The authors would like to thank Mr. Ray Lee, Mr. HC Leung and Mr. Wallace So for their kind assistance in making the light source and powder insufflator; and the Faculty Core Facility for the assistance in animal imaging. The work was supported by the Research Grant Council, Hong Kong (17300319).

The experiments conducted in this study have been approved by the Committee on the Use of Live Animals for Teaching and Research (CULATR), The University of Hong Kong. Dry powder formulations prepared by spray freeze drying (SFD) containing 0.5% of luciferase messenger RNA (mRNA), 5% synthetic peptide PEG12KL4 and 94.5% of mannitol (w/w) are used in this study to demonstrate mRNA expression in the lung16. The mass median aerodynamic diameter (MMAD) of SFD powder is 2.4 &#956;m. Spray dried (SD) mannitol powder are used to investigate the effect of air volume used in powder dispersion16. The MMAD of SD powder is 1.5 &#956;m.
1. Fabrication of dry powder insufflator and loading of dry powder
<ol>
	<li>(Optional)&#160;Neutralize the static charges of dry powder (in a vial) and the 200 &#181;L non-filter round gel-loading pipette tip. Use an anti-static gun or a balance with deionizing function according to the manufacturer&#8217;s instruction.</li>
	<li>Prepare a weighing paper with a size of around 4 cm x 4 cm. Fold the paper in half diagonally and then unfold it.</li>
	<li>Weigh 1-2 mg of dry powder on the weighing paper.</li>
	<li>Fill a gel-loading pipette tip with powder through the wider opening of the tip. Tap gently to pack the powder until the powder forms loose agglomerates near the narrow end of the tip (Figure 1A). Avoid packing the powder too tightly as it may hamper powder dispersion.</li>
	<li>Connect the powder-loaded tip to a 1 mL syringe through a three-way stopcock (Figure 1B). The size of syringe can be changed according to the volume of air used to disperse the powder. Hold the tip and syringe vertically during connection to prevent spillage of powder. If administration is not performed immediately, use parafilm to seal the openings of the tip and store it temporarily under suitable condition until administration.</li>
</ol>
2. Fabrication of intubation device
<ol>
	<li>Light source (Figure 2)
	<ol>
		<li>Prepare a custom-made light source with a light emitting diode (LED) torch and a flexible optical fiber with a diameter of 0.8-1 mm.</li>
		<li>Make a centered orifice on the clear lens of the LED torch with a hand drill or a drill bit so that the optical fiber can barely pass through.</li>
		<li>Insert the optical fiber through the orifice. Switch on the LED torch to adjust the position and the depth of insertion for maximum brightness at the other end of the optical fiber.</li>
		<li>Affix the optical fiber in position with clear epoxy glue.</li>
	</ol>
	</li>
	<li>Guiding cannula (Figure 3)
	<ol>
		<li>Take a 1 mL plastic Pasteur pipette (Figure 3A) and hold the pipette at both ends.</li>
		<li>Use an alcohol lamp (or other heat sources in the laboratory such as a Bunsen burner) to heat the middle of the pipette by placing it at 5-10 cm above the flame (Figure 3B). Rotate the pipette to make sure it is heated evenly.</li>
		<li>When the plastic becomes soft and deformable, move the pipette away from the flame and stretch the pipette gently.</li>
		<li>Cut the stretched pipette in the middle with a pair of scissor into Part A and Part B (Figure 3C-E). Use Part A as a fine tip pipette and Part B as a guiding cannula. To increase the chance of successful intubation with the guiding cannula, make a bevel (not too sharp which may increase the risk of injuring the animal) at the end of Part B (Figure 3F). When a 200 &#181;L gel-loading pipette tip (for powder loading) is inserted into the guiding cannula, it should protrude the cannula by 1-2 mm. 
		NOTE: A guiding cannula (Part B) with the appropriate dimension (internal and external diameter) for intubation could have a 21 gauge needle fit inside it while it can also fit inside a 17 gauge needle. Multiple attempts may be needed in stretching the pipettes to achieve the appropriate dimension.</li>
		<li>(Optional): Cut a small opening at the wider end of the guiding cannula to make it more flexible so that it is easier to hold the optical fiber (Figure 3F). This opening also allows the fitting of a microsprayer for the administration of liquid aerosol.</li>
	</ol>
	</li>
</ol>
3. Intubation
<ol>
	<li>Anaesthetize the mouse (BALB/c, 7-9 weeks) with ketamine (100 mg/kg) and xylazine (10 mg/kg) by intraperitoneal injection.</li>
	<li>Prepare a platform made of Plexiglass and mount it to a stand with a clamp (Figure 4A). Place the anaesthetized mouse on the platform (at around 60&#176; of inclination) in a supine position. The height and angle of inclination of the platform could be adjusted by the position of the clamp on the stand.</li>
	<li>Suspend the mouse by hooking its incisors on a nylon floss (Figure 4B). Secure the position of the mouse by a piece of tape or a rubber band.</li>
	<li>Insert the optical fiber into the guiding cannula before intubation with the tip of the fiber level with the opening of the guiding cannula. Turn on the LED torch to illuminate.</li>
	<li>Gently protrude the tongue of the mouse with a pair of forceps to expose its trachea.</li>
	<li>Use the other hand to hold the guiding cannula with optical fiber inside. Insert them through the oral cavity. With the illumination from the optical fiber, the opening of the trachea can be visualized as an orifice between the vocal cords.</li>
	<li>Align the bevel of the guiding cannula towards the midline of the opening (Figure 5A). Gently intubate the guiding cannula with optical fiber into the trachea by aiming the finest tip of the cannula at the tracheal opening.</li>
	<li>Upon intubation, swiftly remove the optical fiber and leave the guiding cannula inside the trachea (Figure 5B). A normal respiration should be observed.</li>
	<li>Hold the fine tip pipette (Part A) at the opening of the guiding cannula and insufflate a small puff of air (about 0.2 mL) into the lung of the mouse. A slight inflation in the chest of the mouse indicates proper intubation. Remove the fine tip pipette prior to powder administration.</li>
</ol>
4. Powder administration
<ol>
	<li>Hold the powder loaded-tip that is connected to the syringe as described in step 1.5. Ensure the airflow between the syringe and the tip is disconnected.</li>
	<li>Pull the syringe plunger backward to withdraw 0.6 mL of air. 
	NOTE: The volume of air used to disperse the powder is dependent on the properties of the powder and the amount of powder loaded. This is further described in the result section.</li>
	<li>Turn the valve of the three-way stopcock to connect the airflow between the syringe and the powder-loaded tip.</li>
	<li>Insert the powder-loaded tip into the guiding cannula which has already been placed in the trachea of the mouse (Figure 5C). Hold the guiding cannula and push the syringe plunger forcefully in one continuous action to disperse the powder as aerosols into the lung. 
	NOTE: Any forward motion of the device should be minimized to avoid injuring the animal.</li>
	<li>Remove the tip and check if the powder inside the tip has been emptied. If not, repeat step 4.1 to 4.4. 
	NOTE: If the powder is packed too tightly due to excessive tapping, it might not be dispersed properly.</li>
	<li>Once the administration is complete, remove the guiding cannula from the trachea.</li>
	<li>Allow the mouse to recover by positioning it horizontally in a supine position with its tongue half protruded to avoid the blockade of the airways.</li>
</ol>

<ol><li>Newman, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drug+delivery+to+the+lungs%3A+challenges+and+opportunities%5BTitle%5D)+AND+%22Therapeutic+Delivery%22%5BJournal%5D)">Drug delivery to the lungs: challenges and opportunities.</a> Therapeutic Delivery. 8 (8), 647-661 (2017).</li>
<li>Setter, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhaled+dry+powder+insulin+for+the+treatment+of+diabetes+mellitus%5BTitle%5D)+AND+%22Clinical+Therapeutics%22%5BJournal%5D)">Inhaled dry powder insulin for the treatment of diabetes mellitus.</a> Clinical Therapeutics. 29 (5), 795-813 (2007).</li>
<li>Muralidharan, P., Hayes, D., Mansour, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dry+powder+inhalers+in+COPD%2C+lung+inflammation+and+pulmonary+infections%5BTitle%5D)+AND+%22Expert+Opinion+on+Drug+Delivery%22%5BJournal%5D)">Dry powder inhalers in COPD, lung inflammation and pulmonary infections.</a> Expert Opinion on Drug Delivery. 12 (6), 947-962 (2015).</li>
<li>de Boer, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dry+powder+inhalation%3A+past%2C+present+and+future%5BTitle%5D)+AND+%22Expert+Opinion+on+Drug+Delivery%22%5BJournal%5D)">Dry powder inhalation: past, present and future.</a> Expert Opinion on Drug Delivery. 14 (4), 499-512 (2017).</li>
<li>Das, S., Tucker, I., Stewart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhaled+dry+powder+formulations+for+treating+tuberculosis%5BTitle%5D)+AND+%22Current+Drug+Delivery%22%5BJournal%5D)">Inhaled dry powder formulations for treating tuberculosis.</a> Current Drug Delivery. 12 (1), 26-39 (2015).</li>
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<li>Phillips, J. E., Zhang, X., Johnston, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dry+powder+and+nebulized+aerosol+inhalation+of+pharmaceuticals+delivered+to+mice+using+a+nose-only+exposure+system%5BTitle%5D)+AND+%22JoVE+%28Journal+of+Visualized+Experiments%29%22%5BJournal%5D)">Dry powder and nebulized aerosol inhalation of pharmaceuticals delivered to mice using a nose-only exposure system.</a> JoVE (Journal of Visualized Experiments). (122), e55454(2017).</li>
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<li>Price, D. N., Muttil, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Delivery+of+Therapeutics+to+the+Lung%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Delivery of Therapeutics to the Lung.</a> Methods in Molecular Biology. 1809, 415-429 (2018).</li>
<li>Chang, R. Y. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proof-of-Principle+Study+in+a+Murine+Lung+Infection+Model+of+Antipseudomonal+Activity+of+Phage+PEV20+in+a+Dry-Powder+Formulation%5BTitle%5D)+AND+%22Antimicrobial+Agents+and+Chemotherapy%22%5BJournal%5D)">Proof-of-Principle Study in a Murine Lung Infection Model of Antipseudomonal Activity of Phage PEV20 in a Dry-Powder Formulation.</a> Antimicrobial Agents and Chemotherapy. 62 (2), (2018).</li>
<li>Ito, T., Okuda, T., Takayama, R., Okamoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+of+an+Evaluation+Method+for+Gene+Silencing+by+Serial+Pulmonary+Administration+of+siRNA+and+pDNA+Powders%3A+Naked+siRNA+Inhalation+Powder+Suppresses+Luciferase+Gene+Expression+in+the+Lung%5BTitle%5D)+AND+%22Journal+of+pharmaceutical+sciences%22%5BJournal%5D)">Establishment of an Evaluation Method for Gene Silencing by Serial Pulmonary Administration of siRNA and pDNA Powders: Naked siRNA Inhalation Powder Suppresses Luciferase Gene Expression in the Lung.</a> Journal of pharmaceutical sciences. 108 (8), 2661-2667 (2019).</li>
<li>Patil, J. S., Sarasija, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pulmonary+drug+delivery+strategies%3A+A+concise%2C+systematic+review%5BTitle%5D)+AND+%22Lung+India%22%5BJournal%5D)">Pulmonary drug delivery strategies: A concise, systematic review.</a> Lung India. 29 (1), 44-49 (2012).</li>
<li>Ihara, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histological+Quantification+of+Gene+Silencing+by+Intratracheal+Administration+of+Dry+Powdered+Small-Interfering+RNA%2FChitosan+Complexes+in+the+Murine+Lung%5BTitle%5D)+AND+%22Pharmaceutical+Research%22%5BJournal%5D)">Histological Quantification of Gene Silencing by Intratracheal Administration of Dry Powdered Small-Interfering RNA/Chitosan Complexes in the Murine Lung.</a> Pharmaceutical Research. 32 (12), 3877-3885 (2015).</li>
<li>Qiu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effective+mRNA+pulmonary+delivery+by+dry+powder+formulation+of+PEGylated+synthetic+KL4+peptide%5BTitle%5D)+AND+%22Journal+of+Controlled+Release%22%5BJournal%5D)">Effective mRNA pulmonary delivery by dry powder formulation of PEGylated synthetic KL4 peptide.</a> Journal of Controlled Release. 314, 102-115 (2019).</li>
<li>Pfeifer, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dry+powder+aerosols+of+polyethylenimine+%28PEI%29-based+gene+vectors+mediate+efficient+gene+delivery+to+the+lung%5BTitle%5D)+AND+%22Journal+of+Controlled+Release%22%5BJournal%5D)">Dry powder aerosols of polyethylenimine (PEI)-based gene vectors mediate efficient gene delivery to the lung.</a> Journal of Controlled Release. 154 (1), 69-76 (2011).</li>
<li>Kim, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Doxorubicin-loaded+highly+porous+large+PLGA+microparticles+as+a+sustained-+release+inhalation+system+for+the+treatment+of+metastatic+lung+cancer%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Doxorubicin-loaded highly porous large PLGA microparticles as a sustained- release inhalation system for the treatment of metastatic lung cancer.</a> Biomaterials. 33 (22), 5574-5583 (2012).</li>
<li>Tonnis, W. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+aerosol+generator+for+homogenous+distribution+of+powder+over+the+lungs+after+pulmonary+administration+to+small+laboratory+animals%5BTitle%5D)+AND+%22European+Journal+of+Pharmaceutics+and+Biopharmaceutics%22%5BJournal%5D)">A novel aerosol generator for homogenous distribution of powder over the lungs after pulmonary administration to small laboratory animals.</a> European Journal of Pharmaceutics and Biopharmaceutics. 88 (3), 1056-1063 (2014).</li>
<li>Hoppentocht, M., Hoste, C., Hagedoorn, P., Frijlink, H. W., de Boer, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+evaluation+of+the+DP-4M+PennCentury+insufflator%5BTitle%5D)+AND+%22European+Journal+of+Pharmaceutics+and+Biopharmaceutics%22%5BJournal%5D)">In vitro evaluation of the DP-4M PennCentury insufflator.</a> European Journal of Pharmaceutics and Biopharmaceutics. 88 (1), 153-159 (2014).</li>
<li>Liao, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Porous+and+highly+dispersible+voriconazole+dry+powders+produced+by+spray+freeze+drying+for+pulmonary+delivery+with+efficient+lung+deposition%5BTitle%5D)+AND+%22International+Journal+of+Pharmaceutics%22%5BJournal%5D)">Porous and highly dispersible voriconazole dry powders produced by spray freeze drying for pulmonary delivery with efficient lung deposition.</a> International Journal of Pharmaceutics. 560, 144-154 (2019).</li>
<li>Ito, T., Okuda, T., Takashima, Y., Okamoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Naked+pDNA+Inhalation+Powder+Composed+of+Hyaluronic+Acid+Exhibits+High+Gene+Expression+in+the+Lungs%5BTitle%5D)+AND+%22Molecular+Pharmaceutics%22%5BJournal%5D)">Naked pDNA Inhalation Powder Composed of Hyaluronic Acid Exhibits High Gene Expression in the Lungs.</a> Molecular Pharmaceutics. 16 (2), 489-497 (2019).</li>
<li>Chaurasiya, B., Zhou, M., Tu, J., Sun, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Design+and+validation+of+a+simple+device+for+insufflation+of+dry+powders+in+a+mice+model%5BTitle%5D)+AND+%22European+Journal+of+Pharmaceutical+Sciences%22%5BJournal%5D)">Design and validation of a simple device for insufflation of dry powders in a mice model.</a> European Journal of Pharmaceutical Sciences. 123, 495-501 (2018).</li>
</ol>

The authors have no conflicts of interest to disclose.

In this paper, custom-made devices for dry powder insufflation and intratracheal intubation are presented. In the powder loading step, dry powder are loaded into a 200 &#181;L gel-loading pipette tip. It is important to gently tap the tip to allow the loose packing of powder at the narrow end of the tip. However, if the powder are packed too tightly, they will get stuck in the tip and cannot be properly dispersed. It is recommended to neutralize the static charges of the powder and the pipette tip in order to facilitate ...

The pulmonary route of administration offers various benefits in delivering therapeutics for both local and systemic actions. For the treatment of lung diseases, high local drug concentration can be achieved by pulmonary delivery, thereby reducing the required dose and lowering the incidence of systemic side effects. Moreover, the relatively low enzymatic activities in the lung can reduce premature drug metabolism. The lungs are also efficient for drug absorption for systemic action due to the large and well-perfused surface area, the extremely thin epithelial cell layer and the high blood volume in pulmonary capillaries1.
Inhaled dry powder formulations have been widely investigated for the prevention and treatment of various diseases such as asthma, chronic obstructive pulmonary disease, diabetes mellitus and pulmonary vaccination2,3,4. Drugs in the solid state are generally more stable than in the liquid form, and dry powder inhalers are more portable and user-friendly than nebulizers5,6. In the development of inhaled dry powder formulations, the safety, the pharmacokinetic profile and the therapeutic efficacy need to be evaluated in preclinical animal models following pulmonary administration7. Unlike humans who can inhale dry powder actively, pulmonary delivery of dry powder to small animals is challenging. It is necessary to establish an efficient protocol of delivering dry powder to the lungs of animals.
Mice are widely used as research animal models because they are economical and they breed well. They are also easy to handle and many disease models are well-established. There are two major approaches to administer dry powder to the lung&#160;of mouse: inhalation and intratracheal administration. For inhalation, the mouse is placed in a whole-body or nose-only chamber where dry powder is aerosolized and the animals breathe in the aerosol without sedation8,9. Expensive equipment is required and the drug delivery efficiency is low. While the whole-body chamber may be technically less challenging, the nose-only exposure chamber could minimize exposure of drugs to the body surface. Regardless, it is still difficult to precisely control and determine the dose delivered to the lungs. The dry powder is mainly deposited in the nasopharynx region where mucociliary clearance is prominent10. Moreover, mice inside the chamber are under significant stress during the administration process because they are constrained and deprived of food and water supply11. For intratracheal administration, it generally refers to the introduction of the substance directly into the trachea. There are two different techniques to achieve this: tracheotomy and orotracheal intubation. The former requires a surgical procedure that makes an incision in the trachea, which is invasive and seldom used for powder administration. Only the second technique is described here. Compared to the inhalation method, intratracheal administration is the more commonly used method for pulmonary delivery in the mouse because of its high delivery efficiency with minimal drug loss12,13. It is a simple and fast method to precisely deliver a small amount of powder within a few milligrams to the mouse. Although the mouse is anatomically and physiologically distinct to humans and anesthetization is required during the intubation process, intratracheal administration bypasses the upper respiratory tract and offers a more effective way to assess the biological activities of the dry powder formulation such as the pulmonary absorption, bioavailability and therapeutic effects14,15.
To administer dry powder intratracheally, the mouse has to be intubated, which could be challenging. In this paper, the fabrication of a custom-made dry powder insufflator and an intubation device is described. The procedures of intubation and insufflation of dry powder in the lung of the mouse are demonstrated.

<table><tbody><tr><td>BALB/c mouse</td><td></td><td></td><td>Female; 7-9 weeks old; Body weight 20-25 g</td></tr><tr><td>CleanCap Firefly Luciferase mRNA</td><td>TriLink Biotechnology</td><td>L-7602</td><td></td></tr><tr><td>Dry Powder Insufflator</td><td>PennCentury</td><td>Model DP-4M</td><td></td></tr><tr><td>Ketamine 10%</td><td>Alfasan International B.V.</td><td>NA</td><td></td></tr><tr><td>Light emitting diode (LED) torch</td><td>Unilite Internation</td><td>PS-K1</td><td></td></tr><tr><td>Mannitol (Pearlitol 160C)</td><td>Roquette</td><td>450001</td><td></td></tr><tr><td>Non-filter round gel loading pipette tip (200 &amp;micro;L)</td><td>Labcon</td><td>1034-800-000</td><td></td></tr><tr><td>Nylon floss</td><td>Reach</td><td>30017050</td><td></td></tr><tr><td>One milliliter syringe without needle</td><td>Terumo</td><td>SS-01T</td><td></td></tr><tr><td>Optical fibre</td><td>Fibre Data</td><td>OMPF1000</td><td></td></tr><tr><td>PEG12KL4 peptide</td><td>EZ Biolab</td><td></td><td>(PEG12)-KLLLLKLLLLKLLLLKLLLLK-NH2</td></tr><tr><td>Plastic Pasteur fine tip pipette</td><td>Alpha Labotatories</td><td>LW4061</td><td></td></tr><tr><td>Three-way stopcock</td><td>Braun</td><td>D201</td><td></td></tr><tr><td>Xylazine 2%</td><td>Alfasan International B.V.</td><td>NA</td><td></td></tr><tr><td>Zerostat 3 anti-static gun</td><td>MILTY</td><td>5036694022153</td><td></td></tr></tbody></table>

intratracheal administration of dry powder formulation in mice

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper introduces a noninvasive method of intratracheal delivery of dry powder formulation in mice. A dry powder loading device that consists of a 200 &#181;L gel loading pipette tip connected to an 1 mL syringe via a three-way stopcock is presented. A small amount of dry powder (1-2 mg) is loaded into the pipette tip and dispersed by 0.6 mL of air in the syringe. Because pipette tips are disposable and inexpensive, different dry powder formulations can be loaded into different tips in advance. Various formulations can be evaluated in the same animal experiment without device cleaning and dose refilling, thereby saving time and eliminating the risk of cross-contamination from residual powder. The extent of powder dispersion can be inspected by the amount of powder remaining in the pipette tip. A protocol of intubation in mouse with a custom-made light source and a guiding cannula is included. Proper intubation is one of the key factors that influences the intratracheal delivery of dry powder formulation to the deep lung region of the mouse.

When a dry powder insufflator is used to deliver powder aerosol to the lung of an animal, the volume of air used is critical as it affects the safety as well as the powder dispersion efficiency. To optimize the method, different volumes of air (0.3 mL, 0.6 mL and 1.0 mL) were used to disperse the dry powder (1 mg of spray dried mannitol) and the weight of mice was monitored for 48 hours after administration (Figure 6). The use of 0.3 mL and 0.6 mL of air did not cause weight loss of the mice...

Watch this Scientific Journal Video about Intratracheal Administration of Dry Powder Formulation in Mice at JoVE.com

Intratracheal Administration of Dry Powder Formulation in Mice

Dry powder formulations for inhalation have great potential in treating respiratory diseases. Before entering human studies, it is necessary to evaluate the efficacy of the dry powder formulation in preclinical studies. A simple and noninvasive method of the administration of dry powder in mice through the intratracheal route is presented.

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper ...

intratracheal-administration-of-dry-powder-formulation-in-mice

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

10.1K Views.  The University of Hong Kong. Dry powder formulations for inhalation have great potential in treating respiratory diseases. Before entering human studies, it is necessary to evaluate the efficacy of the dry powder formulation in preclinical studies. A simple and noninvasive method of the administration of dry powder in mice through the intratracheal route is presented.

Video: Intratracheal Administration of Dry Powder Formulation in Mice

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an 
appropriate immune response to a variety of immunogens1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be 
administered intranasally1,3,4, intratracheally5 or as aerosols6. Delivery by each route involves distinct technical skills and limitations that need to be 
considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory 
tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)7, potentially complicating interpretation of the results. In addition, swallowing, 
sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may 
be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) 
route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the 
trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t 
instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the 
innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

Immunology and Infection

Direct Tracheal Instillation of Solutes into Mouse Lung

Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice. Using this approach, one can target delivery of test solutes or solids (such as lung therapeutics, surfactants, viruses, and small oligonucleotides) into the distal lung. Tracheal instillations may be the preferred methodology, over inhalation protocols that may primarily target the upper respiratory tract and possibly expose the investigator to potentially hazardous substances. Additionally, in using the tracheal instillation protocol, animals can fully recover from the non-invasive procedure. This allows for making subsequent physiological measurements on test animals, or reinstallation using the same animal. The amount of instillate introduced into the lung must be carefully determined and osmotically balanced to ensure animal recovery. Typically, 30-75 &mu;L instillate volume can be introduced into mouse lung.

Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice.

Intratracheal instillations deliver solutes directly into the lungs.  This procedure targets the delivery of the instillate into the distal regions of the ...

Biology

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences between the murine and human respiratory systems, especially in their upper respiratory tracts (URT). In some models, these differences in the murine nasal cavity can have a significant impact on disease progression and presentation in the lower respiratory tract (LRT) when using intranasal instillation techniques, potentially limiting the usefulness of the mouse model to study these diseases. For these reasons, it would be advantageous to develop a technique to instill bacteria directly into the mouse lungs in order to study LRT disease in the absence of involvement of the URT. We have termed this lung specific delivery technique intubation-mediated intratracheal (IMIT) instillation. This noninvasive technique minimizes the potential for instillation into the bloodstream, which can occur during more invasive traditional surgical intratracheal infection approaches, and limits the possibility of incidental digestive tract delivery. IMIT is a two-step process in which mice are first intubated, with an intermediate step to ensure correct catheter placement into the trachea, followed by insertion of a blunt needle into the catheter to mediate direct delivery of bacteria into the lung. This approach facilitates a &#62;98% efficacy of delivery into the lungs with excellent distribution of reagent throughout the lung. Thus, IMIT represents a novel approach to study LRT disease and therapeutic delivery directly into the lung, improving upon the ability to use mice as surrogates to study human respiratory disease. Furthermore, the accuracy and reproducibility of this delivery system also makes it amenable to Good Laboratory Practice Standards (GLPS), as well as delivery of a wide range of reagents which require high efficiency delivery to the lung.

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

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 ...

Direct Intrabronchial Administration to Improve the Selective Agent Deposition Within the Mouse Lung

Intratracheal (IT) administration of experimental agents is an essential technique in murine models of diffuse lung diseases, such as bleomycin-induced pulmonary fibrosis.&#160; However, distribution of intratracheally-administered agents to the distal mouse lung is often asymmetric, with lung parenchymal concentrations increased in the smaller (but equally accessible) left lung of the mouse.&#160; Described in this report is a novel intrabronchial (IB) approach to cannulate the left and/or right lungs of living mice non-operatively.&#160; It is also demonstrated how this approach can be used to selectively administer agents to one lung or adapted (via dose-adjusted IB delivery) to improve the left-right symmetry of lung delivery of experimental agents, thereby improving models of diffuse lung disease such as bleomycin-induced pulmonary fibrosis.

Intratracheal (IT) administration of experimental agents in mice often results in asymmetric delivery to the distal lungs.&#160; In this report, we describe a direct intrabronchial (IB) approach to cannulate each lung in living mice non-operatively.&#160; This approach can be used to selectively administer agents to one lung or may be adapted to improve the symmetric agent delivery to both lungs.

Intratracheal (IT) administration of experimental agents is an essential technique in murine models of diffuse lung diseases, such as bleomycin-induced ...

A Minimally Invasive Method for Intratracheal Instillation of Drugs in Neonatal Rodents to Treat Lung Disease

Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered drug. This has direct translational impact because surfactant and drugs are administered locally into the lungs. Though the literature has many publications describing minimally invasive transoral intubation of adult mice and rats in therapeutic experiments, this approach in neonatal rat pups is lacking. The small size of orotracheal region/pharynx in the pups makes visualization of laryngeal lumen (vocal cords) difficult, contributing to the variable success rate of intratracheal drug delivery. We hereby demonstrate effective oral intubation of neonatal rat pup - a technique that is non-traumatic and minimally-invasive, so that it can be used for serial administration of drugs. We used an operating otoscope with an illumination system and a magnifying lens to visualize the tracheal opening of the rat neonates. The drug is then instilled using a 1 mL syringe connected to a pipette tip. The accuracy of the delivery method was demonstrated using Evans blue dye administration. This method is easy to get trained in and could serve as an effective way to instill drugs into trachea. This method could also be used for administration of inoculum or agents to simulate disease conditions in animals and, also, for cell-based treatment strategies for various lung diseases.

This technique of instilling drugs directly into the trachea of neonatal rodents is important in studying the impact of locally administered drugs or biologicals on neonatal lung diseases. Additionally, this method can also be used for inducing lung injury in animal models.

Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered ...

Customizable Disposable Dosators to Deliver Dry Powder Aerosol Drugs to Mice

Disposable Dosators Intended for Dry Powder Delivery to Mice

Dry powder inhalers offer numerous advantages for delivering drugs to the lungs, including stable solid-state drug formulations, device portability, bolus metering and dosing, and a propellant-free dispersal mechanism. To develop pharmaceutical dry powder aerosol products, robust in vivo testing is essential. Typically, initial studies involve using a murine model for preliminary evaluation before conducting formal studies in larger animal species. However, a significant limitation in this approach is the lack of suitable device technology to accurately and reproducibly deliver dry powders to small animals, hindering such models&#39; utility. To address these challenges, disposable syringe dosators were developed specifically for intrapulmonary delivery of dry powders in doses appropriate for mice. These dosators load and deliver a predetermined amount of powder obtained from a uniform bulk density powder bed. This discrete control is achieved by inserting a blunt needle to a fixed depth (tamping) into the powder bed, removing a fixed quantity each time. Notably, this dosing pattern has proven effective for a range of spray-dried powders. In experiments involving four different model spray-dried powders, the dosators demonstrated the ability to achieve doses within the range of 30 to 1100 &#181;g. The achieved dose was influenced by factors such as the number of tamps, the size of the dosator needle, and the specific formulation used. One of the key benefits of these dosators is their ease of manufacturing, making them accessible and cost-effective for delivering dry powders to mice during initial proof-of-concept studies. The disposable nature of the dosators facilitates use in animal procedure rooms, where cleaning and refilling reusable systems and weighing materials is inconvenient. Thus, developing disposable syringe dosators has addressed a significant hurdle in murine dry powder delivery for proof-of-concept studies, enabling researchers to conduct more accurate and reproducible preliminary studies in small animal models for pulmonary drug delivery.

Author Spotlight: Developing a Disposable Dosator for Preclinical Testing of Dry Powder Inhalers in Small Animal Models 

Pharmaceutical dry powder development necessitates reliable in vivo testing, often using a murine model. Device technology for accurately and reproducibly delivering dry powder aerosols to mice is restricted. This study presents disposable dosators for pulmonary drug delivery at mouse-relevant doses, aiding initial proof-of-concept research.

Dry powder inhalers offer numerous advantages for delivering drugs to the lungs, including stable solid-state drug formulations, device portability, bolus ...

Noninvasive Intratracheal Lipopolysaccharide Instillation in Mice

The acute lung injury (ALI) mouse model induced by lipopolysaccharide (LPS) or endotoxin is still among the most commonly used models in animal studies of acute lung injury or acute inflammation. The current most commonly used methods in acute lung injury mouse models are an intraperitoneal injection of LPS and tracheostomy for the tracheal infusion of LPS. However, the former method lacks lung targeting and damages other organs, and the latter method induces operative trauma, infection risk, and a low survival rate. Here, we recommend a noninvasive oropharyngeal endotracheal intubation method for LPS instillation in mice. In this method, LPS is noninvasively introduced into the trachea through the oropharyngeal cavity to be instilled into the lung with the help of an apparatus for endotracheal intubation. This method not only assures lung targeting but also avoids damage and the risk of death in the animals. We expect that this approach will become widely used in the field of acute lung injury.

Here, we propose a protocol for intratracheal lipopolysaccharide (LPS) delivery via noninvasive oropharyngeal endotracheal intubation. This method minimizes the trauma of the surgical procedure for the animal and accurately delivers LPS to the trachea and then to the lungs.

The acute lung injury (ALI) mouse model induced by lipopolysaccharide (LPS) or endotoxin is still among the most commonly used models in animal studies of ...

Intrabronchial Delivery: A Technique to Administer an Experimental Agent Selectively Into a Mouse Lung

Source: Liao, S. et. al. <a href="https://jove.unicartagenaproxy.elogim.com/v/59450/direct-intrabronchial-administration-to-improve-selective-agent">Direct Intrabronchial Administration to Improve the Selective Agent Deposition Within the Mouse Lung</a>. J. Vis. Exp. (2019).
This video describes an intrabronchial approach to administer an experimental agent selectively into distal right and left lung. In the featured protocol, we will administer bleomycin intrabronchially into a mouse to obtain a better model for pulmonary fibrosis.

Podawanie dooskrzelowe: technika selektywnego podawania eksperymentalnego środka do płuc myszy

Source: Liao, S. et. al. Direct Intrabronchial Administration to Improve the Selective Agent Deposition Within the Mouse Lung. J. Vis. Exp. (2019).
This ...