new approach for the treatment of cll using … · 2015-11-02 · deliver the therapeutic agents to...

New approach for the treatment of CLL using chlorambucil/hydroxychloroquine-loaded anti-CD20 nanoparticles

Sara Capolla1,§ (*), Nelly Mezzaroba1,§, Sonia Zorzet1, Claudio Tripodo2, Ramiro Mendoza-Maldonado3, Marilena Granzotto4, Francesca Vita1, Ruben Spretz5, Gustavo Larsen5,6, Sandra Noriega5, Eduardo Mansilla7, Michele Dal Bo8, Valter Gattei8, Gabriele Pozzato4, Luis Núñez5,6, and Paolo Macor1,9 (*) Nano Res., Just Accepted Manuscript • DOI: 10.1007/s12274-015-0935-3

http://www.thenanoresearch.com on November. 2, 2015

© Tsinghua University Press 2015

Just Accepted

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TABLE OF CONTENTS (TOC)

Nano Research DOI 10.1007/s12274-015-0935-3

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

64 Nano Res.

New approach for the Treatment of CLL using

Chlorambucil/Hydroxychloroquine-loaded

anti-CD20 Nanoparticles.

Sara Capolla1§*, Nelly Mezzaroba1§ , Sonia Zorzet1,

Claudio Tripodo2, Ramiro Mendoza-Maldonado3,

Marilena Granzotto4, Francesca Vita1, Ruben Spretz5,

Gustavo Larsen5,6, Sandra Noriega5, Eduardo Mansilla7,

Michele Dal Bo8, Valter Gattei8, Gabriele Pozzato4, Luis

Núñez5,6 and Paolo Macor1,9*

1University of Trieste, Italy; 2University of Palermo,

Italy; 3Molecular Oncology Unit, National Laboratory

Consorzio Interuniversitatio per le Biotecnologie (CIB),

Italy; 4University of Trieste, Italy; 5LNK Chemsolutions

LLC, USA; 6Bio-Target Inc., USA; 7Centro Ùnico

Coordinador de Ablacion e Implante Provincia de

Buenos Aires (C.U.C.A.I.B.A.), Argentina; 8Clinical and

Experimental Onco-Hematology Unit, Centro di

Riferimento Oncologico, Istituto di Ricerca e Cura a

Carattere Scientifico (I.R.C.C.S.), Italy; and 9Callerio

Foundation Onlus, Institutes of Biological Researches,

Italy.

We reported a nanoplatform based on the use of biodegradable

chemotherapeutic-loaded immune-nanoparticles for the treatment of

Chronic Lymphocytic Leukemia. Chemotherapeutic-loaded

nanoparticles were specifically targeted inside leukemic cells by an

anti-CD20 antibody thus improving the survival of leukemia-bearing

mice in respect to the same amount of free drugs.

Paolo Macor, https://dsv.units.it/en/research/researchareas/researchgroups/18231

Gustavo Larsen, www.lnkchemsolutions.com

Luis Núñez, www.biotarget-ln.com

New Approach for the Treatment of CLL using Chlorambucil/Hydroxychloroquine-loaded anti-CD20 Nanoparticles

Sara Capolla1§(*), Nelly Mezzaroba1§, Sonia Zorzet1, Claudio Tripodo2, Ramiro Mendoza-Maldonado3, Marilena Granzotto4, Francesca Vita1, Ruben Spretz5, Gustavo Larsen5,6, Sandra Noriega5, Eduardo Mansilla7, Michele Dal Bo8, Valter Gattei8, Gabriele Pozzato4, Luis Núñez5,6 and Paolo Macor1,9(*) 1Department of Life Sciences, University of Trieste, Trieste, Italy 2Department of Human Pathology, University of Palermo, Italy 3Molecular Oncology Unit, National Laboratory Consorzio Interuniversitatio per le Biotecnologie (CIB), Trieste, Italy 4Dipartimento Universitario Clinico di Scienze mediche, Chirurgiche e della Salute, University of Trieste, Trieste, Italy 5LNK Chemsolutions LLC, Lincoln, NE 68521, USA 6Bio-Target Inc., Chicago, IL, USA; 7Centro Ùnico Coordinador de Ablacion e Implante Provincia de Buenos Aires (C.U.C.A.I.B.A.), Ministry of Health, La Plata, Buenos Aires, Argentina 8Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico, Istituto di Ricerca e Cura a Carattere Scientifico (I.R.C.C.S.), Aviano, Italy 9Callerio Foundation Onlus, Institutes of Biological Researches, Trieste, Italy. § These authors contributed equally to this work.

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

© Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2014

KEYWORDS Chronic lymphocytic leukemia, immune targeted nanoparticles, treatment, xenograft model.

ABSTRACT Current approaches for the treatment of chronic lymphocytic leukemia (CLL) have greatly improved the prognosis for survival, but some patients remain refractive to these therapeutic regimens. Hence, there is an urgent need for novel therapeutic strategies for difficult-to-treat leukemia cases, in addition to reducing the long-term side-effects impact of therapeutics for all leukemia patients. Due to the cytotoxicity of drugs, currently the major challenge is to deliver the therapeutic agents to neoplastic cells while preserving the viability of non-malignant cells. In this contribution, we propose a therapeutic approach in which high doses of hydroxychloroquine and chlorambucil were loaded into biodegradable polymeric nanoparticles coated with an anti-CD20 antibody. We firstly demonstrated nanoparticles’ ability to target and internalize in tumor B-cells. Moreover, these nanoparticles were able to kill not only p53 mutated/deleted leukemia cell lines expressing a low amount of CD20, but also circulating primary cells purified from chronic lymphocytic leukemia patients. Their safety was demonstrated in healthy mice, and their therapeutic effects in a new model of aggressive leukemia. These results demonstrated that anti-CD20 nanoparticles containing hydroxychloroquine and chlorambucil can be effective in controlling aggressive leukemia and provided a rationale for adopting this approach for the treatment of other B-cell disorders.

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


2 Nano Res.

1 Introduction

Chronic Lymphocytic Leukemia (CLL) is a heterogeneous disease with highly variable clinical courses and survivals ranging from months to decades. In particular, a subset of patients is affected by a high-risk CLL form that rapidly progresses and develops a disease that requires symptomatic treatment [1]. Over-represented in this group are patients bearing mutations/deletion of the TP53 gene [2]. Moreover, a high-risk CLL patient fraction was confirmed to carry mutations/deletion of other genes, such as NOTCH1, BIRC3 or SF3B1 [3–7]. For years, the standard therapy was based on the use of alkylating agents, with not much (if any) effects on the CLL natural history. The introduction of fludarabine signified an important breakthrough in CLL therapy. The use of monoclonal antibodies (anti-CD20, anti-CD52 and beyond) opened a new perspective overcoming the paradigm to treat only patients with minimal complications and, alone or in combination with chemotherapy, these therapeutics increased significantly the overall survival of the patients [8, 9]. More recently, inhibitors of B-cell receptor signaling showed durable efficacy in a subset of CLL patients [9–11]. Despite combined therapy advancements, CLL remains an incurable disease in most cases since molecular complete remission is unachievable and, as a consequence, the disease relapses invariably after some months or years. In particular, the small subgroup of patients, known as ultra high-risk CLL, shows poor response to chemo-immunotherapy and have a life expectancy of less than 2 to 3 years with conventional regimens [10, 12]. These considerations indicate that new therapeutic approaches are needed to obtain the complete recovery or at least to improve survival of CLL patients. Since most patients are older in age and often have several co-morbidities, any new treatment approaches, in addition to higher efficacy, must be non-toxic to organs and

tissues. The development of nanoparticles, made with biodegradable biopolymers, and loaded with chemotherapeutic agents, is an attractive method to target neoplastic cells [13–15]. In fact, nanoparticles can be designed by attaching specific antibodies on their surface thus they are able to recognize tumor-associated antigens and induce specific homing on the neoplastic cell surface [16, 17]. Therefore, the efficacy of high-dose chemotherapy is associated to the specificity and the low side effects of antibody-based therapy and protective nature of polymeric encapsulated chemotherapeutics. On these principal characteristics, we developed biodegradable nanoparticles (BNPs) coated with an anti-CD20 antibody to target neoplastic B-cells, and loaded with hydroxychloroquine (HCQ) and chlorambucil (CLB) to specifically kill tumor B-cells [18, 19]. For the first time, we demonstrated the safety and therapeutic effects of targeted nanoparticles in a new leukemia xenograft SCID mice model.

2 Experimental

2.1 Cells, antibodies and sera

The CLL-like cell line MEC1 [20] (kindly provided by prof. Josee Golay), carrying both a TP53 mutation (i.e. c.422insC) and the 17p13 deletion, was cultured in RPMI-1640 medium (Sigma-Aldrich, Milan, Italy) supplemented with 10% fetal bovine serum (FBS; GE Healthcare Milan, Italy). Heparinized peripheral blood samples were obtained after written informed consent from untreated CLL patients at the University Hospital in Trieste (B-cells more than 90% of total circulating cells). The study was approved by the IRB of the CRO (IRCCS) of Aviano (IRB-06-2010). The mononuclear cell fractions were isolated by centrifugation on Ficoll-Hypaque (GE Healthcare) density gradients [21]. MEC1 cells were suspended in serum-free RPMI-1640 medium and stained with


3Nano Res.

VybrantTMDiO cell-labeling solution (GE Healthcare) as previously reported [22]. The anti-CD20 chimeric antibody Rituximab (Roche, Milan, Italy) was derived from the clinic (University of Trieste, Italy). The mouse mAb to CD20 and anti-PARP1 antibody were purchased from BioLegend (San Diego, CA) and Bethyl Laboratories, respectively. For the immunophenotypical characterization, anti-human CD5 PE (Immunotools, Friesoythe, Germany), anti-human CD20 (clone L26, Novacastra), anti-human CD45 APC (Invitrogen, Milan, Italy) and anti-human CD19 TC (GE Healthcare) mAbs were used. Anti-LC3, anti-a-tubulin mAbs and all the secondary antibodies were purchased from Sigma-Aldrich (Milan, Italy) or Aczon (Monte San Pietro, Bologna, Italy). Human sera from AB Rh+ blood donors (NHS - normal human serum) were kindly provided by the Blood Transfusion Center (Trieste, Italy) as a source of complement (NHS - normal human serum).

2.2 BNPs preparation

BNPs preparation was performed using chemicals reagent grade or better. Polyethylene glycol (PEG) was purchased from Nektar, San Carlos, CA; hydroxychloroquine sulfate (HCQ) and chlorambucil (CLB) were purchased from ACROS, Gel Belgium and Sigma Aldrich (St Louis, MO), respectively. BNPs, based on carboxylic acid terminated biodegradable polymers (PLA-b-PEG-COOH and PCL-COOH), were prepared with average diameter of 250nm measured by dynamic light scattering (data not shown) in an under class 100 clean room conditions by implementing Bio-Target’s technology at LNK Chemsolutions, LLC laboratories [19, 23]. All BNPs (final concentration of 900µg/ml) were resuspended in PBS buffer (pH=7.4) with 10% BSA. BNPs were diluted in serum-free RPMI-1640 medium and stained with fluorescein isothiocyanate isomer I (Sigma-Aldrich) or FluoroLinkTM Cy5.5 Monofunctional Dye (GE Healthcare).

2.3 Animals

Female SCID mice (4–6 weeks of age) were provided by Charles River (Milan, Italy) and maintained under pathogen-free conditions. Animals were pretreated with cyclophosphamide (200mg/kg), inoculated subcutaneously with 107 MEC1 cells or intravenously with 5x105 MEC1 cells after 24 hours and examined twice weekly up to 125 days. C57/BL mice were obtained from the animal house of the University of Trieste. All the experimental procedures involving animals were done in compliance with the guidelines of the European and the Italian laws and were approved by the Italian Ministry of Health as well as by the Administration of the University Animal House (Prot. 42/2012).

2.4 Cytometric analysis

BNPs’ binding was assayed incubating 10µL of BNPs with 5x105 cells MEC1 cells for 1h at 37°C. MEC1 localization in mouse blood was performed using anti-CD45 APC and anti-CD19 TC antibodies at 28 days after cells’ injection. For these measurements 30000 events were acquired using FACSCalibur (Becton Dickinson, San Jose, CA) flow cytometer and data were analyzed by CELLQuest software (Becton Dickinson) [24].

2.5 Transmission electron microscopy analysis

Samples were fixed for 1h in a solution of 2% glutaraldehyde (Serva, Heidelberg, Germany) in 0.1M cacodylate buffer (pH=7.3) containing 0.03M CaCl2, rinsed three times (10min each wash) and postfixed in 1% osmium tetroxide for 1h at 4°C. Samples were then dehydrated in ascending ethanols to 100 % ethanol and embedded in Dow Epoxy Resin (DER 332; Unione Chimica Europea, Milan, Italy) and DER732 (Serva), as previously described by Zabucchi et al [25]. Ultrathin sections were cut by an ultratome Leica Ultracut UCT8 (Leica, Wirn, Austria), double stained with uranyl acetate and lead citrate and observed in a transmission electron microscope (EM 208, Philips, Eindhoven, The Netherlands). Micrographs were taken with a Morada Camera (Olympus, Munster, Germany).


4 Nano Res.

2.6 Cell viability, apoptosis and autophagy

To investigate the ability of BNPs to affect cell viability, MEC1 cells (2x105) were incubated with different concentrations of BNPs for 48h at 37°C (in a humidified 37°C, 5% CO2 incubator). The amount of residual viable cells was determined by MTT assay [26] and the percentage of dead cells was calculated as: 100 x [(test release – spontaneous release)/(total release – spontaneous release)]. Apoptosis of patient’s B cells was measured using FITC-labeled recombinant human Annexin V assay (Apoptosis detection kit, Immunostep, Spain) according to the manufacturer’s instructions. For each measurement 30000 events were acquired with a standard FACSCalibur (Becton Dickinson) flow cytometer and analysis of data was performed with CellQuest (Becton Dickinson). PARP-1 and LC-3 activation were evaluated via immunoblotting to study apoptosis induction and autophagy impairment, respectively [27].

2.7 Complement-mediated lysis

A previously described procedure of complement-dependent cytotoxicity (CDC) with some modifications was used to evaluate the effect of Rituximab® on complement-mediated killing of tumor B-cells [28]. The number of residual viable cells was estimated by MTT assay.

2.8 Blood analysis

Red and white blood cells and platelets from treated and untreated mice were analyzed using ABX Micros E660 OT/CT (Horiba ABX Diagnostic, Montpellier, France). Other parameters in the animal plasma were analyzed using Integrated System Dx 880 (Beckman Coulter).

2.9 Histopathological and

Immunohistochemical analysis

Liver, spleen, kidneys, brain, spinal cord and bone marrow samples from leukemia-bearing

mice either dead from the tumor or sacrificed at day +120 were obtained at necropsy. For morphologic evaluation, the specimens were fixed in 10% buffered-formalin solution and embedded in paraffin. Four micrometer-thick sections were stained with H&E. Four to 6µm sections were fixed in cold 100% methanol for 15 minutes. Immunohistochemical analysis was done using the avidin-biotin-peroxidase complex method according to standard procedures [29], and the slides were examined under a Leica DM2000 optical microscope.

2.10 Statistical Analysis

The data were expressed as mean ± SD and analyzed for statistical significance by the two-tailed Student’s t test to compare two paired groups of data. The Kaplan-Meier product-limit method was used to estimate survival curves and the log-rank test was adopted to compare different groups of mice.

3 Results and discussion

3.1 Anti-CD20 BNPs Target Tumor B-cells

Current treatment strategies for leukemia involve chemotherapy, immunotherapy, bone marrow transplant, and several new target therapies. These treatments often induce long-term side-effects, resulting in impairment of vital physiological functions among the survivors. This is particularly true for elderly/unhealthy CLL patients. While current treatment approaches have greatly improved the prognosis for survival, some patients remain refractive to current therapeutic regimens. Hence there is an urgent need for novel therapeutic strategies for difficult-to-treat leukemia cases, in addition to reducing the long-term residual side-effects impact of therapeutics for all leukemia patients. Due to the cytotoxicity of drugs, currently the major challenge is to deliver the therapeutic agent to neoplastic cells while preserving the viability of non-malignant cells. Research on the use of nanoparticles as drug carriers has advanced to the point to focus on assessing the safety and efficacy


5Nano Res.

of such drug delivery systems. In this contribution, four different types of polymeric nanoparticles, BNP0, BNP1, BNP2 and BNP3, were prepared as in Figure S-1 in the ESM and characterized as previously described [19]. The BNP0 were made only by polymeric carriers (PLA-b-PEG-COOH and PCL-COOH); BNP1 were prepared conjugating the anti-CD20 chimeric antibody on the surface of BNP0; BNP2 were produced encapsulating HCQ sulfate and CLB inside the core of BNP1 while BNP3 were prepared loading chemotherapeutic drugs inside BNP0. To characterize both untargeted and targeted nanoparticles, TEM and dynamic light scattering were used. In details, TEM showed that untargeted nanoparticles have a core diameter of 110±40nm while antiCD20-conjugated nanoparticles have a core diameter of 90±30nm. For what concerns dynamic light scattering analysis, untargeted nanoparticles have an hydrodynamic diameter of 190±60nm while targeted nanoparticles have a diameter of 230±70nm; moreover, ζ-potential evidenced values of -7.8±0.9 and -6.0±0.6 mV for untargeted and anti-CD20 conjugated BNPs respectively, as previously described [19]. During the experiments, nanoparticles were stored at -20°C and +4°C and than tested. We do not evidenced any significant modification in their morphology and in their capacity to target and kill tumor B cells, both in vitro and in vivo, suggesting their stability for almost 1 year since their production. To characterize the BNP’s effect, the CLL-like MEC1 cell line was used. It was initially purified from a CLL patient [20] and carried a mutation in TP53 gene and the 17p13 deletion, as demonstrated by direct sequencing and FISH analysis (data not shown). Moreover, its immunephenotype was studied by cytometric analysis confirming what previously reported in literature [30]. In fact, more than 95% of MEC1 cells highly expressed human markers like CD20 and CD45 while CD5 expression was not detected (Figure S-2 in the ESM). BNPs’ functional characterization started by evaluating their ability to bind to leukemia cells. To this aim, BNP0 and BNP1 were labeled with FITC and added to MEC1 cells at different incubation times. BNP1 were able to target MEC1

cells in a dose- and time-dependent manner with a maximal uptake after 1h incubation and using 10µL of particles. Under these conditions, 74% of cells appeared tagged by BNP1 (Figure 1a). On the contrary, BNP0 did not demonstrate specific binding after 1h incubation, suggesting the importance of the anti-CD20 antibody in BNPs’ targeting B cells.

Figure 1 Tumor B-cells/BNPs interaction. (a) Binding of

anti-CD20 BNPs to MEC1 cells. MEC1 cells were incubated

with FITC-labeled BNPs for 1 hour at 37°C and analyzed using

FACS. FL1-H, green fluorescence, 530/30 nm bandpass filter.

(b) Internalization of anti-CD20 BNPs to MEC1 cells. MEC1

cells were incubated with BNPB for 1h and analyzed by

transmission electron microscopy: ultrastructural appearance

of MEC1/BNP1 interaction and internalization was

documented. Arrows in bII and bIII indicate typical

cytoplasmatic localization of anti-CD20 BNP. Bars represent

200nm (bI, bIII), 2µm (bII) and 100nm (bIV).

The BNPs’ interaction with MEC1 cells was further confirmed by confocal microscopy images incubating cells and BNPs labeled with FAST-DiO


6 Nano Res.

and Cy5.5, respectively (Figure S-3 in the ESM). Moreover, TEM studies were performed to follow BNPs’ migration into tumor B cells. Two different types of BNPs were prepared as shown in Figure S1, labeled as BNPA and BNPB. BNPA were produced encapsulating gadopentetate dimeglumine (Magnevist H, Bayer HealthCare Pharmaceuticals Inc) while BNPB were prepared conjugating the anti-CD20 antibody to the surface of BNPA. Exploiting the presence of Gd in the particles, BNPs’ migration was followed by TEM analysis, incubating MEC1 cells with these two different types of BNPs (Figure S-1 in the ESM).

In details, BNPA were never seen inside the cells (data not shown) while images showed the binding of BNPB and their interaction with the cell surface. Moreover, BNPs were never documented in the nucleus and the absence of vesicles surrounding them suggested BNPs’ internalization through a process different from endocytosis (Figure 1b). This data confirmed the results previously obtained both in vitro and in vivo and demonstrated the importance of a targeting agent as anti-CD20 recombinant antibody on nanoparticle’s surface [18]. BNPs’ internalization outside endosomes was already demonstrated for other tumor B-cell lines incubated with BNPs [18] which passed through the membrane without causing significantly its disruption.

3.2 BNP2 Induce Tumor B-Cell Cytotoxicity

In this study, CLB and HCQ were loaded inside polymeric nanoparticles because of their synergistic effect against cancer B cells, as we have previously described [18]. In details, CLB is an alkylating agent administered orally whose rate of drug absorption can vary significantly from patient to patient thus causing side effects [31, 32]. Also, most B-cell malignancies will become resistant to CLB at some point, no matter whether it is used at increasing doses or within more aggressive regimens. In resistant situations, it could be important to have a therapeutic system for a better delivery of high amounts of drugs specifically inside malignant B-cells in order to circumvent genetically driven tumor mechanisms of resistance. The combination of an

elevated concentration of CLB intracellularly with another kind of cytotoxic drug not dependent on surviving genes could not only enhance their respective killing activities but perhaps make a resistant leukemia cell sensitive again. HCQ has demonstrated an interesting cytotoxic effect depending on its capacity to block autophagosomes/lysosome fusion. Its anti-neoplastic properties in vitro depend on its concentration, which however is unobtainable in vivo by the usual oral administration route [33–36]. The synergistic effect of HCQ and CLB was previously described by our group [18] and it could be important especially for those CLL patients in an already resistant disease state, or with poor prognostic biological characteristics. These drugs together were able to cause high cytotoxic effect mainly inducing autophagy and apoptosis [18]. In order to evaluate the cytotoxic effect of BNPs, MEC1 cells were incubated with different amounts of BNP0, BNP1, BNP2 and BNP3 for 48h and residual viable cells were measured. Particles loaded with chemotherapeutic drugs, such as BNP2 and BNP3, were able to induce cell cytotoxicity in a dose-dependent manner while empty particles, such as BNP1, were almost ineffective. Furthermore, 2µL of BNP2 or BNP3 were sufficient to kill more than 85% of MEC1 cells suggesting that chemotherapeutic drugs maintained their cytotoxic properties even after encapsulation inside particles. On the contrary, treatment with BNP1 killed less than 20% of cells in this in vitro test, showing a good safety of this approach (Figure 2a). Cell cytotoxicity is due to the pro-apoptotic effect induced by the chemotherapeutic drugs. Forty eight hours incubation of cells and particles loaded with chemotherapeutic drugs caused high percentage of cell destruction, avoiding any possible molecular studies. Thus, only 16h incubations were made to further study apoptosis’ induction and autophagy impairment. In this setting, more than 20% of tumor cells (2x105) incubated with 2ml of BNP2 showed the apoptotic profile in an AnnexinV/7AAD test (Figure 2b). To confirm apoptosis, the poly-(ADP-ribose) polymerase (PARP-1) was visualized. The enzyme is cleaved


7Nano Res.

from a 113KDa molecule to fragments of 89 and 24KDa during apoptosis. The PARP-1 cleavage was detected by western blot assay using cell lysates of MEC1 cells incubated with different amounts of BNP2 for 16h. These apoptotic studies demonstrated that BNP2 were able to induce PARP-1 cleavage, in particular using 2µL of particles with 5x105 cells (Figure 2c).

Figure 2 In vitro characterization of the cytotoxic effect

of BNP2. MEC1 cells (a) were incubated with 0.5, 1, and

2µl of BNPs or HCQ+CLB for 48 hours at 37°C and

residual viable cells were measured. Data are expressed as

mean ± SD. (b) MEC1 cells were incubated with 1µl of

BNPs for only 16 hours at 37°C and apoptotic cells were

analyzed using AnnexinV/7AAD test. (c) Western blot

analysis of activated PARP-1 and LC3 accumulation from

cell lysates obtained from MEC1 cells incubated with: 1.

Saline; 2. 2µl of BNP1; 3. 0.5µl of BNP2; 4. 2µl of BNP2.

The same pattern was demonstrated studying autophagy, which is impaired by HCQ. This mechanism was analyzed taking in consideration the LC3 protein, which is processed to a cytosolic version (LC3-I, 18KDa) and then converted to activate forms commonly used as a marker of autophagosome accumulation. The effect of HCQ on MEC1 cells was evident detecting increased amount of LC3-III in lysates of cells incubated for only 16h with 0.5 or 2µL of BNP2 by western blot

analysis, also in the presence of significant basal level in untreated cells (Figure 2c).

3.3 Comparison Between BNP2 and Rituximab

Cytotoxic Effects

Rituximab is mainly able to activate the complement system and also to induce antibody-dependent cell cytotoxicity (ADCC) but a very low killing effect is due to its ability to activate apoptotic pathways. For this reason, we have compared the killing of MEC1 cells induced by a saturating concentration of Rituximab through complement-dependent killing, or by BNP2 acting through apoptosis/authophagy. MEC1 cells were analyzed using anti-CD20 mAb showing high amount of the antigen on cell surface (Mean Fluorescence Intensity-MFI: 784). In this particular setting, Rituximab killed up to 22% of MEC1 cells while BNP2 killed 87% of this leukemia cell line (Table 1). The BNP2 cytotoxic effect was evident also analyzing purified cells from CLL patients. Circulating CLL B-cells expressed a lower amount of CD20 on their surface with respect to MEC1 cells (MFI: 50.6 vs 784), as we documented in cells purified from 31 different untreated CLL patients, already characterized for the main biological prognosticators (Table S-1 in the ESM). Moreover, as shown in Table 1, the cytotoxic effect of Rituximab on purified CLL cells ranged between 0 and 38%, with a median value of 4.2%. On the contrary, BNP2 killed up to 84% CLL cells, with a median value of 55.1% (BNP2 vs. Rituximab: p<0.00001), and only 1 out of 31 patient did not respond to the treatment. Interestingly, it is also possible to perform these studies in whole blood samples, as a predictive functional test. Apoptosis was evaluated by Annex V/7AAD test after 16h of incubation with BNP2. In all the samples more than 98% of CD5/CD19 positive BNP2-treated cells resulted in an apoptotic state in comparison with only 11% of BNP1-treated cells. These results seem to be independent from CD20 expression or from specific biological features; in fact, TP53 mutated/deleted or NOTCH1 mutated patients’ cells, that usually have poor response to standard therapies (Table S-2 in the ESM), were


8 Nano Res.

anyway killed by BNP2. Table 1 Comparison between BNP2 and Rituximab effects

CD20

(MFI)

BNP2

(% of killing)

RITUXIMAB

(% of killing)

MEC1 784 86.4 22.0

Pt1 73.6 77.4 0.0

Pt2 20.2 26.9 1.4

Pt3 22.2 81.7 8.9

Pt 4 76.2 25.4 38.0

Pt 5 65.9 59.6 9.1

Pt 6 24.1 0.0 0.0

Pt 7 38.4 68.4 0.0

Pt 8 54.8 65.4 1.9

Pt 9 33.5 54.1 0.0

Pt 10 38.9 39.4 10.6

Pt 11 32.7 71.7 3.5

Pt 12 58.0 58.8 6.2

Pt 13 35.4 78.8 0.0

Pt 14 28.0 84.4 0.0

Pt 15 33.0 64.0 0.0

Pt 16 25.3 55.1 9.9

Pt 17 42.4 88.9 31.1

Pt 18 41.4 44.9 18.0

Pt 19 64.5 47.9 21.8

Pt 20 60.5 61.3 2.9

Pt 21 50.6 10.3 2.7

Pt 22 75.5 32.6 0.0

Pt 23 44.5 23.4 0.0

Pt 24 60.0 29.6 12.6

Pt 25 53.6 12.9 9.9

Pt 26 289.1 72.3 9.3

Pt 27 64.1 28.4 0.0

Pt 28 40.2 41.0 25.6

Pt 29 63.9 76.1 5.1

Pt 30 77.7 63.3 4.2

Pt 31 82.2 18.2 10.4

Median (Pt) 50.64 55.1 4.2

CLL patients (Pt); MFI: mean fluorescence intensity: mean;

percentage of killing: mean (n=3).

3.4 BNPs Show a Safe Toxicological Profile

Side effects induced by HCQ and CLB are well described in the literature [37, 38] and were also

evident in our experiments on healthy mice. The reduction of side effects was addressed by including CLB+HCQ drugs in BNPs produced from biocompatible and biodegradable polymers. The development of these nanoparticles with an average diameter of 250nm as drug delivery agents has several advantages, including specific targeting via receptor-mediated mechanisms and effective penetration of the tumor microenvironment [19]. BNP2 in particular can transport and release into tumor B-cells enough amount of drugs to kill cancer cell, overcoming multidrug resistance overexpressed in several B-cell disorders [39]. The toxic effects induced by the intra-peritoneal injection of BNPs were evaluated in groups of five C57/BL mice receiving 8 injections of saline, 8 injections of BNP2 (containing 400µg of CLB and HCQ) or 4 injections free HCQ+CLB (400µg each). We have previously documented that 8 injections of free drugs killed all the animals [18]. Mice were followed for 28 days. Animal survival, total body weight but also circulating cells and several tissue markers in the blood were analyzed. All the animals survived during the experiment but free drugs-treated mice evidenced a strong reduction in their body weight, with a median of about 20%. Blood samples were collected 3 days after the end of the treatments in order to evaluate complete blood count, hemoglobin, urea, aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), lactate-dehydrogenase (LDH), creatine phosphokinase (CPK), creatinine and aldolase concentration (Table 2). We did not evidence any significant differences during all the experiment between controls and animals receiving 8 injections of BNP2; only platelets seem to be increased after the treatments, remaining in a physiological range. On the contrary, animals receiving only 4 times free HCQ+CLB showed a reduction in white blood cells, mainly due to the low number of circulating lymphocytes, and a significant reduction in erythrocytes. Table 2 Toxicological studies

Saline BNP2 HCQ/CLB

Body weight -2.31 -3.29 -20.61 *§


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(% variation)

WBC

(103/mm3)

3.15 ±

1.08

2.13 ±

0.4

0.58 ± 0.33

*§

LYMPHOCYTES

(103/mm3)

2.65 ±

1.03

1.60 ±

0.17

0.30 ± 0.18

*§

MONOCYTES

(103/mm3)

0.18 ±

0.05

0.20 ±

0.17 0.08 ± 0.18

GRANULOCYTES

(103/mm3)

0.33 ±

0.05

0.33 ±

0.06 0.20 ± 0.1

RBC

(106/mm3) 7.42 ± 1

5.52 ±

1.47

4.87 ± 0.52

*

PLT

(103/mm3)

532.25 ±

73.4

688.00 ±

27.22 *§

464.5

± 93.3

Hemoglobin

(g/L) 11 ± 0.6

11 ±

0.4

9.0 ± 1.3

*§

Urea (mg/dL) 29 ± 5 28 ± 9 28 ± 7

Creatinine

(mg/dl)

0.24 ±

0.06

0.20 ±

0.18

0.14 ±

0.02 *

AST

(IU/L)

152 ±

52

146 ±

16

182 ±

35

ALT

(IU/L)

26.8 ±

6.0

17.0 ±

5.3

20.3 ±

9.6

ALP

(IU/L)

34.0 ±

10,2

26.7 ±

10,0

18.5 ± 5.8

*

LDH

(IU/L) 645 ± 82.8

435 ±

98.6

495 ± 41.5

*

CPK

(IU/L) 2084 ± 413

1143 ±

564

1587 ±

223

Aldolase

(IU/L) 9 ± 4 11 ± 2

16 ± 5

*§

Total body weight, red blood cells (RBC), white blood cells

(WBC), platelets (PLT), and other plasma parameters from

treated and untreated mice were compared. *= p<0.05 vs

control; §= p<0.05 vs BNP2.

At the same time, we observed reduced concentration of hemoglobin, creatinine, ALP and LDH, with increased values of aldolase (Table 2).

3.5 Development of a Disseminated Leukemia

Model Using MEC1 Cells

A xenograft model of human CLL was previously described by Bertilaccio et al. [40], who challenged intravenously or subcutaneously Rag-/-γc-/- mice with 107 MEC1 cells. Unfortunately, we were unable to repeat these results in SCID mice; in fact, the intravenous injection of 107 cells rapidly killed all the animals from respiratory problems. On the other hand, cells’ subcutaneous challenging induced only the formation of a localized tumor mass at the site of injection without colonizing other tissues and inducing the death of the animals in about 70 days (Figure 3).

Figure 3 Development of human/SCID leukemia model.

MEC1 were injected subcutaneously (SC, 107 cells) or

intravenously (IV, 5x105 cells) after cyclophosphamide

pre-treatment. Animal survival was studied and reported as

Kaplan Mayer curves.

However, an intravenous injection of only 5x105 MEC1 cells 24 hours after a pre-treatment with cyclophosphamide (200mg/kg intraperitoneally) which reduce immune effects, in particular via NK cells [41, 42], developed a diffuse leukemia model characterized by the colonization of different organs and also the blood. In details, MEC1 cells’ biodistribution pattern was evaluated by immunohistochemical analysis staining tissues with H&E and detecting human B cells with an anti-human CD45 antibody 28 days after MEC1 injection (Figure 4a). MEC1 cells were detected in liver, spleen, kidney, bone marrow, spinal cord and brain. Moreover, the accumulation of MEC1 cells in mice bloodstream was confirmed by cytometric analysis using anti-human CD19 and anti-human CD45 antibodies. Human B cells presence was detected from the 20th day after tumor cells injection (Figure 4b).


10 Nano Res.

Figure 4 Characterization of diffuse leukemia model in

SCID mice. MEC1 (5x105 cells) were injected intravenously in

SCID mice and human tumor cells’ distribution was analyzed

after 28 days by H&E and by exploiting human CD45 (a).

Original magnification 200X. (b) Human tumor B-cells were

also detected in the circulation by FACS analysis using labeled

anti-CD45 and anti-CD19 mAbs.

All animals died between 30 and 37 days after tumor cell injection (Figure 3), as the evidence of a very reproducible and very aggressive leukemia human/SCID model, which was useful for the characterization of the therapeutic effect of targeted nanoparticles but also for the development of new recombinant antibodies, as already performed for other B-cell malignancies [24, 30, 43].

3.6 BNP2 Therapeutic Effect in a Disseminated

Leukemia Human/Mouse Model

The BNP2 demonstrated their ability to target cancer B-cell in vivo and also their potential efficacy in the treatment of tumor-bearing mice, as already evidenced in other B-cell xenograft [18, 19]. To study BNP2 efficacy in the treatment of the human/SCID leukemia model, MEC1 cells were injected in SCID mice, divided into 8 groups of 6–8

animals per group, and followed for 120 days (Figure 5).

Figure 5 Therapeutic effect of BNPs and HCQ+CLB. SCID

mice (n = 6-10 per group) received (5x106) MEC1 cells

intravenously and BNP1, BNP2, BNP3 or HCQ+CLB as

described in the results; animal survival was represented as

Kaplan Mayer curve

Group 1 did not receive any treatment; all mice died within 30-40 days after tumor cell injection with a median survival of 33.5 days. The therapeutic protocol followed our previous data and derived from toxicological profile obtained with free HCQ+CLB. Thus, group 2 and group 3 received both 80µL of BNP2 (corresponding to 400µg of each encapsulated chemotherapeutic agent targeted via anti-CD20 antibody) for 8 times in 17 days from the 1st and the 4th day after MEC1 cell injection, respectively. The overall survival of group 2 was 83 days and 3 mice out of 7 were cured at the end of the study (BNP2x8 (day 1) vs Untreated: p<0.0001; BNP2x8 (day 1) vs BNP1x8: p<0.0002; BNP2x8 (day 1) vs BNP3x8: p<0.0001; BNP2x8 (day 1) vs HCQ+CLB: p<0.01; BNP2x8 (day 1) vs BNP2x4: p<0.03; BNP2x8 (day 1) vs BNP2x8 (day 4): Not Significant). Group 3 received the same treatment but starting from day 4, resulting in a overall survival of 61 days and 1 out of 7 mice was cured (BNP2x8 (day 4) vs Untreated: p<0.0001; BNP2x8 (day 4) vs BNP1x8: p<0.0002; BNP2x8 (day 4) vs BNP3x8: p<0.0001; BNP2x8 (day 4) vs HCQ+CLB: p<0.03; BNP2x8 (day 4) vs BNP2x4: p<0.04). These results demonstrate BNP2 ability to treat this aggressive human/mouse leukemia model with a better outcome when the


11Nano Res.

treatment started at the early stage of the pathology. Group 4 received only 4 injections of 80µL of BNP2 in 8 days starting from the 4th day after cell injection. This treatment improved the overall survival of about 13.5 days (BNP2x4 vs Untreated: p<0.002; BNP2x4 vs HCQ+CLB: p<0.03). Group 5 and group 6 received 8 injections of 80µL of BNP1 and BNP3, respectively. Both these treatments did not significantly increased mice survival demonstrating both BNPs’ safety and the inability of BNP3 to bind cancer cells due to the absence of the anti-CD20 antibody on the surface of these particles, as already demonstrated by our group [19]. In vitro, untargeted nanoparticles (BNP3) evidenced cell cytotoxicity but their effect was not confirmed in vivo. This was probably due to the blood flow (for circulating tumor cells) and reduced residence time of untargeted nanoparticles in tumor microenvironment. Finally, group 7 received 8 injections of HCQ+CLB (400µg each) in 17 days starting from day 1. This treatment improved survival of 2 days and showed that BNP2 (groups 2) were more effective than free drugs in the treatment of this aggressive human/mouse leukemia model. In the group 8, three animals received 8 injections of free drugs but all the mice died for the toxicity of the treatment in less than 20 days, as already described [18]. 4 Conclusions

In conclusion, the results of the present study demonstrated that anti-CD20 nanoparticles containing HCQ+CLB can be effective as a single agent in controlling a new disseminated model of aggressive leukemia. It also provides a rationale for adopting this therapeutic approach for the treatment of other B-cell disorders with BNP2 or different types of tumors, using other monoclonal antibodies to specifically deliver cytotoxic agent-loaded nanoparticles in cancer cells. Acknowledgements

This study has been made possible by research grants from Italian Association for Cancer

Research (AIRC Project n° 12965/2012), Italian Ministry of Health (GR‐2011‐ 02346826 and GR‐2011‐ 02347441), Fondazione Casali – Trieste, Italy and Stiftung Foundation – Liechtenstein. Nanoparticles fabrication at LNK Chemsolutions, USA, was possible in part by Grant 2R44CA135906-02 (SBIR Phase II) from the National Institutes of Health (USA) to Ruben Spretz, Gustavo Larsen, Sandra Noriega and Luis Núñez. Conflict-of-interest disclosure: Ruben Spretz, Gustavo Larsen, Sandra Noriega and Luis Núñez working in Biotarget Inc. and LNK Chemsolutions LLC have commercial interests in the particle systems described in this work. No conflicts of interest for the other authors. Correspondence: Sara Capolla and Paolo Macor, Department of Life Sciences, University of Trieste, via L. Giorgeri, 5 – 34127, Trieste, Italy. Phone: +39 040 5588682; FAX: +39 040 5584023; e-mail: [email protected], [email protected] Electronic Supplementary Material: Supplementary material about CLL patients’ characterization (confocal microscopy, cytometry, sequencing, killing test) is available in the online version of this article at http://dx.doi.org/10.1007/s12274-***-****-*

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Nano Res

1

Electronic Supplementary Material

New Approach for the Treatment of CLL using Chlorambucil/Hydroxychloroquine-loaded anti-CD20 Nanoparticles

Sara Capolla1§(*), Nelly Mezzaroba1§, Sonia Zorzet1, Claudio Tripodo2, Ramiro Mendoza-Maldonado3, Marilena Granzotto4, Francesca Vita1, Ruben Spretz5, Gustavo Larsen5,6, Sandra Noriega5, Eduardo Mansilla7, Michele Dal Bo8, Valter Gattei8, Gabriele Pozzato4, Luis Núñez5,6 and Paolo Macor1,9(*) 1Department of Life Sciences, University of Trieste, Trieste, Italy 2Department of Human Pathology, University of Palermo, Italy 3Molecular Oncology Unit, National Laboratory Consorzio Interuniversitatio per le Biotecnologie (CIB), Trieste, Italy 4Dipartimento Universitario Clinico di Scienze mediche, Chirurgiche e della Salute, University of Trieste, Trieste, Italy 5LNK Chemsolutions LLC, Lincoln, NE 68521, USA 6Bio-Target Inc., Chicago, IL, USA; 7Centro Ùnico Coordinador de Ablacion e Implante Provincia de Buenos Aires (C.U.C.A.I.B.A.), Ministry of Health, La Plata, Buenos Aires, Argentina 8Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico, Istituto di Ricerca e Cura a Carattere Scientifico (I.R.C.C.S.), Aviano, Italy 9Callerio Foundation Onlus, Institutes of Biological Researches, Trieste, Italy.

§ These authors contributed equally to this work.

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

Electronic supplementary materials (ESM) contain three figures and two tables. Figure S-1 showed types of nanoparticles produced and used for this project; Figure S-2 represented the immunophenotype in terms of CD5, CD20 and CD45 expression on the CLL-cell line MEC1; and Figure S-3 confirmed BNPs’ binding on MEC1 cells. Moreover, Table S-1 showed the genetic features of analyzed CLL patients and Table S-2 put in evidence the differences between Rituximab and BNP2 treatments on cells derived from difficult-to-treat CLL-patients’ samples.

Address correspondence to Capolla S., [email protected]; Macor P., [email protected]

2

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,

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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.

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

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

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

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

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

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

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

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

showing the scanning path across an isolated wire.

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

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

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

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

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

temperatures.

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

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

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

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

chosen from the plot in plot (a).

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

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

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

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

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

exaggerated for clarity.

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