Los autores desean agradecer al Sr. Ray Lee, al Sr. HC Leung y al Sr. Wallace So por su amable asistencia en la fabricación de la fuente de luz y el insuflador de polvo; y el Centro Básico de la Facultad para la asistencia en imágenes de animales. El trabajo fue apoyado por el Research Grant Council, Hong Kong (17300319).

Los experimentos realizados en este estudio han sido aprobados por el Comité sobre el Uso de Animales Vivos para la Enseñanza y la Investigación (CULATR), la Universidad de Hong Kong. Las formulaciones en polvo seco preparadas por liofilización por pulverización (SFD) que contienen 0,5% de ARN mensajero de luciferasa (ARNm), péptido sintético PEG12KL4 al 5% y 94,5% de manitol (p/p) se utilizan en este estudio para demostrar la expresión de ARNm en el pulmón16. El diámetro aerodinámico medio de masa (MMAD) del polvo sfd es de 2,4 μm. El polvo de manitol secado por aspersión (SD) se utiliza para investigar el efecto del volumen de aire utilizado en la dispersión de polvo16. El MMAD del polvo SD es de 1,5 μm.
1. Fabricación de insuflador de polvo seco y carga de polvo seco
<ol><li>(Opcional) Neutralice las cargas estáticas del polvo seco (en un vial) y la punta de pipeta redonda sin filtro de 200 μL de carga de gel. Utilice un arma antiestática o una balanza con función desionizante de acuerdo con las instrucciones del fabricante.</li>
	<li>Prepare un papel de pesaje con un tamaño de alrededor de 4 cm x 4 cm. Dobla el papel por la mitad en diagonal y luego desdoblalo.</li>
	<li>Pesar 1-2 mg de polvo seco en el papel de pesaje.</li>
	<li>Llene una punta de pipeta de carga de gel con polvo a través de la abertura más ancha de la punta. Toque suavemente para empacar el polvo hasta que el polvo forme aglomerados sueltos cerca del extremo estrecho de la punta (Figura 1A). Evite empacar el polvo demasiado apretado, ya que puede obstaculizar la dispersión del polvo.</li>
	<li>Conecte la punta cargada de polvo a una jeringa de 1 ml a través de una parada de tres vías (Figura 1B). El tamaño de la jeringa se puede cambiar de acuerdo con el volumen de aire utilizado para dispersar el polvo. Sostenga la punta y la jeringa verticalmente durante la conexión para evitar el derrame de polvo. Si la administración no se realiza inmediatamente, utilice parafilm para sellar las aberturas de la punta y guárdela temporalmente en condiciones adecuadas hasta la administración.</li>
</ol>2. Fabricación del dispositivo de intubación
<ol><li>Fuente de luz (Figura 2)<ol>
<li>Prepare una fuente de luz hecha a medida con una antorcha de diodo emisor de luz (LED) y una fibra óptica flexible con un diámetro de 0,8-1 mm.</li>
		<li>Haga un orificio centrado en la lente transparente de la antorcha LED con un taladro de mano o una broca para que la fibra óptica apenas pueda pasar.</li>
		<li>Inserte la fibra óptica a través del orificio. Encienda la antorcha LED para ajustar la posición y la profundidad de inserción para obtener el brillo máximo en el otro extremo de la fibra óptica.</li>
		<li>Coloque la fibra óptica en su posición con pegamento epoxi transparente.</li>
	</ol>
</li>
	<li>Cánula guía (Figura 3)<ol>
<li>Tome una pipeta Pasteur de plástico de 1 mL(Figura 3A)y sostenga la pipeta en ambos extremos.</li>
		<li>Utilice una lámpara de alcohol (u otras fuentes de calor en el laboratorio, como un quemador Bunsen) para calentar el centro de la pipeta colocándolo a 5-10 cm por encima de la llama(Figura 3B). Gire la pipeta para asegurarse de que se calienta uniformemente.</li>
		<li>Cuando el plástico se vuelva suave y deformable, aleje la pipeta de la llama y estire la pipeta suavemente.</li>
		<li>Corte la pipeta estirada en el medio con un par de tijera en la Parte A y la Parte B (Figura 3C-E). Utilice la Parte A como una pipeta de punta fina y la Parte B como una cánula guía. Para aumentar la probabilidad de intubación exitosa con la cánula guía, haga un bisel (no demasiado agudo que puede aumentar el riesgo de lesionar al animal) al final de la Parte B (Figura 3F). Cuando se inserta una punta de pipeta de carga de gel de 200 μL (para la carga de polvo) en la cánula guía, debe sobresalir de la cánula por 1-2 mm. NOTA: Una cánula guía (Parte B) con la dimensión apropiada (diámetro interno y externo) para la intubación podría tener una aguja de calibre 21 en su interior, mientras que también puede caber dentro de una aguja de calibre 17. Es posible que se necesiten varios intentos de estirar las pipetas para lograr la dimensión adecuada.</li>
		<li>(Opcional): Corte una pequeña abertura en el extremo más ancho de la cánula guía para hacerla más flexible de modo que sea más fácil sostener la fibra óptica (Figura 3F). Esta abertura también permite la instalación de un microsprayer para la administración de aerosol líquido.</li>
	</ol>
</li>
</ol>3. Intubación
<ol><li>Anestesiar el ratón (BALB/c, 7-9 semanas) con ketamina (100 mg/kg) y xilazina (10 mg/kg) mediante inyección intraperitoneal.</li>
	<li>Prepare una plataforma hecha de plexiglás y montarla en un soporte con una abrazadera (Figura 4A). Coloque el ratón anestesiado en la plataforma (a unos 60° de inclinación) en posición supina. La altura y el ángulo de inclinación de la plataforma podrían ajustarse por la posición de la abrazadera en el soporte.</li>
	<li>Suspenda el ratón enganchando sus incisivos a un hilo dental de nylon (Figura 4B). Asegure la posición del ratón con un trozo de cinta adhesiva o una banda elástica.</li>
	<li>Inserte la fibra óptica en la cánula guía antes de la intubación con la punta del nivel de fibra con la abertura de la cánula guía. Encienda la antorcha LED para iluminar.</li>
	<li>Sobresalga suavemente la lengua del ratón con un par de pórceps para exponer su tráquea.</li>
	<li>Utilice la otra mano para sostener la cánula guía con fibra óptica en el interior. Insertarlos a través de la cavidad oral. Con la iluminación de la fibra óptica, la apertura de la tráquea se puede visualizar como un orificio entre las cuerdas vocales.</li>
	<li>Alinee el bisel de la cánula guía hacia la línea media de la abertura (Figura 5A). Intubar suavemente la cánula guía con fibra óptica en la tráquea apuntando la punta más fina de la cánula en la abertura traqueal.</li>
	<li>Tras la intubación, retire rápidamente la fibra óptica y deje la cánula guía dentro de la tráquea (Figura 5B). Se debe observar una respiración normal.</li>
	<li>Sostenga la pipeta de punta fina (Parte A) en la abertura de la cánula guía e insufle una pequeña bocanada de aire (aproximadamente 0,2 mL) en el pulmón del ratón. Una ligera inflación en el pecho del ratón indica una intubación adecuada. Retire la pipeta de punta fina antes de la administración de polvo.</li>
</ol>4. Administración de polvo
<ol><li>Sostenga la punta cargada de polvo que está conectada a la jeringa como se describe en el paso 1.5. Asegúrese de que el flujo de aire entre la jeringa y la punta esté desconectado.</li>
	<li>Tire del émbolo de la jeringa hacia atrás para retirar 0,6 ml de aire. NOTA: El volumen de aire utilizado para dispersar el polvo depende de las propiedades del polvo y la cantidad de polvo cargado. Esto se describe con más detalle en la sección de resultados.</li>
	<li>Gire la válvula de la llave de paso de tres vías para conectar el flujo de aire entre la jeringa y la punta cargada de polvo.</li>
	<li>Inserte la punta cargada de polvo en la cánula guía que ya se ha colocado en la tráquea del ratón (Figura 5C). Sostenga la cánula guía y empuje el émbolo de la jeringa con fuerza en una acción continua para dispersar el polvo como aerosoles en el pulmón. NOTA: Cualquier movimiento hacia adelante del dispositivo debe minimizarse para evitar lesionar al animal.</li>
	<li>Retire la punta y compruebe si el polvo dentro de la punta se ha vaciado. Si no es así, repita los pasos 4.1 a 4.4. NOTA: Si el polvo está demasiado apretado debido a un exceso de tapping, es posible que no se disperse correctamente.</li>
	<li>Una vez que se complete la administración, retire la cánula guía de la tráquea.</li>
	<li>Permita que el ratón se recupere colocándolo horizontalmente en posición supina con la lengua medio sobresalida para evitar el bloqueo de las vías respiratorias.</li>
</ol>

<ol>
	<li>Newman, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+delivery+to+the+lungs:+challenges+and+opportunities.">Drug delivery to the lungs: challenges and opportunities.</a> Therapeutic Delivery. 8 (8), 647-661 (2017).</li><li>Setter, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhaled+dry+powder+insulin+for+the+treatment+of+diabetes+mellitus.">Inhaled dry powder insulin for the treatment of diabetes mellitus.</a> Clinical Therapeutics. 29 (5), 795-813 (2007).</li><li>Muralidharan, P., Hayes, D., Mansour, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dry+powder+inhalers+in+COPD,+lung+inflammation+and+pulmonary+infections.">Dry powder inhalers in COPD, lung inflammation and pulmonary infections.</a> Expert Opinion on Drug Delivery. 12 (6), 947-962 (2015).</li><li>de Boer, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dry+powder+inhalation:+past,+present+and+future.">Dry powder inhalation: past, present and future.</a> Expert Opinion on Drug Delivery. 14 (4), 499-512 (2017).</li><li>Das, S., Tucker, I., Stewart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhaled+dry+powder+formulations+for+treating+tuberculosis.">Inhaled dry powder formulations for treating tuberculosis.</a> Current Drug Delivery. 12 (1), 26-39 (2015).</li><li>Okamoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+chitosan-pDNA+complex+powder+prepared+by+supercritical+carbon+dioxide+process.">Stability of chitosan-pDNA complex powder prepared by supercritical carbon dioxide process.</a> International Journal of Pharmaceutics. 290 (1-2), 73-81 (2005).</li><li>He, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+inhaled+recombinant+human+insulin+dry+powders:+pharmacokinetics,+pharmacodynamics+and+14-day+inhalation.">Evaluation of inhaled recombinant human insulin dry powders: pharmacokinetics, pharmacodynamics and 14-day inhalation.</a> Journal of Pharmacy and Pharmacology. 71 (2), 176-184 (2019).</li><li>Durham, P. G., Young, E. F., Braunstein, M. S., Welch, J. T., Hickey, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dry+powder+combination+of+pyrazinoic+acid+and+its+n-propyl+ester+for+aerosol+administration+to+animals.">A dry powder combination of pyrazinoic acid and its n-propyl ester for aerosol administration to animals.</a> International Journal of Pharmaceutics. 514 (2), 384-391 (2016).</li><li>Phillips, J. E., Zhang, X., Johnston, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dry+powder+and+nebulized+aerosol+inhalation+of+pharmaceuticals+delivered+to+mice+using+a+nose-only+exposure+system.">Dry powder and nebulized aerosol inhalation of pharmaceuticals delivered to mice using a nose-only exposure system.</a> JoVE (Journal of Visualized Experiments). (122), e55454 (2017).</li><li>Nahar, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro,+in+vivo+and+ex+vivo+models+for+studying+particle+deposition+and+drug+absorption+of+inhaled+pharmaceuticals).">In vitro, in vivo and ex vivo models for studying particle deposition and drug absorption of inhaled pharmaceuticals).</a> European Journal of Pharmaceutical Sciences. 49 (5), 805-818 (2013).</li><li>Price, D. N., Muttil, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+Therapeutics+to+the+Lung.">Delivery of Therapeutics to the Lung.</a> Methods in Molecular Biology. 1809, 415-429 (2018).</li><li>Chang, R. Y. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proof-of-Principle+Study+in+a+Murine+Lung+Infection+Model+of+Antipseudomonal+Activity+of+Phage+PEV20+in+a+Dry-Powder+Formulation.">Proof-of-Principle Study in a Murine Lung Infection Model of Antipseudomonal Activity of Phage PEV20 in a Dry-Powder Formulation.</a> Antimicrobial Agents and Chemotherapy. 62 (2), (2018).</li><li>Ito, T., Okuda, T., Takayama, R., Okamoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+an+Evaluation+Method+for+Gene+Silencing+by+Serial+Pulmonary+Administration+of+siRNA+and+pDNA+Powders:+Naked+siRNA+Inhalation+Powder+Suppresses+Luciferase+Gene+Expression+in+the+Lung.">Establishment of an Evaluation Method for Gene Silencing by Serial Pulmonary Administration of siRNA and pDNA Powders: Naked siRNA Inhalation Powder Suppresses Luciferase Gene Expression in the Lung.</a> Journal of pharmaceutical sciences. 108 (8), 2661-2667 (2019).</li><li>Patil, J. S., Sarasija, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+drug+delivery+strategies:+A+concise,+systematic+review.">Pulmonary drug delivery strategies: A concise, systematic review.</a> Lung India. 29 (1), 44-49 (2012).</li><li>Ihara, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+Quantification+of+Gene+Silencing+by+Intratracheal+Administration+of+Dry+Powdered+Small-Interfering+RNA/Chitosan+Complexes+in+the+Murine+Lung.">Histological Quantification of Gene Silencing by Intratracheal Administration of Dry Powdered Small-Interfering RNA/Chitosan Complexes in the Murine Lung.</a> Pharmaceutical Research. 32 (12), 3877-3885 (2015).</li><li>Qiu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+mRNA+pulmonary+delivery+by+dry+powder+formulation+of+PEGylated+synthetic+KL4+peptide.">Effective mRNA pulmonary delivery by dry powder formulation of PEGylated synthetic KL4 peptide.</a> Journal of Controlled Release. 314, 102-115 (2019).</li><li>Pfeifer, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dry+powder+aerosols+of+polyethylenimine+(PEI)-based+gene+vectors+mediate+efficient+gene+delivery+to+the+lung.">Dry powder aerosols of polyethylenimine (PEI)-based gene vectors mediate efficient gene delivery to the lung.</a> Journal of Controlled Release. 154 (1), 69-76 (2011).</li><li>Kim, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxorubicin-loaded+highly+porous+large+PLGA+microparticles+as+a+sustained-+release+inhalation+system+for+the+treatment+of+metastatic+lung+cancer.">Doxorubicin-loaded highly porous large PLGA microparticles as a sustained- release inhalation system for the treatment of metastatic lung cancer.</a> Biomaterials. 33 (22), 5574-5583 (2012).</li><li>Tonnis, W. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+aerosol+generator+for+homogenous+distribution+of+powder+over+the+lungs+after+pulmonary+administration+to+small+laboratory+animals.">A novel aerosol generator for homogenous distribution of powder over the lungs after pulmonary administration to small laboratory animals.</a> European Journal of Pharmaceutics and Biopharmaceutics. 88 (3), 1056-1063 (2014).</li><li>Hoppentocht, M., Hoste, C., Hagedoorn, P., Frijlink, H. W., de Boer, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+evaluation+of+the+DP-4M+PennCentury+insufflator.">In vitro evaluation of the DP-4M PennCentury insufflator.</a> European Journal of Pharmaceutics and Biopharmaceutics. 88 (1), 153-159 (2014).</li><li>Liao, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+and+highly+dispersible+voriconazole+dry+powders+produced+by+spray+freeze+drying+for+pulmonary+delivery+with+efficient+lung+deposition.">Porous and highly dispersible voriconazole dry powders produced by spray freeze drying for pulmonary delivery with efficient lung deposition.</a> International Journal of Pharmaceutics. 560, 144-154 (2019).</li><li>Ito, T., Okuda, T., Takashima, Y., Okamoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naked+pDNA+Inhalation+Powder+Composed+of+Hyaluronic+Acid+Exhibits+High+Gene+Expression+in+the+Lungs.">Naked pDNA Inhalation Powder Composed of Hyaluronic Acid Exhibits High Gene Expression in the Lungs.</a> Molecular Pharmaceutics. 16 (2), 489-497 (2019).</li><li>Chaurasiya, B., Zhou, M., Tu, J., Sun, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+validation+of+a+simple+device+for+insufflation+of+dry+powders+in+a+mice+model.">Design and validation of a simple device for insufflation of dry powders in a mice model.</a> European Journal of Pharmaceutical Sciences. 123, 495-501 (2018).</li></ol>

Los autores no tienen conflictos de intereses que revelar.

En este papel, los dispositivos hechos a medida para la insuflación seca del polvo y la intubación intratraqueal se presentan. En la etapa de carga de polvo, el polvo seco se carga en una punta de pipeta de carga de gel de 200 μL. Es importante tocar suavemente la punta para permitir el embalaje suelto de polvo en el extremo estrecho de la punta. Sin embargo, si el polvo se empaqueta demasiado apretado, se atascarán en la punta y no se pueden dispersar adecuadamente. Se recomienda neutralizar las cargas estáticas de...

La vía pulmonar de administración ofrece diversos beneficios en la entrega de terapias para acciones locales y sistémicas. Para el tratamiento de enfermedades pulmonares, la alta concentración local de la droga se puede alcanzar por la entrega pulmonar, de tal modo reduciendo la dosis requerida y bajando la incidencia de efectos secundarios sistémicos. Por otra parte, las actividades enzimáticas relativamente bajas en el pulmón pueden reducir metabolismo prematuro de la droga. Los pulmones también son eficientes para la absorción de fármacos para la acción sistémica debido a la gran superficie bien perfundida, la capa extremadamente delgada de células epiteliales y el alto volumen de sangre en capilares pulmonares1.
Las formulaciones de polvo seco inhalado han sido ampliamente investigadas para la prevención y el tratamiento de diversas enfermedades como el asma, la enfermedad pulmonar obstructiva crónica, la diabetes mellitus y la vacunación pulmonar2,3,4. Los fármacos en estado sólido son generalmente más estables que en forma líquida, y los inhaladores de polvo seco son más portátiles y fáciles de usar que los nebulizadores5,6. En el desarrollo de formulaciones de polvo seco inhalado, la seguridad, el perfil farmacocinético y la eficacia terapéutica necesitan ser evaluados en modelos animales preclínicos después de la administración pulmonar7. A diferencia de los seres humanos que pueden inhalar polvo seco activamente, la entrega pulmonar de polvo seco a animales pequeños es un desafío. Es necesario establecer un protocolo eficiente de entrega de polvo seco a los pulmones de los animales.
Los ratones son ampliamente utilizados como modelos de animales de investigación porque son económicos y se reproducen bien. También son fáciles de manejar y muchos modelos de enfermedades están bien establecidos. Existen dos enfoques principales para administrar polvo seco al pulmón del ratón: inhalación y administración intratraqueal. Para la inhalación, el ratón se coloca en una cámara de cuerpo entero o sólo de nariz donde se aerosoliza polvo seco y los animales respiran el aerosol sin sedación8,9. Se requiere un equipo costoso y la eficiencia de la administración de medicamentos es baja. Mientras que la cámara de todo el cuerpo puede ser técnicamente menos desafiante, la cámara de exposición solo para la nariz podría minimizar la exposición de los medicamentos a la superficie del cuerpo. En cualquier caso, todavía es difícil controlar y determinar con precisión la dosis administrada a los pulmones. El polvo seco se deposita principalmente en la región nasofaringe donde el aclaramiento mucociliar es prominente10. Además, los ratones dentro de la cámara están bajo un estrés significativo durante el proceso de administración porque están limitados y privados del suministro de alimentos y agua11. Para la administración intratraqueal, generalmente se refiere a la introducción de la sustancia directamente en la tráquea. Existen dos técnicas diferentes para lograrlo: la traqueotomía y la intubación orotraqueal. El primero requiere un procedimiento quirúrgico que hace una incisión en la tráquea, que es invasiva y rara vez se utiliza para la administración de polvo. Solamente la segunda técnica se describe aquí. En comparación con el método de inhalación, la administración intratraqueal es el método más comúnmente utilizado para la administración pulmonar en el ratón debido a su alta eficiencia de administración con una pérdida mínima de fármacos12,13. Es un método simple y rápido para entregar con precisión una pequeña cantidad de polvo dentro de unos pocos miligramos al ratón. Aunque el ratón es anatómica y fisiológicamente distinto a los humanos y se requiere anestesia durante el proceso de intubación, la administración intratraqueal pasa por alto las vías respiratorias superiores y ofrece una forma más efectiva de evaluar las actividades biológicas de la formulación de polvo seco, como la absorción pulmonar, la biodisponibilidad y los efectos terapéuticos14,15.
Para administrar polvo seco por vía intratraqueal, el ratón tiene que ser intubado, lo que podría ser un desafío. En este papel, la fabricación de un insufflator seco hecho a medida del polvo y de un dispositivo de la intubación se describe. Se demuestran los procedimientos de intubación e insuflación de polvo seco en el pulmón del ratón.

<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

En el desarrollo de formulaciones de polvo seco inhalable, es esencial evaluar sus actividades biológicas en modelos animales preclínicos. Este papel introduce un método no invasor de entrega intratraqueal de la formulación seca del polvo en ratones. Se presenta un dispositivo de carga de polvo seco que consiste en una punta de pipeta de carga de gel de 200 μL conectada a una jeringa de 1 mL a través de una llave de paso de tres vías. Una pequeña cantidad de polvo seco (1-2 mg) se carga en la punta de la pipeta y se dispersa por 0.6 ml de aire en la jeringa. Debido a que las puntas de pipeta son desechables y baratas, diferentes formulaciones de polvo seco se pueden cargar en diferentes puntas de antemano. Se pueden evaluar varias formulaciones en el mismo experimento animal sin necesidad de limpiar el dispositivo y rellenar la dosis, ahorrando así tiempo y eliminando el riesgo de contaminación cruzada del polvo residual. El grado de dispersión del polvo se puede inspeccionar por la cantidad de polvo restante en la punta de la pipeta. Se incluye un protocolo de intubación en ratón con una fuente de luz a medida y una cánula guía. La intubación adecuada es uno de los factores clave que influye en la entrega intratraqueal de la formulación de polvo seco a la región pulmonar profunda del ratón.

Cuando se utiliza un insuflador de polvo seco para entregar aerosol en polvo al pulmón de un animal, el volumen de aire utilizado es crítico, ya que afecta la seguridad, así como la eficiencia de la dispersión del polvo. Para optimizar el método, se utilizaron diferentes volúmenes de aire (0,3 mL, 0,6 mL y 1,0 mL) para dispersar el polvo seco (1 mg de manitol secado por aspersión) y se monitorizó el peso de los ratones durante 48 horas después de la administración(Figura 6). El uso...

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

Las formulaciones de polvo seco para inhalación tienen un gran potencial en el tratamiento de enfermedades respiratorias. Antes de entrar en estudios en humanos, es necesario evaluar la eficacia de la formulación de polvo seco en estudios preclínicos. Un método simple y no invasor de la administración del polvo seco en ratones a través de la ruta intratraqueal se presenta.

Administración Intratraqueal De La Formulación De Polvo Seco En Ratones

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper ...

intratracheal-administration-of-dry-powder-formulation-in-mice

Research

JoVE Journal

Medicine

9.7K vistas.  The University of Hong Kong. Las formulaciones de polvo seco para inhalación tienen un gran potencial en el tratamiento de enfermedades respiratorias. Antes de entrar en estudios en humanos, es necesario evaluar la eficacia de la formulación de polvo seco en estudios preclínicos. Un método simple y no invasor de la administración del polvo seco en ratones a través de la ruta intratraqueal se presenta.

Video: Intratracheal Administration of Dry Powder Formulation in Mice

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Un nuevo enfoque quirúrgico para la administración intratraqueal de agentes bioactivos en un modelo de ratón fetal

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Investigación

Medicina

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

Administración intranasal de la terapéutica del SNC para Ratones Awake

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and bronchodilator drugs. Despite the availability of multiple treatments, both diseases are growing public health concerns. The majority of asthma patients are well controlled on current inhaled therapies but a substantial number of patients with severe asthma are not. Asthma affects an estimated 300 million people worldwide and approximately 20 percent have a severe form of the disease. In contrast to asthma, there are few effective therapies for COPD. An estimated 10% of the population has COPD and the trend in death rates is increasing for COPD while decreasing for other major diseases. Although developing drugs for inhaled delivery is challenging, the nose-only inhalation unit enables direct delivery of novel drugs to the lung of rodents for pre-clinical efficacy and safety/toxicology studies. Inhaled drug delivery has multiple advantages for respiratory diseases, where high concentration in the lung improves efficacy and minimizes systemic side effects. Inhaled corticosteroids and bronchodilators benefit from these advantages and inhaled delivery may also hold potential for future biologic therapies. The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs of rodents, key data required before initiating clinical development.

The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs; key data enabling clinical inhaled drug development.

De polvo seco y nebulizada de inhalación de aerosol de productos farmacéuticos entregado a ratones utilizando un sólo Nose sistema de exposición

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and ...

A Pleural Effusion Model in Rats by Intratracheal Instillation of Polyacrylate/Nanosilica

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study these pulmonary diseases. Previous pleural effusion models paid more attention to biological factors rather than nanoparticles in the environment. Here, we introduce a model to make pleural effusion in rats by intratracheal instillation of polyacrylate/nanosilica, and a method of nanoparticle isolation in the pleural effusion. By intratracheal instillation of polyacrylate/nanosilica with concentrations of 3.125, 6.25 and 12.5 mg/kg&#8729;mL, the pleural effusion in rats presented on day 3, peaked at days 7-10 in 6.25 and 12.5 mg/kg&#8729;mL groups, then slowly decreased and disappeared on day 14. When the concentration of polyacrylate/nanosilica increased, the pleural effusion is produced more and faster. This pleural fluid was detected by ultrasound examination or CT chest scanning and confirmed by dissection of rats. Silica nanoparticles were observed in the rats' pleural effusion by transmission electron microscope. These results showed that the exposure to polyacrylate/nanosilica leads to the induction of pleural effusion, which was consistent with our previous report in humans. Additionally, this model is beneficial for the further study of nanotoxicology and the pleural effusion diseases.

Here, we present a protocol to construct a pleural effusion model in rats by intratracheal instillation of polyacrylate/nanosilica.

Un modelo de derrame Pleural en ratas por la instilación intratraqueal de poliacrilato/Nanosílice

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

Inducir lesión pulmonar aguda en ratones por instilación directa de lipopolisacárido intratraal

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

Establecimiento de un modelo de ojos secos severos mediante dacrioadenectomía completa en conejos

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

Intratracheal Instillation of Stem Cells in Term Neonatal Rats

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia (BPD). In premature infants, BPD is a major complication despite early use of surfactant therapy, optimal ventilation strategies, and noninvasive positive pressure ventilation. Because pulmonary inflammation plays a crucial role in the pathogenesis of BPD, corticosteroid use is one potential treatment to prevent it. Nevertheless, systemic corticosteroid treatment is not usually recommended for preterm infants due to long-term adverse effects. Preclinical studies and human phase I clinical trials demonstrated that use of mesenchymal stromal cells (MSCs) in hyperoxia-induced lung injuries and in preterm infants is safe and feasible. Intratracheal and intravenous MSC transplantation has been shown to protect against neonatal hyperoxic lung injury. Therefore, intratracheal administration of stem cells and combined surfactant and glucocorticoid treatment has emerged as a new strategy to treat newborns with respiratory disorders. The developmental stage of rat lungs at birth is equivalent to that in human lungs at 26&#8722;28 week of gestation. Hence, newborn rats are appropriate for studying intratracheal administration to preterm infants with respiratory distress to evaluate its efficacy. This intratracheal instillation technique is a clinically viable option for delivery of stem cells and drugs into the lungs.

Described is a protocol for performing intratracheal transplantation of mesenchymal stromal cells (MSCs) through intratracheal injection in term neonatal rats. This technique is a clinically viable option for delivery of stem cells and drugs into neonatal rat lungs to evaluate their efficacy.

Instilación intratraqueal de células madre en ratas neonatales a término

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia ...

A Minimally Invasive Method for Intratracheal Instillation of Drugs in Neonatal Rodents to Treat Lung Disease

Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered drug. This has direct translational impact because surfactant and drugs are administered locally into the lungs. Though the literature has many publications describing minimally invasive transoral intubation of adult mice and rats in therapeutic experiments, this approach in neonatal rat pups is lacking. The small size of orotracheal region/pharynx in the pups makes visualization of laryngeal lumen (vocal cords) difficult, contributing to the variable success rate of intratracheal drug delivery. We hereby demonstrate effective oral intubation of neonatal rat pup - a technique that is non-traumatic and minimally-invasive, so that it can be used for serial administration of drugs. We used an operating otoscope with an illumination system and a magnifying lens to visualize the tracheal opening of the rat neonates. The drug is then instilled using a 1 mL syringe connected to a pipette tip. The accuracy of the delivery method was demonstrated using Evans blue dye administration. This method is easy to get trained in and could serve as an effective way to instill drugs into trachea. This method could also be used for administration of inoculum or agents to simulate disease conditions in animals and, also, for cell-based treatment strategies for various lung diseases.

This technique of instilling drugs directly into the trachea of neonatal rodents is important in studying the impact of locally administered drugs or biologicals on neonatal lung diseases. Additionally, this method can also be used for inducing lung injury in animal models.

Un método mínimamente invasivo para la instilación intratraqueal de medicamentos en roedores neonatales para tratar la enfermedad pulmonar

Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered ...

Dosificadores desechables personalizables para administrar medicamentos en aerosol en polvo seco a ratones

Disposable Dosators Intended for Dry Powder Delivery to Mice

Dry powder inhalers offer numerous advantages for delivering drugs to the lungs, including stable solid-state drug formulations, device portability, bolus metering and dosing, and a propellant-free dispersal mechanism. To develop pharmaceutical dry powder aerosol products, robust in vivo testing is essential. Typically, initial studies involve using a murine model for preliminary evaluation before conducting formal studies in larger animal species. However, a significant limitation in this approach is the lack of suitable device technology to accurately and reproducibly deliver dry powders to small animals, hindering such models&#39; utility. To address these challenges, disposable syringe dosators were developed specifically for intrapulmonary delivery of dry powders in doses appropriate for mice. These dosators load and deliver a predetermined amount of powder obtained from a uniform bulk density powder bed. This discrete control is achieved by inserting a blunt needle to a fixed depth (tamping) into the powder bed, removing a fixed quantity each time. Notably, this dosing pattern has proven effective for a range of spray-dried powders. In experiments involving four different model spray-dried powders, the dosators demonstrated the ability to achieve doses within the range of 30 to 1100 &#181;g. The achieved dose was influenced by factors such as the number of tamps, the size of the dosator needle, and the specific formulation used. One of the key benefits of these dosators is their ease of manufacturing, making them accessible and cost-effective for delivering dry powders to mice during initial proof-of-concept studies. The disposable nature of the dosators facilitates use in animal procedure rooms, where cleaning and refilling reusable systems and weighing materials is inconvenient. Thus, developing disposable syringe dosators has addressed a significant hurdle in murine dry powder delivery for proof-of-concept studies, enabling researchers to conduct more accurate and reproducible preliminary studies in small animal models for pulmonary drug delivery.

Autor destacado: Desarrollo de un dosificador desechable para pruebas preclínicas de inhaladores de polvo seco en modelos de animales pequeños 

Pharmaceutical dry powder development necessitates reliable in vivo testing, often using a murine model. Device technology for accurately and reproducibly delivering dry powder aerosols to mice is restricted. This study presents disposable dosators for pulmonary drug delivery at mouse-relevant doses, aiding initial proof-of-concept research.

Dosificadores desechables destinados a la entrega de polvo seco a ratones

Dry powder inhalers offer numerous advantages for delivering drugs to the lungs, including stable solid-state drug formulations, device portability, bolus ...