The authors are grateful for the financial support given to this study by EU FP7 project ``Managing Risks of Nanomaterials`` (MARINA) (grant agreement no: 263215).

1. System Preparation
<ol>
	<li>Make sure to use new or well cleaned particle transport tubing and connectors to assemble the system. Ensure that the chamber walls are cleaned and particle-free (refer to the cleaning methods at the end of the protocol).</li>
	<li>To remove potential background particles, connect a filtered dry air flow (5-10 L/min) directly to the mixing chamber (without installing the funnel between positions a and b in Figure 1), for at least 30 min.</li>
	<li>Measure the particle number concentration in the measurement chamber using the SMPS according to manufacturer&#39;s protocol. If the concentration is below 10 #/cm3 after three scans, then consider the environment clean. Note that the flow rate may slow when measuring using the SMPS.</li>
	<li>Stop the air flow and close the sampling tube outlets and the exit tube outlet with a plastic or rubber stopper (position c in Figure 1) to prevent ambient particles from entering the system.</li>
	<li>Prepare and warm up the measurement instruments (SMPS and OPC) and particle samplers for microscopic analysis.</li>
</ol>
2. Material Preparation&#160;
<ol>
	<li>Store test materials in a well-controlled environment with respect to temperature and humidity. This is very important for ensuring repeatable results in follow-up experiments.</li>
	<li>Weigh the powder carefully using an analytical balance or high-precision balance, in a well-ventilated space (e.g., laboratory hood). 
	Note: Weights of 250-500 mg TiO2, SiO2, ZnO and CeO2 nanoparticle powders were tested, and these generally proved to be sufficient for at least 30 min of stable aerosolization. However, the appropriate amount strongly depends on the powder type and may vary significantly for bulk materials, cement or organic powders.</li>
	<li>Fix the aerosol generator vertically, and feed the powder from the top opening of the aerosol generator using a properly cleaned laboratory funnel. Prior to the experiment, rinse the funnel with water and dry out by filtered air, to remove any dust deposition on the inner wall. Gently tap the funnel to ensure all the powder particles are fed into the process. Do not shake the funnel hard in order to avoid significant loss of the material to early aerosolization.
	<ol>
		<li>Make sure that the majority of the powder particles reach the bottom of the generator, rather than falling on the surrounding sloping walls. Gently tap the side walls of the generator to move deposited powder particles down to the bottom.</li>
		<li>Alternatively, use a long funnel that directly deposits the powder particles at the bottom of the generator. For safety reasons, conduct these operations under a ventilation hood or inside a negative pressure chamber.</li>
	</ol>
	</li>
	<li>As unsticky material may slip through the opening at the bottom of the generator, use a 2 mm diameter needle to temporarily block the opening before feeding in the powder.</li>
	<li>Remove the funnel and close the top and bottom openings of the generator in order to avoid particle emission during transfer.</li>
</ol>
3. Aerosolization
<ol>
	<li>Install the aerosol generator, remove the blocks on the inlet and outlet tubing to the funnel, connect its bottom to filtered air supply and its top exit to the mixing chamber (positions a and b respectively in Figure 1), and attach it vertically with a metallic scaffold.</li>
	<li>Remove the blocks on the setup exit (position c in Figure 1).</li>
	<li>Switch on the aerosolization flow. Slowly increase the rate from 0 to 0.3-0.5 L/min using the flow tuner. Do not move to high flow rates too quickly-the aim is to reach a flow rate that can provide stable aerosol generation for at least 30 min.
	<ol>
		<li>To achieve this, do not consume powder quantity significantly during this period of stable aerosolization. As an empirical rule, use a fluidized-bed height of about 1 cm (denoted by H in Figure 1) for generating a robust aerosol flow while maintaining a stable concentration over a relatively long period. If the energy put into the aerosolization process is too strong then the material will be rapidly used up, failing to sustain a steady aerosol generation during the rest of the experiment. Note that the flow rate range can vary for different powders; the values mentioned above were used for the abovementioned nanopowders tested.</li>
	</ol>
	</li>
	<li>Switch on the dilution flow. Slowly increase the rate from 0 to 2 L/min. The total dilution flow needed is determined by the sampling equipment. The SMPS, OPC, and the mini-sampler used in the system presented here require a total flow of 1.6-1.8 L/min.</li>
</ol>
4. Characterization
<ol>
	<li>Start the online measurement instruments simultaneously (here, the SMPS and OPC) as soon as the aerosolization and dilutions flows are introduced. 
	Note: If a stable state of aerosolization is achieved, the aerosol particle number concentration and the size distribution should become stable after approximately 30 min. Use measurements starting from this time point for the comparison of aerosol properties under different conditions (e.g., humidity) and using different powders. Analyze the results from 10 consecutive SMPS scans to calculate average concentrations and size distributions.</li>
	<li>Once the aerosolization is stable, turn on the pump connected to the TEM sampler to start sampling the airborne particles. Use a flow rate of 0.3 L/min using the TEM grid coated with holey carbon film. The thin film on the grid may be damaged if the flow rate is too high. Detailed information on the use of the sampler is available 21. Typically, the sampling process lasts for about 3 min.
	<ol>
		<li>Vary the sampling duration according to the different particle concentrations, and approximate by considering a moderate surface coverage of the TEM grid by particle deposition (e.g., 50%). Thick depositions may modify particle morphology due to on-site agglomeration.</li>
	</ol>
	</li>
</ol>
5. Post-sampling Operations and Clean-up&#160;
<ol>
	<li>After finishing the measurements, switch off the dilution flow and then the aerosolization flow.</li>
	<li>Disconnect the aerosol generator from the system, block its top and bottom openings, and transfer it to the cleaning space. Clean in a well-ventilated cleaning facility or an enclosed space, especially if hazardous materials have been treated.</li>
	<li>Disperse powder residues with water or organic solvents, depending on the hydrophilicity of the particle surface. Pour the solution off into chemical containers for safe recycling. After long experiments, test powders tend to stick firmly onto the glass wall and do not dissolve easily. If this occur, use acids or bases together with an ultrasonic cleaner to dissolve sticky materials.</li>
	<li>In order to remove any moisture content left on the wall and to thoroughly dry the inside of the generator, pass dry air through it for at least 1 hr. Make sure there are no flames or ignition sources when working with organic solvents, and ensure good ventilation of the space.</li>
	<li>Disconnect particle transport tubing and connectors. Rinse them with water or solvents. Wipe the inner walls of the mixing and measurement chambers with a wet paper tissue or cloth. Dry them in an open space for at least a day or with dry air flow for 1 hr prior to next experiment.</li>
	<li>Regularly clean the SMPS impactor (if used).</li>
</ol>

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The authors declare that they have no competing financial interests.

The funnel based aerosolization setup can effectively activate powder particles at a chosen energy input level (may be quantified by air flow velocity during aerosolization). Particle movements and collisions at the generation site reach an equilibrium state, breaking up powder agglomerates and emitting airborne particles of the same size distribution at a constant rate. A stable aerosolization can last from 30 min to up to 2 hr, which is sufficient time for even slow measurement instruments with high size resolutions, such as the SMPS, to produce statistically significant results. The setup only requires small quantities of test materials, which can be an advantage for testing precious materials such as nanoparticle powders.
However, the system&#39;s environment and process parameters can significantly influence the test results. To produce repeatable data, standard operating procedures must be strictly followed throughout the experiments. When conducting aerosolization tests using this system, the following aspects should be carefully considered.
Firstly, to obtain meaningful results, it is critical that the setup&#39;s internal parts provide a clean environment for the tests. Potential sources of contaminants are ambient particles and test materials from previous experiments. The effect of ambient particles usually disappeared relatively quickly, as soon as the aerosolization and dilution flows were introduced. However, interference from residual materials can persist throughout the experiment. As the generated aerosol particles flow through the system, they can deposit on the inner walls of the transport tubes, the bending points and the narrow channels of the connectors, and the inner surfaces of the mixing and the measurement chambers. If these parts are not properly cleaned prior to new experiments, previously deposited materials can be constantly re-suspended into the main stream of the aerosol flow, thus disturbing the test results.
Secondly, the powder filling process should be conducted very carefully. The most significant issue here is the quantity of the powder fed into the setup, especially when very small amounts of materials are used. At a given aerosolization flow rate, smaller amounts of powder generate lower aerosol concentrations, and possibly particles with smaller sizes, due to the higher energy input per unit weight of powder. Moreover, the storage conditions for test materials (e.g., relative humidity and temperature) have been shown to influence powder aerosolization behavior and levels of dustiness 22. Therefore, raw powders should always be kept in the same atmospheric conditions, where possible.
Thirdly, adjustments to the aerosolization flow at the beginning of the experiment greatly affect the test results. Sharp increases in the flow blow large powder particles up into the air and spread them all over the funnel surface, dramatically reducing the amount of material available for the rest of the experiment. The consequences could be a failed test due to insufficient powder.
Because the setup described here is not built using standardized laboratory equipment, when attempting to replicate the core parts of this system, the following aspects should be considered. Standard laboratory separatory funnels can be used as the aerosol generator (note that they should not be used under pressurized conditions). Separatory funnels of different geometries were tested in the experiments, and they provided similar functionality to the tailored funnel. A rubber sealing block with an embedded transport tube can be used as the funnel lid.
Mixing and measurement compartments of different geometries but similar volumes can be used. Note that compartments that are too large will significantly delay the time needed to reach stable aerosol conditions (concentration). The time required can be estimated by taking into account the total air flow rate and the volume of the compartment. Although the process can be accelerated by using a large dilution flow, it should be remembered that the final particle number concentration can be dramatically decreased due to dilution, and this may influence the aerosol size distribution as well as the performance of the measurement instruments (depending on their detection limits). Electrically conductive materials are recommended.
The length of the transport tubing may vary, depending on the general laboratory settings. However, the length should be kept as short as possible in order to avoid significant particle losses during their transport. The particle penetration efficiency can be calculated by taking into account particle diameter, air flow rate, tube diameter and length, and bearing in mind either gravitational deposition or diffusion loss, or both.
Different characterization methods may be employed. However, the air supply (dilution flow) should be adjusted to match the total sampling flow rate. Insufficient air supply will result in negative pressure in the measurement chamber, drawing in ambient particles thus leading to mistakes in the conclusions. Different air supply sources can be used, but ensure that they are particle-free or pre-treat the air with a high-efficiency filter.
One major limitation of this aerosolization method is that it requires good flowability of the test powders in order to maintain stable particle generation over a relatively long period. Sticky materials, such as hydrophilic powders with a high moisture content, often stop flowing at an early stage of the aerosolization process and produce very low particle concentrations. Potential ways of solving this issue could include a pre-treatment of the raw powder-such as drying-so as to improve its flowability. The storage condition of the raw materials after uses should be well maintained, e.g., kept in a dry environment and under suitable temperature. During the experiments, higher aerosolization flow rate (0.5-1 L/min) and larger amounts of raw material (e.g., 500 mg) could be used. Additionally, lowering the dilution flow rate can increase particle concentration in the measurement chamber.
Another limitation of this method is the reproducibility of airborne particle generation rate (thus particle number concentration in the measurement chamber). Certain level of variation still exists. Possible ways of improvement are a better defined feeding process to reduce material losses, and well-controlled aerosolization flow rate.
The system and protocols described here could be used for various applications. The use of relatively small amounts of test materials makes the method potentially valuable as an alternative tool for testing powder dustiness. The ranking of levels of airborne particles generated by our system for some common materials was similar to those observed in existing aerosolization systems 19, such as the rotating drum 15,17, continuous drop 23, and vortex shaker methods 24. Furthermore, the adjustable energy input (air flow rate) can also be used for studying the stability of nanoparticle powder agglomerates. Finally, stable aerosol generation can serve as a reliable source of airborne engineered nanoparticles for in vivo or in vitro toxicological studies. The controllable particle concentration would allow an analysis of dose-dependent biological responses. Compared to other aerosolization methods using liquid suspensions, the presented method avoids potential problems such as material suspendability and modification of physical-chemical properties of particles in suspension (e.g., agglomeration, surface properties).

Nanomaterial powders are widely used in different industrial sectors, as the raw materials for manufacturing new products or as additives for their functional applications 1-4. However, the potential for workers&#39; exposure to nanopowder aerosols has been noted during various occupational handling activities 5-8, and the associated health risks have been investigated in in-vivo and in-vitro toxicological studies 9-12. In order to facilitate the development of effective strategies to protect workers dealing with nanomaterials, occupational health professionals require a better understanding of how nanoparticle aerosols are generated from powdered materials subjected to external energy inputs.
Different laboratory systems have been developed to simulate powder aerosolization behavior in realistic conditions. Among them, two standard procedures are the established reference methods 13 for testing powder dustiness, which is defined as the tendency of a powder, subjected to a given energy input, to release airborne particles. The first method uses a rotating drum as the medium for energy input and the aerosolization of powder particles 14,15. The second method drops a powder at a constant rate through a vertical cylinder and aerosolizes the powder particles by means of an ascending air flow 16. However, these methods require relatively large quantities of test materials (35 cm3 or 500 g), and this can be a problem with nanomaterial powders due to their high cost and the potential exposure risks. A scaled-down test system combining single-drop and rotating drum processes was developed, allowing the use of smaller amounts of test powders (6 g samples) 17. A recently developed aerosolization system based on vortex shakers has also been used for nanopowders, allowing tests down to 1 cm3 of raw materials 18.
Here, we present a novel aerosolization and deagglomeration system for nanopowder testing based on laboratory funnels. It provides a stable aerosol generation process using less than 1 g of test powder. Steady aerosolization can be maintained for sufficiently long durations for robust aerosol characterizations. The system&#39;s performance has been described in detail in two previous publications 19,20.
The test setup is composed of an aerosol generator, mixing and measurement compartments, and characterization instruments, as shown in Figure 1. Particle transport tubing and connectors link these different elements. A flow tuner and two flowmeters control and monitor the air flow conditions in the system. A manometer and a temperature and humidity sensor monitor the environment inside the measurement chamber. Dry compressed air is filtered using a hyperfilter before it enters the system. An elongated, V-shaped, glass aerosol generator is used for powder aerosolization. This geometry facilitates a robust aerosolization process and smooth particle transport into the subsequent compartment. The flow regime at the bottom of the funnel is turbulent due to the interaction with the powder particles, whereas it is laminar in the upper section (Re-number &#60; 15). The thickness of the generator walls was specifically designed to resist the high pressures (up to 400 kPa &#916;P) needed for deagglomeration testing using critical orifices. A high-precision flow tuner controls the flow rate in 0.01 L/min increments. Conductive tubing (6 mm outer diameter, 1 mm thickness) is used to avoid particle losses due to electrostatic deposition during transportation. The tube length is about 50 cm between the aerosol generator and the mixing chamber, 20 cm between the mixing chamber and the measurement chamber, and 100 cm for the sampling tubes. A 1 L metallic bottle is used as the mixing chamber, and a 12 L metallic drum is used as the measurement chamber. Particle samples are drawn from the top of the measurement chamber. An exit port directs the extra flow into a filtration system. The mixing and measurement chambers are electrically grounded to prevent electrostatic losses of particles. The measurement instruments include a scanning mobility particle sizer (SMPS) and an optical particle counter (OPC) for particle number concentration and size distribution, and a transmission electron microscopy (TEM) sampler (MPS) for particle morphology analysis.
The setup&#39;s aerosolization procedure resembles a fluidized-bed process. The air flow enters from the bottom opening into the funnel (2 mm diameter) and aerosolizes the powder. The powder particles move in a similar way to water in a fountain. The aerosol generated meets a dilution flow in the mixing chamber. The dilution air flow can be conditioned for different levels of humidity if the effect of this parameter requires analysis. The chamber&#39;s air also serves as a buffer volume to smoothly mix the aerosol with dry dilution air according to the sampling needs. The aerosol flow is then introduced into the measurement chamber via a normal tube outlet (for aerosolization testing) or a critical orifice (for deagglomeration testing). The orifice can provide different pressure-drop conditions, applying shear forces to the particles passing through it. This mechanism allows the study of their deagglomeration potential (mechanical stability).
<img alt="Figure 1" src="/files/ftp_upload/54414/54414fig1.jpg" /> 
Figure 1. Diagram of the aerosolization and deagglomeration system. By default, a tube connects the mixing chamber with the measurement chamber. The orifice shown is an optional add-on (not described in this protocol). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54414/54414fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>titanium dioxide nanopowder</td><td>JRC</td><td>NM-103/104</td><td>Reference materials provided within EU FP7 MARINA project</td></tr><tr><td>silicon dioxide nanopowder</td><td>AEROSIL</td><td>R974</td><td></td></tr><tr><td>silicon dioxide nanopowder</td><td>JRC</td><td>NM-200</td><td>Reference materials provided within EU FP7 MARINA project</td></tr><tr><td>zinc oxide nanopowder</td><td>JRC</td><td>NM-110/111</td><td>Reference materials provided within EU FP7 MARINA project</td></tr><tr><td>cerium dioxide nanopowder</td><td>JRC</td><td>NM-211/212</td><td>Reference materials provided within EU FP7 MARINA project</td></tr><tr><td>The V-shaped aerosol generator</td><td>Souffleur de verre S.A.</td><td></td><td>Specially made based on conditions required in the experiments (e.g., geometry, thickness)</td></tr><tr><td>scanning mobility particler sizer (SMPS)</td><td>GRIMM</td><td>Model N&amp;deg; 5.403</td><td>Size range: 11.1&amp;ndash;1083.3&amp;nbsp;nm (impactor: d50=1,082&amp;nbsp;nm); composed of a condensation particle counter (CPC) and a dynamic mobility analyzer (DMA); sampling flow ate: 0.3&amp;nbsp;L/min; sheath flow rate: 3.0&amp;nbsp;L/min; with standard multiple charge correction and diffusion loss correction.</td></tr><tr><td>optical particle counter (OPC)</td><td>GRIMM</td><td>Model N&amp;deg; 5.403</td><td>Size range: 0.25-32 &amp;micro;m</td></tr><tr><td>mini-particle sampler (MPS)</td><td>ECOMESURE</td><td></td><td></td></tr><tr><td>transport tubes</td><td>Milian S.A.</td><td>8 mm-conductive</td><td>6 mm inner diameter</td></tr></tbody></table>

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.

Figure 2 shows a typical example of total aerosol particle number concentration and size changes over time, using the above protocols in an aerosolization experiment with hydrophobic SiO2. Particle concentrations started to rise as soon as the aerosolization flow was introduced. The geometric mean size of particles gradually increased as well. After approximately 10 SMPS scans (3.5 min/scan), the aerosol started to enter a steady state, where particle concentration and mean diameter no longer varied by any significant amount. This state lasted more than 30 min, which was sufficient to complete ten 3-min SMPS scans. Figure 3 shows the change in particle concentration in the form of individual size distributions (based on the same data as in Figure 2). The peak rose slowly over time, and once the aerosol became stable, it remained within the same size range throughout the rest of the test.
The very small mean diameter shown at the beginning of the experiment was not due to unstable powder aerosolization. Rather, it was caused by the residual ambient air inside the funnel after the powder filling procedure. This volume of air was the first to flow into the measurement chamber and was sampled by the SMPS during its initial scans (Figure 4). This could be avoided by carrying out all the experiments in a clean room if this was required by the scientific question at hand. Indeed, the size distribution of the first scan was very similar to that of the ambient air. As the powder aerosol particles continued to flow in, the interference from the ambient particles diminished rapidly, and the effect had nearly disappeared after a few SMPS scans.
<img alt="Figure 2" src="/files/ftp_upload/54414/54414fig2.jpg" /> 
Figure 2. Change in total particle number concentration and mean diameter in an aerosolization experiment (241 mg hydrophobic SiO2; aerosolization flow 0.3 L/min). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54414/54414fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54414/54414fig3.jpg" /> 
Figure 3. Change in particle size distribution in an aerosolization experiment. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54414/54414fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54414/54414fig4.jpg" /> 
Figure 4. Aerosol particle size distributions at the beginning of the aerosolization test. Particle concentration is presented on a relative scale (normalized to the total number) in order to compare the spectrum from the first scan in a very low concentration to spectra from later scans in higher concentrations. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54414/54414fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The changes in particle concentration do not always follow the same patterns. Four possibilities can usually be seen in an aerosolization test. In Figure 5A, the concentration slowly increased to a &#34;plateau&#34; region, then remained nearly unchanged for the rest of the experiment. In Figure 5B, the concentration first rose to a maximum point, gradually decreased to a low level, and then remained stable for more than 1.5 hr. In Figure 5C, the concentration continued decreasing to zero. In Figure 5D, the concentration increased to a maximum level, remained there for a certain period, and then decreased again.
Scenario (a) is usually seen when the standard operating procedure is followed. The aerosolization air flow is slowly introduced and finally stabilized within the proper range. The amount of raw material is sufficient with respect to the aerosolization level, and a constant aerosol generation rate can be maintained for a long period of time. Scenario (b) is most likely due to an excessive aerosolization flow throughout the experiment, combined with an insufficient quantity of powder. The powder is rapidly consumed and is not able to sustain stable aerosol generation. Scenario (c) shows a similar drop in particle number concentration to Scenario (b) except that after a short time, the air flow rate was re-adjusted to a suitable range and kept constant throughout the rest of the test. This allowed the particle concentration to gradually reach a stable range. Scenario (d) appears when an insufficient amount of raw material is used. At the latter phase of the experiment, there is no longer enough test powder to generate aerosol particles at a constant rate, as was possible in the early phase of aerosolization. Consequently, the particle concentration in the system decreases.
<img alt="Figure 5" src="/files/ftp_upload/54414/54414fig5.jpg" /> 
Figure 5. Typical patterns for changing total particle concentrations during aerosolization experiments: (A) slowly increase until a plateau is reached; (B) gradually decrease to zero; (C) rapidly reach a peak and then decrease to a stable level; (D) increase to a steady state and maintain for a certain period of time, then decrease. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54414/54414fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Different aerosolization flow rates were tested in order to study their influence on aerosol generation. Flow rates from 0.3-1.1 L/min were used, and the resulting particle size distributions are shown in Figure 6. The peak of the spectrum rose as the flow increased. At the highest flow rate (1.1 L/min), micron-sized airborne particles started to enter the system (the secondary peak). The modal sizes of the aerosol particles stayed similar when under the same aerosolization flow, however, they decreased gradually when air flow increased through the range from 0.3-0.7 L/min (Figure 7). The increasing particle generation rate and diminishing mean particle diameter as flow rates increased suggest that the more dynamic aerosolization process (with significant particle movements and collisions) facilitated deagglomeration of powder particles, resulting in a modified size distribution of the aerosol particles generated.
<img alt="Figure 6" src="/files/ftp_upload/54414/54414fig6.jpg" /> 
Figure 6. Changing particle size distributions with increasing air flow rates (0.3-1.1 L/min). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54414/54414fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/54414/54414fig7.jpg" /> 
Figure 7. Comparison of particle size distributions under different flow rates. The spectra were turned into similar heights in relative scale (normalized to total particle number), which shows better the shift of the peak. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54414/54414fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The particle number concentrations of aerosols generated in replicate tests can vary by up to several folds, but usually well within one order of magnitude. The mean particle size, on the other hand, is highly reproducible. Figure 8 shows an example of the variation in particle size distribution from four replicate tests using the same material. The standard deviation was 39.7% for the total particle concentration and 6.6% for the geometric mean size. The variation of the number concentration could be due to several reasons: 1) different raw material status (e.g., agglomeration level); 2) human factors in powder filling process (influence the powder quantity deposited at the funnel bottom, thus the amount available for aerosolization); or 3) air flow adjustment at the beginning of aerosolization.
<img alt="Figure 8" src="/files/ftp_upload/54414/54414fig8.jpg" /> 
Figure 8. Variation of test results from replicate aerosolization experiments with hydrophobic SiO2. The error bars represent the standard deviation of particle number concentration in individual size channels. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54414/54414fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A System to Create Stable Nanoparticle Aerosols from Nanopowders at JoVE.com

A System to Create Stable Nanoparticle Aerosols from Nanopowders

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 ...

a-system-to-create-stable-nanoparticle-aerosols-from-nanopowders

Universidad San Sebastian

Research

JoVE Journal

Engineering

9.5K Views.  Universities of Lausanne and Geneva. 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). 

Video: A System to Create Stable Nanoparticle Aerosols from Nanopowders

Generation of Zerovalent Metal Core Nanoparticles Using n-(2-aminoethyl)-3-aminosilanetriol

In this work, a facile one-pot reaction for the formation of metal nanoparticles in a water solution through the use of n-(2-aminoethyl)-3-aminosilanetriol is presented. This compound can be used to effectively reduce and complex metal salts into metal core nanoparticles coated with the compound. By controlling the concentrations of salt and silane one is able to control reaction rates, particle size, and nanoparticle coating. The effects of these changes were characterized through transmission electron microscopy (TEM), UV-Vis spectrometry (UV-Vis), Nuclear Magnetic Resonance spectroscopy (NMR) and Fourier Transform Infrared spectroscopy (FTIR). A unique aspect to this reaction is that usually silanes hydrolyze and cross-link in water; however, in this system the silane is water-soluble and stable. It is known that silicon and amino moieties can form complexes with metal salts. The silicon is known to extend its coordination sphere to form penta- or hexa-coordinated species. Furthermore, the silanol group can undergo hydrolysis to form a Si-O-Si silica network, thereby transforming the metal nanoparticles into a functionalized nanocomposites.

Generation of Zerovalent Metal Core Nanoparticles Using n-2-aminoethyl-3-aminosilanetriol

A novel method for metal core nanoparticle synthesis using a water stable silanol is described.

In this work, a facile one-pot reaction for the formation of metal nanoparticles in a water solution through the use of ...

Chemistry

Experimental Protocol to Investigate Particle Aerosolization of a Product Under Abrasion and Under Environmental Weathering

The present article presents an experimental protocol to investigate particle aerosolization of a product under abrasion and under environmental weathering, which is a fundamental element to the approach of nanosafety-by-design of nanostructured products for their durable development. This approach is basically a preemptive one in which the focus is put on minimizing the emission of engineered nanomaterials' aerosols during the usage phase of the product's life cycle. This can be attained by altering its material properties during its design phase without compromising with any of its added benefits. In this article, an experimental protocol is presented to investigate the nanosafety-by-design of three commercial nanostructured products with respect to their mechanical solicitation and environmental weathering. The means chosen for applying the mechanical solicitation is an abrasion process and for the environmental weathering, it is an accelerated UV exposure in the presence of humidity and heat. The eventual emission of engineered nanomaterials is studied in terms of their number concentration, size distribution, morphology and chemical composition. The purpose of the protocol is to study the emission for test samples and experimental conditions which are corresponding to real life situations. It was found that the application of the mechanical stresses alone emits the engineered nanomaterials' aerosols in which the engineered nanomaterial is always embedded inside the product matrix, thus, a representative product element. In such a case, the emitted aerosols comprise of both nanoparticles as well as microparticles. But if the mechanical stresses are coupled with the environmental weathering, the experimental protocol reveals then the eventual deterioration of the product, after a certain weathering duration, may lead to the emission of the free engineered nanomaterial aerosols too.

In this article, an experimental protocol to investigate particle aerosolization of a product under abrasion and under environmental weathering is presented. Results on the emission of engineered nanomaterials, in the form of aerosols are presented. The specific experimental set-up is described in detail.

The present article presents an experimental protocol to investigate particle aerosolization of a product under abrasion and under environmental ...

Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of fabricating micellar nanocrystals based on combining top-down electrospray, bottom-up self-assembly, and solvent-based structure control. This method involves first using electrospray to generate uniform ultrafine liquid droplets, each of which functions as a micro-reactor in which self-assembly reaction occurs forming micellar nanocrystals, with the structures (micelle shape and nanocrystal encapsulation) controlled by the organic solvent used. This method is largely continuous and produces high quality micellar nanocrystal products with an inexpensive structure control approach. By using a water-miscible organic solvent tetrahydrofuran (THF), worm-shaped micellar nanocrystals can be produced due to solvent-induced/facilitated micelle fusion. Compared with the common spherical micellar nanocrystals, worm-shaped micellar nanocrystals can offer minimized non-specific cellular uptake, thus enhancing biological targeting. By co-encapsulating multiple nanocrystals into each micelle, multifunctional or synergistic effects can be achieved. Current limitations of this fabrication method, which will be part of the future work, primarily include imperfect encapsulation in the micellar nanocrystal product and the incompletely continuous nature of the process.

The present work describes a method to fabricate micellar nanocrystals, an emerging major class of nanobiomaterials. This method combines top-down electrospray, bottom-up self-assembly, and solvent-based structure control. The fabrication method is largely continuous, can produce high quality products, and possesses an inexpensive means of structure control.

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of ...

A Continuous-flow Photocatalytic Reactor for the Precisely Controlled Deposition of Metallic Nanoparticles

In this work, a novel photocatalytic reactor for the pulsed and controlled excitation of the photocatalyst and the precise deposition of metallic nanoparticles is developed. Guidelines for the replication of the reactor and its operation are provided in detail. Three different composite systems (Pt/graphene, Pt/TiO2, and Au/TiO2) with monodisperse and uniformly distributed particles are produced by this reactor, and the photodeposition mechanism, as well as the synthesis optimization strategy, are discussed. The synthesis methods and their technical aspects are described comprehensively. The role of the ultraviolet (UV) dose (in each excitation pulse) on the photodeposition process is investigated and the optimum values for each composite system are provided.

For a continuous and scalable synthesis of noble-metal-based nanocomposites, a novel photocatalytic reactor is developed and its structure, operation principles, and product quality optimization strategies are described.

In this work, a novel photocatalytic reactor for the pulsed and controlled excitation of the photocatalyst and the precise deposition of metallic ...

Development of a Nose-only Inhalation Toxicity Test Chamber That Provides Four Exposure Concentrations of Nano-sized Particles

Using a numerical analysis based on computerized fluid dynamics, a nose-only inhalation toxicity chamber with four different exposure concentrations is designed and validated for flow field uniformity and cross-contamination among the exposure ports for each concentration. The designed flow field values are compared with the measured values from exposure ports located horizontally and vertically. For this purpose, nanoscale sodium chloride particles are generated as test particles and introduced to the inhalation chamber to evaluate the cross-contamination and concentration maintenance among the chambers, for each concentration group. The results indicate that the designed multiconcentration inhalation chamber can be used in animal inhalation toxicity testing without cross-contamination among concentration groups. Moreover, the designed multiconcentration inhalation toxicity chamber can also be converted to a single-concentration inhalation chamber. Further testing with gas, organic vapor, or non-nanoscale particles will ensure the use of the chamber in the inhalation testing of other test articles.

A nose-only inhalation toxicity chamber capable of testing inhalation toxicity at four different exposure concentrations was designed and validated for flow field uniformity and cross-contamination between the exposure ports for each concentration. Here, we present a protocol to confirm that the designed chamber is effective for inhalation toxicity testing.

Using a numerical analysis based on computerized fluid dynamics, a nose-only inhalation toxicity chamber with four different exposure concentrations is ...

Preparation of Nanoparticles for ToF-SIMS and XPS Analysis

Nanoparticles have gained increasing attention in recent years due to their potential and application in different fields including medicine, cosmetics, chemistry, and their potential to enable advanced materials. To effectively understand and regulate the physico-chemical properties and potential adverse effects of nanoparticles, validated measurement procedures for the various properties of nanoparticles need to be developed. While procedures for measuring nanoparticle size and size distribution are already established, standardized methods for analysis of their surface chemistry are not yet in place, although the influence of the surface chemistry on nanoparticle properties is undisputed. In particular, storage and preparation of nanoparticles for surface analysis strongly influences the analytical results from various methods, and in order to obtain consistent results, sample preparation must be both optimized and standardized. In this contribution, we present, in detail, some standard procedures for preparing nanoparticles for surface analytics. In principle, nanoparticles can be deposited on a suitable substrate from suspension or as a powder. Silicon (Si) wafers are commonly used as substrate, however, their cleaning is critical to the process. For sample preparation from suspension, we will discuss drop-casting and spin-coating, where not only the cleanliness of the substrate and purity of the suspension but also its concentration play important roles for the success of the preparation methodology. For nanoparticles with sensitive ligand shells or coatings, deposition as powders is more suitable, although this method requires particular care in fixing the sample.

A number of different procedures for preparing nanoparticles for surface analysis are presented (drop casting, spin coating, deposition from powders, and cryofixation). We discuss the challenges, opportunities, and possible applications of each method, particularly regarding the changes in the surface properties caused by the different preparation methods.

Nanoparticles have gained increasing attention in recent years due to their potential and application in different fields including medicine, cosmetics, ...

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging applications. This increased specificity can have significant clinical implications, including decreased side effects and lower dosages with higher potency. Furthermore, the in situ targeting and controlled modulation of specific cell subsets can enhance in vitro and in vivo investigations of basic biological phenomena and probe cell function. Unfortunately, the required expertise in nanoscale science, chemistry and engineering often prohibit laboratories without experience in these fields from fabricating and customizing nanomaterials as tools for their investigations or vehicles for their therapeutic strategies. Here, we provide protocols for the synthesis and scalable assembly of a versatile non-toxic block copolymer system amenable to the facile formation and loading of nanoscale vehicles for biomedical applications. Flash nanoprecipitation is presented as a methodology for rapid fabrication of diverse nanocarriers from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers. These protocols will allow laboratories with a wide range of expertise and resources to easily and reproducibly fabricate advanced nanocarrier delivery systems for their applications. The design and construction of an automated instrument that employs a high-speed syringe pump to facilitate the flash nanoprecipitation process and to allow enhanced control over the homogeneity, size, morphology and loading of polymersome nanocarriers is described.

Nanomaterials provide versatile mechanisms of controlled therapeutic delivery for both basic science and translational applications, but their fabrication often requires expertise that is unavailable in most biomedical laboratories. Here, we present protocols for the scalable fabrication and therapeutic loading of diverse self-assembled nanocarriers using flash nanoprecipitation.

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Bioengineering

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...

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 ...

Medicine

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 ...