1. CAG system assembly
<ol>
	<li>Assembly of CAG
	<ol>
		<li>Place the capillary in the capillary groove of the aluminum heating blocks, with the output end protruding by about 5 mm.</li>
		<li>Lightly tighten the screws of the two halves of the aluminum heating blocks.</li>
		<li>Assemble the heating elements (a) and thermocouple (b) in the aluminum heating blocks (c), with the wires protruding through the aluminum rear cap (d) (Figure 4A).</li>
		<li>Ensure that the wires of the heating elements are connected to an adaptor and ensure that they are straight.</li>
		<li>Assemble the inner PEEK tube (g) with the outer SS tube (e). Ensure that the 2 x 4 mm push-in fittings (f) are tightly secured onto the outer SS tube (e) (Figure 4B).</li>
		<li>Place O-rings (3 x 30 mm) on the two grooves of the inner PEEK tube (g) and insert the inner PEEK tube (g) into the outer SS tube (e) from the front end.</li>
		<li>Place the assembled aluminum heating elements on the SS rear backing (i), with the aluminum rear cap facing the SS rear backing, and slide the inner PEEK/outer SS tube assembly over the aluminum heating elements through to fit tight with the SS rear backing (i) (Figure 4C).</li>
		<li>Place the aluminum front cap (h) over the aluminum heating element, inside the inner PEEK tube. Ensure that the capillary is slightly protruding from the aluminum front cap. Install the three SS lead screws (j) around the SS rear backing and tighten firmly.</li>
		<li>Place the PEEK adaptor (k) over the inner PEEK tube front. Ensure that the PEEK adaptor fits on the front groove of the inner PEEK tube. Place the 25 mm Scheduler (l) over the PEEK adaptor and through the three SS lead screws. Hand-tighten the nuts over the scheduler such that the PEEK adaptor is tight (Figure 4D).</li>
		<li>Connect the heating elements to the temperature controller and the capillary to the peristaltic pump and the test liquid solution.</li>
		<li>Connect the compressed air for heated air flow to the CAG via the 2 x 4 mm push-in fittings (Figure 4B, [f]).</li>
		<li>Assemble the CAG to the glass piece and connect CAG cooling and first dilution air flows (processed air; Figure 3). Add a second dilution flow entry when necessary as well as aerosol sampling ports and a regulatory T-junction (Figure 5).</li>
	</ol>
	</li>
	<li>CAG cleaning procedure
	<ol>
		<li>Remove the CAG from the CAG glass assembly setup and clean the glass with dry wipes until the glass is visibly dry.</li>
		<li>Observe the capillary output from the CAG for obstruction. If particle deposition can be observed on the outlet of the capillary, change the capillary. Similarly, upon noticing reduced aerosol delivery, replace the capillary with a new one.</li>
		<li>Disassemble the CAG following steps 1.1.9 to 1.1.1.</li>
		<li>Reassemble the CAG following the steps 1.1.1 to 1.1.9 once the capillary is changed.</li>
	</ol>
	</li>
</ol>
2. Calculation of CAG aerosol concentration and dilution
<ol>
	<li>Theoretical calculation of TDF
	<ol>
		<li>Calculate the TDF based on the concentration of the liquid formulation (called stock solution/concentration here) and the LFR: 
		<img alt="Equation 1" src="/files/ftp_upload/61021/61021eq01.jpg" style="margin: auto;" /> 
		TDF: total dilution air flow (L/min) 
		CStock: stock concentration 2%, w/w) 
		LFR: liquid Flow Rate (g/min) 
		CTarget: target concentration (&#181;g/L)</li>
		<li>Using a solution with 2% (w/w) nicotine, with a target nicotine aerosol concentration at 15 &#181;g/L and a LFR of 0.35 g/min, assume that 100% yield will be the following: 
		<img alt="Equation 2" src="/files/ftp_upload/61021/61021eq02.jpg" style="margin: auto;" /> 
		<img alt="Equation 3" src="/files/ftp_upload/61021/61021eq03.jpg" style="margin: auto;" /></li>
	</ol>
	</li>
	<li>Theoretical calculation of LFR
	<ol>
		<li>Calculate the LFR based on the concentration of the liquid stock solution and the TDF: 
		<img alt="Equation 4" src="/files/ftp_upload/61021/61021eq04.jpg" style="margin: auto;" /> 
		LFR: liquid flow rate (g/min) 
		CTarget: target concentration (&#181;g/L) 
		TDF: total dilution air flow (L/min) 
		CStock: stock concentration (%, w/w)</li>
		<li>Using a solution with 2% (w/w) nicotine, with a target nicotine aerosol concentration at 15 &#181;g/L and a TDF of 300 L/min, assume that a 100% yield will be the following: 
		<img alt="Equation 5" src="/files/ftp_upload/61021/61021eq05.jpg" style="margin: auto;" /> 
		<img alt="Equation 11" src="/files/ftp_upload/61021/61021eq11.jpg" style="margin: auto;" /></li>
	</ol>
	</li>
	<li>Calculation of actual yield (%) based on experimental data
	<ol>
		<li>Based on the above theoretical calculations, perform the initial engineering runs to quantify the actual aerosol constituent concentration (CActual) and obtain the actual yield (AY) of the CAG aerosol. Perform further fine-tuning of aerosol concentration by using the same calculations for adjustment of TDF or LFR. 
		<img alt="Equation 6" src="/files/ftp_upload/61021/61021eq06.jpg" style="margin: auto;" /> 
		AY: actual yield (%) 
		CActual: actual aerosol constituent concentration (&#181;g/L) 
		TDF: total dilution air flow (L/min) 
		CStock: stock concentration (%, w/w) 
		LFR: liquid flow rate (g/min)</li>
		<li>Using a solution containing 2% (w/w) nicotine, with a measured nicotine aerosol concentration of 15 &#181;g/L, TDF of 320 L/min, and LFR of 0.35 g/min will result in the following nicotine AY: 
		<img alt="Equation 7" src="/files/ftp_upload/61021/61021eq07.jpg" style="margin: auto;" /> 
		<img alt="Equation 8" src="/files/ftp_upload/61021/61021eq08.jpg" style="margin: auto;" /></li>
	</ol>
	</li>
</ol>
3. CAG aerosol generation
<ol>
	<li>Starting aerosol generation
	<ol>
		<li>Weigh and record the value of the test liquid, magnetic stirrer, and the bottle to a nearest 0.01 g. Liquid stock formulations are prepared with components described in Table 1.</li>
		<li>Supply the respective airflow settings (&#177;5%) (Figure 5): 
		Compressed air for heated flow: 2 L/min 
		Cooling flow: 10 L/min 
		First dilution flow: 150 L/min 
		Second dilution flow: 160 L/min 
		Waste flow: 172 L/min</li>
		<li>Set the temperature control set-point on the digital temperature controller to 250 &#176;C and begin heating of the CAG.</li>
		<li>Place the liquid stock solution with a magnetic stir bar on a magnetic stirrer. Place the inlet tube from the peristaltic pump in the test solution.</li>
		<li>Turn on the peristaltic pump and set the flow to the LFR &#177;5% (g/min).</li>
		<li>When the CAG temperature reaches 250 &#177; 1 &#176;C, begin aerosol generation by starting the peristaltic pump to deliver test liquid to the CAG.</li>
		<li>Check whether the aerosol is generated near the capillary tip and record the time as necessary to calculate the mass flow rate. If no aerosol is generated, check all the equipment and settings again. If still no aerosol is generated, it is highly probable that the capillary is blocked and needs to be replaced.</li>
	</ol>
	</li>
	<li>During aerosol generation
	<ol>
		<li>Drain the liquid that condensates in the glass setup every 60 min, to ensure constant and stable aerosol generation.</li>
	</ol>
	</li>
	<li>Stopping aerosol generation
	<ol>
		<li>Remove the tubing from the test solution bottle and switch the test liquid to deionized water and record the time for calculating the mass flow rate.</li>
		<li>Wait until water vapor comes out from the capillary, turn off the temperature controller, and keep the peristaltic pump on for at least 10 min to flush and clean the capillary.</li>
		<li>Weigh and record the value of the testing liquid and bottle to the nearest 0.01 g and calculate the mass flow rate using the following equation: 
		<img alt="Equation 9" src="/files/ftp_upload/61021/61021eq09v2.jpg" style="margin: auto;" /></li>
		<li>Turn off the compressed air used as heated flow.</li>
		<li>If necessary, remove the CAG from the assembly setup and clean the glass tubing with dry wipes and reassemble the CAG.</li>
	</ol>
	</li>
</ol>
4. Analytical determination of constituents
NOTE: Aerosol sampling is performed at two positions: a) on the undiluted aerosol (both first dilution air and second dilution flow are switched off during undiluted sampling) and b) on the diluted aerosol with all dilutions provided (Figure 5). Up to three sampling ports are available at each of the sampling positions, a and b, allowing simultaneous collection of ACM, and other equipment/probes for analysis of aerosol characteristics. The sampling line is installed perpendicular to the aerosol flow direction and connected to a vacuum pump that allows for drawing a certain volume of aerosol (depending on the pump flow rate and sample duration).
<ol>
	<li>Determination of Aerosol Collected Mass (ACM) 
	NOTE: The particulate phase of the aerosol is trapped on a glass-fiber filter pad (diameter: 44 mm, particle size retention: 1.6 &#181;m). ACM weights before and after sampling are measured with filter holders to minimize losses in weighing due to evaporation of volatile components.
	<ol>
		<li>Place a filter in the filter holder and place the filter caps.</li>
		<li>Weigh the filter holder to nearest 0.0001 g with the filter before sample collection and document the weight.</li>
		<li>Connect the filter holder containing the filter to the aerosol flow and start sample collection.</li>
		<li>After sample collection, weigh the filter with the filter holder and caps, and document the end weight.</li>
		<li>Calculate the ACM using the following formula: 
		<img alt="Equation 10" src="/files/ftp_upload/61021/61021eq10.jpg" style="margin: auto;" /> 
		ACM: concentration of ACM (&#181;g/L) 
		Wb: weight of the filter and filter holder before sampling (g) to nearest 0.0001 g 
		Wa: weight of the filter and filter holder after sampling (g) to nearest 0.0001 g 
		Vaerosol: Volume of aerosol (L) passing through the filter, calculated using: 
		Sampling time (min) x sampling flow (L/min)</li>
		<li>Remove the filter pad from the filter holder and deposit it into a 25 mL glass vial containing 5 mL of ethanol. Extract the ACM by shaking the filter pad on a laboratory shaker for 30 min at 400 rpm.</li>
		<li>Centrifuge the 25 mL glass vial for 5 min at 290 x g and collect the supernatant for quantification of PG/VG and the particulate phase of nicotine.</li>
	</ol>
	</li>
	<li>Determination of nicotine (or flavor) concentration 
	NOTE: The aerosol is trapped on a sample column containing specially processed wide-pore diatomaceous earth, a chemically inert matrix for use in a pH range of 1 to 13 (Figure 6).
	<ol>
		<li>Prepare the sample column within 15 min before starting the aerosol sample collection.
		<ol>
			<li>For determining nicotine concentrations, add 2 mL of 0.5 M sulfuric acid. For determining flavors, add 2 mL of isopropanol.</li>
		</ol>
		</li>
		<li>Check the sampling flow.
		<ol>
			<li>Switch on the vacuum pump, and using the calibrated flow equipment that provides accuracy to 1 ccm/min, check the flow rate with a sample column connected to the sampling line.Adjust the flow with the needle valve to the range of 700 ccm/min &#177; 5%.</li>
			<li>Switch off the vacuum pump.</li>
		</ol>
		</li>
		<li>Sample collection
		<ol>
			<li>Add the two adaptors to the sample column according to its inlet and outlet side (Figure 6). Connect the tube to the vacuum sampling line via the outlet adaptor.</li>
			<li>Connect the sample column assembly to the sampling port via the inlet adaptor.</li>
			<li>Start sample collection by switching on the vacuum pump.</li>
			<li>Record the sampling start time.</li>
			<li>After a preset sampling time, 10 min at undiluted sampling point A and 30 min at diluted sampling point B, switch off the vacuum pump and record the time.</li>
			<li>Remove the sample column from the sampling port.</li>
			<li>Remove the adaptors from the sample column and seal the sample column with a film membrane to prevent losses due to evaporation or contamination. Label the sample column according to the corresponding sample name.</li>
			<li>Store the sealed sample column in a fridge (2-8 &#176;C) until analysis.</li>
		</ol>
		</li>
		<li>Determination of carbonyl concentrations 
		NOTE: Carbonyls are trapped on a glass-filter pad connected in series to a micro-impinger filled with 2,4-dinitrophenylhydrazine (DNPH) dissolved in acetonitrile.</li>
	</ol>
	</li>
	<li>Preparation for trapping
	<ol>
		<li>Fill the micro-impinger with 10 mL of 15 mM DNPH in acetonitrile.
		<ol>
			<li>Prepare a filter pad (see paragraph 4.1).</li>
			<li>Check sampling flow</li>
		</ol>
		</li>
		<li>Switch on the vacuum pump and check the flow rate of the sampling line using a calibrated flow equipment that provides an accuracy of 1 ccm/min. Adjust the flow with the needle valve to the range of 700 ccm/min &#177; 5%.
		<ol>
			<li>Switch off the vacuum pump.</li>
		</ol>
		</li>
		<li>Sampling collection
		<ol>
			<li>Connect the filter holder linked to the micro-impinger to the sampling port.</li>
			<li>Connect the vacuum sampling line to the outlet of the micro-impinger.</li>
			<li>Start sample collection by switching on the vacuum pump.</li>
			<li>Record the sampling start time.</li>
			<li>After a preset sampling time, 10 min at undiluted sampling point a and 30 min at diluted sampling point b, switch off the vacuum pump and record the time.</li>
			<li>Disconnect the sampling trap from the sampling port.</li>
			<li>Empty the impinger into a glass vial. Top up the DNPH solution to 10 mL with acetonitrile.</li>
			<li>Determine the weight of the filter pad and extract it in the DNPH-acetonitrile solution by shaking. Discard the filter pad after extraction.</li>
			<li>Take a 1 mL aliquot of the carbonyl-DNPH solution and add 50 &#181;L of pyridine to stabilize the solution.</li>
			<li>Store the aliquots in a freezer at &#8804;-12 &#176;C until analysis.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The method reported here as well as the specific CAG assembly have been developed for evaluation of aerosols generated from e-liquids to fulfill the requirements of in vivo exposure studies. All the authors are employees of Philip Morris International (PMI) or have worked for PMI under contracted agreements. Philip Morris International is the sole source of funding and sponsor of this study.

Generating aerosols with CAG helps reduce the variability of EC-device specific aerosolization processes, allowing for objective and controllable assessment of the aerosolized e-liquid formulation itself. CAG-generated aerosols have been shown to be representative of the aerosols generated by ECs7. They can be reproducibly generated with the same composition and characteristics and are, therefore, particularly suitable for in vivo long-term exposure studies requiring large volumes of aerosol over a long period of time8.
The CAG setup is relatively simple to assemble and easy to maintain. However, the operating parameters, such as liquid flow rate and respective air flow rates remain critical for production of controlled aerosol, which requires method optimization according to the purpose of application of the CAG-generated aerosol.
The results presented in the current study show that cooling airflow rate has a clear effect on aerosol particle size distribution. The cooling airflow has a direct impact not only on the nucleation of the generated vapors but also on condensation, because of the cooling of the internal tubing in which the generated aerosol flows. In addition, the dense aerosol is prone to substantial coagulation effects. Combined, these processes are complex and their interaction and influence on aerosol formation are rather difficult to generalize for the specific e-liquids, temperatures, and flows. Supplementary airflow composition (dry or humidified with a fixed percentage of relative humidity)-in particular, water content-will influence heat and mass exchange, leading to not only modulated condensation growth of aerosol particles but also wall condensation. Thus, modifications to this method&#39;s parameters are deemed for purpose of use in terms of controlling the PSD17,19.
The presence of chemicals with low solubility or high boiling points could limit the effectiveness of CAG-generated aerosol due to precipitation within the capillary and clogging of the capillary over time. Depending on the chemicals present in the aerosol, the temperature for operating the CAG must be adjusted to generate the vapor. In addition, the stability of the liquid formulation should be regularly assessed. Addition of constituents, including flavors, with different boiling points will have an influence on the final aerosol composition14 and gas-liquid partitioning. It might be necessary to adapt the capillary temperature and heating airflow to prevent backflow and liquid deposition near the hot capillary, which could result in generation of uncontrolled products of thermal degradation (such as carbonyls) because of the long duration of retention of the liquid at a high temperature. In addition, controlling the temperature used to generate the vapor in the capillary has an impact on where the vapor starts to form in the capillary-the higher the temperature, the earlier the vapor is formed. With a higher capillary temperature, the vapor coming out of the capillary will take longer to be cooled down by the cooling air flow and will, therefore, start to nucleate and condense into an aerosol further away from the capillary tip, helping avoid a backflow effect19.
Current e-liquid in vivo toxicology studies are limited in reproducing e-cigarette aerosols due to the logistical complexity to meet the scale of aerosol required, such as in an OECD TG 413 study20. The protocol presented in this study gives an overview on the CAG assembly and settings used at Philip Morris International for aerosol generation in in vivo long-term exposure studies18. These data can serve as a good starting point for further fine-tuning in another laboratory environment (e.g., drug delivery systems21) or for adaptation to specific requirements of a particular study.

Common e-liquids contain a mixture of propylene glycol, glycerol, water, nicotine, and selected flavors. The composition of an aerosol generated from an EC device depends not only on the liquid formulation, but also on the material, design, and characteristics of the device. Consequently, many EC devices may introduce a large variability in aerosol output1, including device-specific production of elevated levels of unwanted constituents, puff volume variation, change in airflow due to blocked ventilation holes, and &#34;dry puffing&#34; (when the liquid container is nearly empty, causing overheating of the device because part of the delivered energy is not used for liquid evaporation)2. In addition, charging, refilling, and cleaning EC devices during long-term inhalation studies would become a huge additional constraint in terms of logistics3. For these reasons, other aerosol generators should be considered for large-scale production of aerosols and proper evaluation of liquid formulations while avoiding device-related variations in aerosol composition and decreasing the work load4,5. Nevertheless, device-generated aerosols should remain a part of risk-assessment strategies, because the levels of certain constituents in EC devices might be more elevated than those in laboratory-controlled standardized aerosol generators owing to the heating/cooling specificities of the devices6,7,8.
Owing to the limited information on regulatory requirements currently available, evaluation methods for the potential toxicity of aerosols generated by electronic cigarettes (EC) are still evolving9,10,11. However, accurate in vitro and in vivo evaluation requires the generation of well-characterized and reproducible volumes of aerosol over time. Producing aerosol from an EC device with a controlled puffing regimen would certainly be the most representative process from the perspective of user consumption. For regulatory toxicity studies, considering a variety of possible liquid formulations that users may often prepare by themselves and, at the same time, modifying some device characteristics (e.g., delivered energy), the use of EC devices for performing long-term repeated-exposure toxicology studies are not only challenging but also potentially inadequate.
The capillary aerosol generator (CAG)-developed by Philip Morris12,13 and further refined by Virginia Commonwealth University14-works on the principle of creating a jet of hot vapor flow from an electrically heated capillary, that is subsequently cooled down with ambient air, causing particle nucleus formation and subsequent condensation, leading to aerosol formation. Because the same physical processes lead to aerosol formation in ECs (apart from the delivery of the liquid to the capillary by a pump in the CAG, which, in an EC, is usually replaced by capillary forces acting on the wicking material drawing the liquid from the reservoir in the EC), the characteristics of CAG-generated aerosols are very similar to those of EC aerosols14 (Figure 1). The CAG allows production of large volumes of aerosol, with few handling requirements; it is, therefore, particularly suitable for in vivo inhalation studies.
The CAG is a laboratory device consisting of a heated capillary tube simply connected to a temperature controller and to a liquid reservoir via a peristaltic pump (Figure 2A). The capillary (160 mm, 21 G, stainless steel) is heated by four heating elements, all embedded into an aluminum block (Figure 2B). The temperature is typically set at 250-275 &#176;C to mimic the coil-heating conditions of an EC device15. The liquid pumped through the capillary is heated up and turned into hot vapor exiting from the tip of the capillary. The CAG assembly (Figure 2C) requires additional elements for mixing the generated vapor with cold air and forming an aerosol. The abrupt mixing of the hot supersaturated vapor with a cold air stream results in nucleation and subsequent condensation, leading to aerosol formation (Figure 2C). In our CAG design (Figure 3), an additional heated air flow first cools down the external body and afterward circulates along the heating blocks to heat up the air flow, preventing, at the same time, condensation of the liquid backflow at the tip of the capillary and stabilizing the vapor jet burst. Additionally, it creates unwanted shielding of hot vapors, thus impacting the nucleation process. For this reason, the flow rate applied for this air flow should be minimal and fit the purpose of the application. This air flow will be called &#34;heated airflow&#34; throughout this manuscript, although it must be understood that this stream is heated passively by the heating blocks and not on purpose by the user.
The cooling airflow rate has a strong influence on the size of the generated aerosol particles. In aerosol production for in vivo inhalation studies, the dilution air flow will determine the exposure dose and might have to be further diluted before reaching the exposure chamber. Besides the chemical composition of aerosols, it is essential to characterize aerosol particle size distribution (PSD) to ensure that the generated aerosol is similar to that generated by ECs and within the inhalation particle size range recommended by the OECD guidelines (often parameterized by the assumption of log-normality of PSD with mass median aerodynamic diameter [MMAD] and geometric standard deviation [GSD]).
The MMAD of the generated aerosols can vary widely depending on the device design, physicochemical liquid properties of the formulation (e.g., density, viscosity, and surface tension), air flow rate, and temperature dictating thermodynamic conditions14,16,17. For in vivo exposure experiments, the airflow generally consists of conditioned, filtered air at 22 &#177; 2 &#176;C and 60% &#177; 5% relative humidity. The generated aerosol can then be diluted further depending on the study requirements, to achieve target concentrations in the test atmosphere. It is then delivered via glass piping to the exposure chamber in order to diminish filtration loss. In the results presented here, the temperature and airflow settings are established to demonstrate that the CAG can be used for continuous production of a controlled aerosol with consistent and inhalable PSD and defined concentrations for in vivo inhalation studies.
In the protocol, we will describe how to: 1) assemble the CAG, 2) determine parameters required to generate aerosol from the CAG, 3) perform aerosol generation, and 4) analyze physical and chemical constituents of interest in the aerosol. For these preliminary runs, we consider a liquid solution based on a mixture of aerosol-forming components: propylene glycol (PG), glycerol (VG), water and nicotine at prescribed mass fractions. Finally, we will share example data for assessment of a complex multispecies mixture generated in our experiments (involving the abovementioned constituents mixed with additional flavor constituents). We will discuss the overall results and challenges along with the applicability of this experimental approach for assessment of such mixtures.

<table><tbody><tr><td>Aluminium front cap</td><td>Mecanique Buri S.A., La Chaux-de Fonds, Switzerland</td><td>Custom Built</td><td>Purpose built, 1 x</td></tr><tr><td>Aluminium heating block, groove diameter 0.4mm</td><td>Phil Gunn Machine Co., Inc, VA, USA</td><td>B-505432</td><td>2 x</td></tr><tr><td>Aluminium rear cap</td><td>Mecanique Buri S.A., La Chaux-de Fonds, Switzerland</td><td>Custom Built</td><td>1 x</td></tr><tr><td>Cambridge glass filter pads</td><td>GE Healthcare UK Limited</td><td>9703-9654</td><td>44 mm diameter</td></tr><tr><td>Capillary 21 G SS, 160 mm</td><td>Phil Gunn Machine Co., Inc, VA, USA</td><td>304H21RW</td><td>1 x</td></tr><tr><td>Dry wipes</td><td>Contec Inc. , SC, USA</td><td>Prosat Wipes saturated with isopropyl alcohol</td><td>cleaning material</td></tr><tr><td>Flowmeter</td><td>TSI, Shoreview, MI, USA</td><td>4100 Series, 0-20 L/min</td><td>or equivalent</td></tr><tr><td>Gilibrator-2 calibrator</td><td>Sensidyne, St-Petersburg FL, USA</td><td>Gilian Gilibrator-2</td><td>Air flow calibrator</td></tr><tr><td>Glass Couplings</td><td>Labo Service, Kontich, Belgium</td><td>QVF</td><td></td></tr><tr><td>Glass piping</td><td>Labo Service, Kontich, Belgium</td><td>QVF</td><td>Pipe 25 and 40 mm</td></tr><tr><td>Heating elements</td><td>Phil Gunn Machine Co., Inc, VA, USA</td><td>LDC01864</td><td>4 x</td></tr><tr><td>High heat grease</td><td>Lubriplate Lubricant Company, NJ, USA</td><td>High temperature multipurpose grease</td><td>CAG maintenance</td></tr><tr><td>Inner PEEK tube</td><td>Mecanique Buri S.A., La Chaux-de Fonds, Switzerland</td><td>Custom Built</td><td>1 x</td></tr><tr><td>Magnetic stirrer</td><td>IKA-Werke GmbH &amp;amp; Co. KG, Staufen, Germany</td><td>C-MAG HS 4</td><td>or equivalent</td></tr><tr><td>Micro impingers</td><td>Labo Service, Kontich, Belgium</td><td>Custom Built</td><td></td></tr><tr><td>Outer SS tube</td><td>Mecanique Buri S.A., La Chaux-de Fonds, Switzerland</td><td>Custom Built</td><td>1 x</td></tr><tr><td>PEEK adaptor</td><td>Mecanique Buri S.A., La Chaux-de Fonds, Switzerland</td><td>Custom Built</td><td>Purpose built, 1 x</td></tr><tr><td>Peristaltic pump</td><td>Watson-Marlow Fluid Technology Group, Falmouth, UK</td><td>Watson-Marlow 530 U</td><td>or equivalent</td></tr><tr><td>Push-in fitting</td><td>Festo Pte Ltd</td><td>NPQM-DK-M5-Q4-P10</td><td>1 x</td></tr><tr><td>Sample Column Extrelut NT3 cartridge</td><td>Merk Sigma-Aldrich</td><td>115095</td><td></td></tr><tr><td>SS 25 mm assembly cap</td><td>Mecanique Buri S.A., La Chaux-de Fonds, Switzerland</td><td>Custom Built</td><td>Purpose built, 1 x</td></tr><tr><td>SS M8 lead screw</td><td>Mecanique Buri S.A., La Chaux-de Fonds, Switzerland</td><td>Custom Built</td><td>3 x</td></tr><tr><td>SS M8 nut</td><td>Mecanique Buri S.A., La Chaux-de Fonds, Switzerland</td><td>Custom Built</td><td>3 x</td></tr><tr><td>SS rear backing</td><td>Mecanique Buri S.A., La Chaux-de Fonds, Switzerland</td><td>Custom Built</td><td>Purpose built, 1 x</td></tr><tr><td>Temperature controller</td><td>Cole Parmer GmbH, Wertheim, Germany</td><td>Digi-Sense TC 9600</td><td>or equivalent</td></tr><tr><td>Thermocouple type K</td><td>RS Components GmbH, W&amp;auml;denswil, Switzerland</td><td>814-0147</td><td>1 x</td></tr></tbody></table>

use of capillary aerosol generator in continuous production of controlled aerosol for non-clinical studies

The capillary aerosol generator (CAG) is operated with the principal of thermal liquid evaporation through heating of e-liquid in the initial phase, followed by nucleation and condensation regulated through a mixture of airflow to generate aerosols, such as in an electronic cigarette (EC). The CAG is particularly useful in generating aerosols of large volumes in a continuous manner, for instances such as in vivo inhalation toxicology studies, where usage of ECs is not feasible. The thermal effects of generating aerosol from the CAG are similar in terms of temperature applied in an EC, thus allowing investigators to assess the vapors of e-liquids at scale and reproducibility. As the operation of the CAG allows users to control critical parameters such as the flow rate of e-liquid, heating temperatures and dilution air flows, it allows investigators to test various e-liquid formulations in a well-controlled device. Properties, such as aerosol particle size, are demonstrated to be regulated with the air flow rate with respect to the e-liquid flow and e-liquid composition. The CAG, however, is limited in assessing common EC-related issues, such as overheating of its elements. We seek to demonstrate that the CAG can generate aerosol that is reproducible and continuous, by assessing the chemical and physical aerosol characteristics with a chosen e-liquid formulation. The protocol describes the operating parameters of liquid flow rate, dilution air-flow rates and operating procedures needing to optimize the aerosol concentration and particle size required for an in vivo toxicology study. Presenting the representative results from the protocol and discussing the challenges and applications of working with a CAG, we demonstrate that CAG can be used in a reproducible fashion. The technology and protocol, that has been developed from prior work, serve as a foundation for future innovations for laboratory-controlled aerosol generation investigations.

Reproducibility of CAG aerosols 
To demonstrate the reproducibility of the CAG-generated aerosol, a base liquid solution containing PG, VG, nicotine, water, and ethanol (71.72%, 17.93%, 2%, 5.85%, and 2.5%, respectively) was used over 10 separate aerosol generation runs. The aerosolization and sampling parameters are summarized in Table 2. Chemical characterization of the CAG-generated aerosols confirmed the high degree of reproducibility of the results obtained using the system. Under the same heating, cooling, and dilution air flows as well as the same sampling conditions, the concentrations of ACM, nicotine, VG, and PG were stable over the aerosol generation runs, with the relative standard deviation of 2.48%, 3.28%, 3.43%, and 3.34% of ACM, Nicotine, VG, and PG respectively (Figure 7).
The concentrations of eight carbonyls-namely, acetaldehyde, acetone, acrolein, butyraldehyde, crotonaldehyde, formaldehyde, methyl ethyl ketone, and propionaldehyde-were measured during three consecutive CAG-aerosol generation runs. As expected with aerosols generated at constant controlled conditions, the yields of all carbonyl analytes remained low (Table 3), not reaching the limits of quantification (LOQ) of the analytical method for most compounds. Only acetaldehyde and formaldehyde had yields above the LOQ. Formaldehyde concentrations in the diluted aerosol sample showed high variability (&#177;32%) owing to the volatility of this analyte as well as yields close to the LOQ. The data confirmed the absence of liquid thermal degradation products in CAG-generated aerosols. Addition of a mixture of flavors had an influence on the carbonyl composition of the aerosol. In the present case, acetaldehyde and butyraldehyde yields were drastically increased, from values close to the LOQ to 2.06 and 1.56 &#181;g/L, respectively, in the diluted aerosol meant to enter the exposure chamber. These data highlight the effect of the composition of the flavor mixture on aerosol composition, and stress the need for investigating the potential toxicity of certain flavoring substances in an e-liquid formulation at an early stage, before final assessment in in vivo long-term exposure studies.
PSD of the CAG-generated aerosols 
The PSD of the CAG-generated aerosols was measured under different cooling and first dilution flows to evaluate the impact of these conditions on the physical characteristics of the aerosol generated from the base liquid solution containing PG, VG, water, and nicotine only. This procedure is essential for identifying appropriate conditions for producing aerosols with particle sizes in the respirable range.
In the present study, cooling and first dilution flows were modified in steps of 10 L/min to maintain the same total volume of aerosol flow (Table 4). The liquid flow (0.5 mL/min), heated flow (2 L/min), and second dilution flow (150 L/min) were kept constant. Aerosol samples were taken from the diluted sampling point b (Figure 5). PSD was determined by using an aerodynamic particle sizer that measure particle sizes from 0.5 to 20 &#181;m, at a sample flow rate of 5 L/min and diluted appropriately to use with equipment. The MMAD and GSD were reported by the aerodynamic particle sizer for each aerosol generation run.
The increase in cooling flow and simultaneous decrease in first dilution flow had an impact on aerosol particle size (Table 4). The greatest influence on particle size was observed when changing the cooling flow from 10 to 20 L/min and the first dilution flow from 160 to 150 L/min. The MMAD more than doubled under these conditions from 1.47 to 4.03 &#181;m. The average aerosol particle size continued to grow with the increasing cooling flow rates, albeit at lower ratios than those observed between 10 and 20 L/min. The distribution of the aerodynamic diameter of the aerosol particles was clearly shifted toward larger diameters when comparing aerosols generated at 10 L/min cooling flow with those generated at 20-50 L/min (Figure 8).
Trapping efficiency of e-liquid flavors 
As discussed earlier, owing to their volatility, various liquid constituents are continuously prone to gas-liquid mass transfer depending on local thermodynamic conditions. In addition, analytical methods have a certain ability to trap such constituents. Actual yield measurements allow us to measure the ability of chemical methods for accurate detection and quantification of selected constituents (for example, because of their condensation potential or reactions, some constituents might not reach their destination, i.e., the exposure chamber in case of inhalation studies). Thus, when assessing various flavored e-liquid formulations, it is essential to be able to determine the most efficient trapping method for chemical assessment of the aerosol. Subsequently, this allows us to measure the transfer rate for each constituent, which is dictated by the often-present losses due to aerosol transport from the place of generation to the exposure chamber. In the present case, an additional study was performed with a liquid containing a mix of flavoring substances. Aerosol was generated with the CAG parameters listed in Table 2 and trapped after dilution (position b, Figure 5), with the sampling flow rate set at 0.7 L/min for 30 min. Trapping was performed on sampling columns preconditioned with 2 mL of isopropanol. The cartridges were eluted with isopropanol shortly after completion of the trapping period, until 20 mL of the solution was recovered. We found that trapping efficiency should generally be investigated and determined for each flavor constituent.
For 70% of the investigated flavor constituents, we had recovery rates &#62;60%, which was well correlated with the boiling points (volatility) of the flavors. This fact implies that inhalation toxicology studies containing complex mixtures should be performed with special attention to the transfer and delivery of aerosol to the exposure site.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61021/61021fig01.jpg" /> 
Figure 1: Functioning principle of the capillary aerosol generator (CAG). The liquid is pumped into an electrically heated capillary delivering bursts of hot supersaturated vapors, which are cooled down by the air flow, causing sudden nucleation and condensation, leading to aerosol formation. <a href="https://www.jove.com/files/ftp_upload/61021/61021fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61021/61021fig02.jpg" /> 
Figure 2: Typical CAG experimental setup and key elements. (A) General view of the CAG assembly, showing the peristaltic pump linking the liquid stock solution to the CAG, dilution air duct, and aerosol formation process. (B) Detailed view of the CAG, with capillary and heating elements. (C) Cross-sectional view of the CAG assembly aerosol generation setup. Details of the cooling and diluting air flows. The glass tubing has two separate compartments. The cooling flow is pushed toward the CAG and enters in contact with the liquid-generated vapor to produce the aerosol. The dilution flow is pushed toward the formed aerosol to dilute the latter. <a href="https://www.jove.com/files/ftp_upload/61021/61021fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61021/61021fig03.jpg" /> 
Figure 3: CAG device details: cross-sectional view. The heating flow is introduced around the heating elements for cooling the external CAG body, preventing condensation of the liquid backflow at the tip of the capillary, and for stabilizing the vapor jet burst. <a href="https://www.jove.com/files/ftp_upload/61021/61021fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61021/61021fig04.jpg" /> 
Figure 4: CAG assembly. The capillary and heating element (A) are inserted into an inner PEEK tube, and this assembly is slipped into an outer stainless-steel tube (B). The assembly is capped and tightly fixed on a support using stainless steel lead screws (C,D). The capillary protruding from the rear end is linked via tubing to the peristaltic pump and liquid formulation. Abbreviations: SS, stainless steel. <a href="https://www.jove.com/files/ftp_upload/61021/61021fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61021/61021fig05.jpg" /> 
Figure 5: CAG aerosol generation settings for in vivo exposure experiments. Aerosol sampling&#160;for analysis takes place at two positions: (a) undiluted aerosol-the first dilution step is switched off during sampling; (b) diluted aerosol, just before entering the exposure chamber. <a href="https://www.jove.com/files/ftp_upload/61021/61021fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61021/61021fig06.jpg" /> 
Figure 6: Sample column with attached adaptors. Before sampling, the sample column is preconditioned with 0.5 M sulfuric acid for nicotine analysis or isopropanol for flavor analysis. The inlet adaptor is connected to the CAG-generated aerosol flow and the outlet adapter to the vacuum pump. <a href="https://www.jove.com/files/ftp_upload/61021/61021fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61021/61021fig07.jpg" /> 
Figure 7: CAG-generated aerosol characterization and reproducibility. Concentration of ACM, nicotine, PG, and VG concentrations over 10 separate experimental aerosol generation runs with the same liquid base solution. ACM, 1105.45 &#177; 27.4 &#181;g/L; Nicotine, 20.16 &#177; 0.7 &#181;g/L; VG, 227.15 &#177; 7.8 &#181;g/L; PG, 656.59 &#177; 22.0 &#181;g/L. Error bars represent standard deviation. Abbreviations: ACM, aerosol collected mass; PG, propylene glycol; VG, glycerol. <a href="https://www.jove.com/files/ftp_upload/61021/61021fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/61021/61021fig08.jpg" /> 
Figure 8: Alterations in particle size distribution of aerosol generated under various cooling flow rates. <a href="https://www.jove.com/files/ftp_upload/61021/61021fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>BASE (PG/VG/N)</td>
			<td>FLAVOUR (PG/VG/N/F)</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>PG/VG/N (g/1000g)</td>
			<td>PG/VG/N/F (g/1000g)</td>
		</tr>
		<tr>
			<td>Benzoic Acid</td>
			<td>3.33</td>
			<td>3.33</td>
		</tr>
		<tr>
			<td>PG</td>
			<td>240.00</td>
			<td>238.91</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>150.00</td>
			<td>150.00</td>
		</tr>
		<tr>
			<td>Lactic Acid</td>
			<td>3.33</td>
			<td>3.33</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>3.33</td>
			<td>3.33</td>
		</tr>
		<tr>
			<td>Blended Flavor mix</td>
			<td>0.00</td>
			<td>1.20</td>
		</tr>
		<tr>
			<td>Glycerine</td>
			<td>560.01</td>
			<td>559.90</td>
		</tr>
		<tr>
			<td>Nicotine</td>
			<td>40.00</td>
			<td>40.00</td>
		</tr>
		<tr>
			<td>Sum</td>
			<td>1000.00</td>
			<td>1000.00</td>
		</tr>
	</tbody>
</table>
Table 1: E-liquid stock formulation components18
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Aerosolization protocol</td>
			<td colspan="3">Sampling protocol</td>
		</tr>
		<tr>
			<td>Parameters</td>
			<td>Undiluted</td>
			<td>Diluted</td>
			<td>Parameters</td>
			<td>Undiluted Location A</td>
			<td>Diluted Location B</td>
		</tr>
		<tr>
			<td>CAG temperature (&#176;C)</td>
			<td colspan="2">250</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pump flow (mL/min)</td>
			<td>0.5</td>
			<td>0.5</td>
			<td>Sampling time (min)</td>
			<td>10</td>
			<td>30</td>
		</tr>
		<tr>
			<td>Heated air flow (L/min)</td>
			<td>2</td>
			<td>2</td>
			<td>Sampling flow (ACM) (L/min)</td>
			<td>0.7</td>
			<td>1.5</td>
		</tr>
		<tr>
			<td>Cooling air flow (L/min)</td>
			<td>10</td>
			<td>10</td>
			<td>Sampling flow Extrelut (L/min)</td>
			<td>0.7</td>
			<td>0.7</td>
		</tr>
		<tr>
			<td>1st air dilution (L/min)</td>
			<td>NA</td>
			<td>150</td>
			<td>Sampling flow Carbonyls (L/min)</td>
			<td>0.7</td>
			<td>0.7</td>
		</tr>
		<tr>
			<td>2nd air dilution (L/min)</td>
			<td>NA</td>
			<td>160</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Waste (L/min)</td>
			<td>NA</td>
			<td>172</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Parameters of aerosol generation, dilution, and sampling
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Carbonyls</td>
			<td colspan="2">Base liquid (PG/VG/Nicotine)</td>
			<td colspan="2">Flavor stock solution high concentration with nicotine 
			(PG/VG/Nicotine/Flavors)</td>
		</tr>
		<tr>
			<td>Undiluted aerosol sample &#181;g/L</td>
			<td>Diluted aerosol sample &#181;g/L</td>
			<td>Undiluted aerosol sample &#181;g/L</td>
			<td>Diluted aerosol sample &#181;g/L</td>
		</tr>
		<tr>
			<td>Acetaldehyde</td>
			<td>0.834 &#177; 0.096</td>
			<td>0.119*</td>
			<td>45.346 &#177; 1.134</td>
			<td>2.058 &#177; 0.202</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>&#60; LOQ</td>
			<td>&#60; LOQ</td>
			<td>&#60; LOQ</td>
			<td>&#60; LOQ</td>
		</tr>
		<tr>
			<td>Acrolein</td>
			<td>&#60; LOQ</td>
			<td>&#60; LOQ</td>
			<td>&#60; LOQ</td>
			<td>&#60; LOQ</td>
		</tr>
		<tr>
			<td>Butyraldehyde</td>
			<td>&#60; LOQ</td>
			<td>&#60; LOQ</td>
			<td>36.475 &#177; 0.996</td>
			<td>1.557 &#177; 0.179</td>
		</tr>
		<tr>
			<td>Crotonaldehyde</td>
			<td>&#60; LOQ</td>
			<td>&#60; LOQ</td>
			<td>0.052 &#177; 0.001</td>
			<td>&#60; LOQ</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>0.731 &#177; 0.072</td>
			<td>0.072 &#177; 0.023</td>
			<td>0.158 &#177; 0.007</td>
			<td>0.026 &#177; 0.004</td>
		</tr>
		<tr>
			<td>Methyl Ethyl Ketone</td>
			<td>&#60; LOQ</td>
			<td>&#60; LOQ</td>
			<td>0.570 &#177; 0.015</td>
			<td>&#60; LOQ</td>
		</tr>
		<tr>
			<td>Propionaldehyde</td>
			<td>&#60; LOQ</td>
			<td>&#60; LOQ</td>
			<td>0.085 &#177; 0.001</td>
			<td>&#60; LOQ</td>
		</tr>
	</tbody>
</table>
Table 3: Determination of carbonyls in the CAG-generated aerosol. Average values from three aerosol generation runs with the same liquid base solution alone and with a flavor mixture. Only one sample over three runs had values greater than the lower limit of quantification (LOQ) of the method.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">Settings (L/min)</td>
			<td colspan="2">Aerosol droplet diameter</td>
		</tr>
		<tr>
			<td>Cooling flow</td>
			<td>1st dilution flow</td>
			<td>MMAD (&#181;m)</td>
			<td>GSD</td>
		</tr>
		<tr>
			<td>10</td>
			<td>160</td>
			<td>1.47 &#177; 0.04</td>
			<td>2.07&#160; &#177; 0.01</td>
		</tr>
		<tr>
			<td>20</td>
			<td>150</td>
			<td>4.03&#160; &#177; 0.18</td>
			<td>2.13&#160; &#177; 0.04</td>
		</tr>
		<tr>
			<td>30</td>
			<td>140</td>
			<td>4.74&#160; &#177; 0.04</td>
			<td>1.89&#160; &#177; 0.02</td>
		</tr>
		<tr>
			<td>40</td>
			<td>130</td>
			<td>5.35&#160; &#177; 0.04</td>
			<td>1.80&#160; &#177; 0.01</td>
		</tr>
		<tr>
			<td>50</td>
			<td>120</td>
			<td>5.23&#160; &#177; 0.03</td>
			<td>1.76&#160; &#177; 0.01</td>
		</tr>
	</tbody>
</table>
Table 4: Determination of aerosol particle size (droplet diameter) under different air flow conditions.&#160;Abbreviations: MMAD, mass median aerodynamic diameter; GSD, geometric standard deviation.

Watch this Scientific Journal Video about Use of Capillary Aerosol Generator in Continuous Production of Controlled Aerosol for Non-Clinical Studies at JoVE.com

Use of Capillary Aerosol Generator in Continuous Production of Controlled Aerosol for Non-Clinical Studies

The protocol describes the settings and use of a capillary aerosol generator for continuous production of controlled aerosol from a multispecies liquid solution, suitable for steady large-volume aerosol delivery (e.g., in vivo inhalation studies).

The capillary aerosol generator (CAG) is operated with the principal of thermal liquid evaporation through heating of e-liquid in the initial phase, ...

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Research

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Engineering

3.4K Views.  Philip Morris Products S.A.. The protocol describes the settings and use of a capillary aerosol generator for continuous production of controlled aerosol from a multispecies liquid solution, suitable for steady large-volume aerosol delivery (e.g., in vivo inhalation studies).

Video: Use of Capillary Aerosol Generator in Continuous Production of Controlled Aerosol for Non-Clinical Studies

A System to Create Stable Nanoparticle Aerosols from Nanopowders

Nanoparticle aerosols released from nanopowders in workplaces are associated with human exposure and health risks. We developed a novel system, requiring minimal amounts of test materials (min. 200 mg), for studying powder aerosolization behavior and aerosol properties. The aerosolization procedure follows the concept of the fluidized-bed process, but occurs in the modified volume of a V-shaped aerosol generator. The airborne particle number concentration is adjustable by controlling the air flow rate. The system supplied stable aerosol generation rates and particle size distributions over long periods (0.5-2 hr and possibly longer), which are important, for example, to study aerosol behavior, but also for toxicological studies. Strict adherence to the operating procedures during the aerosolization experiments ensures the generation of reproducible test results. The critical steps in the standard protocol are the preparation of the material and setup, and the aerosolization operations themselves. The system can be used for experiments requiring stable aerosol concentrations and may also be an alternative method for testing dustiness. The controlled aerosolization made possible with this setup occurs using energy inputs (may be characterized by aerosolization air velocity) that are within the ranges commonly found in occupational environments where nanomaterial powders are handled. This setup and its operating protocol are thus helpful for human exposure and risk assessment.

We designed and developed an effective nanopowder aerosolization setup and operating protocol. The system generated nanoparticle aerosols with stable number concentrations and size distributions for long durations, requiring only small quantities of test material (min. 200 mg).

Nanoparticle aerosols released from nanopowders in workplaces are associated with human exposure and health risks. We developed a novel system, requiring ...

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger.

An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols.

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid ...

Environment

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) in vitro exposure systems are often large and bulky, making transport to the field and operation at the source of emission or within the breathing zone difficult. Through miniaturization of these systems, the lab can be brought to the field, expediting processing time and providing a more appropriate exposure method that does not alter the aerosol prior to contacting the cells. The Portable In vitro Exposure Cassette (PIVEC) adapts a 37 mm filter cassette to allow for in vitro toxicity testing outside of a traditional laboratory setting. The PIVEC was characterized using three sizes of copper nanoparticles to determine deposition efficiency based on gravimetric and particle number concentration analysis. Initial cytotoxicity experiments were performed with exposed lung cells to determine the ability of the system to deposit particles while maintaining cell viability. The PIVEC provides a similar or increased deposition efficiency when comparing to available perpendicular flow in vitro exposure devices. Despite the lower sample throughput, the small size gives some advantages to the current in vitro ALI exposure systems. These include the ability to be worn for personal monitoring, mobility from the laboratory to the source of emission, and the option to set-up multiple systems for spatial resolution while maintaining a lower user cost. The PIVEC is a system capable of collecting aerosols in the field and within the breathing zone onto an air-interfaced, in vitro model.

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

Here, we present a protocol to perform portable cellular aerosol exposures and measure cellular response. The method uses cells, grown at the air-liquid interface, mimicking in vivo physiology. Cellular response to copper nanoparticle aerosols was observed as oxidative stress through reactive oxygen species generation and cytotoxicity as lactate dehydrogenase release.

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) ...

Generation of Electronic Cigarette Aerosol by a Third-Generation Machine-Vaping Device: Application to Toxicological Studies

Electronic-cigarette (e-cig) devices use heat to produce an inhalable aerosol from a liquid (e-liquid) composed mainly of humectants, nicotine, and flavoring chemicals. The aerosol produced includes fine and ultrafine particles, and potentially nicotine and aldehydes, which can be harmful to human health. E-cig users inhale these aerosols and, with the third-generation of e-cig devices, control design features (resistance and voltage) in addition to the choice of e-liquids, and the puffing profile. These are key factors that can significantly impact the toxicity of the inhaled aerosols. E-cig research, however, is challenging and complex mostly due to the absence of standardized assessments and to the numerous varieties of e-cig models and brands, as well as e-liquid flavors and solvents that are available on the market. These considerations highlight the urgent need to harmonize e-cig research protocols, starting with e-cig aerosol generation and characterization techniques. The current study focuses on this challenge by describing a detailed step-by-step e-cig aerosol generation technique with specific experimental parameters that are thought to be realistic and representative of real-life exposure scenarios. The methodology is divided into four sections: preparation, exposure, post-exposure analysis, plus cleaning and maintenance of the device. Representative results from using two types of e-liquid and various voltages are presented in terms of mass concentration, particle size distribution, chemical composition and cotinine levels in mice. These data demonstrate the versatility of the e-cig exposure system used, aside from its value for toxicological studies, as it allows for a broad range of computer-controlled exposure scenarios, including automated representative vaping topography profiles.

Electronic cigarette (e-cig) users are increasing worldwide. Little, however, is known about the health effects induced by inhaled e-cig aerosols. This article describes an e-cig aerosol generation technique suitable for animal exposures and subsequent toxicological studies. Such protocols are required to establish experimentally reproducible and standardized e-cig exposure systems.

Electronic-cigarette (e-cig) devices use heat to produce an inhalable aerosol from a liquid (e-liquid) composed mainly of humectants, nicotine, and ...

Chemistry

Additive Manufacturing-Enabled Low-Cost Particle Detector

As particles with a size of 1 &#181;m or smaller pose a severe health risk to the human body, the detection and regulation of particle emissions are of great importance. A large share of particulate emissions are emitted by the transport sector. Most of the commercially available particle detectors are bulky, very expensive, and need additional equipment. This paper presents a protocol to build and test a standalone particle detector that is small and cost-efficient. 
The focus of this paper lies in the description of the detailed construction manual with video and the sensor evaluation procedure. The computer-aided design model of the sensor is included in the supplemental material. The manual explains all the construction steps, from 3D printing to the fully operational sensor. The sensor can detect charged particles and is therefore suitable for a wide range of applications. A possible field of application would be soot detection from power plants, wildfires, industries, and automobiles.

Here, we present a protocol on how to build and test a simple but efficient low-cost particle detector.

As particles with a size of 1 &#181;m or smaller pose a severe health risk to the human body, the detection and regulation of particle emissions are of ...

Disposable Dosators for Pulmonary Insufflation of Therapeutic Agents to Small Animals

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver drugs locally and systemically. Evaluation of dry powder aerosols necessitates an efficient dispersion mechanism to maintain high concentrations in an exposure chamber or for direct endotracheal administration. While solutions exist to expose animals by passive inhalation to dry powder aerosols, most require masses of powder in large excess of the dose delivered. This precludes conducting early feasibility studies as insufficient drug is available at the research or early development stage to support the dose delivery requirements for conventional aerosol delivery systems. When designing an aerosol drug product, aerodynamic performance can relate directly to delivery efficiency and efficacy. Dispersion of powder into an aerosol requires energy input sufficient to overcome interparticulate forces, and particle engineering approaches can substantially improve aerosol performance. We have developed a dispersion system (dosator) which can aerosolize engineered dry powder aerosols efficiently for the purpose of direct pulmonary insufflation, dispersion into an exposure system or generation for analytical purposes.

During development of drugs for pulmonary delivery, it is necessary to evaluate pharmacokinetics and efficacy in an animal model. We present a method to build a disposable aerosol dispersion system from of-the-shelf components that can be used to administer intrapulmonary dry powder aerosol to rodents.

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver ...

Medicine

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and bronchodilator drugs. Despite the availability of multiple treatments, both diseases are growing public health concerns. The majority of asthma patients are well controlled on current inhaled therapies but a substantial number of patients with severe asthma are not. Asthma affects an estimated 300 million people worldwide and approximately 20 percent have a severe form of the disease. In contrast to asthma, there are few effective therapies for COPD. An estimated 10% of the population has COPD and the trend in death rates is increasing for COPD while decreasing for other major diseases. Although developing drugs for inhaled delivery is challenging, the nose-only inhalation unit enables direct delivery of novel drugs to the lung of rodents for pre-clinical efficacy and safety/toxicology studies. Inhaled drug delivery has multiple advantages for respiratory diseases, where high concentration in the lung improves efficacy and minimizes systemic side effects. Inhaled corticosteroids and bronchodilators benefit from these advantages and inhaled delivery may also hold potential for future biologic therapies. The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs of rodents, key data required before initiating clinical development.

The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs; key data enabling clinical inhaled drug development.

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and ...

Whole-Body Nanoparticle Aerosol Inhalation Exposures

Inhalation is the most likely exposure route for individuals working with aerosolizable engineered nano-materials (ENM). To properly perform nanoparticle inhalation toxicology studies, the aerosols in a chamber housing the experimental animals must have: 1) a steady concentration maintained at a desired level for the entire exposure period; 2) a homogenous composition free of contaminants; and 3) a stable size distribution with a geometric mean diameter &lt; 200 nm and a geometric standard deviation &sigma;g &lt; 2.5 5. The generation of aerosols containing nanoparticles is quite challenging because nanoparticles easily agglomerate. This is largely due to very strong inter-particle forces and the formation of large fractal structures in tens or hundreds of microns in size 6, which are difficult to be broken up. Several common aerosol generators, including nebulizers, fluidized beds, Venturi aspirators and the Wright dust feed, were tested; however, none were able to produce nanoparticle aerosols which satisfy all criteria 5.
A whole-body nanoparticle aerosol inhalation exposure system was fabricated, validated and utilized for nano-TiO2 inhalation toxicology studies. Critical components: 1) novel nano-TiO2 aerosol generator; 2) 0.5 m3 whole-body inhalation exposure chamber; and 3) monitor and control system. Nano-TiO2 aerosols generated from bulk dry nano-TiO2 powders (primary diameter of 21 nm, bulk density of 3.8 g/cm3) were delivered into the exposure chamber at a flow rate of 90 LPM (10.8 air changes/hr). Particle size distribution and mass concentration profiles were measured continuously with a scanning mobility particle sizer (SMPS), and an electric low pressure impactor (ELPI). The aerosol mass concentration (C) was verified gravimetrically (mg/m3). The mass (M) of the collected particles was determined as M = (Mpost-Mpre), where Mpre and Mpost are masses of the filter before and after sampling (mg). The mass concentration was calculated as C = M/(Q*t), where Q is sampling flowrate (m3/min), and t is the sampling time (minute). The chamber pressure, temperature, relative humidity (RH), O2 and CO2 concentrations were monitored and controlled continuously. Nano-TiO2 aerosols collected on Nuclepore filters were analyzed with a scanning electron microscope (SEM) and energy dispersive X-ray (EDX) analysis.
In summary, we report that the nano-particle aerosols generated and delivered to our exposure chamber have: 1) steady mass concentration; 2) homogenous composition free of contaminants; 3) stable particle size distributions with a count-median aerodynamic diameter of 157 nm during aerosol generation. This system reliably and repeatedly creates test atmospheres that simulate occupational, environmental or domestic ENM aerosol exposures.

A whole-body nanoparticle aerosol inhalation exposure facility was constructed for nano-sized titanium dioxide (TiO2) inhalation toxicology studies. This system provides nano-TiO2 aerosol test atmospheres that have: 1) a steady mass concentration; 2) a homogenous composition free of contaminants; and 3) a stable particle size distribution during aerosol generation.

Inhalation is the most likely exposure route for individuals working with aerosolizable engineered nano-materials (ENM). To properly perform nanoparticle ...

Biology

Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to gases, particles or complex atmospheres (e.g., cigarette smoke), thus providing realistic physiological exposure of the apical surface of the human alveolar region to air. In contrast to sequential exposure models with linear aerosol guidance, the modular design of the radial flow system meets all requirements for the continuous generation and transport of the test atmosphere to the cells, a homogenous distribution and deposition of the particles and the continuous removal of the atmosphere. This exposure method is primarily designed for the exposure of cells to airborne particles, but can be adapted to the exposure of liquid aerosols and highly toxic and aggressive gases depending on the aerosol generation method and the material of the exposure modules.
Within the framework of a recently completed validation study, this exposure system was proven as a transferable, reproducible and predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles, thereby potentially reducing or replacing animal experiments that would normally provide this toxicological assessment.

We present a robust, transferable and predictive in vitro exposure system for the screening and monitoring of airborne particles concerning their acute pulmonary cytotoxicity by exposing cultivated human lung cells at the air-liquid interface (ALI).

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...

Use of Capillary Aerosol Generator in Continuous Production of Controlled Aerosol for Non-Clinical Studies

Summary

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Chapters in this video

A System to Create Stable Nanoparticle Aerosols from Nanopowders

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

Generation of Electronic Cigarette Aerosol by a Third-Generation Machine-Vaping Device: Application to Toxicological Studies

Additive Manufacturing-Enabled Low-Cost Particle Detector

Disposable Dosators for Pulmonary Insufflation of Therapeutic Agents to Small Animals

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

Whole-Body Nanoparticle Aerosol Inhalation Exposures

Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing