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.
