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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 dr.......

Representative 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 .......

Acknowledgements

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).

....

Materials

NameCompanyCatalog NumberComments
BALB/c mouseFemale; 7-9 weeks old; Body weight 20-25 g
CleanCap Firefly Luciferase mRNATriLink BiotechnologyL-7602
Dry Powder InsufflatorPennCenturyModel DP-4M
Ketamine 10%Alfasan International B.V.NA
Light emitting diode (LED) torchUnilite InternationPS-K1
Mannitol (Pearlitol 160C)Roquette450001
Non-filter round gel loading pipette tip (200 µL)Labcon1034-800-000
Nylon flossReach30017050
One milliliter syringe without needleTerumoSS-01T
Optical fibreFibre DataOMPF1000
PEG12KL4 peptideEZ Biolab(PEG12)-KLLLLKLLLLKLLLLKLLLLK-NH2
Plastic Pasteur fine tip pipetteAlpha LabotatoriesLW4061
Three-way stopcockBraunD201
Xylazine 2%Alfasan International B.V.NA
Zerostat 3 anti-static gunMILTY5036694022153

References

  1. Newman, S. P. Drug delivery to the lungs: challenges and opportunities. Therapeutic Delivery. 8 (8), 647-661 (2017).
  2. Setter, S. M., et al. Inhaled dry powder insulin for the treatment of diabetes mellitus.

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Intratracheal AdministrationDry Powder FormulationMiceInsufflatorPulmonary AbsorptionBioavailabilityTherapeutic EffectsPre clinical Animal ModelsIntubationStatic ChargesGel Loading Pipette TipWeighing PaperSyringeAnesthesiaBALB c MouseOptical FiberGuiding Cannula

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