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Nano Res

1

Self-delivery of peptide based prodrug for tumor targeting therapy

Mengyun Peng1,§, Siyong Qin1,2,§, Huizhen Jia1, Diwei Zheng1,3, Lei Rong1, and Xianzheng Zhang1 (*) Nano Res., Just Accepted Manuscript • DOI: 10.1007/s12274-015-0945-1

http://www.thenanoresearch.com on November. 16, 2015

© Tsinghua University Press 2015

Just Accepted

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Nano Research DOI 10.1007/s12274-015-0945-1

www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research

64 Nano Res.

TABLE OF CONTENTS (TOC)

Self-delivery of peptide based prodrug for tumor

targeting therapy

Meng-Yun Peng,a,‡ Si-Yong Qin,a,b,‡ Hui-Zhen Jia,a

Di-Wei Zheng,a,c Lei Rong,a Xian-Zheng Zhanga*

a Key Laboratory of Biomedical Polymers of Ministry

of Education & Department of Chemistry, Wuhan

University, Wuhan 430072, P. R. China b School of Chemistry and Materials Science,

South-Central University for Nationalities, Wuhan

430074, China c Hubei Collaborative Innovation Center for Advanced

Organic Chemical Materials; Key Laboratory for the

Green Preparation and Application of Functional

Materials of Ministry of Education, Hubei

University, Wuhan, Hubei 430062, P. R. China ‡ These authors contributed equally to this work.

* To whom correspondence should be addressed.

Tel. & Fax: 86-27-68754509.

E-mail address: [email protected] (X. Z. Zhang).

A self-assembled nanofibrous prodrug was fabricated, which could

target tumor cells specifically and inhibit tumor growth efficiently via

peptide ligand mediated targeting therapy.

Self-delivery of peptide based prodrug for tumor targeting therapy

Meng-Yun Peng,a,‡ Si-Yong Qin,a,b,‡ Hui-Zhen Jia,a Di-Wei Zheng,a,c Lei Rong,a Xian-Zheng Zhanga (*)

a Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072, P. R.

China b School of Chemistry and Materials Science, South-Central University for Nationalities, Wuhan 430074, China c Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials; Key Laboratory for the Green Preparation and

Application of Functional Materials of Ministry of Education, Hubei University, Wuhan, Hubei 430062, P. R. China ‡ These authors contributed equally to this work.

Received: day month year Revised: day month year Accepted: day month year

© Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2014

KEYWORDS Self-assembly; self-delivery; prodrug; Tumor targeting therapy

ABSTRACT A novel self-delivered prodrug system was fabricated for tumor targetingtherapy. In this nano-system, RGDS tetrapeptide was used to improve the therapeutic index to integrin-overexpressed tumor cells. The anti-tumorous CPT drug was further appended to the ε-amino group of lysine by a 20-O-succinyl linkage and controllably released via the hydrolytic cleavage. Prodrug molecules self-assembled into fibrillar nano-architectures and achieved self-delivery modality after injected subcutaneously. Introduction ofhydrophobic myristic acid was in favour of the self-assembly and enhanced cell internalization. In vitro and in vivo studies demonstrated that self-assemblednanofibers could effectively target the tumorous cells with over-expressedintegrins and inhibit the tumor growth via RGD-mediated specific targeting.Traditional idea that fibrillar structures hold low therapeutic efficacy due to poor cell uptake may be challenged.

Introduction 1

Arising from the rapid development in 2 biomaterials and nanotechnology, the drug 3 delivery systems (DDSs) have been well 4 established for tumor therapy in recent years. The 5 success of these available DDSs mainly relies on the 6 creation of carriers for the effective delivery of 7 cargos into tumor lesions. A representative 8 example is the emergence of nano-carriers, which 9 could protect the therapeutic agents from 10 disruption by encapsulating them into liposomes 11 [1], polymeric micelles [2,3], inorganic [4-6] and 12

organic nanomaterials [7]. These nano-carriers 13 capitalized on the enhanced permeability and 14 retention (EPR) effect via the passive tumor 15 targeting delivery [8]. However, their wide 16 applications were limited by inherent limitations 17 such as the low drug loading efficiency [9] and 18 toxicity from the carriers themselves. 19

To address those drawbacks, an alternative 20 strategy focused on fabricating prodrugs, which 21 involved coupling the drugs with small molecules 22 [9,10] or biocompatible polymer [11]. Peptides due 23 to their excellent biodegradability, bioactivity, 24 environmental sensitivity, and active sites for 25

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


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chemical modification were widely exploited to 1 fabricate the desirable prodrugs [12,13]. 2 Peptide-based prodrug system as a potent platform 3 exhibited many merits for drug delivery, including 4 enhanced cell endocytosis, improved drug 5 availability, high loading efficiency, as well as 6 temporally or spatially controllable drug release 7 [12-15]. Furthermore, prodrugs modified with 8 low-molecular-weight (LMW) peptides are 9 achievable in manipulating the quantitative and 10 high drug loading per carrier. Nevertheless, LMW 11 prodrugs may be subject to the rapid clearance and 12 premature degradation [14]. Very recently, 13 self-delivered LMW prodrug systems have been 14 proposed, in which prodrugs self-assembled into 15 supramolecular nanostructures such as 16 nanocapsule [9] and nanoparticle [16, 17], and were 17 able to enter tumor cells/tissues without the need of 18 any additional carriers. However, most reported 19 systems are lack of positive selectivity to tumorous 20 cells, and prodrug systems usually present the 21 spherical shape. It was predicted that nanofibers 22 with high aspect ratio might exhibit a therapeutic 23 advantage over traditional spherical nanoparticles 24 due to prolonged circulation time in vivo [18, 19]. 25 Hence, fabricating the fibrillar self-delivered 26 prodrug systems with positive targeting function is 27 desirable for tumor therapy. 28

Here, we report a self-delivered fibrillar prodrug 29 for the tumor targeting therapy. Briefly, the 30 prodrug of 31 C13H27-CONH-Arg-Gly-Asp-Ser-Lys(CPT) (namely 32 RGD-prodrug) is composed of three parts: a 33 conjugated antitumor drug, tumor cell targeting 34 moiety, and a hydrophobic tail (Scheme 1A). A 35 monoterpene indole alkaloid camptothecin (CPT) 36 was used as the antitumor drug. The hydroxyl 37 group in CPT provides a feasible site for covalent 38 modification, and the π-conjugated property 39 endues the active drug as a suitable unit for 40 participating in self-assembly [20]. The targeting 41 moiety used here is a tetrapeptide RGDS, which 42 can be recognized by integrin receptors associated 43 with various tumor [21,22]. The critical aggregation 44 concentration (CAC) required by prodrug for 45 self-assembly is important for self-delivery systems. 46 The hydrophobic myristic acid was introduced into 47 the N-terminus of RGDS peptide, which assisted 48 self-assembly at a low CAC and enhanced cell 49 endocytosis. 50

51

Scheme 1. Self-delivered prodrug nanofibers for the tumor 52 targeting therapy. (A) Chemical structure of the designed 53 amphiphilic prodrug; (B) Schematic illustration of the prodrug 54 self-assembling into nanofibers: driven by the π-π stacking 55 interaction, free prodrug begun to arrange via J-type 56 self-aggregation; (C) Self-delivered prodrug nanofibers 57 exerting the therapeutic action by inhibiting the tumor growth 58 in mice. 59

Experimental 60

2.1 Materials 61

N-Fluorenyl-9-methoxycarbonyl (Fmoc) amino 62 acids, Rink Amide-AM resin (100-200 mesh, 63 loading: 0.77 mmol/g), N-hydroxy benzotriazole 64 (HOBt), 65 benzotriazole-N,N,N’,N’-tetramethyluroniumhexa- 66 fluorophosphate (HBTU), Triisopropylsilane (TIS) 67 and piperidine were purchased from GL Biochem 68 (Shanghai) Ltd. (China) and used as received. 69 Trifluorocaetic acid (TFA), N, 70 N-dimethylformamide (DMF), N, N-Diisopropyl 71 ethylamine (DIEA) were obtained from Shanghai 72 Reagent Chemical Co. and used after redistillation. 73 (S)-(+)-camptothecin (CPT), succinic anhydride and 74 1,8-Diazabicycloundec-7-ene (DBU) were 75 purchased from Heowns Biochemical Technology 76 (Tianjing) Co., Ltd and used as received. 77 Dulbecco’s modified Eagle medium (DMEM), 78 RPMI 1640 medium, fetal bovine serum (FBS), 79 trypsin, penicillin, streptomycin, 80 3-[4,5-dimethylthiazol-2-yl] 81 -2,5-diphenyltetra-zoliumbromide (MTT), and 82 Dulbecco’s phosphate-buffered saline (PBS) were 83 purchased from Invitrogen. All other solvents and 84 reagents were used directly. 85

2.2 Synthesis of CPT-COOH 86


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(S)-(+)-camptothecin (CPT, 348.4 mg, 1.0 mmol) and 1 succinic anhydride (300.3 mg, 3.0 mmol) were put 2 into a 100 mL round-bottom flask and suspended 3 with 30 mL of dichloromethane. Then, 0.44 mL of 1, 4 8-Diazabicycloundec-7-ene (DBU, 3.0 mmol) was 5 dropped slowly into the mixture at 0 °C. The 6 reaction was stirred for 4 h at room temperature, 7 and stopped by 20 mL of water. 1% aqueous HCl 8 solution was used to acidize the mixture. The 9 yellow precipitate was collected and washed with 10 1% aqueous HCl solution (10 mL × 3 times) and 11 H2O (10 mL × 3 times). The precipitate was 12 recrystallized with methanol, and the product was 13 obtained as the yellow green powder and 14 characterized by ESI-MS and NMR spectrum (Fig. 15 S2-3). 16

2.3 Synthesis of free peptide and prodrugs 17

C13H27-CONH-Arg-Gly-Asp-Ser-COOH (free 18 peptide), C13H27-CONH-Arg-Gly-Asp-Ser-Lys(CPT) 19 (RGD-prodrug) and 20 C13H27-CONH-Asp-Arg-Gly-Ser- Lys(CPT) 21 (DRG-prodrug) were prepared manually according 22 to the Fmoc solid phase peptide synthesis method 23 [23]. Simply, 20% (v/v) piperidine/DMF was used 24 for Fmoc deprotection of Rink Amide-AM resin. 25 Fmoc-protected amino acids were linked to the 26 Rink Amide-AM resin progressively in a DMF 27 solution containing HBTU/HOBt/DIEA, using 10% 28 ninhydrin in methanol for coupling efficacy 29 monitoring. As for prodrug synthesis, 2% TFA in 30 dichloromethane was used to remove the Mtt 31 group of Lys side chain. CPT-COOH was linked to 32 the N-terminal of Lys via the same coupling 33 method as amino acids and the mixture was 34 allowed to react overnight. Cleavage of the 35 prodrug from the resin was achieved by the recipe 36 of TFA/H2O/TIS (95/1/4 by vol) for 2 h at room 37 temperature. The prodrug was precipitated in cold 38 ether and further characterized by ESI, NMR and 39 HPLC (Fig. S4-8). 40

2.4 Transmission electron microscopy (TEM) 41

Morphology observation of self-assemblies were 42 performed on a JEM-100CX II instrument operating 43 at an acceleration voltage of 100 kV. Samples with 44 concentrations of 80 µM were prepared by 45 dropping the liquid onto a formvar film modified 46 copper grid. Excess solution in the grid was blotted 47 away and dried in air. 48

2.5 CAC determination of prodrugs 49

The critical aggregation concentration (CAC) of 50 RGD-prodrug and DRG-prodrug was determined 51 using Nile red as a hydrophobic fluorescent probe 52 [24]. 50 µL of acetone solution containing Nile red 53 (10-6 M) was added to several vials and allowed to 54 stand overnight for solvent removal, respectively. 55 Then, 2 mL of prodrug solution with different 56 concentrations was added to each of Nile red 57 containing vial and sonicated for 30 min. 58 Fluorescence spectrum of Nile red was recorded 59 upon excitation at 485 nm and fluorescence 60 intensity of Nile red at 725 nm was recorded (Fig. 61 S9). 62

2.6 Drug release 63

The self-assembled RGD-prodrug (25 µM) was 64 prepared and incubated at room temperature for 4 65 h. 3 mL of the prodrug solution was added into a 66 dialysis tube (MWCO 2000 Da) and dialyzed 67 against 10 mL of aqueous solution with different 68 pHs (pH 7.0 and 5.2) for 12 d. The amount of 69 released CPT was measured by detecting the 70 fluorescence of the dialysate, and the drug release 71 ratio was calculated with the initial amount of CPT. 72

2.7 In vitro cytotoxicity assay 73

The in vitro cytotoxicity of prodrugs and peptide 74 against H22, HeLa and COS-7 cells was examined 75 by MTT assay following the literature procedures 76 [25]. The H22/HeLa/COS-7 cells were seeded in 77 96-well plates at a density of around 1.2×104 78 cells/well. HeLa and COS-7 cells were cultured 79 with 100 µL of DMEM and H22 was cultured with 80 100 µL of RPMI 1640 medium. Both medium were 81 supplemented with 1% antibiotics 82 (penicillin/streptomycin, 10,000 U/mL) and 10% 83 FBS. Cells were cultured in a humidified 84 atmosphere containing 5% CO2 at 37 °C for 24 h 85 before adding the material. In the following step, 86 100 µL of fresh medium containing the material 87 was added and incubated with cells for another 48 88 h. For HeLa and COS-7 cells, each well was added 89 with 20 µL of 5 mg/mL MTT solution and 90 incubated for another 4 h. After that, the medium 91 was replaced by 150 µL of DMSO. For H22 cells, 92 cytotoxicity assay was carried out with 20 µL of 93 Cell Counting Kit-8 each well and incubated 94 another 4 h. The optical density (OD) was 95


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measured at 570 nm for HeLa/COS-7 and 450 nm 1 for H22 using a microplate reader (Bio-Rad, Model 2 550, USA). The relative cell viability of the material 3 was calculated according to the formula: cell 4 viability (%) = (OD (sample)/OD (control)) × 100%. The 5 toxicity assay of DRG-prodrug, free peptide and 6 free CPT were carried out in the same way. 7

2.8 Confocal laser scanning microscopy 8

The ability of the RGD-prodrug specifically 9 targeting H22/HeLa/COS-7 cells was evaluated by 10 confocal laser scanning microscopy (CLSM). Each 11 well of the 6-well plate was seeded with 12 H22/HeLa/COS-7 at a cell number of 1×105 13 respectively and cultured for 24 h. 12.5 µM 14 RGD-prodrug and DRG-prodrug were added to 15 the wells and incubated with H22, HeLa and 16 COS-7 cells for 20 min, respectively. After the 17 incubation, the materials were removed and the 18 cells were washed with PBS for three times. For 19 RGD blocking on the cell membrane of HeLa, 20 excess free peptide (25 µM) was pre-incubated with 21 HeLa cells for 30 min, then 12.5 µM RGD-prodrug 22 was added and treated for 20 min. The fluorescence 23 was observed with a confocal laser scanning 24 microscopy (C1-Si, Nikon, Japan) which was 25 equipped with a 405 nm diode for CPT 26 fluorescence measurements. 27

2.9 In vivo experiment 28

As a widely used strategy for tumor formation, the 29 transplantable murine hepatoma22 (H22) model 30 was chosen to evaluate the antitumor efficiency of 31 the prodrug. The H22 tumor-bearing mice were 32 obtained from Zhongnan Hospital (Wuhan, China) 33 and the animal experiments were approved by the 34 local ethical committee and all the experiments 35 were performed according to the National 36 Institutes of Health Guide for the Care and Use of 37 Laboratory Animals. The animals were divided 38 into four groups (6 mice in each group) with 39 similar starting weight (about 20 g) randomly. Each 40 group respectively received an equivalent volume 41 (200 µL) of PBS, free CPT (10 mg/kg), 42 RGD-prodrug and DRG-prodrug (10 mg CPT 43 equiv/kg) every 2 days. Tumor regions were 44 shaved to reduce the influence of hair on the tumor 45 surface. Body weight and the subcutaneous tumor 46 size (estimated by formulas 1/2×L×W2) were 47 recorded throughout the 14 days experimental 48

period. The tumor growth ratio was defined as 49 V/V0 (V0 was the initial tumor volume without any 50 treatment). Antitumor activity of CPT against H22 51 hepatocellular carcinoma implanted in BALB/C 52 mice was evaluated by a growth-inhibition analysis. 53 Mice were killed upon completion of 14 days 54 treatment. Tumor and organs were sacrificed, and 55 immersed in a buffered solution of 4% 56 paraformaldehyde. The tumor and organs were 57 imaged (Ex: 450 nm, Em: 500-650 nm) using an in 58 vivo imaging system (Maestro, CRi Inc., Woburn, 59 MA, USA). For H&E staining assay, the specimen 60 was embedded in paraffin and sectioned serially at 61 4 mm thickness and stained by hematoxylin and 62 eosin (H&E). These histological sections were 63 evaluated by optical microscopy. 64

Results and discussion 65

3.1 Characterization of prodrug self-assembly 66

Self-assembly of RGD-prodrug was studied by 67 TEM in phosphate buffered saline (PBS, 10 mM). 68 As shown in Fig. 1A, RGD-prodrug self-organized 69 into well-defined nanofibers with the diameter of 70 around 20 nm at 80 µM. The formation of 71 nanofibers may be ascribed to π-π stacking from 72 quinoline rings within CPT, the hydrophobic 73 interaction from alkyl groups and hydrogen 74 bonding from peptide backbones (Scheme 1B). To 75 validate the participation of hydrophobic 76 interaction from alkyl groups for the self-assembly, 77 the self-assembled behavior of free peptide was 78 also investigated and a similar nanofiber bundling 79 was observed by TEM (Fig. 1B). The formation of 80 nanofibers was mainly driven by the hydrophobic 81 interaction and intermolecular hydrogen bonding 82 [26]. The nanofibers were more 83

84

Figure 1 TEM images of self-assembled nanofibers from 85 RGD-prodrug (A) and free peptide (B) at 80 µM, 86 respectively. 87

robust than that from RGD-prodrug. Difference in 88 widths could be attributed to the fact that 89


5Nano Res.

introduction of CPT to some extent hindered the 1 well organization of alkyl group within 2 RGD-prodrug. 3

To validate π-π stacking for RGD-prodrug 4 self-assembly, the intermolecular interactions 5 involved in the nanofibers were investigated by 6 spectroscopic analysis. The UV/Vis spectrum of 7 RGD-prodrug displayed two bands at 364 nm and 8 379 nm in DMSO, where the units were expected to 9 exist in a minimally aggregated state (Fig. 2A). The 10 lower extinction coefficients of two main peaks 11 measured in PBS and a lower ratio of intensities of 12 the peaks at 355 nm and 368 nm indicated the 13 formation of J-aggregated CPT chromophores in 14 the nanofibers [27, 28]. It was also reported that 15 aggregated π-conjugated systems were frequently 16 associated with the bisignate cotton effect [14]. 17 Therefore, CD spectra of prodrug were collected 18 from 240 to 400 nm in DMSO and PBS, which 19 exhibited two bisignate CD signals centered at 260 20 nm and 360 nm in PBS (Fig. 2B), resulting from the 21 excition coupling between adjacent quinoline rings 22 within CPT. With the increase in RGD-prodrug 23 concentration, the red shift of the central peak from 24 443 nm to 450 nm in fluorescence spectra further 25 indicated π-π-stacking interaction for the 26 self-assembly (Fig. 2C) [29, 30]. 27

28

Figure 2 UV/Vis spectra (A) and CD spectra (B) of 29 RGD-prodrug in DMSO and PBS; (C) Fluorescence 30 emission spectra of RGD-prodrug in PBS with different 31 concentrations. 32

33

Figure 3 Determination of CAC of RGD-prodrug (A) and 34 DRG-prodrug (B); The fluorescent intensity of Nile red was 35 plotted against RGD-prodrug concentration. CAC was 36 determined from inflection point of the plot; (C) 37 RGD-prodrug release profiles of nanofibers at different pH 38 values at 37 oC. Data are given as mean±s.d. (n= 3). 39

3.2 CAC determination and drug release 40

CAC values of RGD-prodrug and DRG-prodrug 41 were determined using Nile red as a fluorescent 42 probe, giving low values of 12 µM and 13 µM, 43 respectively (Fig. 3A, B). The method was based on 44 the fact that the fluorescence intensity of Nile red 45 was low in water due to low solubility, while 46 increased quickly when it was entrapped in 47 hydrophobic fibrillar core. Self-delivery system 48 with a low CAC could effectively reduce toxicity to 49 normal cells. The higher concentration is needed 50 for self-delivery, the higher toxicity would be 51 caused to normal tissues. To investigate the 52 controlled release behavior of self-delivered 53 nanofibers, drug release profiles of the 54 RGD-prodrug at pH 5.2 and 7.0 were recorded. As 55 shown in Fig. 3C, both profiles exhibited sustained 56 release due to the slow disassembly of nanofibers. 57 Under acid condition, nanofibers displayed a faster 58 drug release rate, which was ascribed to the charge 59 repulsion from ionized guanidino groups and the 60 accelerated cleavage of 20-O-succinyl linkage for 61 CPT release. At pH 7.0, this self-delivered prodrug 62 system could effectively sequester active CPT 63 molecules within the hydrophobic fibrillar core and 64 protect the bioactive drug from rapid lactone 65 hydrolysis. 66

3.3 In vitro cytotoxicity assay 67


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1

Figure 4 Cell cytotoxicities of RGD-prodrug (A) and 2 DRG-prodrug (B) against H22/HeLa/COS-7 cells. 3

The antitumor efficiency of RGD-prodrug and 4 DRG-prodrug was firstly evaluated by cell 5 viability protocol to assess the cytotoxicity 6 against three different cell lines of tumorous H22, 7 HeLa cells and normal COS-7 cells. The 8 cytotoxicity of free peptide and free CPT was 9 investigated as control (Fig. S10). As shown in Fig. 10 4, prodrugs exhibited dose-dependent 11 cytotoxicity against both tumorous and normal 12 cells. Data showed an enhanced cytotoxicity 13 against RGD-receptor over-expressed H22 and 14 HeLa cells compared with normal COS-7 cells, 15 which was reflected by the difference in the IC50 16 values of 0.8 µM, 3.0 µM and 4.8 µM, respectively 17 (Fig. S11). Attractively, the scrambled 18 DRG-prodrug showed lower cytotoxicity than 19 RGD-prodrug, which was particularly evident in 20 HeLa cells (Fig. 4B). The result indicated that 21 RGD-mediated cell targeting increased the 22 cellular uptake effectively and therefore enhanced 23 cytotoxicity. 24

3.4 Evaluation of selective tumor targeting of 25 prodrug 26

The RGD-mediated targeting antitumor activity 27 of RGD-prodrug was further intuitively 28 evaluated by confocal microscopy. Confocal 29 images showed a strong fluorescence signal 30 in/around H22 and HeLa cells after the 31 incubation with RGD-prodrug for 20 min (Fig. 5A, 32 C), but pale fluorescence was observed in cells 33 incubated with the scrambled DRG-prodrug for 34 the same time (Fig. 5B, D). On the contrary, 35 COS-7 cells incubated with RGD-prodrug and 36 DRG-prodrug showed almost no fluorescence 37 (Fig. 5F, G). The difference in blue fluorescence 38 intensity suggested that RGD-prodrug could 39 more easily interact with H22 and HeLa cells but 40 scrambled DRG-prodrug failed to capture those 41 cells due to the lack of targeting group. The rapid 42 fluorescent imaging to H22 and HeLa cells 43 indicated the effective targeting of RGD-prodrug 44 to these RGD-receptor over-expressed tumorous 45 cells. Furthermore, the integrin receptor mediated 46 specific targeting was also confirmed by a 47 competition assay. Before the addition of 48 RGD-prodrug, HeLa cells were pre-incubated 49 with excess free peptide for RGD-receptor 50 blocking on cell membranes. Only dim 51 fluorescence was observed in Hela cells even 52 though incubated with RGD-prodrug for 20 min 53 (Fig. 5E), It was ascribed 54

Figure 5 Confocal images of prodrugs against H22, HeLa and COS-7 cells. (A, B) H22 cells incubated with RGD-prodrug and DRG-prodrug, respectively, at a concentration of 12.5 µM for 20 min; (C, D) HeLa cells incubated with RGD-prodrug and DRG-prodrug at the same condition; (E) HeLa cells pre-incubated with 25 µM free peptide for 30 min and incubated with 12.5 µM RGD-prodrug for 20 min; (F, G) COS-7 incubated with RGD-prodrug and DRG-prodrug at a concentration of 12.5 µM for 20 min.


7Nano Res.

to the successful capping of free peptide to the 1 integrin receptors on HeLa cells and the 2 pre-incubation blocked the receptor mediated 3 specific targeting. Those findings indicated that 4 the RGD-prodrug could selectively target 5 tumorous cells with over-expressed integrin 6 receptors. 7

3.5 In vivo antitumor efficiency 8

H22 tumor-bearing mice were used as the animal 9 model to demonstrate the antitumor effect of 10 RGD-prodrug in vivo. As shown in Fig. 6A, mice 11 administrated with RGD-prodrug and 12 DRG-prodrug showed a similar weight increment 13 to the PBS group. In contrast, CPT group showed a 14 largely negative growth in weight, indicating 15 highly non-specific systemic toxicity. Prodrugs 16 decorated with biocompatible peptide functioned 17 mildly on the mice body, suggesting that the 18 introduction of peptide could decrease the side 19 effect. The relevant tumor volume growth tendency 20 was shown in Fig. 6B, in which RGD-prodrug 21 exhibited a significant tumor suppression effect 22 compared with other three groups. The difference 23 in antitumor efficiency was attributed to RGDS 24 peptide, which enhanced the internalization of 25 drug by the tumor [31]. Fig. 6C showed the images 26 of the ultimately sacrificed tumor tissues. It was 27 obvious that tumor volume in RGD-prodrug group 28 was much smaller than that in PBS group, and also 29 smaller than DRG-prodrug and CPT groups. The 30 fluorescence distribution of tumor and organs after 31

32

Figure 6 In vivo antitumor efficiency study. (A) Recorded 33 mice weight over time during the treatment; (B) Relative 34 tumor growth ratio of three groups as a function of time. (C) 35 Images of sacrificed tumor tissues; (D) Fluorescence 36 distribution of prodrugs in tumor and organs. 37

24 h subcutaneously injection was imaged and 38 presented. As shown in Fig. 6D, RGD-prodrug 39 displayed a higher fluorescence intensity 40 distribution in tumor compared with DRG-prodrug, 41 suggesting that RGD-prodrug accumulated in the 42 tumor due to RGDS targeting moiety. Additionally, 43 there was also some fluorescence in the heart, liver, 44 and kidney. This was because the subcutaneously 45 injected materials first entered lymph circulation 46 and then circulated through the whole body [32]. 47 Some aggregated nanofibers were filtered by the 48 heart and liver, resulting in the accumulation and 49 showing a fluorescent signal [33]. The in vivo 50 antitumor experiment demonstrated the well 51 biocompatibility and RGD-mediated selective 52 targeting therapy of self-delivered prodrug. 53

3.6 H&E staining assay 54

Furthermore, tumor tissues and other organs 55 were sliced and stained via the standard 56 hematoxylin and eosin (H&E) method. As shown 57 in Fig. 7, tumor cells were almost killed in mice 58 treated with RGD-prodrug compared to that 59 treated with PBS. As a negative control, there was 60 no obvious tumor necrosis found in histological 61 section of PBS groups. On the contrary, apparent 62 karyolysis and necrotic were observed in free 63 CPT and DRG-prodrug groups. With the 64 RGD-prodrug treatment, there was nearly no 65 intact nucleus survived, which indicated the 66 prodrug displayed a highly tumor suppression 67 effect. Negligible physiological morphology 68 changes of spleen, heart, lung and kidney were 69 found in drug treated groups when 70

71

Figure 7 H&E staining images of tumor tissues and other 72 organs sacrificed at day 14 after treatment of four groups. 73

compared with PBS group. Damage on the liver in 74


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free CPT group was found obviously, since free 1 CPT was a nonspecific anti-tumor drug and easy to 2 accumulate in the liver [34]. In contrast, peptide 3 decorated prodrugs performed a less harmful effect 4 on liver cells although the prodrug would also 5 accumulate in liver , which was attributed to 6 self-delivery modality that nanofibers prevented 7 the drug from contacting with normal organs. The 8 results indicated that the self-delivered 9 RGD-prodrug nanofibers accomplished a 10 selectively and highly antitumor efficiency in living 11 mice. 12

Conclusions 13

In summary, we reported a novel prodrug 14 self-delivered nanosystem for tumor therapy. The 15 prodrug self-assembled into nanofibers, which 16 sequestered active CPT molecules within the 17 hydrophobic cores and therefore reduced the 18 nonspecific toxicity of free drug to normal cells 19 and organs. The self-delivered nanofibers 20 enabled a fixed and high drug loading and 21 achieved CPT delivery in mouse without the 22 need of additional carriers. The in vitro and in 23 vivo results demonstrated that this self-delivery 24 system could effectively inhibit tumor growth 25 based on RGDS tetrapeptide mediated positive 26 targeting and nanoscale-assistant passive 27 delivery. The findings not only suggest that 28 fibrillar nano-architecture as an alternative of 29 traditional spherical nanoparticle can provide a 30 promise carrier for drug delivery, but also give 31 the insight in designing new carrier-free systems 32 for tumor therapy. 33

Acknowledgements 34

This work was supported by the National 35 Natural Science Foundation of China (51125014, 36 51503227 and 51233003) and Natural Science 37 Foundation of Hubei Province of China 38 (2014CFB696 and 2013CFA003). 39

Electronic Supplementary Material: 40 Supplementary material (peptide synthesis and 41 characterization) is available in the online version 42 of this article at 43 http://dx.doi.org/10.1007/s12274-***-****-*. 44

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88

Electronic Supplementary Material

10

Self-delivery of peptide based prodrug for tumor

targeting therapy

Meng-Yun Peng,a,‡ Si-Yong Qin,a,b,‡ Hui-Zhen Jia,a Di-Wei Zheng,a,c Lei Rong,a Xian-Zheng Zhang (*)

a Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072, P. R.

China b School of Chemistry and Materials Science, South-Central University for Nationalities, Wuhan 430074, China c Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials; Key Laboratory for the Green Preparation and

Application of Functional Materials of Ministry of Education, Hubei University, Wuhan, Hubei 430062, P. R. China

‡ These authors contributed equally to this work.

Supporting information to DOI 10.1007/s12274-****-****-* (automatically inserted by the publisher)

11

Figure S1 Structures of the peptide, RGD-prodrug and DRG-prodrug.

12

Figure S2 ESI-MS spectrum of CPT-COOH.

13

Figure S3 1H NMR spectra of CPT and CPT-COOH. 1H NMR (300 MHz; DMSO-d6) for CPT-COOH, δ:

12.28 (1H, Hb), 8.68 (2H), 8.11-8.20 (2H), 7.85-7.89 (1H), 7.69-7.73 (1H), 7.04 (2H), 5.44-5.53 (2H), 5.23-5.33

(2H), 2.68-2.85 (2H), 2.09-2.20 (2H), 0.87-0.94 (3H).

14

Figure S4 ESI-MS spectrum of free peptide.

15

Figure S5 ESI-MS spectrum of RGD-prodrug.

16

Figure S6 ESI-MS spectrum of DRG-prodrug.

17

Figure S7 1H NMR spectra of RGD-prodrug (A) and DRG-prodrug (B). RGD-prodrug: 1H NMR, (300

MHz, DMSO-d6 (ppm)): 7.1-8.7 (-NH-CO-; the aryl hydrogens in CPT), 5.28-5.52 (the methylene

hydrogens in CPT), 4.02, 4.23 and 4.60 (-NH-CH2-CO-; -NH-CH(CH2)3-NH-C(NH)NH2-CO-;

NH-CH(CH2-OH)-CO-), 1.35-3.15 (the hydrogens in the amino acid residues); 1.18-1.26 (the methylene

hydrogens in the alkyl chain); 0.81-0.93 (the methyl hydrogens in the alkyl chain and CPT). DRG-prodrug

shows a similar 1H NMR spectrum with that of RGD-prodrug.

18

Figure S8 The HPLC chromatograms of the RGD-prodrug (A) and DRG-prodrug (B). The purity of the

RGD-prodrug and DRG-prodrug was 94% and 90%, respectively. Prodrug purity was assessed by

analytical reverse-phase HPLC (1 mL/min) with CH3CN/water (1:1).

19

Figure S9 CAC determination of RGD-prodrug (A) and DRG-prodrug (B) using the Nile Red as a hydrophobic

fluorescence probe. Fluorescence intensity of Nile Red changed with the prodrug concentration increasing,

intensity at 725 nm was recorded.

20

Figure S10 Cell cytotoxicities of free CPT (A) and free peptide (B) against H22/HeLa/COS-7 cells. Cell

viability values were estimated by MTT and Cell Counting Kit-8 assay versus incubation with different

concentrations for 48 h.

21

Figure S11 Cell cytotoxicities of RGD-prodrug against H22/HeLa/COS-7 cells. The IC50 values of

H22/HeLa/COS-7 cells were 0.8 µM, 3.0 µM and 4.8 µM, respectively. Cell viability values were estimated by

MTT and Cell Counting Kit-8 assay versus incubation with different concentrations for 48 h.

22

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

23

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).

24

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.

25

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

26

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,

27

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.

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31

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.

32

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.

33

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.

34

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.

35

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).

36

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.

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