AREA DRUGS & THERAPEUTICS COMMITTEE: 8 DECEMBER 2014 ADTC(M) 14/05 Minutes: 62 - 75 NHS GREATER GLASGOW AND CLYDE Minutes of a Meeting of the Area Drugs and Therapeutics Committee held in the Boardroom, JB Russell House on Monday, 8 December 2014 at 2.00 p.m. Dr J Gravil (in the Chair) Miss F Qureshi . Observer
Microsoft word - nano res-tup-research.doc
DOI 10.1007/s12274-015-0935-3 New approach for the treatment of CLL using
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
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. TABLE OF CONTENTS (TOC)
New approach for the Treatment of CLL using
Sara Capolla1 § *, Nelly Mezzaroba1 § , Sonia Zorzet1, 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* We reported a nanoplatform based on the use of biodegradable 1University of Trieste, Italy; 2University of Palermo, chemotherapeutic-loaded immune-nanoparticles for the treatment of Italy; 3Molecular Oncology Unit, National Laboratory Chemotherapeutic-loaded Consorzio Interuniversitatio per le Biotecnologie (CIB), nanoparticles were specifically targeted inside leukemic cells by an Italy; 4University of Trieste, Italy; 5LNK Chemsolutions anti-CD20 antibody thus improving the survival of leukemia-bearing LLC, USA; 6Bio-Target Inc., USA; 7Centro Ùnico mice in respect to the same amount of free drugs. 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, Gustavo Larsen, www.lnkchemsolutions.com Luis Núñez, www.biotarget-ln.com
DOI (automatically inserted by the publisher) Research Article New Approach for the Treatment of CLL using
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 9Cal erio Foundation Onlus, Institutes of Biological Researches, Trieste, Italy. § These authors contributed equal y to this work. Received: day month year
Revised: day month year
Current approaches for the treatment of chronic lymphocytic leukemia (CLL) Accepted: day month year
have greatly improved the prognosis for survival, but some patients remain (automatically inserted by 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 Tsinghua University Press in which high doses of hydroxychloroquine and chlorambucil were loaded into and Springer-Verlag Berlin 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. KEYWORDS
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. tissues. The development of nanoparticles, made 1 Introduction
with biodegradable biopolymers, and loaded with chemotherapeutic agents, is an attractive method Chronic Lymphocytic Leukemia (CLL) is a to target neoplastic cells [13–15]. In fact, heterogeneous disease with highly variable clinical nanoparticles can be designed by attaching courses and survivals ranging from months to specific antibodies on their surface thus they are decades. In particular, a subset of patients is able to recognize tumor-associated antigens and affected by a high-risk CLL form that rapidly induce specific homing on the neoplastic cell progresses and develops a disease that requires surface [16, 17]. Therefore, the efficacy of symptomatic treatment . Over-represented in high-dose chemotherapy is associated to the this group are patients bearing mutations/deletion specificity and the low side effects of of the TP53 gene . Moreover, a high-risk CLL antibody-based therapy and protective nature of patient fraction was confirmed to carry polymeric encapsulated chemotherapeutics. mutations/deletion of other genes, such as On these principal characteristics, we developed NOTCH1, BIRC3 or SF3B1 [3–7]. biodegradable nanoparticles (BNPs) coated with For years, the standard therapy was based on the an anti-CD20 antibody to target neoplastic B-cells, use of alkylating agents, with not much (if any) and loaded with hydroxychloroquine (HCQ) and effects on the CLL natural history. The chlorambucil (CLB) to specifically kill tumor introduction of fludarabine signified an important B-cells [18, 19]. For the first time, we breakthrough in CLL therapy. The use of demonstrated the safety and therapeutic effects monoclonal antibodies (anti-CD20, anti-CD52 and of targeted nanoparticles in a new leukemia beyond) opened a new perspective overcoming xenograft SCID mice model. the paradigm to treat only patients with minimal complications and, alone or in combination with 2 Experimental
chemotherapy, these therapeutics increased significantly the overall survival of the patients [8, 2.1 Cells, antibodies and sera
9]. More recently, inhibitors of B-cell receptor signaling showed durable efficacy in a subset of The CLL-like cell line MEC1  (kindly CLL patients [9–11]. Despite combined therapy provided by prof. Josee Golay), carrying both a advancements, CLL remains an incurable disease TP53 mutation (i.e. c.422insC) and the 17p13 in most cases since molecular complete remission deletion, was cultured in RPMI-1640 medium is unachievable and, as a consequence, the disease (Sigma-Aldrich, Milan, Italy) supplemented with relapses invariably after some months or years. In 10% fetal bovine serum (FBS; GE Healthcare particular, the small subgroup of patients, known Milan, Italy). Heparinized peripheral blood as ultra high-risk CLL, shows poor response to samples were obtained after written informed chemo-immunotherapy and have a life expectancy consent from untreated CLL patients at the of less than 2 to 3 years with conventional University Hospital in Trieste (B-cells more than regimens [10, 12]. These considerations indicate 90% of total circulating cells). The study was that new therapeutic approaches are needed to approved by the IRB of the CRO (IRCCS) of obtain the complete recovery or at least to improve Aviano (IRB-06-2010). The mononuclear cell survival of CLL patients. Since most patients are fractions were isolated by centrifugation on older in age and often have several co-morbidities, any new treatment approaches, in addition to gradients . MEC1 cells were suspended in higher efficacy, must be non-toxic to organs and serum-free RPMI-1640 medium and stained with Female SCID mice (4–6 weeks of age) were Healthcare) as previously reported . The provided by Charles River (Milan, Italy) and anti-CD20 chimeric antibody Rituximab (Roche, maintained under pathogen-free conditions. Milan, Italy) was derived from the clinic Animals were pretreated with cyclophosphamide (University of Trieste, Italy). The mouse mAb to (200mg/kg), inoculated subcutaneously with 107 CD20 and anti-PARP1 antibody were purchased MEC1 cells or intravenously with 5x105 MEC1 from BioLegend (San Diego, CA) and Bethyl cells after 24 hours and examined twice weekly up to 125 days. C57/BL mice were obtained from immunophenotypical characterization, the animal house of the University of Trieste. All anti-human CD5 PE (Immunotools, Friesoythe, the experimental procedures involving animals Germany), anti-human CD20 (clone L26, were done in compliance with the guidelines of Novacastra), anti-human CD45 APC (Invitrogen, the European and the Italian laws and were Milan, Italy) and anti-human CD19 TC (GE approved by the Italian Ministry of Health as well Healthcare) mAbs were used. Anti-LC3, as by the Administration of the University anti-a-tubulin mAbs and all the secondary Animal House (Prot. 42/2012). antibodies were purchased from Sigma-Aldrich (Milan, Italy) or Aczon (Monte San Pietro, 2.4 Cytometric analysis
Bologna, Italy). Human sera from AB Rh+ blood donors (NHS - normal human serum) were BNPs' binding was assayed incubating 10µL of kindly provided by the Blood Transfusion Center BNPs with 5x105 cells MEC1 cells for 1h at 37°C. (Trieste, Italy) as a source of complement (NHS - MEC1 localization in mouse blood was performed normal human serum). using anti-CD45 APC and anti-CD19 TC antibodies at 28 days after cells' injection. For 2.2 BNPs preparation
these measurements 30000 events were acquired using FACSCalibur (Becton Dickinson, San Jose, BNPs preparation was performed using CA) flow cytometer and data were analyzed by chemicals reagent grade or better. Polyethylene CELLQuest software (Becton Dickinson) . glycol (PEG) was purchased from Nektar, San Carlos, CA; hydroxychloroquine sulfate (HCQ) 2.5 Transmission electron microscopy analysis
and chlorambucil (CLB) were purchased from ACROS, Gel Belgium and Sigma Aldrich (St Louis, Samples were fixed for 1h in a solution of 2% MO), respectively. BNPs, based on carboxylic acid glutaraldehyde (Serva, Heidelberg, Germany) in 0.1M cacodylate buffer (pH=7.3) containing 0.03M (PLA-b-PEG-COOH and PCL-COOH), were CaCl2, rinsed three times (10min each wash) and prepared with average diameter of 250nm postfixed in 1% osmium tetroxide for 1h at 4°C. measured by dynamic light scattering (data not Samples were then dehydrated in ascending shown) in an under class 100 clean room ethanols to 100 % ethanol and embedded in Dow conditions by implementing Bio-Target's Epoxy Resin (DER 332; Unione Chimica Europea, technology at LNK Chemsolutions, LLC Milan, Italy) and DER732 (Serva), as previously laboratories [19, 23]. All BNPs (final concentration described by Zabucchi et al . Ultrathin of 900µg/ml) were resuspended in PBS buffer sections were cut by an ultratome Leica Ultracut (pH=7.4) with 10% BSA. BNPs were diluted in UCT8 (Leica, Wirn, Austria), double stained with serum-free RPMI-1640 medium and stained with uranyl acetate and lead citrate and observed in a transmission electron microscope (EM 208, Monofunctional Dye (GE Healthcare). Micrographs were taken with a Morada Camera (Olympus, Munster, Germany). 2.3 Animals
mice either dead from the tumor or sacrificed at day +120 were obtained at necropsy. For 2.6 Cell viability, apoptosis and autophagy
morphologic evaluation, the specimens were fixed in 10% buffered-formalin solution and To investigate the ability of BNPs to affect cell embedded in paraffin. Four micrometer-thick viability, MEC1 cells (2x105) were incubated with sections were stained with H&E. Four to 6µm different concentrations of BNPs for 48h at 37°C sections were fixed in cold 100% methanol for 15 (in a humidified 37°C, 5% CO2 incubator). The minutes. Immunohistochemical analysis was amount of residual viable cells was determined done using the avidin-biotin-peroxidase complex by MTT assay  and the percentage of dead method according to standard procedures , cells was calculated as: 100 x [(test release – and the slides were examined under a Leica spontaneous release)/(total release – spontaneous DM2000 optical microscope. release)]. Apoptosis of patient's B cells was measured using FITC-labeled recombinant 2.10 Statistical Analysis
human Annexin V assay (Apoptosis detection kit, Immunostep, The data were expressed as mean ± SD and analyzed for statistical significance by the measurement 30000 events were acquired with a two-tailed Student's t test to compare two paired standard FACSCalibur (Becton Dickinson) flow groups of data. The Kaplan-Meier product-limit cytometer and analysis of data was performed method was used to estimate survival curves and with CellQuest (Becton Dickinson). PARP-1 and the log-rank test was adopted to compare different groups of mice. immunoblotting to study apoptosis induction 3 Results and discussion
and autophagy impairment, respectively . 2.7 Complement-mediated lysis
3.1 Anti-CD20 BNPs Target Tumor B-cells
Current treatment strategies for leukemia involve complement-dependent cytotoxicity (CDC) with chemotherapy, immunotherapy, bone marrow some modifications was used to evaluate the transplant, and several new target therapies. These effect of Rituximab® on complement-mediated treatments often induce long-term side-effects, killing of tumor B-cells . The number of resulting in impairment of vital physiological residual viable cells was estimated by MTT assay. functions among the survivors. This is particularly true for elderly/unhealthy CLL patients. While 2.8 Blood analysis
current treatment approaches have greatly improved the prognosis for survival, some patients Red and white blood cells and platelets from remain refractive to current therapeutic regimens. treated and untreated mice were analyzed using Hence there is an urgent need for novel ABX Micros E660 OT/CT (Horiba ABX Diagnostic, difficult-to-treat Montpellier, France). Other parameters in the leukemia cases, in addition to reducing the animal plasma were analyzed using Integrated long-term residual side-effects impact of System Dx 880 (Beckman Coulter). therapeutics for all leukemia patients. Due to the cytotoxicity of drugs, currently the major challenge is to deliver the therapeutic agent to neoplastic Immunohistochemical analysis
cells while preserving the viability of non-malignant cells. Research on the use of Liver, spleen, kidneys, brain, spinal cord and nanoparticles as drug carriers has advanced to the bone marrow samples from leukemia-bearing point to focus on assessing the safety and efficacy of such drug delivery systems. In this contribution, cells in a dose- and time-dependent manner with a four different types of polymeric nanoparticles, maximal uptake after 1h incubation and using BNP0, BNP1, BNP2 and BNP3, were prepared as in 10µL of particles. Under these conditions, 74% of Figure S-1 in the ESM and characterized as cells appeared tagged by BNP1 (Figure 1a). On the previously described . The BNP0 were made contrary, BNP0 did not demonstrate specific only by polymeric carriers (PLA-b-PEG-COOH binding after 1h incubation, suggesting the and PCL-COOH); BNP1 were prepared importance of the anti-CD20 antibody in BNPs' conjugating the anti-CD20 chimeric antibody on targeting B cells. 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 . 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 Figure 1 Tumor B-cells/BNPs interaction. (a) Binding of
from a CLL patient  and carried a mutation in anti-CD20 BNPs to MEC1 cells. MEC1 cells were incubated TP53 gene and the 17p13 deletion, as demonstrated with FITC-labeled BNPs for 1 hour at 37°C and analyzed using by direct sequencing and FISH analysis (data not FACS. FL1-H, green fluorescence, 530/30 nm bandpass filter. shown). Moreover, its immunephenotype was (b) Internalization of anti-CD20 BNPs to MEC1 cells. MEC1 studied by cytometric analysis confirming what cells were incubated with BNPB for 1h and analyzed by previously reported in literature . In fact, more transmission electron microscopy: ultrastructural appearance than 95% of MEC1 cells highly expressed human of MEC1/BNP1 interaction and internalization was markers like CD20 and CD45 while CD5 documented. Arrows in bII and bIII indicate typical expression was not detected (Figure S-2 in the cytoplasmatic localization of anti-CD20 BNP. Bars represent ESM). BNPs' functional characterization started by 200nm (bI, bIII), 2µm (bII) and 100nm (bIV). evaluating their ability to bind to leukemia cells. To this aim, BNP0 and BNP1 were labeled with The BNPs' interaction with MEC1 cells was further FITC and added to MEC1 cells at different confirmed by confocal microscopy images incubation times. BNP1 were able to target MEC1 incubating cells and BNPs labeled with FAST-DiO and Cy5.5, respectively (Figure S-3 in the ESM). elevated concentration of CLB intracellularly Moreover, TEM studies were performed to follow with another kind of cytotoxic drug not BNPs' migration into tumor B cells. Two different dependent on surviving genes could not only types of BNPs were prepared as shown in Figure enhance their respective killing activities but S1, labeled as BNPA and BNPB. BNPA were perhaps make a resistant leukemia cell sensitive again. HCQ has demonstrated an interesting dimeglumine (Magnevist H, Bayer HealthCare cytotoxic effect depending on its capacity to block Pharmaceuticals Inc) while BNPB were prepared autophagosomes/lysosome conjugating the anti-CD20 antibody to the surface anti-neoplastic properties in vitro depend on its of BNPA. Exploiting the presence of Gd in the concentration, which however is unobtainable in particles, BNPs' migration was followed by TEM vivo by the usual oral administration route analysis, incubating MEC1 cells with these two [33–36]. The synergistic effect of HCQ and CLB different types of BNPs (Figure S-1 in the ESM). was previously described by our group  and In details, BNPA were never seen inside the cells it could be important especially for those CLL (data not shown) while images showed the patients in an already resistant disease state, or binding of BNPB and their interaction with the cell with poor prognostic biological characteristics. surface. Moreover, BNPs were never documented These drugs together were able to cause high in the nucleus and the absence of vesicles cytotoxic effect mainly inducing autophagy and surrounding them suggested BNPs' internalization apoptosis . In order to evaluate the cytotoxic through a process different from endocytosis effect of BNPs, MEC1 cells were incubated with (Figure 1b). This data confirmed the results different amounts of BNP0, BNP1, BNP2 and previously obtained both in vitro and in vivo and BNP3 for 48h and residual viable cells were demonstrated the importance of a targeting agent chemotherapeutic drugs, such as BNP2 and nanoparticle's surface . BNPs' internalization BNP3, were able to induce cell cytotoxicity in a outside endosomes was already demonstrated for dose-dependent manner while empty particles, other tumor B-cell lines incubated with BNPs  such as BNP1, were almost ineffective. which passed through the membrane without Furthermore, 2µL of BNP2 or BNP3 were causing significantly its disruption. sufficient to kill more than 85% of MEC1 cells suggesting chemotherapeutic 3.2 BNP2 Induce Tumor B-Cell Cytotoxicity
maintained their cytotoxic properties even after encapsulation inside particles. On the contrary, In this study, CLB and HCQ were loaded inside treatment with BNP1 killed less than 20% of cells polymeric nanoparticles because of their in this in vitro test, showing a good safety of this synergistic effect against cancer B cells, as we approach (Figure 2a). Cell cytotoxicity is due to have previously described . In details, CLB is the pro-apoptotic effect induced by the an alkylating agent administered orally whose chemotherapeutic drugs. Forty eight hours rate of drug absorption can vary significantly incubation of cells and particles loaded with from patient to patient thus causing side effects chemotherapeutic drugs caused high percentage [31, 32]. Also, most B-cell malignancies will of cell destruction, avoiding any possible become resistant to CLB at some point, no matter molecular studies. Thus, only 16h incubations whether it is used at increasing doses or within were made to further study apoptosis' induction more aggressive regimens. In resistant situations, and autophagy impairment. In this setting, more it could be important to have a therapeutic than 20% of tumor cells (2x105) incubated with system for a better delivery of high amounts of 2ml of BNP2 showed the apoptotic profile in an drugs specifically inside malignant B-cells in AnnexinV/7AAD test (Figure 2b). To confirm order to circumvent genetically driven tumor apoptosis, the poly-(ADP-ribose) polymerase mechanisms of resistance. The combination of an (PARP-1) was visualized. The enzyme is cleaved from a 113KDa molecule to fragments of 89 and analysis, also in the presence of significant basal 24KDa during apoptosis. The PARP-1 cleavage level in untreated cells (Figure 2c). was detected by western blot assay using cell lysates of MEC1 cells incubated with different 3.3 Comparison Between BNP2 and Rituximab
amounts of BNP2 for 16h. These apoptotic Cytotoxic Effects
studies demonstrated that BNP2 were able to induce PARP-1 cleavage, in particular using 2µL Rituximab is mainly able to activate the of particles with 5x105 cells (Figure 2c). 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 Figure 2 In vitro characterization of the cytotoxic effect
of BNP2. MEC1 cells (a) were incubated with 0.5, 1, and prognosticators (Table S-1 in the ESM). Moreover, 2µl of BNPs or HCQ+CLB for 48 hours at 37°C and as shown in Table 1, the cytotoxic effect of residual viable cells were measured. Data are expressed as Rituximab on purified CLL cells ranged between mean ± SD. (b) MEC1 cells were incubated with 1µl of 0 and 38%, with a median value of 4.2%. On the BNPs for only 16 hours at 37°C and apoptotic cells were contrary, BNP2 killed up to 84% CLL cells, with a analyzed using AnnexinV/7AAD test. (c) Western blot median value of 55.1% (BNP2 vs. Rituximab: analysis of activated PARP-1 and LC3 accumulation from p<0.00001), and only 1 out of 31 patient did not cell lysates obtained from MEC1 cells incubated with: 1. respond to the treatment. Interestingly, it is also Saline; 2. 2µl of BNP1; 3. 0.5µl of BNP2; 4. 2µl of BNP2. possible to perform these studies in whole blood samples, as a predictive functional test. Apoptosis The same pattern was demonstrated studying was evaluated by Annex V/7AAD test after 16h of autophagy, which is impaired by HCQ. This incubation with BNP2. In all the samples more mechanism was analyzed taking in consideration than 98% of CD5/CD19 positive BNP2-treated the LC3 protein, which is processed to a cytosolic cells resulted in an apoptotic state in comparison version (LC3-I, 18KDa) and then converted to with only 11% of BNP1-treated cells. These activate forms commonly used as a marker of results seem to be independent from CD20 autophagosome accumulation. The effect of HCQ expression or from specific biological features; in on MEC1 cells was evident detecting increased fact, TP53 mutated/deleted or NOTCH1 mutated amount of LC3-III in lysates of cells incubated for patients' cells, that usually have poor response to only 16h with 0.5 or 2µL of BNP2 by western blot standard therapies (Table S-2 in the ESM), were anyway killed by BNP2. evident in our experiments on healthy mice. The reduction of side effects was addressed by Table 1 Comparison between BNP2 and Rituximab effects
including CLB+HCQ drugs in BNPs produced RITUXIMAB
from biocompatible and biodegradable polymers. (% of killing)
(% of killing)
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 microenvironment . 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 . 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 . 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, transaminase (ALT), alkaline phosphatase (ALP), lactate-dehydrogenase 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 Median (Pt)
physiological range. On the contrary, animals CLL patients (Pt); MFI: mean fluorescence intensity: mean; receiving only 4 times free HCQ+CLB showed a percentage of killing: mean (n=3). reduction in white blood cells, mainly due to the low number of circulating lymphocytes, and a significant reduction in erythrocytes. 3.4 BNPs Show a Safe Toxicological Profile
Table 2 Toxicological studies
Side effects induced by HCQ and CLB are well described in the literature [37, 38] and were also Body weight
A xenograft model of human CLL was previously described by Bertilaccio et al. , who challenged intravenously or subcutaneously Rag-/-γc-/- mice (103/mm3)
with 107 MEC1 cells. Unfortunately, we were LYMPHOCYTES
unable to repeat these results in SCID mice; in fact, (103/mm3)
the intravenous injection of 107 cells rapidly killed MONOCYTES
all the animals from respiratory problems. On the other hand, cells' subcutaneous challenging (103/mm3)
induced only the formation of a localized tumor mass at the site of injection without colonizing (103/mm3)
other tissues and inducing the death of the animals in about 70 days (Figure 3). (106/mm3)
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 Aldolase
organs and also the blood. In details, MEC1 cells' biodistribution pattern was evaluated by Total body weight, red blood cells (RBC), white blood cells immunohistochemical analysis staining tissues (WBC), platelets (PLT), and other plasma parameters from with H&E and detecting human B cells with an treated and untreated mice were compared. *= p<0.05 vs anti-human CD45 antibody 28 days after MEC1 control; §= p<0.05 vs BNP2. injection (Figure 4a). MEC1 cells were detected in liver, spleen, kidney, bone marrow, spinal cord At the same time, we observed reduced and brain. Moreover, the accumulation of MEC1 concentration of hemoglobin, creatinine, ALP and cells in mice bloodstream was confirmed by LDH, with increased values of aldolase (Table 2).
cytometric analysis using anti-human CD19 and anti-human CD45 antibodies. Human B cells 3.5 Development of a Disseminated Leukemia
presence was detected from the 20th day after Model Using MEC1 Cells
tumor cells injection (Figure 4b). 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 Figure 4 Characterization of diffuse leukemia model in
Kaplan Mayer curve SCID mice. MEC1 (5x105 cells) were injected intravenously in SCID mice and human tumor cells' distribution was analyzed Group 1 did not receive any treatment; all mice after 28 days by H&E and by exploiting human CD45 (a). died within 30-40 days after tumor cell injection Original magnification 200X. (b) Human tumor B-cells were with a median survival of 33.5 days. The also detected in the circulation by FACS analysis using labeled therapeutic protocol followed our previous data anti-CD45 and anti-CD19 mAbs. and derived from toxicological profile obtained with free HCQ+CLB. Thus, group 2 and group 3 All animals died between 30 and 37 days after received both 80µL of BNP2 (corresponding to tumor cell injection (Figure 3), as the evidence of a 400µg of each encapsulated chemotherapeutic very reproducible and very aggressive leukemia agent targeted via anti-CD20 antibody) for 8 times human/SCID model, which was useful for the in 17 days from the 1st and the 4th day after MEC1 characterization of the therapeutic effect of cell injection, respectively. The overall survival of targeted nanoparticles but also for the group 2 was 83 days and 3 mice out of 7 were development of new recombinant antibodies, as cured at the end of the study (BNP2x8 (day 1) vs already performed for other B-cell malignancies 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 3.6 BNP2 Therapeutic Effect in a Disseminated
(day 1) vs BNP2x4: p<0.03; BNP2x8 (day 1) vs Leukemia Human/Mouse Model
BNP2x8 (day 4): Not Significant). Group 3 received the same treatment but starting from day 4, The BNP2 demonstrated their ability to target resulting in a overall survival of 61 days and 1 out cancer B-cell in vivo and also their potential efficacy of 7 mice was cured (BNP2x8 (day 4) vs Untreated: in the treatment of tumor-bearing mice, as already p<0.0001; BNP2x8 (day 4) vs BNP1x8: p<0.0002; evidenced in other B-cell xenograft [18, 19]. To BNP2x8 (day 4) vs BNP3x8: p<0.0001; BNP2x8 (day study BNP2 efficacy in the treatment of the 4) vs HCQ+CLB: p<0.03; BNP2x8 (day 4) vs human/SCID leukemia model, MEC1 cells were BNP2x4: p<0.04). These results demonstrate BNP2 injected in SCID mice, divided into 8 groups of 6–8 ability to treat this aggressive human/mouse leukemia model with a better outcome when the treatment started at the early stage of the Research (AIRC Project n° 12965/2012), Italian Ministry of Health (GR‐2011‐ 02346826 and Group 4 received only 4 injections of 80µL of BNP2 GR‐2011‐ 02347441), Fondazione Casali – Trieste, in 8 days starting from the 4th day after cell Italy and Stiftung Foundation – Liechtenstein. injection. This treatment improved the overall Nanoparticles fabrication at LNK Chemsolutions, survival of about 13.5 days (BNP2x4 vs Untreated: USA, was possible in part by Grant p<0.002; BNP2x4 vs HCQ+CLB: p<0.03). Group 5 and group 6 received 8 injections of 80µL 2R44CA135906-02 (SBIR Phase II) from the of BNP1 and BNP3, respectively. Both these National Institutes of Health (USA) to Ruben treatments did not significantly increased mice Spretz, Gustavo Larsen, Sandra Noriega and Luis survival demonstrating both BNPs' safety and the inability of BNP3 to bind cancer cells due to the Conflict-of-interest disclosure: Ruben Spretz, absence of the anti-CD20 antibody on the surface Gustavo Larsen, Sandra Noriega and Luis Núñez of these particles, as already demonstrated by our working in Biotarget Inc. group . In vitro, untargeted nanoparticles Chemsolutions LLC have commercial interests in (BNP3) evidenced cell cytotoxicity but their effect the particle systems described in this work. No was not confirmed in vivo. This was probably due conflicts of interest for the other authors. to the blood flow (for circulating tumor cells) and reduced Correspondence: Sara Capolla and Paolo Macor, nanoparticles in tumor microenvironment. Department of Life Sciences, University of Trieste, Finally, group 7 received 8 injections of via L. Giorgeri, 5 – 34127, Trieste, Italy. Phone: HCQ+CLB (400µg each) in 17 days starting from +39 040 5588682; FAX: +39 040 5584023; e-mail: day 1. This treatment improved survival of 2 days [email protected], [email protected] and showed that BNP2 (groups 2) were more effective than free drugs in the treatment of this Electronic
aggressive human/mouse leukemia model. Supplementary material about CLL patients' In the group 8, three animals received 8 injections characterization (confocal microscopy, cytometry, of free drugs but all the mice died for the toxicity sequencing, killing test) is available in the online of the treatment in less than 20 days, as already 4 Conclusions
In conclusion, the results of the present study demonstrated that anti-CD20 nanoparticles References
containing HCQ+CLB can be effective as a single agent in controlling a new disseminated model of aggressive leukemia. It also provides a rationale Caligaris-Cappio, F.; Dighiero, G.; Döhner, H.; for adopting this therapeutic approach for the Hillmen, P.; Keating, M.J.; Montserrat, E.; Rai, K.R.; treatment of other B-cell disorders with BNP2 or Kipps, T.J., Guidelines for the diagnosis and treatment different types of tumors, using other monoclonal of chronic lymphocytic leukemia: A report from the antibodies to specifically deliver cytotoxic International Workshop on Chronic Lymphocytic agent-loaded nanoparticles in cancer cells.
Institute-Working Group 1996 guidelines. Blood 2008, 111, 5446–5456. This study has been made possible by research Zenz, T.; Eichhorst, B.; Busch, R.; Denzel, T.; Häbe, grants from Italian Association for Cancer S.; Winkler, D.; Bühler, A.; Edelmann, J.; Bergmann, M.; Hopfinger, G.; Hensel, M.; Hallek, M.; Döhner, H.; identifies new prognostic subgroups in chronic Stilgenbauer, S., TP53 mutation and survival in lymphocytic leukemia. Blood 2013, 121, 1403–1412. chronic lymphocytic leukemia. Journal of Clinical  Rossi, D.; Rasi, S.; Fabbri, G.; Spina, V.; Fangazio, M.; Oncology : Official Journal of the American Society of Forconi, F.; Marasca, R.; Laurenti, L.; Bruscaggin, A.; Clinical Oncology 2010, 28, 4473–4479. Cerri, M.; Monti, S.; Cresta, S.; Famà, R.; De Paoli, L.; Puente, X.S.; Pinyol, M.; Quesada, V.; Conde, L.; Bulian, P.; Gattei, V.; Guarini, A.; Deaglio, S.; Capello, Ordóñez, G.R.; Villamor, N.; Escaramis, G.; Jares, P.; D.; Rabadan, R.; Pasqualucci, L.; Dalla-Favera, R.; Beà, S.; González-Díaz, M.; Bassaganyas, L.; Foà, R.; Gaidano, G., Mutations of NOTCH1 are an Baumann, T.; Juan, M.; López-Guerra, M.; Colomer, D.; Tubío, J.M.C.; López, C.; Navarro, A.; Tornador, lymphocytic leukemia. Blood 2012, 119, 521–529. C.; Aymerich, M.; Rozman, M.; Hernández, J.M.; Wang, L.; Lawrence, M.S.; Wan, Y.; Stojanov, P.; Sougnez, C.; Stevenson, K.; Werner, L.; Sivachenko, Gutiérrez-Fernández, A.; Costa, D.; Carrió, A.; A.; DeLuca, D.S.; Zhang, L.; Zhang, W.; Vartanov, Guijarro, S.; Enjuanes, A.; Hernández, L.; Yagüe, J.; A.R.; Fernandes, S.M.; Goldstein, N.R.; Folco, E.G.; Nicolás, P.; Romeo-Casabona, C.M.; Himmelbauer, H.; Cibulskis, K.; Tesar, B.; Sievers, Q.L.; Shefler, E.; Castillo, E.; Dohm, J.C.; de Sanjosé, S.; Piris, M.A.; de Gabriel, S.; Hacohen, N.; Reed, R.; Meyerson, M.; Alava, E.; San Miguel, J.; Royo, R.; Gelpí, J.L.; Golub, T.R.; Lander, E.S.; Neuberg, D.; Brown, J.R.; Torrents, D.; Orozco, M.; Pisano, D.G.; Valencia, A.; Getz, G.; Wu, C.J., SF3B1 and other novel cancer Guigó, R.; Bayés, M.; Heath, S.; Gut, M.; Klatt, P.; genes in chronic lymphocytic leukemia. The New Marshall, J.; Raine, K.; Stebbings, L.A.; Futreal, P.A.; England Journal of Medicine 2011, 365, 2497–506. López-Guillermo, A.; Estivill, X.; Montserrat, E.; Dreger, P.; Schetelig, J.; Andersen, N.; Corradini, P.; Gelder, M. van; Gribben, J.; Kimby, E.; Michallet, M.; sequencing identifies recurrent mutations in chronic Moreno, C.; Stilgenbauer, S.; Montserrat, E., lymphocytic leukaemia. Nature 2011, 475, 101–105. Managing high-risk CLL during transition to a new treatment era: stem cell transplantation or novel agents? Fabbri, G.; Rasi, S.; Rossi, D.; Trifonov, V.; Blood 2014, 124, 3841–3849. Khiabanian, H.; Ma, J.; Grunn, A.; Fangazio, M.; Capello, D.; Monti, S.; Cresta, S.; Gargiulo, E.; Siddiqi, T.; Rosen, S., Novel Biologic Agents for Forconi, F.; Guarini, A.; Arcaini, L.; Paulli, M.; Non-Hodgkin Lymphoma and Chronic Lymphocytic Laurenti, L.; Larocca, L.M.; Marasca, R.; Gattei, V.; Leukemia - Part 1. Oncology (Williston Park) 2015, 29, Oscier, D.; Bertoni, F.; Mullighan, C.G.; Foá, R.; Pasqualucci, L.; Rabadan, R.; Dalla-Favera, R.;  Woyach, J.; Johnson, A., Targeted therapies in CLL: Gaidano, G., Analysis of the chronic lymphocytic leukemia coding genome: role of NOTCH1 mutational management. Blood 2015. activation. The Journal of Experimental Medicine  Jain, N.; O'Brien, S., Initial treatment of CLL: 2011, 208, 1389–1401. integrating biology and functional status. Blood 2015, Rossi, D.; Rasi, S.; Spina, V.; Bruscaggin, A.; Monti, 126, 463–70. S.; Ciardullo, C.; Deambrogi, C.; Khiabanian, H.;  Hallek, M., Chronic lymphocytic leukemia: 2013 Serra, R.; Bertoni, F.; Forconi, F.; Laurenti, L.; update on diagnosis, risk stratification and treatment. Marasca, R.; Dal-Bo, M.; Rossi, F.M.; Bulian, P.; American Journal of Hematology 2013, 88, 803–816. Nomdedeu, J.; Del Poeta, G.; Gattei, V.; Pasqualucci, L.; Rabadan, R.; Foà, R.; Dalla-Favera, R.; Gaidano,  Dawidczyk, C.M.; Kim, C.; Park, J.H.; Russell, L.M.; G., Integrated mutational and cytogenetic analysis State-of-the-art in design rules for drug delivery B-chronic lymphocytic leukaemia in prolymphocytoid platforms: Lessons learned from FDA-approved nanomedicines. Journal of Controlled Release 2014, 187, 133–144.  Dereani, S.; Macor, P.; D'Agaro, T.; Mezzaroba, N.;  Bartlett, D.W.; Su, H.; Hildebrandt, I.J.; Weber, W.A.; Dal-Bo, M.; Capolla, S.; Zucchetto, A.; Tissino, E.; Davis, M.E., Impact of tumor-specific targeting on the Del Poeta, G.; Zorzet, S.; Gattei, V.; Bomben, R., biodistribution and efficacy of siRNA nanoparticles Potential therapeutic role of antagomiR17 for the measured by multimodality in vivo imaging. treatment of chronic lymphocytic leukemia. J Hematol Proceedings of the National Academy of Sciences of Oncol 2014, 7, 79. the United States of America 2007, 104, 15549–15554.  Biffi, S.; Garrovo, C.; Macor, P.; Tripodo, C.; Zorzet,  Sanna, V.; Pala, N.; Sechi, M., Targeted therapy using S.; Secco, E., In Vivo Biodistribution and Lifetime nanotechnology: Focus on cancer. International Analysis of Cy5.5-Conjugated Rituximab in Mice Journal of Nanomedicine 2014, 9, 467–483. Bearing Near-Infrared Optical Imaging. Molecular Imaging 2008, 7, 272–282.  Chattopadhyay, N.; Fonge, H.; Cai, Z.; Scollard, D.; Lechtman, E.; Done, S.J.; Pignol, J.P.; Reilly, R.M.,  Marín, G.H.; Mansilla, E.; Mezzaroba, N.; Zorzet, S.; Role of antibody-mediated tumor targeting and route Núñez, L.; Larsen, G.; Tau, J.M.; Maceira, A.; Spretz, of administration in nanoparticle tumor accumulation R.; Mertz, C.; Ingrao, S.; Tripodo, C.; Tedesco, F.; Molecular Pharmaceutics Macor, P., Exploratory study on the effects of biodegradable nanoparticles with drugs on malignant B cells and on a human/mouse model of Burkitt lymphoma. Current Clinical Pharmacology 2010, 5, Nanoparticle Delivery of Cancer Drugs. Annual Review of Medicine 2012, 63, 185–198.  Macor, P.; Secco, E.; Mezzaroba, N.; Zorzet, S.;  Mezzaroba, N.; Zorzet, S.; Secco, E.; Biffi, S.; Tripodo, Durigutto, P.; Gaiotto, T.; De Maso, L.; Biffi, S.; C.; Calvaruso, M.; Mendoza-Maldonado, R.; Capolla, Garrovo, C.; Capolla, S.; Tripodo, C.; Gattei, V.; S.; Granzotto, M.; Spretz, R.; Larsen, G.; Noriega, S.; Marzari, R.; Tedesco, F.; Sblattero, D., Bispecific Lucafò, M.; Mansilla, E.; Garrovo, C.; Marìn, G.H.; antibodies targeting tumor-associated antigens and Baj, G.; Gattei, V.; Pozzato, G.; Nunez, L.; Macor, P., neutralizing complement regulators increase the New Potential Therapeutic Approach for the Treatment efficacy of antibody-based immunotherapy in mice. Leukemia 2015, 29, 406–414. Anti-CD20 Nanoparticles. PLoS ONE 2013, 8, e74216.  Zabucchi, G.; Soranzo, M.R.; Menegazzi, R.; Vecchio, M.; Knowles, A.; Piccinini, C.; Spessotto, P.; Patriarca,  Capolla, S.; Garrovo, C.; Zorzet, S.; Lorenzon, A.; P., Eosinophil peroxidase deficiency: morphological Rampazzo, E.; Spretz, R.; Pozzato, G.; Nunez, L.; immunocytochemical Macor, P.; Biffi, S., Targeted tumor imaging of eosinophil-specific anti-CD20-polymeric nanoparticles developed for the diagnosis of B-cell malignancies. International Journal of Nanomedicine 2015, 10, 4099–109.  Tripodo, C.; Florena, A.M.; Macor, P.; Di Bernardo, A.; Porcasi, R.; Guarnotta, C.; Ingrao, S.; Zerilli, M.;  Stacchini, A.; Aragno, M.; Vallario, A.; Alfarano, A.; Secco, E.; Todaro, M.; Tedesco, F.; Franco, V., Circosta, P.; Gottardi, D.; Faldella, A.; Rege-Cambrin, P-selectin glycoprotein ligand-1 as a potential target G.; Thunberg, U.; Nilsson, K.; Caligaris-Cappio, F., for humoral immunotherapy of multiple myeloma. MEC1 and MEC2: Two new cell lines derived from Current Cancer Drug Targets 2009, 9, 617–625.  Mendoza-Maldonado, R.; Paolinelli, R.; Galbiati, L.; Giadrossi, S.; Giacca, M., Interaction of the hydroxychloroquine, delivered in a biodegradable retinoblastoma protein with orc1 and its recruitment to nanoparticle system, overcomes drug resistance of human origins of DNA replication. PLoS ONE 2010, 5, B-chronic lymphocytic leukemia cells in vitro. Cancer Biotherapy & Radiopharmaceuticals 2010, 25,  Macor, P.; Tripodo, C.; Zorzet, S.; Piovan, E.; Bossi, F.; Marzari, R.; Amadori, A.; Tedesco, F., In vivo Hydroxychloroquine, targeting of human neutralizing antibodies against chloroquine, and all-trans retinoic acid regulate growth, CD55 and CD59 to lymphoma cells increases the survival, and histone acetylation in breast cancer cells. antitumor activity of rituximab. Cancer Research 2007, Anti-Cancer Drugs 2009, 20, 736–745. 67, 10556–10563.  Tehrani, R.; Ostrowski, R.A.; Hariman, R.; Jay, W.M.,  Florena, A.M.; Tripodo, C.; Iannitto, E.; Porcasi, R.; Ocular toxicity of hydroxychloroquine. Seminars in Ingrao, S.; Franco, V., Value of bone marrow biopsy in Ophthalmology 2008, 23, 201–9. thrombocythemia.  Stein, M.; Bell, M.J.; Ang, L.C., Hydroxychloroquine Haematologica 2004, 89, 911–919. neuromyotoxicity. J Rheumatol 2000, 27, 2927–2931.  Bertilaccio, M.T.S.; Scielzo, C.; Simonetti, G.; Hacken,  Rao, D.A.; Forrest, M.L.; Alani, A.W.; Kwon, G.S.; E.T.; Apollonio, B.; Ghia, P.; Caligaris-Cappio, F., Xenograft models of chronic lymphocytic leukemia: nanoparticles for sustained regional lymphatic drug problems, pitfalls and future directions. Leukemia 2013, delivery. J Pharm Sci 2010, 99, 2018–2031.  Bertilaccio, M.T.S.; Scielzo, C.; Simonetti, G.;  Kalil, N.; Cheson, B.D., Management of chronic Ponzoni, M.; Apollonio, B.; Fazi, C.; Scarfò, L.; lymphocytic leukaemia. Drugs and Aging 2000, 16, Rocchi, M.; Muzio, M.; Caligaris-Cappio, F.; Ghia, P., A novel Rag2-/-gammac-/--xenograft model of human  Zhou, Y.; Hileman, E.O.; Plunkett, W.; Keating, M.J.; CLL. Blood 2010, 115, 1605–1609. Huang, P., Free radical stress in chronic lymphocytic  Nonaka, Y.; Ishibashi, H.; Nakai, M.; Shibata, H.; Kiso, leukemia cells and its role in cellular sensitivity to Y.; Abe, S., Effects of the antlered form of Ganoderma ROS-generating anticancer agents. Blood 2003, 101, lucidum on tumor growth and metastasis in Bioscience,  Amaravadi, R.K.; Lippincott-Schwartz, J.; Yin, X.-M.; Biotechnology, Biochemistry Weiss, W.A.; Takebe, N.; Timmer, W.; DiPaola, R.S.; Lotze, M.T.; White, E., Principles and current  Zorzet, S.; Perissin, L.; Rapozzi, V.; Giraldi, T., strategies for targeting autophagy for cancer treatment. Restraint Stress Reduces the Antitumor Efficacy of Clin Cancer Res. 2011, 17, 654–666. Ciclophosphamide in Tumor-Bearing Mice. Brain,  Lagneaux, L.; Delforge, A.; Dejeneffe, M.; Massy, M.; Behavior, and Immunity 1998, 12, 23–33. Bernier, M.; Bron, D., Hydroxychloroquine-induced  Macor, P.; Secco, E.; Zorzet, S.; Tripodo, C.; apoptosis of chronic lymphocytic leukemia involves Celeghini, C.; Tedesco, F., An update on the xenograft and mouse models suitable for investigating new Bcl-2/bax/ratio. therapeutic compounds for the treatment of B-cell malignancies. Current Pharmaceutical Design 2008,  Mansilla, E.; Marin, G.H.; Nuñez, L.; Drago, H.; 14, 2023–2039. Sturla, F.; Mertz, C.; Rivera, L.; Ichim, T.; Riordan, N.; Electronic Supplementary Material
New Approach for the Treatment of CLL using
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 9Cal erio 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] 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 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 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
Silver nanowires (AgNWs) are long, thin, and possess conductivity values on the same order of magnitude as bulk silver (Ag) . 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).
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 , 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. 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 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,
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. The authors would like to acknowledge the use of the Electron Imaging Center for Nanomachines (EICN) located in the California NanoSystems Institute at UCLA. REFERENCES
Sun, Y.; Gates, B.; Mayers, B.; Xia, Y., Crystalline silver nanowires by soft solution processing. Nano Lett. 2002, 2, 165-168.
Kim, T.; Kim, Y. W.; Lee, H. S.; Kim, H.; Yang, W. S.; Suh, K. S., Uniformly interconnected silver-nanowire networks for transparent film heaters. Adv. Funct. Mater. 2013, 23, 1250-1255.
Hu, L.; Wu, H.; Cui, Y., Metal nanogrids, nanowires, and nanofibers for transparent electrodes. MRS Bull. 2011, 36, 760-765.
van de Groep, J.; Spinelli, P.; Polman, A., Transparent conducting silver nanowire networks. Nano Lett. 2012, 12, 3138-3144.
Yang, L.; Zhang, T.; Zhou, H.; Price, S. C.; Wiley, B. J.; You, W., Solution-processed flexible polymer solar cells with silver nanowire electrodes. ACS Appl. Mater. Interfaces 2011, 3, 4075-4084.
Scardaci, V.; Coull, R.; Lyons, P. E.; Rickard, D.; Coleman, J. N., Spray deposition of highly transparent, low-resistance networks of silver nanowires over large areas. Small 2011, 7, 2621-2628.
Wiley, B.; Sun, Y.; Xia, Y., Synthesis of silver nanostructures with controlled shapes and properties. Acc. Chem. Res. 2007, 40, 1067-1076.
Korte, K. E.; Skrabalak, S. E.; Xia, Y., Rapid synthesis of silver nanowires through a cucl- or cucl2-mediated polyol process. J. Mater. Chem. 2008, 18, 437-441.
Anuj, R. M.; Akshay, K.; Chongwu, Z., Large scale, highly conductive and patterned transparent films of silver nanowires on arbitrary substrates and their application in touch screens. Nanotechnology 2011, 22, 245201.
 Lee, J.-Y.; Connor, S. T.; Cui, Y.; Peumans, P., Solution-processed metal nanowire mesh transparent electrodes. Nano Lett. 2008, 8, 689-692.
 Zhu, R.; Chung, C.-H.; Cha, K. C.; Yang, W.; Zheng, Y. B.; Zhou, H.; Song, T.-B.; Chen, C.-C.; Weiss, P. S.; Li, G.; Yang, Y., Fused silver nanowires with metal oxide nanoparticles and organic polymers for highly transparent conductors. ACS Nano 2011,
5, 9877-9882.  Chung, C.-H.; Song, T.-B.; Bob, B.; Zhu, R.; Duan, H.-S.; Yang, Y., Silver nanowire composite window layers for fully solution-deposited thin-film photovoltaic devices. Adv. Mater. 2012, 24, 5499-5504.
 Kim, A.; Won, Y.; Woo, K.; Kim, C.-H.; Moon, J., Highly transparent low resistance zno/ag nanowire/zno composite electrode for thin film solar cells. ACS Nano 2013, 7,
 Ajuria, J.; Ugarte, I.; Cambarau, W.; Etxebarria, I.; Tena-Zaera, R. n.; Pacios, R., Insights on the working principles of flexible and efficient ito-free organic solar cells based on solution processed ag nanowire electrodes. Sol. Energy Mater. Sol. Cells 2012, 102, 148-152.
 Tokuno, T.; Nogi, M.; Karakawa, M.; Jiu, J.; Nge, T.; Aso, Y.; Suganuma, K., Fabrication of silver nanowire transparent electrodes at room temperature. Nano Res. 2011, 4, 1215-1222.
 Lim, J.-W.; Cho, D.-Y.; Jihoon, K.; Na, S.-I.; Kim, H.-K., Simple brush-painting of flexible and transparent ag nanowire network electrodes as an alternative ito anode for cost-efficient flexible organic solar cells. Sol. Energy Mater. Sol. Cells 2012, 107,
 De, S.; Higgins, T. M.; Lyons, P. E.; Doherty, E. M.; Nirmalraj, P. N.; Blau, W. J.; Boland, J. J.; Coleman, J. N., Silver nanowire networks as flexible, transparent, conducting films: Extremely high dc to optical conductivity ratios. ACS Nano 2009, 3,
 Hu, L.; Kim, H. S.; Lee, J.-Y.; Peumans, P.; Cui, Y., Scalable coating and properties of transparent, flexible, silver nanowire electrodes. ACS Nano 2010, 4, 2955-2963.
 Garnett, E. C.; Cai, W.; Cha, J. J.; Mahmood, F.; Connor, S. T.; Greyson Christoforo, M.; Cui, Y.; McGehee, M. D.; Brongersma, M. L., Self-limited plasmonic welding of silver nanowire junctions. Nat. Mater. 2012, 11, 241-249.
 Yu, Z.; Zhang, Q.; Li, L.; Chen, Q.; Niu, X.; Liu, J.; Pei, Q., Highly flexible silver nanowire electrodes for shape-memory polymer light-emitting diodes. Adv. Mater. 2011, 23, 664-668.
 Song, T.-B.; Chen, Y.; Chung, C.-H.; Yang, Y.; Bob, B.; Duan, H.-S.; Li, G.; Tu, K.-N.; Huang, Y., Nanoscale joule heating and electromigration enhanced ripening of silver nanowire contacts. ACS Nano 2014, 8, 2804-2811.
 Lee, P.; Lee, J.; Lee, H.; Yeo, J.; Hong, S.; Nam, K. H.; Lee, D.; Lee, S. S.; Ko, S. H., Highly stretchable and highly conductive metal electrode by very long metal nanowire percolation network. Adv. Mater. 2012, 24, 3326-3332.
 Lee, J. H.; Lee, P.; Lee, D.; Lee, S. S.; Ko, S. H., Large-scale synthesis and characterization of very long silver nanowires via successive multistep growth. Cryst. Growth Des. 2012, 12, 5598-5605.
 Bob, B.; Song, T.-B.; Chen, C.-C.; Xu, Z.; Yang, Y., Nanoscale dispersions of gelled Sno2: Material properties and device applications. Chem. Mater. 2013, 25, 4725-4730.
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.
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.
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. 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 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). 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.
Guideline for oral healthcare of adults with Huntington's disease Graham Manley1, Helen Lane1, Annette Carlsson2, Bitte Ahlborg2, Åsa Mårtensson2, Monica B Nilsson2, Sheila A Simpson3,4 & Daniela Rae*3,4; On behalf of the contributing members of the European Huntington's Disease Networks Standards of Care Dental Care Group A preventive dentistry regime should be implemented at the earliest possible opportunity and maintained throughout development of the condition. The use of high fluoride toothpaste is essential.