Name: intratracheal administration of dry powder formulation in mice
Uploaded: 2020-07-25T15:00:00
Description: Watch this Scientific Journal Video about Intratracheal Administration of Dry Powder Formulation in Mice at JoVE.com

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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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 ...

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