DOI: 10.17033/data.00000139
title: BIOCOMPATIBILITY of carbon nanoparticles with tissues of the neuromuscular system: NMS-CNT

description: NMS-CNT project focuses on solving a universal health problem, namely, the repair of extensive damage of peripheral nerves. The initial aim of the project is to undertake a rigorous evaluation of the biocompatibility of carbon nanoparticles with neural and skeletal muscles tissues. This is an essential prelude to the consortium's medium to long-term goal of developing marketable novel carbon nanotube-based implants for nerve repair. The most promising nerve repair implants have two major limitations. First, the growth of regenerating nerves within them is often random resulting in inappropriate tissue reinnervation. The recovery of tissue function is consequently poor. Second, current implants cannot support nerve regeneration over distances greater than 4cm. Carbon nanotubes (CNTs) support neuronal growth and possess a unique combination of physico-chemical properties that are ideal for modulating and optimising stages of nerve repair. CNT incorporation into current implants could improve nerve repair technology significantly by supporting and guiding the growth of regenerating axons through lesions greater than 4cm. The increasing research effort focussed on achieving this goal assumes biocompatibilty of CNTs with neural tissue and associated skeletal muscle tissue. However, to date, fundamental information concerning the impact on neuronal health of exposure to CNTs is surprisingly lacking. The consortium partners seeks to fill this considerable knowledge gap through the following objectives: 1. Prepare high quality, purified, water soluble CNTs including SW-CNTs, MW-CNTs, ultra-long-single-wall CNTs (ULSW-CNTs) and chemically functionalised preparations of each 2. Determination of catalyst content via atomic absorption spectometry, characterisation of CNTs via FT-IR and FT Raman spectroscopy 3. Identify CNT preparations (from 1) that are generally biocompatible with neurons and glial cells of the peripheral nervous system (PNS) 4. Identify the CNT preparations (from 1) that are biocompatible with adult skeletal muscle tissue and peripheral nerves 5. Study the pharmacokinetic profiles of the CNTs administered intramuscularly, intrathecally, and implanted near peripheral nerves 6. Disseminate insights from the proposed study to relevant stakeholders such as the scientific and pharmaceutical technology communities including other relevant SMEs
description: Data collection methodology as reported in Fraczek-Szczypta et al., J Nanopart Res. 2012 Oct; 14(10): 1181; doi: 10.1007/s11051-012-1181-1: Three types of as-prepared and functionalised MWCNTs were used in this study. The as-prepared MWCNTs examined in this study were provided by NanoAmor USA. The pristine MWCNTs had diameters in the range of 10–30 nm and were 1–2 lm long. The nanotubes were chemically oxidised in a mixture of concentrated H2SO4 and HNO3 acids, according to the procedures described in detail elsewhere (Fraczek-Szczypta et al. 2011). The nanotubes prepared in such a way were referred to as MWCNT-F. The aim of this process was the removal of metallic catalysts and chemical modification of nanotubes by introducing carboxyl acid groups on their surface. Simultaneously, during the oxidation process significant differences in their morphology and length were observed. The possible physical and chemical reactions taking place during the oxidative treatment of CNTs are schematically shown in Fig. 1. The next step after the chemical oxidation of the MWCNT-F was their functionalisation in ethylenediamine (C2H4(NH2)2). Amino-functionalisation of CNTs was performed to verify their influence on cell response. The literature states that the functionalisation of CNTs significantly reduces their cytotoxicity and improves cell growth (Vukovic et al. 2010; Hu et al. 2004). This process was performed according to the procedure described elsewhere (Chen et al. 1998; Shen et al. 2007) (Fig. 2). In the first step, the MWCNT-F were treated in a mixture of SOCl2 (thionyl chloride): dimethylformamide (DMF) (20:1) for 24 h at 70 C, followed by cooling them to room temperature and finally the nanotubes were washed several times using tetrahydrofuran (THF) to remove excess SOCl2. The aim of this step was to generate acyl chloride groups and facilitate functionalisation of CNTs with amines. Subsequently, such prepared CNTs were treated with ethylenediamine (C2H4(NH2)2) for 48 h at a 95 C. After this treatment, the CNTs were washed in ethanol and dried under vacuum. CNTs prepared in such a way were denoted as MWCNT-NHs. The morphology of MWCNTs before and after chemical oxidation was analysed using transmission electron microscopy (TEM) (Tecnai G2 F20 (200 kV) and Joel). The absolute zeta potential (f) and size distribution of CNTs before and after acid oxidation were performed in phosphate buffer (PBS), using combination of electrophoresis and the LDV technique (Laser Doppler Velocimetry, Malvern Zetasizer Nano ZS) in the range of the particle size from 5 nm to 10 lm, with the laser light source of wavelength k = 520 nm. The degree of purification of CNTs was determined using inductively coupled plasma optical emission spectrometry (ICP-OES) (Multiwave 3000, Perkin Elmer Co.). Evaluation of the functionalisation of CNTs was performed using Fourier transformation infrared spectroscopy (FTIR) (Bio-Rad FTS60 V spectrometer). The transmission of FTIR spectra was registered in the range of 800–3800 cm-1 using KBr pellets. The study of composition and chemical state of selected elements was made using X-ray photoelectron spectroscopy (XPS) (Vacuum Systems Workshop Ltd., England). Depth of analysis was about 5 nm. Mg Ka X-ray radiation with 200 W energy was used as the excitation source. Electron energy analyser was set to FAT mode with pass energy 22 eV. The shift of the binding energy due to surface charging effect was calibrated by assuming binding energy of C1s to be always 284.6 eV. The murine macrophage RAW 264.7 cell line (ATCC, GB) was used in this study. The cells were cultured in 75 cm2 tissue culture flasks (Nunc, Denmark) in Dulbecco’s modified Eagle’s medium (DMEM; PAA, Austria) supplemented with antibiotics (penicillin G 100 U/ml, streptomycin 10 lg/ml (Sigma-Aldrich, Germany)) and 10 % bovine foetal serum (PAA, Austria). The flasks of cultured cells were incubated at 37 C in humidified 95 % air and 5 % CO2. Cells were routinely processed by harvesting using a cell scraper and replicated in tissue culture flasks at a ratio of 1:5. Before incubation with cells (in vitro tests), each type of CNTs was sonicated for 2 min using a tip sonicator (PALMER INSTRUMENTS, Model: CP 130 PB, 130 W power, 20 kHz) in PBS, with a dual concentration of CNT 38 and 80 lg/ml, respectively. Subsequently, CNTs were sterilised by the UV method for 0.5 h. The interaction of nanotubes with RAW 264.7 macrophages was observed using an inverted microscope (Olympus CKX41, Germany) and scanning electron microscopy (SEM, Nova NanoSEM 200, FEI). To determine cytotoxicity of CNTs in contact with macrophages, ToxiLight�BioAssay Kit and ViaLight� Plus Kit tests (LONZA Rockland, Inc.) were used. The ViaLight� Plus Kit is intended for rapid and safe detection of proliferation and cytotoxicity of mammalian cells and cell lines in culture by determination of their adenosine triphosphate (ATP) levels. The ToxiLight� BioAssay is a non-destructive bioluminescent cytotoxicity assay designed to measure toxicity in mammalian cells and cell lines in culture. The kit quantitatively measures the release of adenylate kinase (AK) from damaged cells. The cytotoxicity was measured on the third day after seeding. The results were expressed as mean ± SD obtained from 8 to 11 samples for each experimental group. Significant effects (p &lt; 0.05) were determined using the unpaired Student’s t test. Data collection methodology as reported in Fraczek-Szczypta et al., 2015; Materials Science and Engineering C; https://doi.org/10.1016/j.msec.2014.10.036: Three kinds of pristine and functionalized multi-walled carbon nanotubes (MWCNTs) were used in this work. As-prepared MWCNTs were provided by NanoAmor USA. The MWCNTs synthesized by CVD method with the use of metallic catalyst (Ni) had diameters in the range of 10–30 nm and were 1–2μm long. ICP-OES analysis of the as-prepared MWCNTs indicated the presence of nickel (1.2 wt.%). The nanotubes were chemically oxidized in a mixture of concentrated H2SO4 and HNO3 acids, according to the procedures described in detail elsewhere [13] . The nanotubes after such a treatment were denoted asMWCNT-F. The aim of this stage was the removal of metallic residues from MWCNTs and receiving their hydrophilic character by the introduction of carboxylic and hydroxyl groups on their surface[14].The concentration of nickel in CNTs after oxidation (MWCNT-F) is 0.1 wt.%. The oxidation is also required for future functionalization by using ethylenediamine (C2H4(NH2)2). Amino-functionalization of MWCNT-Fs was performed to verify their influence on cell response, especially on nerve cells. As it is described in the literature the functionalization of CNTs significantly reduces their cytotoxicity and enhances cell growth, especially nerve cells[15,16]. The nanotubes prepared in such a way were referred to as MWCNT-NH. The procedure of functionalization was previously described elsewhere [14,17,18].Multi-walled carbon nanotubes before and after functionalization were investigated with muscle cells in vitro and also this material was implanted into muscle tissue of rat. Histochemical studies allowed for observation of processes connected with muscle fiber regeneration. Muscle fiber function recovery in contact with MWCNTs is associated with the restoration of innervation as a result of production of neuro-muscular synapses. To evaluate direct influence of CNTs on cell response during in vitro study the appropriate sample preparation is required. All kinds of CNTs should be uniformly distributed over the whole surface of the culture dish in which the experiment is conducted. For this purpose, three kinds of MWCNTs were deposited on PTFE membrane filters (Mem-brane Filters (PTFE supported) Whatman®) using filtration under pressure. These filters have diameter Ø = 47 mm and pore size Ø = 0.2μm. Before deposition of CNTs on PTFE membrane, each type of carbon nanotubes was sonicated for 5 min using a tip sonicator (PALMER INSTRUMENTS, Model: CP 130 PB, 130 W power, 20 kHz) in ethanol (96% CZDA, CAS: 64-17-5 POCH Co.) in a concentration of CNT0.4 mg/mL. Four milliliters of each solution was used to obtain nanotubes deposited evenly on the filter surface. The preparation process of PTFE filters with CNTs is schematically shown in Fig. 1. After the deposition, the membranes with CNTs were dried and cut into disks with a diameter fitting to the size of culture plate wells. The degree of purification and functionalization of CNTs was estimated using inductively coupled plasma optical emission spectrometry(ICP-OES) (Multiwave 3000, Perkin Elmer Co.) and Fourier transformation infrared spectroscopy (FTIR) (Bio-Rad FTS60 V spectrometer), respectively, described in detail in previous articles [13,14]. Morphology and microstructure of CNTs on a membrane were determined using scanning electron microscopy (SEM, Nova NanoSEM 200, FEI). The contact angle of CNTs on membrane was measured by sessile drop method using an automatic drop shape analysis system DSA 10 MK2 (Kruss, Germany). UHQ-water (produced by Pure lab UHQ, Elga, Germany)drops of the volume of 0.2μl were put on each sample and the contact angle was calculated by averaging the results of 10–11 measurements. The surface energy (γs) of CNTs was calculated using the Owens–Wendt method[19] . It allows for determining two components of the surface energy, i.e. the dispersive (γds) and polar (γps) components. For this purpose, two liquids with known values of γdl and γpl of the components are used. In this work it was water (γp= 51 mJ/m2 and γd= 21.8 mJ/m2) and ethylene glycol (γp= 19 mJ/m2 and γd=29 mJ/m2).2.1. In vitro study: Cell culture Murine adherent myoblast cell line C2C12 (ATCC, GB), which may differentiate into muscle cells was used. The cells were cultured in75 cm2 tissue culture flasks (Nunc, Denmark) in Dulbecco's modified Eagle's medium (ATCC, GB) supplemented with antibiotics (100 U/ml penicillin G and 100μg/ml streptomycin (Sigma-Aldrich, Germany) and 10% bovine fetal serum (ATCC, GB). The flasks of cultured cells were incubated at 37 °C in humidified 95% air and 5% CO2. Cells were routinely processed by harvesting using 5% trypsin-EDTA solution and replicated in tissue culture flasks at a ratio of 1:6. For in vitro tests membranes with deposited MWCNTs were immersed in 70% ethanol and sterilized with UV radiation for 0.5 h each side. Sterile disks cut-out from membranes were placed into 48-well culture plates and C2C12 cells were seeded on their surface at a density of 2 × 105/ml per well. The cells were cultured in contact with nanotubes for 7 days. The interaction of nanotubes with C2C12 myoblasts, their spreading and tendency to differentiate into myocytes were observed under a fluorescence microscope (Olympus, Japan) and by scanning electron microscope (SEM, Nova NanoSEM 200, FEI). To determine cytotoxic effect of nanotubes, cell viability and adhesion, ToxiLight_BioAssay Kit,ViaLight_Plus Kit and ToxiLight™100% lysis reagent set (Lonza, USA) were used. The ToxiLight_BioAssay is a non-destructive bioluminescent cytotoxicity assay designed to measure toxicity in mammalian cells and cell lines in culture. The ToxiLight™100% lysis reagent used with the ToxiLight™ cytotoxicity assay provides a total adenylate kinase control proportional to the total cell number. The ViaLight Plus Kit is intended for detection of viability and proliferation of mammalian cells by the determination of their adenosine triphosphate levels. The results were expressed as mean ± SD obtained from 5 to 8 samples for each experimental group. Significant effects (p &lt; 0.05) were determined using the unpaired Student's t test.2.2. In vivo study: The experiment was performed according to the EU ISO 10993-6guidelines. The study protocol was approved by the 1st Local Bioethics Committee in Krakow, Poland (no. 45/2010). Before the implantation, the specimens of nanotubes were sterilized using plasma method at 50 °C. The nanotubes were implanted under sterile conditions into the gluteal muscles of adult Wistar rats with a mean weight of 180 g. The animals were anesthetized with an intraperitoneal injection of ketamine and xylazine (100 mg/kg and 5 mg/kg, respectively) (Biowet, Poland). The skin at the site of the surgery was shaved and disinfected with iodine. A small incision in the skin and the underlying muscle was made to create a 4 mm deep pouch. The nanotubes were inserted into the bottom of the pouch. Each animal received a 1 mg portion of the “powder” into the left muscle. The muscle and skin wounds were closed with 5/0 PDS II(polydioxanone) monofilament absorbable sutures. All animals survived the surgery. No wound-healing complications were observed after the surgery or during the whole experiment. Before and after the surgery the animals were maintained under standard conditions with free access to food and water. After three months from the surgery animals were sacrificed by de-capitation and tissue specimens containing the implanted material were excised. Samples were immediately frozen in liquid nitrogen and cut into 8μm thick slides in a cryostat microtome. To estimate the pro-cesses of tissue regeneration, histochemical reactions were carried out on the obtained slides. The activity of enzymes (histochemical reactions): cytochrome C oxidase (CCO), acid phosphatase (FK) and non-specificesterase(NSE) in the tissues surrounding implants was examined by the methods de-scribed by Pearse, Goldberg and Barka, and Kiernan, respectively [20–22] . The main aim of these procedures was to investigate the tissue reaction to the presence of nanotubes, the extent of inflammation in tis-sues around the implant and regeneration of muscle fibers. Additionally, as the neuromuscular junctions are positive for esterases, their presence can be observed in the slides treated for the NSE activity. The neuromuscular junction sare demonstrated by dark red–brown deposits at the rim of the fibers.