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Tailoring minimalist self-assembling peptides for localised viral vector gene delivery

Alexandra L. Rodriguez1, Ting-Yi Wang2, Kiara F. Bruggeman1, Rui Li3, Richard J. Williams4, Clare L. Parish2

(*), and David R. Nisbet1 (*) Nano Res., Just Accepted Manuscript • DOI: 10.1007/s12274-015-0946-0

http://www.thenanoresearch.com on November. 16, 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/s12274-015-0946-0

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Tailoring minimalist self-assembling peptides for localised viral vector gene delivery

Alexandra L Rodriguez1, Ting-Yi Wang2, Kiara F Bruggeman1, Rui Li3, Richard J Williams4,

Clare L Parish2* & David R Nisbet1*

*equal contribution & corresponding authors

Email: david.nisbet@anu.edu.au

1 Research School of Engineering, The Australian National University, Canberra, ACT 2601,

Australia; 2 Florey Institute of Neuroscience & Mental Health, The University of Melbourne,

Parkville, VIC 3010, Australia; 3 Centre for Chemistry and Biotechnology, Deakin University,

Waurn Ponds, VIC 3217, Australia; 4 School of Aerospace, Mechanical and Manufacturing

Engineering and the Health Innovations, Research Institute, RMIT University, Melbourne, VIC

3001, Australia

Keywords: viral vectors, gene therapy, self-assembling peptides, biomaterials

Abstract Viral vector gene delivery is a promising technique for the therapeutic administration of proteins to damaged tissue in order to improve regeneration outcomes in a variety of disease settings including brain and spinal cord injury as well as autoimmune diseases. Though promising results have been demonstrated, limitations of viral vectors, including spread of virus to distant sites, neutralisation by the host immune system and low transduction efficiencies have stimulated the investigation of biomaterials as gene delivery vehicles for improved protein expression at an injury site. Here, we show how N-fluorenylmethyloxycarbonyl (Fmoc) self-assembling peptide (SAP) hydrogels, designed for tissue specific central nervous system (CNS) applications via incorporation of the laminin peptide sequence, isoleucine–lysine–valine–alanine–valine (IKVAV), are effective as biocompatible, localised viral vector gene delivery vehicles in vivo. Through the addition of a C-terminal lysine (K) residue, we show that

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increased electrostatic interactions, provided by the additional amine side-chain, allows the effective immobilisation of lentiviral vector particles, thereby constraining their activity exclusively to the site of injection and enabling focal gene delivery in vivo in a tissue specific manner. When the C-terminal lysine was absent there was no difference between the number of transfected cells, the volume of tissue transfected or the transfection efficiency with and without the Fmoc-SAP. Importantly, immobilisation of the virus only effected transfection cell number and volume with no impact observed on transfection efficiency. This hydrogel allows the sustained and targeted delivery of growth factors post injury. We have established Fmoc-SAPs as a versatile platform for enhanced biomaterial design for a range of tissue engineering applications.

1. Introduction

Viral vector gene delivery is playing a significant role in the development of therapeutic treatments for a variety of diseases including cancer, autoimmune diseases and neurodegenerative diseases [1, 2]. In particular, with limited capacity for self-repair in the brain and spinal cord, the delivery of neurotrophins to damaged CNS has shown significant potential in delaying disease progression and promoting regeneration [3, 4]. Neurotrophic support provided to cells in vivo by various growth factors and cell signalling molecules (such as brain-derived neutrophic factor [5], nerve growth factor [6] and glial cell-derived neurotrophic factor [7]) can slow progressive neurodegeneration in disorders including Parkinson’s disease and motor neuron disease. Evidence suggests that this approach can also limit the size of the injured area by protecting the surrounding parenchyma from secondary degeneration in acute insults such as stroke or traumatic brain injury, thereby improving the overall capacity for CNS regeneration [3]. Current strategies in growth factor delivery, however, are challenged by their short half-life, difficulty in penetrating the blood brain barrier and susceptibility to enzyme degradation; thereby requiring high concentrations of growth factors to achieve any therapeutic benefit [8]. These limitations make the delivery of soluble growth factors, typically by invasive and cumbersome infusion kits, both inefficient and expensive [9]. As a result, viral vector gene delivery has been investigated as a means to achieve long-term delivery of neurotrophic factors at a site of injury through the introduction of DNA into cells for localised protein expression [10-14]. This overcomes the challenges presented by the delivery of soluble proteins, providing a targeted source of growth factors for the neuroprotection of cells in and surrounding the injury site. Though there has been success in delivering genetic material for targeted therapeutic protein expression using viral vectors [3], there is a need to improve on this technology with a localised and efficient gene delivery method for future clinical applications [1, 15]. For this reason, the use of biomaterials as sophisticated tools to overcome these issues associated with viral vectors has acquired significant attention [1]. Rationally designed biomaterials are powerful tools for regenerative medicine [16], primarily as adjuvant scaffolds that mimic the physical and biochemical properties of the extracellular microenvironment [17]; such as those required to support neural cells for CNS repair [18]. Furthermore, they can be engineered to incorporate viral vectors for delivery of genetic material through encapsulation methods, including the use of nanoparticles and microspheres [19, 20], and/or attached via chemical functionalisation [21, 22]. The covalent and/or non-covalent attachment of viral vectors [23-25] encourages binding of the vector to the material in order to limit vector spread, shield the vector from neutralisation by the host immune system and increase transduction efficiency [1]. Previous research has demonstrated that amine (-NH2) moieties can enhance virus adsorption and cell attachment to the biomaterial surface, resulting in improved transduction efficiency [26]. This was attributed to the presence of positively charged amine (-NH3

+) moieties reducing the electrostatic repulsion between the negatively charged membranes on the viral capsid and cell surface, enhancing the ability of the virus to attach to the cell membrane and deliver its genetic material [26]. Such examples highlight the utility of biomaterials as viral vector delivery vehicles. Recently, SAPs have also been demonstrated to effectively deliver genetic material to human mesenchymal stem cells in culture via recombinant adeno-associated virus [27]. However, the RADA16 SAP [28] used present no biofunctionality, nor any capacity to immobilise virus for controlled release. Here, we design a multifunctional biomaterial for both integrated tissue specificity and superior vector delivery ensuring improved overall tissue regenerative outcomes. Fmoc-peptides are a class of SAPs known to self-assemble through a robust mechanism driven by the intermolecular sharing of π electrons between Fmoc protecting groups, and stabilised by the anti-parallel β-sheet interactions of the protected peptide sequence; a mechanism known as π-β self-assembly [29]. These assemblies take the form of hollow fibrils, ~10-30 nm in diameter and microns in length, that orientate themselves longitudinally into a network of interconnected bundles, forming a structural mimetic of the extracellular matrix (ECM) [30]. We have shown that the mechanical properties of these Fmoc-SAPs can be easily tailored for tissue specific applications through adjustment of the rate of assembly as well as the ionic concentration, without disturbing the nanofibrous morphology [31]. The biocompatibility of Fmoc-SAPs has been demonstrated across a range of sequences, and we have shown that optimal biocompatibility and functionality of the Fmoc-SAPs requires the incorporation of bioactive peptide sequences [30, 32-34]. Previously, in a demonstration of the utility of these materials in a neural context, we incorporated the laminin-based IKVAV sequence (known to promote neuron survival, neural differentiation and neurite extension [35]) into an Fmoc-SAP system as was done here [36] and validated its performance in vivo [34]. This capacity to concomitantly present a bioactive peptide sequence at high density for directing cell behaviour, whilst also providing structural support through the self-assembled matrix, make Fmoc-SAP scaffolds a highly sophisticated class of biomaterials, ideal for tissue engineering in the CNS.

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In this study, we hypothesised that these scaffolds could be used to non-specifically immobilise viral vectors via reversible, non-covalent interactions between the SAP scaffold and the viral membrane by taking advantage of the chemical flexibility afforded via careful selection of the amino-acids within the peptide sequence. As proof of principle, we used the previously designed Fmoc-DDIKVAV to deliver mCherry lentivirus in vivo, investigating its capacity for localised viral vector gene delivery. We subsequently improved on this design with the development of a second Fmoc-SAP, Fmoc-DDIKVAVK, engineered for the high-density presentation of -NH2 functional groups at the C-terminus of the peptide. We show that by increasing the electrostatic interactions available for binding, we can enable focal gene delivery by containing viral activity to the site of the hydrogel injection. Such technology could hold significant potential for the sustained and targeted delivery of growth factors to aide in tissue repair.

2. Materials and methods 2.1 Solid-phase peptide synthesis Fmoc-SAPs were synthesised manually by solid phase peptide synthesis (SPPS) as previously described [34]. SPPS was performed in a rotating glass reactor vessel at 0.4 mmol scale. Fmoc protected amino acids, Hydroxybenzotriazole (HOBt), O-Benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate (HBTU) and Wang based resins were purchased from GL Biochem (China). All other chemicals were purchased from Sigma-Aldrich. 2.2 Fmoc-self-assembling peptide hydrogel preparation To prepare the Fmoc-SAP hydrogels, 10 mg of peptide was dissolved in 100 µL deionised water and 75 µL 0.5 M sodium hydroxide (NaOH). 0.1 M hydrochloric acid (HCl) was then used to reduce the pH of the peptide solution. HCl was added dropwise until the solution reached ~ pH 7.4 and formed a self-supporting hydrogel. The final volume of HCl added varied between 100 and 150 µL. Finally, the gel was made up to a final concentration of 20 mg/mL by addition of phosphate buffered saline (PBS). The solution was vortexed throughout the entire procedure. 2.3 Transmission electron microscopy Transmission electron microscopy (TEM) images were obtained using a HITACHI HA7100 TEM. A LaB6 filament at 100kV was used. Negative stains were performed to image the Fmoc-SAP samples as previously described [34]. 2.4 Atomic force microscopy Atomic force microscopy (AFM) was performed using a Multimolde 8 (Bruker BioSciences Corporation, USA) operated in peak force QNM with a scan size of 10 µm. Highly ordered pyrolytic graphite (HOPG) substrates (SPI, USA) were used for sample preparation. 15 µL of Fmoc-SAP hydrogel was applied on the HOPG substrates. Scanasyst-air probes with silicon tip on nitride lever (Bruker BioSciences Corporation, USA) were used. 2.5 Fourier transform infrared spectroscopy For Fourier transform infrared spectroscopy (FTIR), 30 µL of Fmoc-SAP hydrogel sample at 20 mg/mL was placed on the single reflection diamond. An Alpha Platinum Attenuated Total Reflectance FTIR (Bruker Optics) was used to obtain scans from 1550 – 1750 cm-1, corresponding to the amide I region. A background scan of deionised water was subtracted from the hydrogel data. 2.6 Circular dichroism Fmoc-SAP hydrogels, prepared as described above at 20 mg/mL, were diluted at 1:200 in deionised water for circular dichroism (CD). Approximately 400 µL was placed in the cuvette. Deionised water was used as the background scan and subtracted from the hydrogel CD data. All scans were taken from 350 to 170 nm at a step size of 1 and 1 nm bandwidth using a Chirascan CD Spectrometer (Applied Photophysics Limited). 2.7 Rheology A Kinexus Pro+ Rheometer (Malvern) was used to assess the viscoelastic properties of the previously prepared Fmoc-SAP hydrogels at 20 mg/mL with a 20 mm rough flat plate and solvent trap geometry. Approximately 200 µL of the hydrogel sample was placed on the geometry. A frequency sweep was performed from 0.1 – 100 Hz using an oscillatory strain of 0.1 % at a 0.2 mm gap size. 2.8 Determination of lentiviral release and immobilisation To determine successful immobilisation of the mCherry lentivirus, release of mCherry lentivirus from the two Fmoc-SAP hydrogels was compared. 100 µL samples of Fmoc-SAP hydrogels at 20 mg/mL were mixed with 1 µL of mCherry lentivirus (titre at 9.28 x 1010 copies/mL; GeneCopoeia) and placed in a 96 well plate. 200 µL of PBS was placed on top of the gel and the plate placed in an incubator (37 °C, 5 % CO2). The PBS was then collected and replaced with fresh PBS every 24 hours for 5 days. The PBS supernatant was stored in a freezer until ready for analysis by an enzyme linked immunosorbent assay (ELISA). A HIV Type 1 p24 Antigen ELISA 2.0 kit (ZeptoMetrix) was used to measure the amount of mCherry released. The ELISA was performed as per the manufacturer’s instructions. 2.9 Implantation of m-Cherry virus with Fmoc-self-assembling peptide

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The Florey Institute of Neuroscience and Mental Health animal ethics committee approved all animal experiments with procedures conducted in accordance with the Australian National Health and Medical Research Council’s published Code of Practice for the Use of Animals in Research. Adult Swiss mice were housed on a 12 h light/dark cycle with ad libitum access to food and water. Mice received stereotaxic implants of mCherry lentivirus alone (n = 6) or mCherry virus in the presence of Fmoc-DDIKVAV (n = 6) or Fmoc-DDIKVAVK (n = 6). 5 % isoflurane was used to induce anaesthesia. 2 % isoflurane was maintained for the duration of surgery. Mice were placed into a stereotaxic frame and a craniotomy performed for microinjections into the underlying striatum (co-ordinates: anterior +1.0 mm and lateral -2.3 mm, relative to Bregma). A fine pulled micropipette coupled to a Hamilton syringe was used to inject virus or virus with Fmoc-SAP at a depth of 3.2 mm below the dura surface. For control animals, 1 µL of mCherry (109 UI/ml) was diluted in 1 µL sterile PBS and the total 2 µL suspension implanted. Alternatively, the virus was mixed at a 1:1 ratio with either of the Fmoc-SAPs. After 21 days, mice were killed by an overdose of sodium pentobarbitone (100 mg/kg) and transcardially perfused with warm saline followed by 4 % paraformaldehyde (PFA). After removal, mouse brains were post-fixed for 2 hours in 4 % PFA. Following fixation, brains were cryo-preserved in a 30 % sucrose solution overnight. The brains were coronally sectioned (40 µm at a 1:12 series) and immunohistochemistry against red fluorescent protein (rabbit anti-RFP, 1:1000, Rockland, USA) was performed to amplify the mCherry signal, using previously described methods [37]. Cell counts for mCherry virally infected cells (mCherry+), transduction volume and density measurements were carried out on a Leica microscope (Leica CTR6000) using LAS software. One-way ANOVAs with Tukey post-hoc tests were used to identify statistically significant changes between groups. Statistical significance was set at P < 0.05. Data represents mean + standard error of the mean (SEM).

3. Results and discussion 3.1 Materials characterisation of the lysine functionalised self-assembling peptide An initial titration was carried out to investigate any shifts in the apparent pKa of the newly derived Fmoc-DDIKVAVK resulting from the addition of a K residue at the C-terminus, and the effect this could have on the pH of gelation (Figure 1a) [36]. Although the addition of the K residue was observed to shift the pKa (from ~7.0 to ~7.8), it indicated that self-assembly of this SAP could take place under physiological conditions (pH ~7.4). This was confirmed when both Fmoc-DDIKVAV and Fmoc-DDIKVAVK formed clear, self-supporting hydrogels at pH ~7.4 using the pH switch method previously described (Figure 1b) [34].

Figure 1. Fmoc-DDIKVAVK forms a hydrogel under physiological conditions. a, Titration curve

for Fmoc-DDIKVAV (open circles) and Fmoc-DDIKVAVK (closed circles) showing a slight

shift in pKa (Fmoc-DDIKVAV – dashed line; Fmoc-DDIKVAVK – solid line) that correlates to

the expected pH range of self-assembly. b, An inversion test demonstrates the gelation of the two

Fmoc-SAPs using a pH switch. Rheological analysis for c, Fmoc-DDIKVAV and d,

Fmoc-DDIKVAVK. Closed circles = G’; open circles = G”.

The mechanical properties of both hydrogels were compared using parallel plate rheometry. Here, both gels demonstrated viscoelastic properties characteristic of previously described Fmoc-SAP hydrogels, where the storage modulus (G’) is greater than the loss modulus (G”) and shows only a weak dependence on frequency (Figures 1c & d) [36]. Fmoc-DDIKVAVK appeared to be a slightly stronger material than Fmoc-DDIKVAV (G’ = 10 500 Pa and G’ = 6500 Pa respectively); most likely the consequence of increased electrostatic interactions. The higher pKa Fmoc-DDIKVAVK required more HCl addition to achieve gelation at physiological pH. The positively charged lysine groups attract negatively charged chloride ions from solution, which themselves attract further lysine groups attached to other fibrils. This increased electrostatic interaction enables stronger supramolecular interactions between the aligned fibrils, increasing the effective points of entanglement and resulting in a more rigid nanofibrous network [31]. AFM demonstrated a microscale network was formed by both hydrogels, confirming that although the addition of a K residue shifted the pKa slightly, self-assembly of the nanofibrous structures that underpinned the hydrogel was maintained at physiological pH (Figures 2a & d). This modification in supramolecular ordering was further visualised using TEM, with Fmoc-DDIKVAVK exhibiting a higher degree of alignment of the nanofibres compared to the more branched arrangement of Fmoc-DDIKVAV (Figures 2b & e).

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Figure 2. Microscopy images of Fmoc-SAPs show the nanofibrous structure of the gels, with

spectroscopy confirming that the underlying π-β self-assembly mechanism is maintained with the

addition of a K residue. A cumulative release profile confirms lentivirus immobilisation in the

presence of K. Fmoc-DDIKVAV nanofibrous network shown in a, AFM image and b, TEM

image. c, TEM image of lentivirus (green) embedded within the branched arrangement of the

individual fibres, inset shows lentivirus alone. d-f, Corresponding images for Fmoc-DDIKVAVK.

Scale for AFM = 1 µm, TEM = 200 nm. g, FTIR and h, CD for Fmoc-DDIKVAV (solid line)

and Fmoc-DDIKVAVK (dashed line). i, A cumulative release profile for Fmoc-DDIKVAVK

(closed circles) and Fmoc-DDIKVAV (open circles) over 5 days.

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Finally, the effect of the addition of a K residue on the π-β assembly mechanism was characterised spectroscopically, using FTIR and CD. The FTIR spectra showed major peaks at 1630 cm-1 and minor peaks at 1690 cm-1, representative of anti-parallel β-sheets formed by hydrogen bonding between the peptides sequences (Figure 2g) [36]. The CD spectra further supported the FTIR data, with a transition at 200 nm indicative of β-sheets in both Fmoc-DDIKVAV and Fmoc-DDIKVAVK (Figure 2h) [29]. Importantly, the wavelength of the minima was not affected but increased in magnitude, indicating that the addition of the K residue was not affecting the intermolecular π-β interactions, but was promoting increased supramolecular ordering of the fibrils, as observed by TEM and AFM [36].

3.2 Assessment of virus retention in self-assembling hydrogels Satisfied that the additional K residue was not adversely impinging on the assembly process, we proceeded to evaluate the material as a viral vector gene delivery vehicle. We blended lentiviruses into preformed Fmoc-SAP hydrogels, and subsequently allowed them to reform gels at room temperature. Initial TEM images showed the lentiviral particles (80 – 100 nm in diameter) visibly embedded within the nanofibrous assembly of both Fmoc-SAPs (Figures 2c & f). To investigate the retention of the lentiviral particles within the Fmoc-SAP scaffolds, and whether increased retention was induced by the additional positively charged residue, the release of the virus from the gels (in a static, physiological environment) was measured using a HIV-1 p24 antigen ELISA. Measured absorbance values were interpolated using a calibration curve of standard concentrations within the detection limits of the instrument and data above or below these limits were approximated as the minimum or maximum of the detection range respectively. Fmoc-SAP hydrogels (100 µL) were again mixed with the lentivirus and placed in a 96-well plate with 200 µL of PBS placed on top of each gel-virus sample. The plate was left in an incubator (37 °C, 5 % CO2) and the 200 µL phosphate buffered saline (PBS) removed and replaced every 24 hours for 5 days. The isolated supernatant at each time point was analysed with ELISA. The results showed an increase in lentiviral particle retention in the K-functionalised peptide hydrogel compared to Fmoc-DDIKVAV over 5 days, as revealed by the reduced detection of HIV-1 p24 (viral particles) in the supernatant, (Figure 2i). This indicates that the addition of a positively charged K residue resulted in effective immobilisation of the majority of the viral particles within the Fmoc-DDIKVAVK hydrogel (Figure 3). This is important for the broad applicability of this method. Here we have used a lentiviral vector as proof of concept, however, immobilisation via electrostatic interactions with the negatively charged viral capsule could be ultilised for any viral capsule such as adeno- and retroviruses.

Figure 3. Charge-based immobilisation of lentivirus within Fmoc self-assembling peptides is achieved through peptide functionalisation with a K residue. When a K residue is added to the laminin based self-assembling peptide sequence, Fmoc-DDIKVAV, the positive charges from the –NH2 side groups immobilise the lentiviral vector via electrostatic interactions. This results in retention of the vector within the nanofibrous Fmoc-SAP network resulting in localised gene delivery to cells at the injection site. When no K is present, the virus is released from the Fmoc-SAP hydrogel. (TEM scale bar = 200 nm)

3.3 In vivo assessment of self-assembling hydrogels as viral vector gene delivery vehicles Upon confirmation of viral vector immobilisation in Fmoc-DDIKVAVK hydrogels, we investigated the capacity for the Fmoc-SAP to localise viral vector delivery in vivo. mCherry lentivirus was loaded into the Fmoc-DDIKVAV and Fmoc-DDIKVAVK hydrogels, prior to stereotaxic injection into the mouse brain (striatum) and compared to delivery of virus alone. The Fmoc-SAP hydrogels were injected at a concentration of 10 mg/mL, and the amount of viral vector mediated transfection was assessed after three weeks. A cell count of transduced (mCherry+) cells showed that delivery of the lentivirus in the absence of the Fmoc-SAPs resulted in the highest number of transduced cells with a statistically significant difference in the level of transduction between the Fmoc-DDIKVAVK and both other groups (Figure 4a).

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Figure 4. a, Focal delivery of the mCherry lentivirus within a Fmoc-DDIKVAVK gel

significantly reduced the number of mCherry+ in the host striatum. b, Similarly, the volume of

mCherry+ cells within the striatum was reduced with the addition of a K residue demonstrating

the ability of Fmoc-DDIKVAVK to immobilise the lentivirus and subsequently localise delivery

of genetic material. c, Consequently, the density of the mCherry+ cells was similar across all

groups, demonstrating that the transduction efficiency of the delivered virus was not

compromised by the presence of the Fmoc-SAPs. Data represents mean + SEM. *P < 0.05, **P <

0.01. Chromogenic stain of mCherry+ cells in the mouse brain, illustrating the transduction

efficiency following d, direct viral vector delivery, e, viral vector delivery with Fmoc-DDIKVAV

and f, viral vector delivery with Fmoc-DDIKVAVK. Note the reduced volume of transduction

with the addition of a K residue (5 x magnifcation) d’- f’, high power (20 x) images of d-f,

illustrating transduced mCherry.

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Additionally, a difference in volume of the mCherry+ area was observed with Fmoc-DDIKVAVK having the least mCherry coverage (Figure 4b). There was no significant difference observed in the density of mCherry+ cells at the site of delivery between the three groups (Figure 4c). Importantly, this indicates that the capacity for transfection by the mCherry lentivirus was not compromised by the Fmoc-SAP hydrogels but simply achieved localised DNA delivery to the microenvironment surrounding the material without affecting the tropism of the virus. In particular, the introduction of a K residue resulted in the most focal delivery due to immobilisation of the mCherry virus (Figure 4d). This is noteworthy, as we have enhanced the properties of this sophisticated minimalist Fmoc-SAP system for our desired application in viral vector gene delivery for the CNS. The development of this adjuvant scaffold now offers the potential to deliver biologically relevant DNA (via viral vectors within SAPs) directly at the edge of a lesion (for example post traumatic brain injury or stroke) to enhance cell survival within the penumbra, reducing its size and encouraging reinnervation. Delivery within the Fmoc-SAP hydrogel will also avoid neurite misrouting, antibody neurtralisation and immune responses that increase as a result vector diffusion, as our scaffold will protect the virus from the host immune response through localised delivery. In addition to the Fmoc-SAP’s ability to support cells through its nanofibrous architecture, provide high-density presentation of bioactive peptides to direct cell behaviour and capacity for injection into a void, making intimate contact with the surrounding tissue, the designed hydrogel can now limit the spread of the viral vector to non-target tissue and deliver the desired genetic material to cells, providing long-term protein expression for therapeutic benefit. 4. Conclusion Though viral vectors have emerged as useful tools for the delivery of genetic material, there is a need for a delivery mechanism that will shield the viral vector from the host immune system, improving transduction efficiency, and achieve localised long-term therapeutic protein expression. In this work, we established the suitability of Fmoc-SAPs to focally deliver a viral vector into the brain without compromising the infectivity of the virus. The addition of a K residue demonstrated that the additional -NH2 groups resulted in immobilisation of the virus within the material, achieving localised transduction of the cells surrounding the injected Fmoc-SAP. With the added versatility of tissue specification through the high-density presentation of the laminin based IKVAV sequence, such a material holds exciting promise as a valuable tool for continuing advances in gene therapy, particularly viral vector gene delivery for tissue engineering of the CNS.

Acknowledgements

Access to the facilities of the Centre for Advanced Microscopy (CAM) with funding through the

Australian Microscopy and Microanalysis Research Facility (AMMRF) is gratefully

acknowledged. We acknowledge NanoScope – Scientific Graphics and Illustration for kindly

designing the virus immobilization schematic in Figure 3. We would also like to thank Conor

Horgan, Francesca Maclean and Anitha Parnneerselvan for thorough proof reading. Funding for

this research was obtained from the National Health and Medical Research Council, Australia

(NHMRC, APP1050684), and the Australian Research Council (ARC, DP130103131). ALR was

supported by an Australian Postgraduate Award; RJW was funded via an Alfred Deakin Research

Fellowship; CLP was supported by Senior Medical Research Fellowship provided by the Viertel

charitable Foundation, Australia; DRN was supported by an ARC Australian Postdoctoral

Fellowship, followed by an NHMRC Career Development Fellowship. The Florey Institute of

Neuroscience and Mental Health acknowledges the support from the Victorian Government’s

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Operational Infrastructure Support Grant. DR Nisbet and CL Parish contributed equally to the

work.

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16

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,

21

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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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.

27

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.