Non-invasive pulmonary aerosol delivery in mice by the endotracheal route

doi:10.1016/j.ejpb.2005.04.009

European Journal of Pharmaceutics and Biopharmaceutics

Volume 61, Issue 3, October 2005, Pages 214-218

https://doi.org/10.1016/j.ejpb.2005.04.009 Get rights and content

Abstract

In this report we present in detail a non-invasive pulmonary application method that can be a useful tool in studying drug and vaccine delivery to the lower airways. In this method the formulation is sprayed directly into the lungs of mice via the endotracheal route using a MicroSprayer™ aerolizer. Mean droplet size produced was 8 μm, appropriate for deposition in the large airways. Endotracheal application of suspension of fluorescent nanospheres, 200 nm in size, by this method resulted in nanoparticle deposition in the smaller airways (bronchi and bronchioles). Mice showed full recovery one day after administration of 50 μl of formulation. Furthermore, no mortality was observed as a result of the technique. We conclude that this endotracheal application is a useful tool for studying pulmonary drug delivery in mice. The technique is especially useful for the pulmonary application of vaccines, since it enables multiple administrations without a need for analgesics.

Introduction

The pulmonary route for local and systemic drug delivery is constantly being investigated for the purpose of targeting drugs to specific lung cell populations and for non-parenteral systemic delivery of macromolecular drugs. In addition, research exploring the possibility of using the pulmonary route to establish a mucosal as well as systemic immune response against airborne pathogens is on the rise [1], [2], [3].

Animal testing plays a very important role in the assessment of aerosol delivery with respect to efficiency, reproducibility, and safety of drugs and vaccines. Even though pulmonary application in small laboratory animals such as mice is considered complicated and often discouraging, it appears to be mandatory in pulmonary disease models and in vaccination studies [4]. The most common method of pulmonary drug application is the intratracheal instillation. This method has been used for the application of subunit vaccines, cationic lipoplexes and polymeric microspheres into mice lungs [3], [5], [6], [7], [8]. The advantages of intratracheal instillation are direct application of the drug into the lungs accompanied by minimal drug loss in the nose, throat and upper airways, the dose given is quantifiable, the application itself takes a short time and costs are relatively low. Nevertheless, this method results in a poor distribution of the formulation in the lung, mice can tolerate only small volumes, and the most important disadvantage is that it requires invasive surgery. The latter represents a problem when multiple administrations over longer experimental periods of time have to be performed, as is the case in vaccination studies. It is accompanied by pain and discomfort to the animal and requires treatment with analgesics to comply with the guidelines of most national ethical committees. Such analgesic treatment is bound to influence immunologic readout, and therefore, could be problematic in vaccination studies. Another method for aerosol application is the use of inhalation chambers. These chambers are used for whole body or nose only exposure and are designed for restrained, unrestrained or anesthetized animals. The aerosol is generated by a nebuliser connected to the exposure chamber and the animal is exposed for a period of time according to the dose required [9], [10], [11], [12]. This method is non-invasive and results in good peripheral distribution of the agent in the lungs [9]. However, it is difficult to determine the exact dose that reaches the lungs since there is the possibility of drug loss in the chamber, on the animal's skin as well as in the nose and throat. This represents a drawback when investigating dose dependent effects of drugs and vaccines. Furthermore, using a nebuliser requires a lot of time, especially when large groups of animals are used for the study.

The mouse is the animal model of choice for testing vaccines against several diseases such as tuberculosis, mainly because of its well-characterized immune system and the possibility to create strains showing specific immunologic characteristics (e.g. knock-out mice, different HLA-type mice). The methods for pulmonary delivery in mice described in literature (as discussed above) are not optimal for vaccination experiments where multiple administrations of specified doses of the vaccine are needed.

Here we describe in details an improved non-invasive method for pulmonary delivery in mice. It is our intention to share our experience with this technique to guide and to foster work in this particular interesting area of drug and vaccine delivery.

Section snippets

Animals

Female Balb/c and C57BL/6 mice (6–12 weeks old; Charles River, Sulzfeld, Germany) were used for in vivo pulmonary administration. Animal experiments were performed according to the ‘Principles of Laboratory Animal Care’ as defined by NIH and were permitted by the Ethical Committee of Leiden University (Leiden, The Netherlands).

Aerosolisation of solutions and suspensions

Aerosolisation was performed using a MicroSprayer™ aerosoliser (IA-1C; Penn-Century, Philadelphia, PA, USA) suitable for mice, attached to a high-pressure syringe

Aerosol droplet size

Measurements of droplets sizes obtained by spraying from the Penn-century Microsprayer™ were performed by using the time-of-flight principle. Mean mass aerodynamic diameter (MMAD) of the droplets was 8.01±0.57 μm with a geometric standard deviation (GSD) of 1.1.

Method validation

Pulmonary delivery of India ink solution as an aerosol confirmed that the aerosol was applied through the trachea, avoiding drug loss by intragastric application via the esophagus (Fig. 3). India ink was clearly visualized in the airways

Discussion and conclusions

The pulmonary application method described here offers the opportunity of multiple administrations of drugs or vaccines in mice avoiding any surgical procedure. The main challenge of endotracheal application in small animals such as mice is the visualization of the trachea, which is essential for reliable lung administration. Our experimental set-up facilitated tracheal visualization and enabled easy intubation regardless of the applicator used. The experiments using India ink solution verified

Acknowledgements

We would like to acknowledge Kees Geerse from Technical University of Delft for the Aerosizer measurements and Hans de Bont from the Division of Toxicology at LACDR for assisting with the fluorescent microscope.

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European Journal of Pharmaceutics and Biopharmaceutics

Abstract

Introduction

Section snippets

Animals

Aerosolisation of solutions and suspensions

Aerosol droplet size

Method validation

Discussion and conclusions

Acknowledgements

References (18)

Aerogenic vaccination of mice with Mycobacterium bovis BCG

Tubercle

BCG-induced protection in guinea pigs vaccinated and challenged via the respiratory route

Tuber. Lung Dis.

Analysis of local and systemic immunological responses after intra-tracheal, intra-nasal and intra-muscular administration of microsphere co-encapsulated Yersinia pestis sub-unit vaccines

Vaccine

Disease model: pulmonary tuberculosis

Trends Mol. Med.

Protection studies following bronchopulmonary and intramuscular immunisation with yersinia pestis F1 and V subunit vaccines coencapsulated in biodegradable microspheres: a comparison of efficacy

Vaccine

Airway delivery of cationic lipid: DNA complexes for cystic fibrosis

Adv. Drug Deliv. Rev.

Lack of genotoxicity of bitumen fumes in transgenic mouse lung

Toxicology

Pulmonary delivery of chitosan-DNA nanoparticles enhances the immunogenicity of a DNA vaccine encoding HLA-A0201-restricted T-cell epitopes of Mycobacterium tuberculosis*

Vaccine

Mechanism of lipoplex gene delivery in mouse lung: binding and internalization of fluorescent lipid and DNA components

Gene Ther.

Cited by (173)

A Novel Chitosan-coated Leciplex Loaded with Ambrisentan as a Possible Pulmonary Nanosystem: Optimization, Characterization, and Pharmacokinetics Assessments

Construction of an inhalable recombinant M2e-FP-expressing Bacillus subtilis spores-based vaccine and evaluation of its protection efficacy against influenza in a mouse model

Lessons from the history of inorganic nanoparticles for inhalable diagnostics and therapeutics

Influence of P-glycoprotein on pulmonary disposition of the model substrate [<sup>11</sup>C]metoclopramide assessed by PET imaging in rats

Novel trehalose-based excipients for stabilizing nebulized anti-SARS-CoV-2 antibody

Leucine improves the aerosol performance of dry powder inhaler formulations of siRNA-loaded nanoparticles

NoteNon-invasive pulmonary aerosol delivery in mice by the endotracheal route

Abstract

Introduction

Section snippets

Animals

Aerosolisation of solutions and suspensions

Aerosol droplet size

Method validation

Discussion and conclusions

Acknowledgements

Tubercle

Tuber. Lung Dis.

Vaccine

Trends Mol. Med.

Vaccine

Adv. Drug Deliv. Rev.

Toxicology

Vaccine

Mechanism of lipoplex gene delivery in mouse lung: binding and internalization of fluorescent lipid and DNA components

Gene Ther.

Note
Non-invasive pulmonary aerosol delivery in mice by the endotracheal route