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 dr...

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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=Proof-of-Principle+Study+in+a+Murine+Lung+Infection+Model+of+Antipseudomonal+Activity+of+Phage+PEV20+in+a+Dry-Powder+Formulation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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. 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F., 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+novel+aerosol+generator+for+homogenous+distribution+of+powder+over+the+lungs+after+pulmonary+administration+to+small+laboratory+animals.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+evaluation+of+the+DP-4M+PennCentury+insufflator.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+and+highly+dispersible+voriconazole+dry+powders+produced+by+spray+freeze+drying+for+pulmonary+delivery+with+efficient+lung+deposition.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naked+pDNA+Inhalation+Powder+Composed+of+Hyaluronic+Acid+Exhibits+High+Gene+Expression+in+the+Lungs.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+validation+of+a+simple+device+for+insufflation+of+dry+powders+in+a+mice+model.">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 &#181;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 is essential to evaluate the performance and biological activities of an inhaled powder formulation, such as pulmonary absorption, bioavailability, and therapeutic effects in pre-clinical animal models. The intubation process is non-invasive and could deliver powder formulation to the mice safely and accurately. The custom-made dry powder insufflator is disposable, inexpensive, and efficient in disbursing powder formulations.The insufflators could be used in evaluating different formulations on multiple mice in the same experiments without a risk of cross-contamination from residual powder. The intubation process is challenging for non-experienced researchers. The ability to visualize and aim the finest tip of the cannula at the opening of the trachea is crucial for correct insertion of the guiding cannula.This technique requires practice to minimize improper insertion. My postdoc Carol and my PhD student Rachel are going to demonstrate the preparation and intubation procedure. To begin, neutralize the static charges of the dry powder and the 200 microliter non-filter round gel loading pipette tip with an anti-static gun or a balance with deionizing function.Prepare a weighing paper with an approximate size of four by four centimeters. Fold the paper in half diagonally and then unfold it. Then use it to weigh one to two milligrams of dry powder.Fill a gel loading pipette tip with powder through the wider opening and tap it gently to pack the powder until it forms loose agglomerates near the narrow end of the tip. Avoid packing the powder too tightly as it may hamper dispersion. Connect the powder loaded tip to a one milliliter syringe through a three-way stopcock holding the tip and syringe vertically during connection to prevent spillage of powder.The size of the syringe can be changed according to the volume of air used to disperse the powder. If administration is not performed immediately, use parafilm to seal the openings of the tip and store it temporarily under suitable conditions until administration. After anesthetizing the seven to nine-week-old BALB/c mouse with ketamine and xylazine, place it on a plexiglass platform mounted to a stand.Suspend the mouse by hooking its incisors on a nylon floss and secure its position with a piece of tape or a rubber band. Adjust the height and angle of the platform. Insert the optical fiber into the guiding cannula with the tip of the fiber level with the opening of the cannula.Then turn on the LED torch to illuminate. Gently protrude the tongue of the mouse with a pair of forceps to expose its trachea. Use the other hand to hold the guiding cannula with optical fiber and 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. Align the bevel of the guiding cannula towards the midline of the opening and gently intubate it with the optical fiber into the trachea by aiming the finest tip of the cannula at the tracheal opening. Upon intubation, swiftly remove the optical fiber and leave the guiding cannula inside the trachea.Normal restoration should be observed. Hold the fine tip pipette at the opening of the guiding cannula and insufflate a small puff of air into the lung of the mouse. A slight inflation in the chest indicates proper intubation.Remove the fine tip pipette prior to powder administration. Hold the power loaded tip that is connected to the syringe, making sure that the air flow between the syringe and the tip is disconnected. Pull the syringe plunger backward to withdraw 0.6 milliliters of air.Turn the valve of the three-way stopcock to connect the air flow between the syringe and the power loaded tip. Then insert the power loaded tip into the guiding cannula which has already been placed in the trachea of the mouse. Hold the guiding cannula and push the syringe plunger forcefully in one continuous action to disperse the powder as aerosols into the lung minimizing any forward motion to avoid injury to the animal.Remove the tip and check if the powder has been emptied. Once the administration is complete, remove the guiding cannula from the trachea. Allow the mouse to recover by positioning it horizontally in a supine position with its tongue half protruded to avoid a blockage of the airways.To optimize the method, different volumes of air were used to disperse one milligram of the spray dried mannitol and the weight of mice was monitored. The use of 0.3 and 0.6 milliliters of air did not cause weight loss of the mice up to 48 hours post-administration. Dispersing the powder with one milliliter of air resulted in over 5%weight loss within 24 hours, which was not fully recovered after 48 hours.The mice were intratracheally administered with one milligram of SFD powder containing five micrograms of mRNA and the luciferase expression in the lungs was evaluated at 24 hours post-administration using an in vivo imaging system. The SFD powder was dispersed in the deep lung and luciferase expression was observed. As a comparison, the powder was reconstituted in water and administered with a micro sprayer using the same intubation procedure.The luciferase expression of the reconstituted formulation was significantly higher than the dry powder formulation, which could be due to the powder disillusion issue or different pharmacokinetic profile between powder and liquid form. The histological characteristics of the lungs treated with mRNA dry powder aerosol were compared with untreated control and LPS-treated groups. The lung without any treatment illustrated a healthy presentation while the lung treated with LPS showed irregular distribution of airspace and inflammatory cell infiltration into the interstitial and alveolar spaces.The lungs treated with SFD powder did not show any signs of inflammation. For successful powder dispersion, the powder in the loading tip should be tapped gently and not packed too tightly in the tip. This intubation setting can also be adapted to administer liquid formulations either with a pipette or a micro sprayer.

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9.4K 조회수.  The University of Hong Kong. Intratracheal administration of dry powder is essential to evaluate the performance and biological activities of an inhaled powder formulation, such as pulmonary absorption, bioavailability, and therapeutic effects in pre-clinical animal models. The intubation process is non-invasive and could deliver powder formulation to the mice safely and accurately. The custom-made dry powder insufflator is disposable, inexpensive, and efficient in disbursing powder formulations.The insufflators c...