Os autores gostariam de agradecer ao Sr. Ray Lee, Sr. HC Leung e Sr. Wallace So por sua gentil ajuda na fabricação da fonte de luz e do insufrador de pó; e o Centro de Atendimento à Faculdade para a assistência em imagens de animais. O trabalho foi apoiado pelo Research Grant Council, Hong Kong (17300319).

Os experimentos realizados neste estudo foram aprovados pelo Comitê de Uso de Animais Vivos para Ensino e Pesquisa (CULATR), da Universidade de Hong Kong. Formulações de pó seco preparadas pela secagem de congelamento de spray (SFD) contendo 0,5% do RNA mensageiro da luciferase (mRNA), 5% de peptídeo sintético PEG12KL4 e 94,5% de mannitol (w/w) são utilizadas neste estudo para demonstrar a expressão mRNA no pulmão16. O diâmetro aerodinâmico médio de massa (MMAD) do pó SFD é de 2,4 μm. Pó de manitol seco (SD) são usados para investigar o efeito do volume de ar usado na dispersão de pó16. O pó de SD de SD é de 1,5 μm.
1. Fabricação de insufrante de pó seco e carregamento de pó seco
<ol><li>(Opcional) Neutralizar as cargas estáticas de pó seco (em um frasco) e a ponta de pipeta de carregamento de gel redondo de 200 μL. Use uma arma antiestática ou um equilíbrio com função desionizante de acordo com a instrução do fabricante.</li>
	<li>Prepare um papel de pesagem com um tamanho de cerca de 4 cm x 4 cm. Dobre o papel ao meio diagonalmente e, em seguida, desdobre-o.</li>
	<li>Pesar 1-2 mg de pó seco no papel de pesagem.</li>
	<li>Encha uma ponta de pipeta de carregamento de gel com pó através da abertura mais ampla da ponta. Bata suavemente para embalar o pó até que o pó se forme agglomerados soltos perto da extremidade estreita da ponta(Figura 1A). Evite embalar o pó muito fortemente, pois pode dificultar a dispersão do pó.</li>
	<li>Conecte a ponta carregada em pó a uma seringa de 1 mL através de uma torneira de três vias(Figura 1B). O tamanho da seringa pode ser alterado de acordo com o volume de ar usado para dispersar o pó. Segure a ponta e a seringa verticalmente durante a conexão para evitar derramamento de pó. Se a administração não for realizada imediatamente, use o parafilm para selar as aberturas da ponta e armazená-la temporariamente em condições adequadas até a administração.</li>
</ol>2. Fabricação de dispositivo de intubação
<ol><li>Fonte de luz(Figura 2)<ol>
<li>Prepare uma fonte de luz personalizada com uma tocha de diodo emissor de luz (LED) e uma fibra óptica flexível com diâmetro de 0,8-1 mm.</li>
		<li>Faça um orifício centrado na lente clara da tocha LED com uma broca de mão ou uma broca para que a fibra óptica mal possa passar.</li>
		<li>Insira a fibra óptica através do orifício. Ligue a tocha LED para ajustar a posição e a profundidade de inserção para o brilho máximo na outra extremidade da fibra óptica.</li>
		<li>Fixar a fibra óptica em posição com cola epóxi clara.</li>
	</ol>
</li>
	<li>Cânula orientadora (Figura 3)<ol>
<li>Pegue uma pipeta Pasteur de plástico de 1 mL(Figura 3A)e segure a pipeta em ambas as extremidades.</li>
		<li>Use uma lâmpada de álcool (ou outras fontes de calor no laboratório, como um queimador de Bunsen) para aquecer o meio da pipeta colocando-a a 5-10 cm acima da chama(Figura 3B). Gire a pipeta para ter certeza de que está aquecida uniformemente.</li>
		<li>Quando o plástico ficar macio e deformado, mova a pipeta para longe da chama e estique a pipeta suavemente.</li>
		<li>Corte a pipeta esticada no meio com um par de tesouras na Parte A e Parte B(Figura 3C-E). Use a peça A como uma pipeta de ponta fina e parte B como uma cânula guia. Para aumentar a chance de intubação bem sucedida com a cânula guia, faça um bisbigo (não muito afiado que pode aumentar o risco de ferir o animal) no final da Parte B (Figura 3F). Quando uma ponta de pipeta de carregamento de gel de 200 μL (para carregamento em pó) é inserida na cânula guia, ela deve projetar a cânula por 1-2 mm. NOTA: Uma cânula guia (Parte B) com a dimensão apropriada (diâmetro interno e externo) para intubação poderia ter uma agulha de calibre 21 dentro dela, enquanto também pode caber dentro de uma agulha de calibre 17. Podem ser necessárias múltiplas tentativas no alongamento das pipetas para alcançar a dimensão apropriada.</li>
		<li>(Opcional): Corte uma pequena abertura na extremidade mais larga da cânula guia para torná-la mais flexível para que seja mais fácil segurar a fibra óptica(Figura 3F). Esta abertura também permite a montagem de um microsprayer para a administração de aerossol líquido.</li>
	</ol>
</li>
</ol>3. Intubação
<ol><li>Anestesiar o rato (BALB/c, 7-9 semanas) com cetamina (100 mg/kg) e xilazina (10 mg/kg) por injeção intraperitoneal.</li>
	<li>Prepare uma plataforma feita de Plexiglass e monte-a em um suporte com um grampo(Figura 4A). Coloque o mouse anestesiado na plataforma (em torno de 60° de inclinação) em uma posição supina. A altura e o ângulo de inclinação da plataforma poderiam ser ajustados pela posição do grampo no suporte.</li>
	<li>Suspenda o mouse enganchando seus incisivos em um fio dental de nylon(Figura 4B). Fixar a posição do mouse por um pedaço de fita ou um elástico.</li>
	<li>Insira a fibra óptica na cânula guia antes da intubação com a ponta do nível de fibra com a abertura da cânula guia. Ligue a tocha led para iluminar.</li>
	<li>Saliente suavemente a língua do rato com um par de fórceps para expor sua traqueia.</li>
	<li>Use a outra mão para segurar a cânula guia com fibra óptica dentro. Insira-os através da cavidade oral. Com a iluminação da fibra óptica, a abertura da traqueia pode ser visualizada como um orifício entre as cordas vocais.</li>
	<li>Alinhe o chanfrado da cânula guia para a linha média da abertura (Figura 5A). Entuba suavemente a cânula guia com fibra óptica na traqueia, apontando a melhor ponta da cânula na abertura traqueal.</li>
	<li>Após a intubação, remova rapidamente a fibra óptica e deixe a cânula guia dentro da traqueia (Figura 5B). Uma respiração normal deve ser observada.</li>
	<li>Segure a pipeta de ponta fina (Parte A) na abertura da cânula guia e insufre um pequeno sopro de ar (cerca de 0,2 mL) no pulmão do camundongo. Uma ligeira inflação no peito do mouse indica intubação adequada. Remova a pipeta de ponta fina antes da administração do pó.</li>
</ol>4. Administração de pó
<ol><li>Segure a ponta carregada em pó que está conectada à seringa conforme descrito na etapa 1.5. Certifique-se de que o fluxo de ar entre a seringa e a ponta esteja desconectado.</li>
	<li>Puxe o êmbolo da seringa para trás para retirar 0,6 mL de ar. NOTA: O volume de ar utilizado para dispersar o pó depende das propriedades do pó e da quantidade de pó carregado. Isso é descrito ainda na seção de resultados.</li>
	<li>Gire a válvula da torneira de três vias para conectar o fluxo de ar entre a seringa e a ponta carregada em pó.</li>
	<li>Insira a ponta carregada em pó na cânula guia que já foi colocada na traqueia do mouse(Figura 5C). Segure a cânula guia e empurre o êmbolo da seringa com força em uma ação contínua para dispersar o pó enquanto aerossóis para dentro do pulmão. NOTA: Qualquer movimento dianteiro do dispositivo deve ser minimizado para evitar ferir o animal.</li>
	<li>Retire a ponta e verifique se o pó dentro da ponta foi esvaziado. Se não, repita o passo 4.1 para 4.4. NOTA: Se o pó estiver muito apertado devido ao toque excessivo, pode não ser dispersado corretamente.</li>
	<li>Uma vez que a administração esteja completa, remova a cânula guia da traqueia.</li>
	<li>Deixe o mouse se recuperar posicionando-o horizontalmente em uma posição supina com a língua meio salientes para evitar o bloqueio das vias aéreas.</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>

Os autores não têm conflitos de interesse para divulgar.

Neste artigo, são apresentados dispositivos personalizados para insuficiência de pó seco e intubação intratraqueal. Na etapa de carga em pó, o pó seco é carregado em uma ponta de pipeta de carregamento de gel de 200 μL. É importante tocar suavemente a ponta para permitir a embalagem solta de pó na extremidade estreita da ponta. No entanto, se o pó estiver muito apertado, eles ficarão presos na ponta e não poderão ser devidamente dispersos. Recomenda-se neutralizar as cargas estáticas do pó e da ponta da ...

A rota pulmonar da administração oferece diversos benefícios na entrega de terapêuticas para ações locais e sistêmicas. Para o tratamento de doenças pulmonares, a alta concentração de medicamentos locais pode ser alcançada por parto pulmonar, reduzindo assim a dose necessária e diminuindo a incidência de efeitos colaterais sistêmicos. Além disso, as atividades enzimáticas relativamente baixas no pulmão podem reduzir o metabolismo de drogas prematuras. Os pulmões também são eficientes para a absorção de medicamentos para ação sistêmica devido à grande e bem perfusada superfície, a camada de célula epitelial extremamente fina e o alto volume sanguíneo em capilares pulmonares1.
Formulações em pó seco inaladas têm sido amplamente investigadas para a prevenção e tratamento de diversas doenças como asma, doença pulmonar obstrutiva crônica, diabetes mellitus evacinaçãopulmonar 2,3,4. As drogas no estado sólido são geralmente mais estáveis do que na forma líquida, e os inaladores de pó seco são mais portáteis e fáceis de usar do que os nebulizadores5,6. No desenvolvimento de formulações de pó seco inalado, a segurança, o perfil farmacocinético e a eficácia terapêutica precisam ser avaliados em modelos animais pré-clínicos após a administração pulmonar7. Ao contrário dos humanos que podem inalar pó seco ativamente, a entrega pulmonar de pó seco para animais de pequeno porte é um desafio. É necessário estabelecer um protocolo eficiente de entrega de pó seco aos pulmões dos animais.
Os camundongos são amplamente utilizados como modelos animais de pesquisa porque são econômicos e se reproduzem bem. Eles também são fáceis de manusear e muitos modelos de doenças são bem estabelecidos. Existem duas abordagens principais para administrar pó seco no pulmão do camundongo: inalação e administração intratraqueal. Para inalação, o camundongo é colocado em uma câmara de corpo inteiro ou nariz, onde o pó seco é aerossolizado e os animais respiram no aerossol sem sedação8,9. Equipamentos caros são necessários e a eficiência de entrega de medicamentos é baixa. Embora a câmara do corpo inteiro possa ser tecnicamente menos desafiadora, a câmara de exposição apenas para nariz poderia minimizar a exposição de drogas à superfície do corpo. Independentemente disso, ainda é difícil controlar com precisão e determinar a dose entregue aos pulmões. O pó seco é depositado principalmente na região da nasofaringe, onde o despejo mucociliar é proeminente10. Além disso, os camundongos dentro da câmara estão sob estresse significativo durante o processo de administração porque são constrangidos e privados de alimentos e abastecimento de água11. Para a administração intratraqueal, geralmente refere-se à introdução da substância diretamente na traqueia. Existem duas técnicas diferentes para conseguir isso: traqueotomia e intubação orotraqueal. O primeiro requer um procedimento cirúrgico que faça uma incisão na traqueia, que é invasiva e raramente utilizada para administração de pó. Apenas a segunda técnica é descrita aqui. Em comparação com o método de inalação, a administração intratracheal é o método mais utilizado para a entrega pulmonar no camundongo devido à sua alta eficiência de entrega com perda mínima de medicamentos12,13. É um método simples e rápido para entregar precisamente uma pequena quantidade de pó dentro de alguns miligramas para o mouse. Embora o camundongo seja anatomicamente e fisiologicamente distinto dos seres humanos e a anestesia seja necessária durante o processo de intubação, a administração intratraqueal contorna o trato respiratório superior e oferece uma maneira mais eficaz de avaliar as atividades biológicas da formulação de pó seco, como a absorção pulmonar, a biodisponibilidade e os efeitos terapêuticos14,15.
Para administrar pó seco intratracheicamente, o rato tem que ser entubado, o que pode ser desafiador. Neste artigo, descreve-se a fabricação de um insuflê de pó seco feito sob medida e um dispositivo de intubação. Os procedimentos de intubação e insuficiência de pó seco no pulmão do camundongo são demonstrados.

<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

No desenvolvimento de formulações inaláveis de pó seco, é essencial avaliar suas atividades biológicas em modelos animais pré-clínicos. Este artigo introduz um método não invasivo de entrega intratraqueal de formulação de pó seco em camundongos. Um dispositivo de carregamento de pó seco que consiste em uma ponta de pipeta de carregamento de gel de 200 μL conectada a uma seringa de 1 mL através de uma torneira de três vias é apresentado. Uma pequena quantidade de pó seco (1-2 mg) é carregada na ponta da pipeta e dispersada por 0,6 mL de ar na seringa. Como as pontas da pipeta são descartáveis e baratas, diferentes formulações de pó seco podem ser carregadas em diferentes pontas com antecedência. Várias formulações podem ser avaliadas no mesmo experimento animal sem limpeza de dispositivos e recarga de dose, economizando tempo e eliminando o risco de contaminação cruzada do pó residual. A extensão da dispersão do pó pode ser inspecionada pela quantidade de pó restante na ponta da pipeta. Um protocolo de entubação no mouse com uma fonte de luz personalizada e uma cânula orientadora está incluído. A intubação adequada é um dos fatores-chave que influencia a entrega intratraqueal da formulação de pó seco para a região pulmonar profunda do camundongo.

Quando um insuflê de pó seco é usado para fornecer aerossol em pó para o pulmão de um animal, o volume de ar utilizado é crítico, pois afeta a segurança, bem como a eficiência de dispersão em pó. Para otimizar o método, foram utilizados diferentes volumes de ar (0,3 mL, 0,6 mL e 1,0 mL) para dispersar o pó seco (1 mg de mannil seco com spray) e o peso dos camundongos foi monitorado por 48 horas após a administração(Figura 6). O uso de 0,3 mL e 0,6 mL de ar não causou perda d...

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

Formulações de pó seco para inalação têm grande potencial no tratamento de doenças respiratórias. Antes de entrar em estudos humanos, é necessário avaliar a eficácia da formulação de pó seco em estudos pré-clínicos. É apresentado um método simples e não invasivo de administração de pó seco em camundongos através da rota intratraqueal.

Administração Intratraqueal de Formulação de Pó Seco em Camundongos

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 visualizações.  The University of Hong Kong. Formulações de pó seco para inalação têm grande potencial no tratamento de doenças respiratórias. Antes de entrar em estudos humanos, é necessário avaliar a eficácia da formulação de pó seco em estudos pré-clínicos. É apresentado um método simples e não invasivo de administração de pó seco em camundongos através da rota intratraqueal.

Vídeo: 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.

Uma nova abordagem cirúrgica para administração intratraqueal de agentes bioativos em um modelo de camundongo Fetal

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

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.

A administração intranasal de Terapêutica do SNC para Ratos 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.

Pó seco e nebulizada de inalação de aerossóis de produtos farmacêuticos entregue no Ratos Usando um nariz somente Sistema de Exposição

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.

Um modelo de derrame Pleural em ratos por instilação intratraqueal de poliacrilato/Nanosilica

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.

Induzindo lesão pulmonar aguda em camundongos por instilação de lipopolissacárido intratraqueal direto

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.

Estabelecimento de um modelo severo do olho seco usando dacryoadenectomy completo nos coelhos

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.

Instilação intratraqueal de células-tronco em ratos neonatais de termo

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.

Um método minimamente invasivo para a instilação intratraqueal de drogas em roedores neonatais para tratar doenças pulmonares

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 ...

Customizable Disposable Dosators to Deliver Dry Powder Aerosol Drugs to Mice

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.

Author Spotlight: Developing a Disposable Dosator for Preclinical Testing of Dry Powder Inhalers in Small Animal Models 

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.

Dosadores descartáveis destinados à entrega de pó seco a ratos

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