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1 Protocol Project Title: Multi-walled carbon nanotubes (MWCNT) dispersion protocol using BSA plus DPPC CEIN Principal Investigator: Andre Nel Theme# 2 Version Number: 1.0 Production Start Date: 10/28/2011 Version 1.0 Date: 10/28/2011 Authors: Xiang Wang, Tian Xia, Andre Nel Department: UC-CEIN Contact Phone # s: xiangwang@ucla.edu Reviewed/Revised by: Andre Nel This protocol has been published in whole or in part in the following journal articles: 1.) Wang, X.; Xia, T.; Ntim, S. A.; Ji, Z. X.; George, S.; Meng, H. A.; Zhang, H. Y.; Castranova, V.; Mitra, S.; Nel, A. E. Quantitative Techniques for Assessing and Controlling the Dispersion and Biological Effects of Multiwalled Carbon Nanotubes in Mammalian Tissue Culture Cells. ACS Nano (12): ) Wang, X.; Xia, T.; Ntim, S. A.; Ji, Z. X.; Lin, S,; Meng, H,; Chung, C.; George, S.; Zhang, H. Y.; Wang, M.; Li, N.; Yang, Y,; Castranova, V.; Bonner, J C.; Mitra, S.; Nel, A. E. Dispersal State of Multiwalled Carbon Nanotubes Influences Pro-fibrogenic Epithelial and Macrophages Responses that Correlate with the Extent of Pulmonary Fibrosis. ACS Nano. dx.doi.org/ /nn Summary This protocol describes the background, procedures, and execution parameters for dispersion of carbon nanotubes (CNTs) by using a combination of lung lining fluid dispersants and sonication to prepare the materials for tissue experiments and lung instillation studies. Background and Project Goals This protocol describes the use bovine serum albumin (BSA) and dipalmitoylphosphatidylcholine (DPPC) to disperse and stabilize CNTs in tissue culture and other physiological media. 1/7

2 In order to accomplish reproducible and reliable experimental outcomes, it is important to control the state of CNT dispersion due to its impact on biological outcome in cells and the rodent lung. For instance, agglomerated CNTs are more prone to be deposited in proximal airways where they induce granulomatous inflammation as compared to better dispersed tubes that spread more distally to alveoli and interstitial spaces, where they may elicit interstitial fibrosis. It is important to develop a method to disperse CNTs in tissue culture medium and physiological media. We developed an effective dispersal method based on previous observation by Sager et al. (Nanotoxicology, 2007, 1: ). BSA plus DPPC provide effective tube dispersal for tissue culture as well as instillation studies in the rodent lung. In order to demonstrate the utility of this technique, we used a small series of MWCNTs that were labeled as-prepared MWNTs (AP-MWNTs), purified MWNTs (PD-MWNTs) and carboxyl modified MWNTs (COOH-MWNTs). Table 1 Physicochemical Characterization of the AP-MWCNT, PD-MWCNT and COOH-MWCNT AP-MWCNT PD-MWCNT COOH-MWCNT Outer Diameter (nm) Inner Diameter (nm) Length (µm) Purity around 94 % > 95 % > 95 % Impurities (wt. %) 4.49 % Ni, 0.76 % Fe 1.80 % Ni, 0.08 % Fe 0.18 % S Cat. #: AP-MWCNT: 112; PD-MWCNT: 113; COOH-MWCNT: 114. Materials & Reagents Materials/Reagents/Equipment Disposables 1.7 ml micocentrifuge tubes Vendor BioExpress Catalog Number C /7

3 15 ml centrifuge tubes 50 ml centrifuge tubes Reagents 1 PBS Sterile deionizd ddh 2 O Fetal Bovine Serum (FBS) Bovine serum albumin (BSA) Dipalmitoylphosphatidylcholine absolute Ethanol (100 %) Bronchial Epithelial growth media Dulbeco s Modified Eagles Medium Equipment sensitive scale (sensitivity at 0.1 mg) water sonicator bath UV-vis spectrometry Laboratory Safety Precautions Falcon Fisherbrand Invitrogen Irvine Scientific Gemini Bio Products Gemini Bio-Products Sigma-Aldrich Sigma-Aldrich Lonza Invitrogen Mettler Toledo Branson, Danbury Molecular Devices Corp P P0763 E7023 CC3171&CC AL54 Model 2510 SpectroMax M5e Nanoparticles (dry powders) handling has to be done in chemical fume hood and with N95 filter mask. Scientists performing this procedure must wear a lab coat and gloves. In situations where there might be a chance of an accidental splash to the eyes, safety glasses must be worn. Please refer to the Nanotoolkit produced by the California Nanosafety Consortium of Higher Education for recommendations regarding safe handling and disposal of nanomaterials. Prior to suspension of the nanoparticles, use engineering controls, work practices, and PPE as specified for Category 2 (Moderate Exposure Potential); after suspension, use use engineering controls, work practices, and PPE as specified for Category 1 (Low Exposure Potential) as specified in the Nanotoolkit. As described in the Nanotoolkit, NIOSH has determined that workers may be at risk of developing adverse respiratory health effects if exposed to certain nanomaterials for a working lifetime at the upper limit of quantitation (LOQ) using NIOSH Method 5040, which is currently the recommended analytical method for measuring airborne CNTs. The LOQ for CNTs using NIOSH Method 5040 is 7 μg/m3. Animal data-based risk estimates from NIOSH indicate that workers may have >10% excess risk of developing early stage pulmonary fibrosis if exposed over a full working lifetime at the upper LOQ for NIOSH Method Until improved sampling and analytical methods can be developed, and until data become available to determine if an alternative exposure metric to mass may be more biologically relevant, NIOSH is recommending a REL of 7 μg/m3 elemental carbon (EC) as an 8-hr TWA respirable mass airborne concentration. a Likewise, NIOSH recommends airborne exposure limits of 2.4 mg/m3 for fine TiO2 and 0.3 mg/m3 for ultrafine (including engineered nanoscale) TiO2, as time-weighted average (TWA) concentrations for up to 10 hr/day during a 40-hour work week. These recommendations represent levels that over a working lifetime are estimated to reduce risks of lung cancer to below 1 in 3/7

4 1,000. The recommendations are based on using chronic inhalation studies in rats to predict lung tumor risks in humans. b Citations: a NIOSH. (2010). Occupational Exposure to Carbon Nanotubes and Nanofiber. Current Intelligence Bulletin. b NIOSH. (2011). Occupational Exposure to Titanium Dioxide. Current Intelligence Bulletin. n/a Workflow Calibration Check 1) Weigh out 5 mg MWCNT powder into a glass sample vial in a chemical fume hood, add 1 ml sterile deionzied (DI) H 2 O to achieve a 5 mg/ml stock solution; 2) Prepare the BSA and DPPC dispersion medium using tissue culture media (e.g. BEGM, DMEM, and RPMI 1640) or PBS (carrier for pulmonary instillation studies). 3) Add the MWCNT stock solution to the dispersion medium (containing BSA and DPPC) and sonicate to achieve MWCNT dispersion; 4) Verifying the state of the MWCNT dispersion by determining the suspension stability index. Reagent/Stock Preparation 1) Prepare a 4 wt % low-endotoxin bovine serum albumin (BSA) stock solution: weigh out the 1 g of BSA powder using a sensitive scale (e.g. Mettler Toledo Cat.# AL54) to make a 4 wt % BSA stock solution at in sterile DI H 2 O (Irvine Scientific, Cat.# 9309); 2) Prepare a 10 mg/ml dipalmitoylphosphatidylcholine (DPPC) stock solution: add 1 ml absolute ethanol to 10 mg DPPC to make the DPPC stock solution at 10 mg/ml; 3) Prepare a 5 mg/ml MWCNT stock solution: weigh out 5 mg MWCNT powder using a sensitive scale (e.g. Mettler Toledo Cat.# AL54) in a chemical fume hood. Add the powder directly to 1 ml sterile DI H 2 O in a glass sample vial to achieve a 5 mg/ml stock solution. Sonicate the stock 4/7

5 solution for 15 min in a water bath sonicator (Branson, Model 2510, 100 W output power; 42 khz frequency). Procedure 1. Prepare a 5 mg/ml of MWCNT stock solution 1) To make stock solutions, the MWCNT dry powder is weighed in a chemical fume hood by using a sensitive (e.g. Mettler Toledo Cat.# AL54) scale that can detect 0.1 mg quantities of material. 2) The MWCNT powder is brought to a concentration of 5 mg/ml using sterile DI H 2 O (Irvine Scientific, Cat.# 9309) to obtain a MWCNT stock solution. All the working MWCNT solutions are prepared fresh from the stock solution. 2. Prepare a 10mL of BSA and DPPC dispersion medium 1) Prepare 10 ml of a 0.6 mg/ml BSA solution in the selected tissue culture medium (e.g. BEGM, DMEM, and RPMI 1640) or PBS by adding 150 μl 4 wt % of BSA solution into a sterile centrifuge tube containing 9.85 ml tissue culture medium or PBS; 2) Prepare 1 ml of 10 mg/ml dipalmitoylphosphatidylcholine (DPPC) stock solution by adding the 1 ml absolute ethanol to 10 mg DPPC powder; 3) Add 10 μl 10 mg/ml DPPC stock solution to the solution prepared in 2-1) to obtain 10 ml dispersion medium containing 0.01 mg/ml DPPC and 0.6 mg/ml BSA. This is known as the fully reconstituted dispersion medium. Culture medium or PBS without BSA and DPPC is labeled the non-reconstituted medium. 5/7

6 3. Prepare MWCNT dispersion in BSA and DPPC dispersion medium 1) The 5 mg/ml MWCNT stock solution is vortexed for 5 seconds before sonication for 15 min using a water bath sonicator (Branson, Model 2510, 100 W output power; 42 khz frequency). Add 20 μl of MWCNT stock solution to 980 μl of the fully reconstituted dispersion tissue culture or PBS dispersion medium to achieve a 100 μg/ml MWCNT working solution for experiments. This MWCNT working solution should be sonicated for 15 min before further dilution and treatment. 4. Characterization of the state of MWCNT dispersion using the suspension stability index 1) Turn on the UV-vis spectrometry (SpectroMax M5e, Molecular Devices Corp., Sunnyvale, CA, USA) for 10 min, allowing the temperature to stabilize before use. The power switch is located at the back right of the instrument; 2) For comparison of the MWCNT dispersion in fully reconstituted dispersion medium, prepare 1 ml MWCNT suspension at 50 µg/ml in the desired tissue culture medium or PBS in 1.7 ml micocentrifuge tubes following the same procedures as in section 1; also prepare 1 ml MWCNTs suspended in non-reconstituted tissue culture medium or PBS at 50 µg/ml; 3) Sonicate the fully reconstituted MWCNT suspension in a water bath sonicator (Branson, Model 2510, 100 W output power; 42 khz frequency) for 15 min and then transfer the solution into a 1 ml cuvette by using a syringe or pipette; 4) Load the cuvette (containing the MWCNT suspension) into the cuvette holder. Make sure the cuvette is placed flat and firmly in the holder before closing the lid; 6/7

7 5) Launch the SpectroMax M5e software and set the absorbance wavelength to 550 nm (λ=550 nm) for the measurement, and click READ to obtain the first absorbance at t=0 hr with follow up reading at 1 hr time intervals for 4-6 hr and then again at 24 hr; 6) For comparison of the non-reconstituted MWCNT suspension, follow the same steps from 3) to 5) by using non-reconstituted tissue culture medium or PBS; 7) The suspension stability index of the MWCNT at 50 µg/ml is expressed as the % of the initial absorbance (λ=550 nm) for tubes incubated for 24 hr after sonication in the desired tissue culture medium or PBS. SOP Approval DEPARTMENT APPROVED BY DATE Principle Investigator Andre Nel MSSR 7/7