the relatively large nanowire diameters, which scatter a noticeable amount of light even when the conditions required for current percolation are just barely met. Nonetheless, the sheet resistance and transmission of the completed nanocomposite networks place them within an acceptable range for applications in a variety of optoelectronic devices. Figure 5(b) shows the wavelength dependent transmission spectra of several nanowire networks, which transmit light well out into the infrared region. The presence of high transmission values out to wavelengths well above 1300 nm, where ITO or other conductive oxide layers would typically begin to show parasitic absorption, is due to the use of semiconducting SnO2 ligands, which is complimentary to the broad spectrum transmission of the silver nanowire network itself.

Avoiding the use of highly doped nanoparticles has the potential to provide optical advantages, but can create difficulties when attempting to make electrical contact to neighboring device layers. In order to investigate their functionality in thin film devices, we have incorporated AgNW/SnO2 nanocomposite films as electrodes in amorphous silicon (a-Si) solar cells. Two contact structures were used during fabrication: one with the nanocomposite film directly in contact with the p-i-n absorber structure and one with a 10 nm Al:ZnO (AZO) layer present to assist in forming Ohmic contact with the device. The I-V characteristics of the resulting devices are shown in Figure 6(a).

The thin AZO contact layers typically show sheet resistance values greater than 2.5 kΩ/⧠, and so cannot be responsible for long range lateral current transport within the electrode structure. However, their presence is clearly beneficial in improving contact between the nanocomposite electrode and the absorber material, as the SnO2 matrix material is evidently not conductive enough to form a high quality contact with the p-type side of the a-Si stack. We hope that future modifications to the AgNW/SnO2 composite, or perhaps the use of islands of high conductivity material such as a discontinuous layer of doped nanoparticles will allow for the deposition of completed electrode stacks that provide both rapid fabrication and good performance.

Figure 6(b) contains the top view image of a completed device. The enhanced viscosity of the nanowire/sol-gel composite inks allows for films to be blade coated onto substrates with a variety of surface properties without reductions in network uniformity. In contrast with traditional back electrodes deposited in vacuum environments, the nanocomposite can be blade coated into place in a single pass under atmospheric conditions and dried within moments. We anticipate that the use of sol-gel mixtures to enhance wetting and dispersibility may prove useful in the formulation of other varieties of semiconducting and metallic inks for deposition onto a variety of substrate structures.

3. Conclusions

In summary, we have successfully exchanged the insulating ligands that normally surround as-synthesized AgNWs with shells of substantially more conductive SnO2 nanoparticles. The exchange of one set of ligands for the other is mediated by

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the presence of ammonia during the mixing process, which appears to be necessary for the effective removal of the PVP ligands that initially cover the nanowire surface. The resulting nanowire/nanoparticle mixtures allow for the deposition of nanocomposite films that require no annealing or other post-treatments to function as high quality transparent conductors with transmission and sheet resistance values of 85% and 10 Ω/⧠, respectively. Networks formed in this manner can be deposited quickly and easily in open air, and have been demonstrated as an effective n-type electrode in a-Si solar cells when a thin interfacial layer is deposited first to ensure good electronic contact with the rest of the device. The ligand management strategy described here could potentially be useful in any number of material systems that presently suffer from highly insulating materials that reside on the surface of otherwise high performance nano and microstructures.

4. Experimental Details

Tin oxide nanoparticle synthesis. Tin chloride pentahydrate was dissolved in ethylene glycol by

stirring for several hours at a concentration of 10 grams per 80 mL to serve as a stock solution. In a typical

synthesis reaction, 10 mL of the SnCl4·5H2O stock solution is added to a 100 mL flask and stirred at room

temperature. Still at room temperature, 250 mg ammonium acetate and 500 mg ammonium acetate were

added in powder form to regulate the solution pH and to serve as coordinating agents for the growing

oxide nanoparticles. 30 ml of water was then added, and the flask was heated to 90 °C for 1 to 2 hours in

an oil bath, during which the solution took on a cloudy white color. The gelled nanoparticles were then

washed twice in ethanol in order to keep trace amounts of ammonia present in the solution. Additional

washing cycles would deactivate the SnO2, and then require the addition of ammonia to coordinate with

as-synthesized AgNWs.

Silver nanowire synthesis. Copper(ii) chloride dihydrate was first dissolved in ethylene glycol at

1 mg/ml to serve as a stock solution for nanowire seed formation. 20 ml of ethylene glycol was then added

into a 100 ml flask, along with 200 µL of copper chloride solution. the mixture was then heated to 150 °C

while stirring at 325 rpm, and .35g of PVP (MW 55,000) was added. In a small separate flask, .25 grams of

silver nitrate was dissolved in 10 ml ethylene glycol by sonicating for approximately 2 minutes, similar to

the method described here.22 The silver nitrate solution was then injected into the larger flask over

approximately 15 minutes, and the reaction was allowed to progress for 2 hours. After the reaction had

reached completion, the various steps were repeated without cooling down. 200 µL of copper chloride

solution and .35g PVP were added in a similar manner to the first reaction cycle, and another .25g silver

nitrate were dissolved via ultrasonics and injected over 15 minutes. The second reaction cycle was allowed

to progress for another 2 hours, before the flask was cooled and the reaction products were collected and

washed three times in ethanol.

Nanocomposite ink formation. After the synthesis of the two types of nanostructures is complete,

29

the double washed SnO2 nanoparticles and triple-washed nanowires can be combined at a variety of weight

ratios to form the completed nanocomposite ink. The dispersibility of the mixture is improved when more

SnO2 is used, although the sheet resistance of the final networks will begin to increase if they contain

excessive SnO2. AgNW agglomeration during mixing is most easily avoided if the SnO2 and AgNW

solutions are first diluted to the range of 10 to 20 mg/ml in ethanol, with the SnO2 solution being added

first to an empty vial and the AgNW solution added afterwards. The dilute mixture was then be allowed to

settle overnight, and the excess solvent removed to concentrate the wires to a concentration that is

appropriate for blade coating.

Film and electrode deposition. The completed nanocomposite ink was deposited onto any desired

substrates using a razor blade and scotch tape spacer. The majority of the substrates used in this study were

Corning soda lime glass, but the combined inks also deposited well on silicon, SiO2, and any other

substrates tested. Electrode deposition onto a-Si substrates was accomplished by masking off the desired

cell area with tape, and then depositing over the entire region. The p-i-n a-Si stacks and 10 nm AZO

contact layers were deposited using PECVD and sputtering, respectively.

ACKNOWLEDGMENTS The authors would like to acknowledge the use of the Electron Imaging Center for Nanomachines

(EICN) located in the California NanoSystems Institute at UCLA.

REFERENCES [1] Sun, Y.; Gates, B.; Mayers, B.; Xia, Y., Crystalline silver nanowires by soft solution

processing. Nano Lett. 2002, 2, 165-168.

[2] Kim, T.; Kim, Y. W.; Lee, H. S.; Kim, H.; Yang, W. S.; Suh, K. S., Uniformly

interconnected silver-nanowire networks for transparent film heaters. Adv. Funct.

Mater. 2013, 23, 1250-1255.

[3] Hu, L.; Wu, H.; Cui, Y., Metal nanogrids, nanowires, and nanofibers for transparent

electrodes. MRS Bull. 2011, 36, 760-765.

30

[4] van de Groep, J.; Spinelli, P.; Polman, A., Transparent conducting silver nanowire

networks. Nano Lett. 2012, 12, 3138-3144.

[5] Yang, L.; Zhang, T.; Zhou, H.; Price, S. C.; Wiley, B. J.; You, W., Solution-processed

flexible polymer solar cells with silver nanowire electrodes. ACS Appl. Mater.

Interfaces 2011, 3, 4075-4084.

[6] Scardaci, V.; Coull, R.; Lyons, P. E.; Rickard, D.; Coleman, J. N., Spray deposition of

highly transparent, low-resistance networks of silver nanowires over large areas. Small

2011, 7, 2621-2628.

[7] Wiley, B.; Sun, Y.; Xia, Y., Synthesis of silver nanostructures with controlled shapes

and properties. Acc. Chem. Res. 2007, 40, 1067-1076.

[8] Korte, K. E.; Skrabalak, S. E.; Xia, Y., Rapid synthesis of silver nanowires through a

cucl- or cucl2-mediated polyol process. J. Mater. Chem. 2008, 18, 437-441.

[9] Anuj, R. M.; Akshay, K.; Chongwu, Z., Large scale, highly conductive and patterned

transparent films of silver nanowires on arbitrary substrates and their application in

touch screens. Nanotechnology 2011, 22, 245201.

[10] Lee, J.-Y.; Connor, S. T.; Cui, Y.; Peumans, P., Solution-processed metal nanowire

mesh transparent electrodes. Nano Lett. 2008, 8, 689-692.

[11] Zhu, R.; Chung, C.-H.; Cha, K. C.; Yang, W.; Zheng, Y. B.; Zhou, H.; Song, T.-B.;

Chen, C.-C.; Weiss, P. S.; Li, G.; Yang, Y., Fused silver nanowires with metal oxide

nanoparticles and organic polymers for highly transparent conductors. ACS Nano 2011,

5, 9877-9882.

[12] Chung, C.-H.; Song, T.-B.; Bob, B.; Zhu, R.; Duan, H.-S.; Yang, Y., Silver nanowire

composite window layers for fully solution-deposited thin-film photovoltaic devices.

Adv. Mater. 2012, 24, 5499-5504.

31

[13] Kim, A.; Won, Y.; Woo, K.; Kim, C.-H.; Moon, J., Highly transparent low resistance

zno/ag nanowire/zno composite electrode for thin film solar cells. ACS Nano 2013, 7,

1081-1091.

[14] Ajuria, J.; Ugarte, I.; Cambarau, W.; Etxebarria, I.; Tena-Zaera, R. n.; Pacios, R.,

Insights on the working principles of flexible and efficient ito-free organic solar cells

based on solution processed ag nanowire electrodes. Sol. Energy Mater. Sol. Cells

2012, 102, 148-152.

[15] Tokuno, T.; Nogi, M.; Karakawa, M.; Jiu, J.; Nge, T.; Aso, Y.; Suganuma, K.,

Fabrication of silver nanowire transparent electrodes at room temperature. Nano Res.

2011, 4, 1215-1222.

[16] Lim, J.-W.; Cho, D.-Y.; Jihoon, K.; Na, S.-I.; Kim, H.-K., Simple brush-painting of

flexible and transparent ag nanowire network electrodes as an alternative ito anode for

cost-efficient flexible organic solar cells. Sol. Energy Mater. Sol. Cells 2012, 107,

348-354.

[17] De, S.; Higgins, T. M.; Lyons, P. E.; Doherty, E. M.; Nirmalraj, P. N.; Blau, W. J.;

Boland, J. J.; Coleman, J. N., Silver nanowire networks as flexible, transparent,

conducting films: Extremely high dc to optical conductivity ratios. ACS Nano 2009, 3,

1767-1774.

[18] Hu, L.; Kim, H. S.; Lee, J.-Y.; Peumans, P.; Cui, Y., Scalable coating and properties of

transparent, flexible, silver nanowire electrodes. ACS Nano 2010, 4, 2955-2963.

[19] Garnett, E. C.; Cai, W.; Cha, J. J.; Mahmood, F.; Connor, S. T.; Greyson Christoforo,

M.; Cui, Y.; McGehee, M. D.; Brongersma, M. L., Self-limited plasmonic welding of

silver nanowire junctions. Nat. Mater. 2012, 11, 241-249.

32

[20] Yu, Z.; Zhang, Q.; Li, L.; Chen, Q.; Niu, X.; Liu, J.; Pei, Q., Highly flexible silver

nanowire electrodes for shape-memory polymer light-emitting diodes. Adv. Mater.

2011, 23, 664-668.

[21] Song, T.-B.; Chen, Y.; Chung, C.-H.; Yang, Y.; Bob, B.; Duan, H.-S.; Li, G.; Tu,

K.-N.; Huang, Y., Nanoscale joule heating and electromigration enhanced ripening of

silver nanowire contacts. ACS Nano 2014, 8, 2804-2811.

[22] Lee, P.; Lee, J.; Lee, H.; Yeo, J.; Hong, S.; Nam, K. H.; Lee, D.; Lee, S. S.; Ko, S. H.,

Highly stretchable and highly conductive metal electrode by very long metal nanowire

percolation network. Adv. Mater. 2012, 24, 3326-3332.

[23] Lee, J. H.; Lee, P.; Lee, D.; Lee, S. S.; Ko, S. H., Large-scale synthesis and

characterization of very long silver nanowires via successive multistep growth. Cryst.

Growth Des. 2012, 12, 5598-5605.

[24] Bob, B.; Song, T.-B.; Chen, C.-C.; Xu, Z.; Yang, Y., Nanoscale dispersions of gelled

Sno2: Material properties and device applications. Chem. Mater. 2013, 25, 4725-4730.

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Figure 1. Process flow diagram showing the synthesis of AgNWs and SnO2 nanoparticles followed

by stirring in the presence of ammonium salts to create the final nanocomposite ink. Transparent

conducting films were produced by blade coating the completed inks onto the desired substrate.

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Figure 2. (a,c,d) SEM images of as-synthesized AgNWs at various magnifications. (b,e,f) SEM

images of nanocomposite films, showing the tendency of the SnO2 nanoparticles to coat the entire

outer surface of the AgNWs, increasing their apparent diameter and giving them a soft appearance.

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Figure 3. Schematic diagrams and TEM images of (a) a single untreated AgNW, (b) an AgNW in the

presence of uncoupled SnO2 (all ammonium ions removed), and (c) an AgNW with a coordinating

SnO2 shell. Scale bars in images (a), (b), and (c) are 300 nm, 400 nm, and 600 nm, respectively. (d,e)

Optical images of AgNW and SnO2 nanoparticle dispersions mixed in varying amounts (d) before and

(e) after settling for 24 hours. The numbers associated with each solution represent the AgNW:SnO2

concentrations in mg/ml. The uncoupled solution contains AgNWs and non-coordinating SnO2

nanoparticles, and shows settling behavior similar to the pure AgNW and pure SnO2 solutions. (f)

Normalized Ag and Sn EDX signal mapped across the diameter of a single nanowire, with the inset

showing the scanning path across an isolated wire.

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Figure 4. Sheet resistance versus temperature for films deposited using (red) AgNWs that have been

washed three times in ethanol and (blue) mixtures of AgNW and SnO2 with weight ratio of 2:1. The

annealing time at each temperature value was approximately 10 minutes. The large sheet resistance

values of the bare AgNWs when annealed below 200 °C is typical for nanowires fabricated using

copper chloride seeds, which clearly illustrate the impact of SnO2 coordination at low treatment

temperatures.

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Figure 5. (a) Sheet resistance and transmission data for samples deposited from solutions of varying

nanostructure concentration. Each of these samples were fabricated starting from the same

nanocomposite ink, which was then diluted to a range of concentrations while maintaining the same

AgNW to SnO2 weight ratio. (b) Transmission spectra of several transparent conducting networks

chosen from the plot in plot (a).

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Figure 6. (a) I-V characteristics of devices made with AgNW/SnO2 rear electrodes with (blue) and

without (red) a 10 nm AZO contact layer. The dramatic double diode effect is likely a result of a

significant barrier to charge injection at the electrode/a-Si interface. (b) Top view SEM image of the

AgNW/SnO2 composite films on top of the textured a-Si absorber. (c) Schematic cross section of the

a-Si device architecture used in solar cell fabrication. The thickness of the thin AZO contact layer is

exaggerated for clarity.