Name: intratracheal administration of dry powder formulation in mice
Uploaded: 2020-07-25T15:00:00
Description: Watch this Scientific Journal Video about Intratracheal Administration of Dry Powder Formulation in Mice at JoVE.com

저자는 레이 리 씨, HC 렁 씨와 월리스 소 씨에게 광원과 분말 인수프플레이터를 만드는 데 친절한 도움을 준 것에 대해 감사드립니다. 동물 화상 진찰에 대한 지원을위한 교수 핵심 시설. 이 작품은 홍콩 연구 보조금 위원회(17300319)의 지원을 받았습니다.

이 연구에서 수행 된 실험은 홍콩 대학 인 교육 및 연구를위한 살아있는 동물 사용위원회 (CULATR)의 승인을 받았습니다. 루시파라기 메신저 RNA(mRNA)의 0.5%를 함유한 스프레이 동결 건조(SFD)에 의해 제조된 건식 분말 제형은, 5% 합성 펩타이드 PEG12KL4 및 94.5%의 매니톨(w/w)을 함유하여 폐16에서mRNA 발현을 입증하기 위해 이 연구에서 사용된다. SFD 분말의 질량 중앙공기역학적 직...

<ol>
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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>

이 논문에서는 건조 분말 부피 및 장내 삽관을 위한 맞춤형 장치가 제공됩니다. 분말 적재 단계에서, 건조 분말은 200 μL 젤 로딩 파이펫 팁에로드됩니다. 팁의 좁은 끝에 분말의 느슨한 포장을 허용 하기 위해 팁을 부드럽게 탭 하는 것이 중요 하다. 그러나 분말이 너무 단단히 포장되면 팁에 갇히고 제대로 분산 될 수 없습니다. 분말 로딩을 용이하게 하기 위해 분말 및 파이펫 팁의 정적 충전을 중...

투여의 폐 경로는 지역 및 조직 적 행동 모두에 대한 치료를 제공하는 다양한 혜택을 제공합니다. 폐 질환의 치료를 위해, 높은 국부 약물 농도는 폐 전달에 의해 달성 될 수있다, 따라서 필요한 용량을 감소시키고 전신 부작용의 발생률을 낮추는. 더욱이, 폐에서 상대적으로 낮은 효소 활동은 조기 약물 대사를 줄일 수 있습니다. 폐는 또한 크고 잘 퍼진 표면적, 매우 얇은 상피 세포 층 및 폐 모세혈관1의높은 혈액 부피로 인한 전신 작용에 대한 약물 흡수에 효율적입니다.
흡입건조분말제제는 천식, 만성폐쇄성 폐질환, 당뇨병 및 폐예방접종2,3,4등 다양한 질환의 예방 및 치료를 위해 널리 조사되고있다. 고체 상태의 약물은 일반적으로 액체 형태보다 더 안정하며, 건조 분말 흡입기는 분무기5,6보다휴대성이 뛰어나고 사용자 친화적이다. 흡입건조분말제형의 개발에서, 안전성, 약동성 프로파일 및 치료효능은 폐투여에 따른 전임상 동물모델에서 평가될 필요가있다 7. 건조 분말을 적극적으로 흡입할 수 있는 인간과 달리, 작은 동물에게 건조 분말을 폐로 전달하는 것은 어려운 일입니다. 동물의 폐에 건조 분말을 전달하는 효율적인 프로토콜을 확립 할 필요가있다.
마우스는 경제적이기 때문에 연구 동물 모델로 널리 사용되며 잘 번식합니다. 그들은 또한 취급하기 쉽고 많은 질병 모형이 잘 설치되어 있습니다. 마우스의 폐에 건조 분말을 관리 하는 두 가지 주요 접근 이 있다: 흡입 및 장 내 관리. 흡입을 위해 마우스는 건조 분말이 에어로졸화되고 동물이 체증 없이 에어로졸에서 호흡하는 전신 또는 코 전용챔버에배치됩니다8,9. 고가의 장비가 필요하며 약물 전달 효율이 낮습니다. 전신 챔버는 기술적으로 덜 어려울 수 있지만, 코 전용 노출 챔버는 신체 표면에 약물의 노출을 최소화 할 수 있습니다. 에 관계없이, 폐에 전달 된 복용량을 정확하게 제어하고 결정하는 것은 여전히 어렵습니다. 건조 분말은 주로 점막 간격이 눈에 띄는 비소하린스 지역에 증착된다10. 더욱이, 챔버 내부의 마우스는 식량 및급수(11)를제한하고 박탈당하기 때문에 투여 과정에서 상당한 스트레스를 받고 있다. 장내 투여를 위해, 일반적으로 기관내로 직접 물질의 도입을 의미한다. 이를 달성하기 위한 두 가지 기술이 있습니다: 기관 절제술과 오로트라세이션 삽관. 전자는 침입적이고 분말 투여에 거의 사용되지 않는 기관에서 절개를하는 수술 절차가 필요합니다. 두 번째 기술만 여기에 설명되어 있습니다. 흡입 방법에 비해, 내막 투여는 최소한의 약물 손실로 높은 전달 효율때문에 마우스내 폐 전달을 위해 보다 일반적으로 사용되는방법이다(12,13). 그것은 정확 하 게 마우스에 몇 밀리 그램 내에서 분말의 작은 금액을 제공 하는 간단 하 고 빠른 방법. 마우스는 해부학적으로 인간과 생리적으로 구별되지만, 관적 과정에서 마취가 요구되지만, 내내 투여는 상부 호흡기를 우회하고 폐 흡수, 생체 이용률 및 치료효과(14,15)와같은 건조 분말 제형의 생물학적 활성을 평가하는 보다 효과적인 방법을 제공한다.
건조 분말을 장내 관리하려면 마우스를 삽관해야 하므로 어려울 수 있습니다. 이 논문에서는 맞춤형 건조 분말 인수프플레이터 및 삽관 장치의 제조가 설명되어 있습니다. 마우스의 폐에 있는 건식 분말의 삽관 및 부당의 절차가 입증됩니다.

<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

흡입 할 수없는 건조 분말 제제의 개발에서, 전임상 동물 모델에서 자신의 생물학적 활동을 평가하는 것이 필수적이다. 본 논문은 마우스에서 건식 분말 제형의 비침습적 전달 방법을 소개합니다. 3방향 스톱콕을 통해 1mL 주사기에 연결된 200 μL 젤 로딩 파이펫 팁으로 구성된 드라이 파우더 로딩 장치가 제시됩니다. 소량의 건조 분말(1-2 mg)이 파이펫 팁에 적재되어 주사기에 0.6mL의 공기가 분산됩니다. 파이펫 팁은 일회용이고 저렴하기 때문에 다른 건조 분말 제형은 사전에 다른 팁으로 로드 할 수 있습니다. 다양한 제형은 장치 세척 및 용량 리필 없이 동일한 동물 실험에서 평가될 수 있으므로 시간을 절약하고 잔류 분말로부터 의 교차 오염의 위험을 제거합니다. 분말 분산의 정도는 파이펫 팁에 남아있는 분말의 양에 의해 검사 될 수있다. 사용자 정의 광원과 안내 캐뉼라가있는 마우스에 삽관 프로토콜이 포함되어 있습니다. 적절한 관착은 마우스의 깊은 폐 부위에 건조 분말 제형의 장내 전달에 영향을 미치는 주요 요인 중 하나입니다.

건조 분말 insufflator동물의 폐에 분말 에어로졸을 전달하는 데 사용되는 경우, 사용되는 공기의 양은 분말 분산 효율뿐만 아니라 안전에 영향을 미치기 때문에 매우 중요합니다. 이를 최적화하기 위해, 다양한 공기량(0.3mL, 0.6mL 및 1.0mL)을 사용하여 건조 분말(1 mg의 분무 건조 매니톨)을 분산시키고 마우스의 무게를 투여 후 48시간 동안 모니터링하였다(도6). 0.3 mL 및 0.6 mL의 공?...

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

흡입을 위한 건식 분말 제형은 호흡기 질환 치료에 큰 잠재력을 가지고 있습니다. 인간 연구에 들어가기 전에, 전임상 연구에서 건식 분말 제형의 효능을 평가할 필요가 있다. 간 내 막 경로를 통해 마우스에서 건조 분말을 투여하는 간단하고 비침습적 방법이 제시된다.

쥐에서 건조 분말 제형의 장내 투여

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 is essential to evaluate the performance and biological activities of an inhaled powder formulation, such as pulmonary absorption, bioavailability, and therapeutic effects in pre-clinical animal models. The intubation process is non-invasive and could deliver powder formulation to the mice safely and accurately. The custom-made dry powder insufflator is disposable, inexpensive, and efficient in disbursing powder formulations.The insufflators could be used in evaluating different formulations on multiple mice in the same experiments without a risk of cross-contamination from residual powder. The intubation process is challenging for non-experienced researchers. The ability to visualize and aim the finest tip of the cannula at the opening of the trachea is crucial for correct insertion of the guiding cannula.This technique requires practice to minimize improper insertion. My postdoc Carol and my PhD student Rachel are going to demonstrate the preparation and intubation procedure. To begin, neutralize the static charges of the dry powder and the 200 microliter non-filter round gel loading pipette tip with an anti-static gun or a balance with deionizing function.Prepare a weighing paper with an approximate size of four by four centimeters. Fold the paper in half diagonally and then unfold it. Then use it to weigh one to two milligrams of dry powder.Fill a gel loading pipette tip with powder through the wider opening and tap it gently to pack the powder until it forms loose agglomerates near the narrow end of the tip. Avoid packing the powder too tightly as it may hamper dispersion. Connect the powder loaded tip to a one milliliter syringe through a three-way stopcock holding the tip and syringe vertically during connection to prevent spillage of powder.The size of the syringe can be changed according to the volume of air used to disperse the powder. If administration is not performed immediately, use parafilm to seal the openings of the tip and store it temporarily under suitable conditions until administration. After anesthetizing the seven to nine-week-old BALB/c mouse with ketamine and xylazine, place it on a plexiglass platform mounted to a stand.Suspend the mouse by hooking its incisors on a nylon floss and secure its position with a piece of tape or a rubber band. Adjust the height and angle of the platform. Insert the optical fiber into the guiding cannula with the tip of the fiber level with the opening of the cannula.Then turn on the LED torch to illuminate. Gently protrude the tongue of the mouse with a pair of forceps to expose its trachea. Use the other hand to hold the guiding cannula with optical fiber and insert them through the oral cavity.With the illumination from the optical fiber, the opening of the trachea can be visualized as an orifice between the vocal cords. Align the bevel of the guiding cannula towards the midline of the opening and gently intubate it with the optical fiber into the trachea by aiming the finest tip of the cannula at the tracheal opening. Upon intubation, swiftly remove the optical fiber and leave the guiding cannula inside the trachea.Normal restoration should be observed. Hold the fine tip pipette at the opening of the guiding cannula and insufflate a small puff of air into the lung of the mouse. A slight inflation in the chest indicates proper intubation.Remove the fine tip pipette prior to powder administration. Hold the power loaded tip that is connected to the syringe, making sure that the air flow between the syringe and the tip is disconnected. Pull the syringe plunger backward to withdraw 0.6 milliliters of air.Turn the valve of the three-way stopcock to connect the air flow between the syringe and the power loaded tip. Then insert the power loaded tip into the guiding cannula which has already been placed in the trachea of the mouse. Hold the guiding cannula and push the syringe plunger forcefully in one continuous action to disperse the powder as aerosols into the lung minimizing any forward motion to avoid injury to the animal.Remove the tip and check if the powder has been emptied. Once the administration is complete, remove the guiding cannula from the trachea. Allow the mouse to recover by positioning it horizontally in a supine position with its tongue half protruded to avoid a blockage of the airways.To optimize the method, different volumes of air were used to disperse one milligram of the spray dried mannitol and the weight of mice was monitored. The use of 0.3 and 0.6 milliliters of air did not cause weight loss of the mice up to 48 hours post-administration. Dispersing the powder with one milliliter of air resulted in over 5%weight loss within 24 hours, which was not fully recovered after 48 hours.The mice were intratracheally administered with one milligram of SFD powder containing five micrograms of mRNA and the luciferase expression in the lungs was evaluated at 24 hours post-administration using an in vivo imaging system. The SFD powder was dispersed in the deep lung and luciferase expression was observed. As a comparison, the powder was reconstituted in water and administered with a micro sprayer using the same intubation procedure.The luciferase expression of the reconstituted formulation was significantly higher than the dry powder formulation, which could be due to the powder disillusion issue or different pharmacokinetic profile between powder and liquid form. The histological characteristics of the lungs treated with mRNA dry powder aerosol were compared with untreated control and LPS-treated groups. The lung without any treatment illustrated a healthy presentation while the lung treated with LPS showed irregular distribution of airspace and inflammatory cell infiltration into the interstitial and alveolar spaces.The lungs treated with SFD powder did not show any signs of inflammation. For successful powder dispersion, the powder in the loading tip should be tapped gently and not packed too tightly in the tip. This intubation setting can also be adapted to administer liquid formulations either with a pipette or a micro sprayer.

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