Intratracheal Administration of Dry Powder Formulation in Mice

Summary

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.

Abstract

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 µL 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.

Introduction

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.

Protocol

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 μm. Spray dried (SD) mannitol powder are used to investigate the effect of air volume used in powder dispersion¹⁶. The MMAD of SD powder i....

Results

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.......

Discussion

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 µL 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 .......

Materials

List of materials used in this article

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