Name: evaluating regional pulmonary deposition using patient-specific 3d printed lung models
Uploaded: 2020-11-11T13:00:00
Description: Watch this Scientific Journal Video about Evaluating Regional Pulmonary Deposition using Patient-Specific 3D Printed Lung Models at JoVE.com

The authors thank Professor Yu Feng, Dr. Jenna Briddell, Ian Woodward, and Lucas Attia for their helpful discussions.

1. Preparation of 3D printed experimental components
NOTE: All software used in the protocol are indicated in the Table of Materials. Additionally, the slicing software utilized is specific to the 3D printer listed in the Table of Materials; however, this protocol can be extended to a wide range of stereolithography (SLA) 3D printers.
<ol>
	<li>Convert patient CT scans to 3D objects (.stl files). 
	NOTE: For a more detailed discussion of the geometric&#...

<ol>
	<li>Goel, A., Baboota, S., Sahni, J. K., Ali, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+targeted+pulmonary+delivery+for+treatment+of+lung+cancer.">Exploring targeted pulmonary delivery for treatment of lung cancer.</a> International Journal of Pharmaceutical Investigation. 3 (1), 8-14 (2013).</li><li>Kleinstreuer, C., Zhang, Z., Li, Z., Roberts, W. L., Rojas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+methodology+for+targeting+drug-aerosols+in+the+human+respiratory+system.">A new methodology for targeting drug-aerosols in the human respiratory system.</a> International Journal of Heat and Mass Transfer. 51 (23), 5578-5589 (2008).</li><li>Feng, Y., Chen, X., Yang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+In+Silico+Investigation+of+a+Lobe-Specific+Targeted+Pulmonary+Drug+Delivery+Method.">An In Silico Investigation of a Lobe-Specific Targeted Pulmonary Drug Delivery Method.</a> Design of Medical Devices Conference. , (2018).</li><li>Marple, V. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next+generation+pharmaceutical+impactor+(a+new+impactor+for+pharmaceutical+inhaler+testing).+Part+I:+Design.">Next generation pharmaceutical impactor (a new impactor for pharmaceutical inhaler testing). Part I: Design.</a> Journal of Aerosol Medicine. 16 (3), 283-299 (2003).</li><li>Feng, Y., Zhao, J., Chen, X., Lin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+In+Silico+Subject-Variability+Study+of+Upper+Airway+Morphological+Influence+on+the+Airflow+Regime+in+a+Tracheobronchial+Tree.">An In Silico Subject-Variability Study of Upper Airway Morphological Influence on the Airflow Regime in a Tracheobronchial Tree.</a> Bioengineering. 4 (4), 90 (2017).</li><li>Huynh, B. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Realizing+Lobe-Specific+Aerosol+Targeting+in+a+3D-Printed+In+Vitro+Lung+Model.">Realizing Lobe-Specific Aerosol Targeting in a 3D-Printed In Vitro Lung Model.</a> Journal of Aerosol Medicine and Pulmonary Drug Delivery. , (2020).</li><li>Sul, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+Airflow+Sensitivity+to+Healthy+and+Diseased+Lung+Conditions+in+a+Computational+Fluid+Dynamics+Model+Validated+In+Vitro.">Assessing Airflow Sensitivity to Healthy and Diseased Lung Conditions in a Computational Fluid Dynamics Model Validated In Vitro.</a> Journal of Biomechanical Engineering. 140 (5), (2018).</li><li>Martonen, T. B., Katz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deposition+Patterns+of+Polydisperse+Aerosols+Within+Human+Lungs.">Deposition Patterns of Polydisperse Aerosols Within Human Lungs.</a> Journal of Aerosol Medicine. 6 (4), 251-274 (1993).</li><li>Nahar, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro,+in+vivo+and+ex+vivo+models+for+studying+particle+deposition+and+drug+absorption+of+inhaled+pharmaceuticals.">In vitro, in vivo and ex vivo models for studying particle deposition and drug absorption of inhaled pharmaceuticals.</a> European Journal of Pharmaceutical Sciences. 49 (5), 805-818 (2013).</li><li>Nichols, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Multi-laboratory+in+Vitro+Study+to+Compare+Data+from+Abbreviated+and+Pharmacopeial+Impactor+Measurements+for+Orally+Inhaled+Products:+a+Report+of+the+European+Aerosol+Group+(EPAG).">A Multi-laboratory in Vitro Study to Compare Data from Abbreviated and Pharmacopeial Impactor Measurements for Orally Inhaled Products: a Report of the European Aerosol Group (EPAG).</a> AAPS PharmSciTech. 17 (6), 1383-1392 (2016).</li><li>Yoshida, H., Kuwana, A., Shibata, H., Izutsu, K. I., Goda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Aerodynamic+Particle+Size+Distribution+Between+a+Next+Generation+Impactor+and+a+Cascade+Impactor+at+a+Range+of+Flow+Rates.">Comparison of Aerodynamic Particle Size Distribution Between a Next Generation Impactor and a Cascade Impactor at a Range of Flow Rates.</a> AAPS PharmSciTech. 18 (3), 646-653 (2017).</li><li>Feng, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+silico+inter-subject+variability+study+of+extra-thoracic+morphology+effects+on+inhaled+particle+transport+and+deposition.">An in silico inter-subject variability study of extra-thoracic morphology effects on inhaled particle transport and deposition.</a> Journal of Aerosol Science. 123, 185-207 (2018).</li><li>Kleinstreuer, C., Seelecke, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhaler+system+for+targeted+maximum+drug-aerosol+delivery.">Inhaler system for targeted maximum drug-aerosol delivery.</a> United States patent. , (2005).</li><li>. How Medical 3D Printing is Gaining Ground in Top Hospitals Available from: <a target="_blank" href="https://www.materialise.com/en/blog/3d-printing-hospitals">https://www.materialise.com/en/blog/3d-printing-hospitals</a> (2019)</li><li>Weber, P. W., Price, O. T., McClellan, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demographic+Variability+of+Inhalation+Mechanics:+A+Review.">Demographic Variability of Inhalation Mechanics: A Review.</a> Defense Threat Reduction Agency. , (2016).</li><li>Jiang, Y. Y., Xu, X., Su, H. L., Liu, D. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gender-related+difference+in+the+upper+airway+dimensions+and+hyoid+bone+position+in+Chinese+Han+children+and+adolescents+aged+6-18+years+using+cone+beam+computed+tomography.">Gender-related difference in the upper airway dimensions and hyoid bone position in Chinese Han children and adolescents aged 6-18 years using cone beam computed tomography.</a> Acta Odontologica Scandinavica. 73 (5), 391-400 (2015).</li><li>Martin, S. E., Mathur, R., Marshall, I., Douglas, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+age,+sex,+obesity+and+posture+on+upper+airway+size.">The effect of age, sex, obesity and posture on upper airway size.</a> European Respiratory Journal. 10 (9), 2087 (1997).</li><li>Xi, J., Longest, P. W., Martonen, T. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+laryngeal+jet+on+nano-+and+microparticle+transport+and+deposition+in+an+approximate+model+of+the+upper+tracheobronchial+airways.">Effects of the laryngeal jet on nano- and microparticle transport and deposition in an approximate model of the upper tracheobronchial airways.</a> Journal of Applied Physiology. 104 (6), 1761-1777 (2008).</li><li>Zhao, J., Feng, Y., Fromen, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glottis+motion+effects+on+the+particle+transport+and+deposition+in+a+subject-specific+mouth-to-trachea+model:+A+CFPD+study.">Glottis motion effects on the particle transport and deposition in a subject-specific mouth-to-trachea model: A CFPD study.</a> Computers in Biology and Medicine. 116, 103532 (2020).</li><li>Kim, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+obstructive+pulmonary+disease:+lobe-based+visual+assessment+of+volumetric+CT+by+Using+standard+images--comparison+with+quantitative+CT+and+pulmonary+function+test+in+the+COPDGene+study.">Chronic obstructive pulmonary disease: lobe-based visual assessment of volumetric CT by Using standard images--comparison with quantitative CT and pulmonary function test in the COPDGene study.</a> Radiology. 266 (2), 626-635 (2013).</li><li>. The Cancer Imaging Archive Available from: <a target="_blank" href="https://www.cancerimagingarchive.net/">https://www.cancerimagingarchive.net/</a> (2020)</li><li>Li, A., Ahmadi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer+Simulation+of+Deposition+of+Aerosols+in+a+Turbulent+Channel+Flow+with+Rough+Walls.">Computer Simulation of Deposition of Aerosols in a Turbulent Channel Flow with Rough Walls.</a> Aerosol Science and Technology. 18 (1), 11-24 (1993).</li><li>Khalili, S. F., Ghanbarzadeh, S., Nokhodchi, A., Hamishehkar, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+different+coating+materials+on+the+prevention+of+powder+bounce+in+the+next+generation+impactor.">The effect of different coating materials on the prevention of powder bounce in the next generation impactor.</a> Research in Pharmaceutical Sciences. 13 (3), 283-287 (2018).</li><li>Galliger, Z., Vogt, C. D., Panoskaltsis-Mortari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+bioprinting+for+lungs+and+hollow+organs.">3D bioprinting for lungs and hollow organs.</a> Translational Research. 211, 19-34 (2019).</li><li>Schwarz, K., Biller, H., Windt, H., Koch, W., Hohlfeld, J. 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The current state-of-the-art device for pulmonary pharmaceutical testing of a complete inhalation dose is the Next Generator Impactor (NGI), which measures the aerodynamic diameter of an aerosol4. This sizing data is then used to predict the lung generation at which the aerosol will deposit based on a correlation developed for a healthy adult male11. Unfortunately, this method is limited in its ability to assess differences in regional lung deposition, determine the effects...

Many pulmonary diseases such as lung cancer and chronic obstructive pulmonary disease (COPD) exhibit regional differences in disease characteristics; however, there are a lack of therapeutic techniques available to target drug delivery to only diseased regions of the lung1. Multiple computational fluid dynamic (CFD) models have demonstrated that it is possible to modulate drug deposition profiles by identifying specific streamlines in the lung2,3. Development of both inhalers and endotracheal (ET) tube adaptors with regional targeting capabilities are on-going in our lab to control aerosol distribution to diseased lung regions. Extension of these principles to clinical use is limited by current preclinical testing capacity. The precise location a drug deposits within the lung is known to be the best predictor of efficacy; however, current pharmaceutical assessments of inhalable therapeutics are most often predicted using in vitro-in vivo correlations of particle size to merely approximate deposition4. This technique does not allow for any spatial analysis to determine the effects of different airway geometries on regional distribution through the various lobes of the lung. Additionally, this testing lacks anatomically accurate lung geometries, which researchers have shown can have a significant impact on deposition profiles5. Some efforts have been made to incorporate patient-specific lung geometries into testing protocols through the addition of the upper airways; however, most of these approaches sample aerosol delivery to various generations of the lung rather than each lung lobe6,7,8. The following protocol presents a high-throughput method of generating patient-specific lung models with the capacity to quantify relative particle deposition in each of the five lobes of the lung9.
Anatomically accurate model lungs are generated by 3D printing patient computed tomography (CT) scans. When used in conjunction with an easily assembled flow system, the relative flow rates through each of the model lung&#8217;s lobes can be independently controlled and tailored to mimic those of different patient demographics and/or disease states. With this method, researchers can test the efficacy of potential therapeutic methods in a relevant lung geometry and correlate each method&#8217;s performance with the progression of diseased morphology. Here, two device designs developed in our lab are tested for their ability to increase deposition in a desired lung lobe by controlling the location of aerosol release in the mouth or trachea. This protocol also has the potential to significantly impact the development of personalized procedures for patients by facilitating the rapid prediction of treatment efficacy in a model lung specific to that patient&#8217;s CT scan data.

<table>
	<tbody>
		<tr>
			<td>1/4&#34; Plastic Barbed Tube Fitting</td>
			<td>McMaster Carr</td>
			<td>5372K111</td>
			<td></td>
		</tr>
		<tr>
			<td>10 um Filter Paper</td>
			<td>Fisher</td>
			<td>1093-110</td>
			<td></td>
		</tr>
		<tr>
			<td>1um Fluorescent Polystyrene Particles</td>
			<td>Polysciences</td>
			<td>15702-10</td>
			<td></td>
		</tr>
		<tr>
			<td>1um Non-Fluorescent Polystyrene Particles</td>
			<td>Polysciences</td>
			<td>8226</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Fisher</td>
			<td>A516-4</td>
			<td>Referred to in protocol as &#34;IPA&#34;</td>
		</tr>
		<tr>
			<td>3/8&#34; Plastic Barbed Tube Fitting</td>
			<td>McMaster Carr</td>
			<td>5372K117</td>
			<td></td>
		</tr>
		<tr>
			<td>Air Flow Meter (1 - 280 mL/min)</td>
			<td>McMaster Carr</td>
			<td>41695K32</td>
			<td>Referred to in protocol as &#34;flow meter&#34;</td>
		</tr>
		<tr>
			<td>Carbon M1 3D Printer</td>
			<td>Carbon 3D</td>
			<td></td>
			<td>https://www.carbon3d.com/, Associated software referred to in protocol as &#34;slicing software&#34;</td>
		</tr>
		<tr>
			<td>Collison Jet Nebulizer</td>
			<td>CH Technologies</td>
			<td>ARGCNB0008 (CN-25)</td>
			<td>6 Jet MRE style horizontal collision with glass jar, Referred to in protocol as &#34;nebulizer&#34;, http://chtechusa.com/Manuals/MRE_Collison_Manual.pdf</td>
		</tr>
		<tr>
			<td>Convection Oven</td>
			<td>Yamato</td>
			<td>DKN602</td>
			<td></td>
		</tr>
		<tr>
			<td>Copley Critical Flow Controller TPK2000 Reve 120V</td>
			<td>MSP Corp</td>
			<td>0001-01-9810</td>
			<td>Referred to in protocol as &#34;flow controller&#34;</td>
		</tr>
		<tr>
			<td>Copley High Capacity Pump Model HCP5</td>
			<td>MSP Corp</td>
			<td>0001-01-9982</td>
			<td>Referred to in protocol as &#34;vacuum pump&#34;</td>
		</tr>
		<tr>
			<td>Cytation</td>
			<td>BioTek</td>
			<td>CYT5MPV</td>
			<td>Multifunctional Spectrophotometer/Fluorescent imager equiped with 4x/20x/40x objectives and DAPI/GFP/TexasRed laser/filter cubes</td>
		</tr>
		<tr>
			<td>EPU40 Resin</td>
			<td>Carbon 3D</td>
			<td></td>
			<td>https://www.carbon3d.com/materials/epu-elastomeric-polyurethane/, Referred to in protocol as &#34;soft resin&#34;</td>
		</tr>
		<tr>
			<td>Filter for vacuum pump</td>
			<td>Whatman</td>
			<td>6722-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Meter Model DFM 2000</td>
			<td>MSP Corp</td>
			<td>0001-01-8764</td>
			<td>Referred to in protocol as &#34;electronic flow meter&#34;</td>
		</tr>
		<tr>
			<td>ImageJ Software</td>
			<td>ImageJ</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/download.html</td>
		</tr>
		<tr>
			<td>Inline Air Flow Control Valve (Push-to-Connect)</td>
			<td>McMaster Carr</td>
			<td>62005K333</td>
			<td>Referred to in protocol as &#34;valve&#34;</td>
		</tr>
		<tr>
			<td>Inline Filter Devices</td>
			<td>Whatman</td>
			<td>WHA67225000</td>
			<td></td>
		</tr>
		<tr>
			<td>Marine-Grade Plywood Sheet</td>
			<td>McMaster Carr</td>
			<td>62005K333</td>
			<td>Referred to in protocol as &#34;wooden board&#34;</td>
		</tr>
		<tr>
			<td>Materialise Mimics Software</td>
			<td>Materialise</td>
			<td></td>
			<td>https://www.materialise.com/en/medical/mimics-innovation-suite, Referred to in protocol as &#34;CT scan software&#34;</td>
		</tr>
		<tr>
			<td>Meshmixer Software</td>
			<td>Autodesk</td>
			<td></td>
			<td>http://www.meshmixer.com/, Referred to in protocol as &#34;mesh editing software&#34;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>A454-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Opticure LED Cube</td>
			<td>APM Technica</td>
			<td>102843</td>
			<td>Referred to in protocol as &#34;UV oven&#34;</td>
		</tr>
		<tr>
			<td>PR25 Resin</td>
			<td>Carbon 3D</td>
			<td></td>
			<td>https://www.carbon3d.com/materials/uma-urethanemethacrylate, /Referred to in protocol as &#34;hard resin&#34;</td>
		</tr>
		<tr>
			<td>PVC Tube for Chemicals</td>
			<td>McMaster Carr</td>
			<td>5231K161</td>
			<td>1/4&#34; ID</td>
		</tr>
		<tr>
			<td>Screws</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SolidWorks Software</td>
			<td>Dassault Syst&#232;mes&#160;SolidWorks Corporation</td>
			<td></td>
			<td>https://www.solidworks.com/, Referred to in protocol as &#34;3D modeling software&#34;</td>
		</tr>
		<tr>
			<td>Straight Flow Rectangular Manifold</td>
			<td>McMaster Carr</td>
			<td>1125T31</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing to Flow Controller</td>
			<td>McMaster Carr</td>
			<td>5233K65</td>
			<td>3/8&#34; ID</td>
		</tr>
		<tr>
			<td>Wire</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluating regional pulmonary deposition using patient-specific 3d printed lung models

Development of targeted therapies for pulmonary diseases is limited by the availability of preclinical testing methods with the ability to predict regional aerosol delivery. Leveraging 3D printing to generate patient-specific lung models, we outline the design of a high-throughput, in vitro experimental setup for quantifying lobular pulmonary deposition. This system is made with a combination of commercially available and 3D printed components and allows the flow rate through each lobe of the lung to be independently controlled. Delivery of fluorescent aerosols to each lobe is measured using fluorescence microscopy. This protocol has the potential to promote the growth of personalized medicine for respiratory diseases through its ability to model a wide range of patient demographics and disease states. Both the geometry of the 3D printed lung model and the air flow profile setting can be easily modulated to reflect clinical data for patients with varying age, race, and gender. Clinically relevant drug delivery devices, such as the endotracheal tube shown here, can be incorporated into the testing setup to more accurately predict a device&#8217;s capacity to target therapeutic delivery to a diseased region of the lung. The versatility of this experimental setup allows it to be customized to reflect a multitude of inhalation conditions, enhancing the rigor of preclinical therapeutic testing.

Particles in this size range (1-5 &#956;m) and flow conditions (1-10 L/min) follow the fluid stream lines based on both their theoretical Stokes number and in vivo data; therefore, in the absence of a targeted delivery device, particles released into the lung model are expected to deposit according to the percentage of total airflow diverted to each lobe. The relative amounts of particle delivery to each lobe can then be compared to clinical lobe flow rate data obtained through analyzing patient-specific high-resolution ...

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Evaluating Regional Pulmonary Deposition using Patient-Specific 3D Printed Lung Models

We present a high-throughput, in vitro method for quantifying regional pulmonary deposition at the lobe level using CT scan-derived, 3D printed lung models with tunable air flow profiles.

Development of targeted therapies for pulmonary diseases is limited by the availability of preclinical testing methods with the ability to predict ...

This protocol has the potential to drive the development of new targeted pulmonary therapeutics by enabling the pre-clinical predictions of regional deposition. This technique incorporates anatomically accurate lung models, 3D printed from patient's CT scans for the rapid generation of personalized predictive results regarding the efficacy of potential treatments. This technique can be used to develop targeted therapies that minimize off target effects for diseases that are characterized by regional area obstructions such as lung cancer or COPD.After printing the experimental components and completing post-processing as per the manufacturer's instructions, carefully wash the parts printed in soft resin with at least 99%purity isopropyl alcohol to remove any excess uncured resin before thermal curing the parts in a convection oven for eight hours according to the manufacturer's specifications. Then, wash the parts printed in hard resin with the alcohol to remove any excess uncured resin and cure the parts in a UV oven for one minute per side. For lobe outlet cap assembly, insert one end of the oval barbed tubing connection base into the cap before carefully stretching the flexible cap over the other end of the oval base, taking special care not to crack the thin base and with the nozzle protruding through the opening in the cap base.Next, cut 10 microliter filter paper to a size slightly larger than the outlet area and fold the filter paper over the lobe outlet, holding the paper in place with one hand. Then use tweezers in the other hand, to stretch the cap with the barbed tubing connection over the outlet and press the cap down until the notch matches the corresponding notch on the lobe outlet. Before each experimental run, connect each lung model lobe outlet to the tubing of the corresponding flow meter and valve taking care not to apply too much lateral pressure to the barbed tubing connection.Attach the electronic flow meter to the lung model mouth inlet to measure the total airflow rate to the lung model and turn on the flow controller and vacuum pump. In the flow controller, select the test setup setting and slowly increase the flow rate until the electronic flow meter displays the desired total flow rate. Use the valves to adjust the flow rate through the right upper, right middle, right lower, left upper and left lower lung lobes.Once the load flow rates shown on the flow meters are steady at the desired value, check the overall flow rate again on the electronic flow meter to verify that there are no leaks in the system. Then, exit the test setup in the flow controller leaving the vacuum pump on. For aerosol delivery to the lung model, fill a nebulizer with a solution of the desired fluorescent particles and connect the nebulizer to the lung model inlet.To measure the efficacy of the targeting device, insert the device into the lung model and connect the nebulizer to the device. Connect the compressed airline to the nebulizer. Set the flow controller to run for one ten second trial and open the compressed air valve slightly to begin generating an aerosol within the nebulizer.Press start on the flow controller and immediately open the compressed air valve fully. When the flow controller reaches about nine seconds, begin closing the compressed air valve and close the fume hood sash as much as possible. Once the valve is fully closed, disconnect the nebulizer from the compressed airline.Fully close the fume hood sash, shut off the vacuum pump and let any aerosols clear from the fume hood. After about 10 minutes, disconnect the lung model from the tubing system, taking care not to crack the barbed tubing connections and run a pair of tweezers under the edge of each lobe outlet cap to remove the caps from the outlets. Then transfer the filter paper from each cap into individual wells of a 24-well plate particle depositions side down.When all of the filter paper has been collected, place the plate onto the stage of a digital fluorescence microscope and set the microscope to a 4X magnification and the appropriate fluorescence channel. Then take at least three images of the filter paper from each lobe at random locations and save the images as TIF files. Under one liter per minute collection conditions in a healthy lung, ended along effected by COPD, the experimentally determined deposition profile is not statistically different from the clinical data demonstrating that the setup accurately mimics the distribution of air flow to each of the lung lobes.Compared to the non-targeted particle deposition profile, the use of a modified endotracheal tube generates a nearly four fold increase in left lower lobe delivery. In addition to diverting over 96%of the delivered particles to the left lung. Altering the release location setting to target the right lower lobe, this device generates more than double the particle delivery to the right lobe and diverts 94%of the delivered particles to the right lung.Compared to the non-targeted particle deposition profile, the concentric cylinder device causes a nearly three fold increase in left upper lobe delivery in addition to diverting over 87%of the delivered particles to the left lung. Targeting efficiency can also be observed qualitatively by comparing the images of the target lobe filter to the other outlet filters. As illustrated, the most effective targeting method will yield a high particle deposition at the intended lobe of interest and a low deposition at the remaining lobe outlets.It is essential to make sure that components are properly connected to prevent leaks within the system as leaks will impact deposition results. This protocol permits researchers to test potential drug delivery devices for targeting specific regions of the lung prior to clinical trials reducing the cost associated with drug development and efficacy enhancement in human patients.

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