Name: in vitro permeation of fitc-loaded ferritins across a rat blood-brain barrier: a model to study the delivery of nanoformulated molecules
Uploaded: 2016-08-22T15:00:00
Description: Watch this Scientific Journal Video about In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules at JoVE.com

The authors acknowledge Assessorato alla Sanit&#224;, Regione Lombardia and Sacco Hospital (NanoMeDia Project) for research funding.

1. BBB 모델 구축 참고 : 우리가 시판 차 RBMECs와 RCA 단자를 사용하는 것이 좋습니다 BBB를 모델을 구축하십시오. 모든 단계를 층류 후드에서 멸균 처리 시약 및 일회용으로 수행되어야한다. <ol><li> 세포 배양 <ol><li> 폴리 -L- 라이신 100 μg의 / ㎖ (RT에서 1 시간) 또는 피브로넥틴 50 ㎍ / ㎖ (37 ° C에서 1 시간)와 코트 세포 배양 플라스크는 각각 RCA 단자 또는 RBMECs의 부착을 촉진. 이어서, 5 % 소 태아...

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여기에 설명 된 체외 방법은 나노 입자와 nanoformulation에 따라 형광 분자의 트랜스 BBB 전달을 연구하는 데 유용한 검증 된 접근 방식을 나타냅니다. 여기에서 우리는 BBB에서화물 분자의 전위를 연구 할 수있는 좋은 후보를 나타냅니다 FNN를 사용합니다. 그것은 구체적으로 BMECs의 내강 막에 표현 수용체 매개 내재화 경로를 사용하여 나노 입자의 흡수를 매개하는 TfR1 수용체에 의해 인식되기 ?...

약물 요법에 대한 중추 신경계의 저항 (CNS) 질환 (예. 암, 간질, 우울증, 정신 분열증 및 HIV 관련 신경 장애)로 인해 뇌 혈관 장벽을 통과 곤란한 약물 투과 포함하는 다양한 다른 메커니즘이다 (BBB) . BBB를 혈액 순환 물질로부터 뇌 조직을 분리 경계이다. 이 배리어 내 혈관 주위 세포와 성상 세포의 endfeet 지원 뇌 미세 혈관 내피 세포 (BMECs)의 층은, 분자량이 400 이상인 다 1 hydrosoluble 이들 약물에 BBB의 높은 선택성을 담당한다. 또 다른 약물 관련 저항기구는 CNS에 약물 침투를 감소시키고 뇌 (2)로부터 자신의 압출을 용이하게하기 위해 협력 약물 유출 컨베이어 (P 당 단백질 및 약제 내성 단백질)의 BMECs에 존재 연결된다. 지난 10 년간, 나노 기술 방법 중 다수는 BBB 3-6 걸쳐 약물 전달의 임상 적 및 생물학적 과제를 해결하기 위해 개발되었다. 이러한 상황에서, 나노 페리틴 (FNN)는 완전히 혁신적이고 유망한 솔루션을 나타낸다. FNN 8 nm의 내경 중공 구형 구조로 배열되어 자기 조립 (24) 페리틴 (FN) 모노머, 12 nm의 구형이다. 페리틴 소단위는 산성 pH에서 분해 및 다양한 유기 분자를 캡슐화 할 수 있도록 중립성 pH를 가져와 형상 기억 방식으로 조립 될 수있다. 따라서, FNN 다기능 약물 전달 시스템 7,8의 개발이 흥미있는 모델을 나타낸다. 또한, FNN이 셀 (9)의 내강 막 상에 발현되는 트랜스페린 수용체 (TFR) (1)의 구체적인 포상 BMECs 감사와 상호 작용할 수있다. 지금까지, BBB의 시험 관내 모델이 다른 순으로 하였다 개발되어왔다R은 다양한 약물의 독성 BBB 향해 또는 유출 수송차와 분자의 상호 작용 트랜스 BBB 투과성 해명. 사실,이 모델은 생체 내 연구를 진행하기 전에 활성 분자의 신속한 검사를 위해 접근 체외에서 유효한 것으로 간주됩니다. 이 모델 동물 (쥐, 마우스, 돼지와 소) 또는 인간의 세포 라인 10,11,12에서 얻은 BMECs 또는 공동 배양 BMECs 및 성상 세포 (드물게 혈관 주위 세포)의 단일 내피 층으로 구성되어 있습니다. TransEndothelial 전기 저항 (봉사자)과 정의 분자량 추적자의 명백한 침투성 (P 응용 프로그램)은 체외 모델의 품질을 결정하는 데 사용되는 두 가지 중요한 매개 변수를 나타냅니다. 여기에 우리가 쥐 BMECs (RBMECs)의 공동 문화를 기반으로 BBB 체외 모델의 고용을 설명하고 쥐 대뇌 피질 성상 세포 (RCA 단자)는 형광 isothi을 캡슐화 페리틴 nanocages의 트랜스 BBB 투과를 공부ocyanate (FITC).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Rat Brain Microvascular Endothelial Cells&#160;</td>
			<td>Innoprot</td>
			<td>P10308</td>
			<td>isolated from Sprague Dawley rat brain tissue, cryopreserved at passage one and delivered frozen</td>
		</tr>
		<tr>
			<td>Cortical Astrocytes&#160;</td>
			<td>Innoprot</td>
			<td>P10202</td>
			<td>isolated from 2 days rat brain tissue, cryopreserved at passage one and delivered frozen.</td>
		</tr>
		<tr>
			<td>Endothelial Cell Medium kit</td>
			<td>Innoprot</td>
			<td>P60104</td>
			<td>ECM (500 ml) and fetal bovin serum (25 ml), endothelial cell growth supplement (5 ml) and penicillin/streptomycin (5 ml). Warm in 37 &#176;C water bath before use and protect from light</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA without Phenol Red</td>
			<td>EuroClone</td>
			<td>ECM0920D</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate-dextran 40000</td>
			<td>Sigma</td>
			<td>FD40S</td>
			<td>protect from light</td>
		</tr>
		<tr>
			<td>paraformaldehyde&#160;</td>
			<td>Sigma</td>
			<td>158127</td>
			<td>diluition in chemical hood</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffer saline w/o Ca and Mg</td>
			<td>EuroClone</td>
			<td>ECB4004L</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton&#160;X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum albumin&#160;</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>goat serum&#160;</td>
			<td>EuroClone</td>
			<td>ECS0200D</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse monoclonal anti-Von Willebrand Factor</td>
			<td>Dako</td>
			<td>M0616</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 546-conjugated antibody against mouse IgGs</td>
			<td>ThermoFischer Scientific</td>
			<td>A-11003</td>
			<td>protect from light</td>
		</tr>
		<tr>
			<td>DAPI (4&#8217; ,6-diamidino-2-phenylindole)&#160;</td>
			<td>ThermoFischer Scientific</td>
			<td>D1306</td>
			<td>protect from light</td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>ThermoFischer Scientific</td>
			<td>P36934</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine Hydrobromide</td>
			<td>Sigma</td>
			<td>P1274</td>
			<td>the same solution can be used several times</td>
		</tr>
		<tr>
			<td>fibronectin from bovine plasma&#160;</td>
			<td>Sigma</td>
			<td>F1141</td>
			<td>the same solution can be used several times</td>
		</tr>
		<tr>
			<td>Polyethylene terephthalate (PET) inserts</td>
			<td>Falcon</td>
			<td>F3090</td>
			<td>Transparent Polyethylene terephthalate (PET) membranes; surface area: 4.2 cm2; pore size 0.4 &#181;m/surface area</td>
		</tr>
		<tr>
			<td>T75 Primo TC flask</td>
			<td>EuroClone</td>
			<td>ET7076</td>
			<td></td>
		</tr>
		<tr>
			<td>T175 Primo TC flask</td>
			<td>EuroClone</td>
			<td>ET7181</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOM2 Epithelial Tissue Volt/Ohmmeter&#160;&#160;&#160;</td>
			<td>World Precision Instruments Germany</td>
			<td>EVOM2</td>
			<td></td>
		</tr>
		<tr>
			<td>Endohm- 24SNAP cup</td>
			<td>World Precision Instruments Germany</td>
			<td>ENDOHM-24SNAP</td>
			<td></td>
		</tr>
		<tr>
			<td>Light/fluorescence microscope with camera</td>
			<td>Leica Microsystems</td>
			<td>DM IL LED Fluo/ ICC50 W Camera Module</td>
			<td>&#160;inverted microscope for live cells with camera&#160;</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Leica Microsystems</td>
			<td>TCS SPE</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro permeation of fitc-loaded ferritins across a rat blood-brain barrier: a model to study the delivery of nanoformulated molecules

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs through the blood-brain barrier (BBB), causing difficulties for effective pharmacological therapies. In recent years, different strategies for promoting brain targeting have aimed to improve drug delivery and activity at this site, including innovative nanosystems for drug delivery across the BBB. In this context, an in vitro approach based on a simplified cellular model of the BBB provides a useful tool to investigate the effect of nanoformulations on the trans-BBB permeation of molecules. This study describes the development of a double-layer BBB, consisting of co-cultured commercially available primary rat brain microvascular endothelial cells and astrocytes. A multiparametric approach for the validation of the model, based on the measurement of the transendothelial electrical resistance and the apparent permeability of a high molecular weight dextran, is also described. As proof of concept for the employment of this BBB model to study the effect of different nanoformulations on the translocation of fluorescent molecules across the barrier, we describe the use of fluorescein isothiocyanate (FITC), loaded into ferritin nanoparticles. The ability of ferritins to improve the trans-BBB permeation of FITC was demonstrated by flux measurements and confocal microscopy analyses. The results suggest this is a useful system for validating nanosystems for delivery of drugs across the BBB.

인서트의 BBB 모델, 세포 부착 및 성장의 확립 중에 PET 막의 특성상 투명 광학 현미경 감사를 사용하여 모니터링 할 수있다. 35,000 세포 / cm 2, RT (도 2A)에서 배양 4 시간 후, 인서트의 바닥면에 효과적으로 부착의 밀도로 시딩 RCA 단자와, 스핀들 형상의 형태를 가지고, 일에 막 표면을 덮도록 성장 (그림 2B). 60,000 세포 / ㎠의 밀도로 접...

Watch this Scientific Journal Video about In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules at JoVE.com

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

A method to establish an in vitro model of blood-brain barrier based on a co-culture of rat brain microvascular endothelial cells and astrocytes is described and validated. This system proved to be a valid tool to study the effect of nanoformulation on the trans-barrier permeation of fluorescent molecules.

쥐 뇌 혈관 벽의 맞은 편에 FITC로드 Ferritins의 생체 외 투과 년 : 모델 Nanoformulated 분자의 배달을 공부하기

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs ...

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In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

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Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

복원 된 인간의 각막 모양의 상피 (RHCE) 조직 모델을 사용하여 눈에 자극성 화학 물질의 위험 식별을위한 눈 자극 시험 (EIT)

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

전기 방사를 적응하는 것은 트라이 계층에 대한 세 가지 독특한 환경을 제공하기 위해<em&gt; 체외</em&gt;기도 벽의 모델

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Endosomolytic 시약 dfTAT와 배양 라이브 세포에 단백질, 펩타이드 또는 세포 투과성 작은 ​​분자의 배달

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

Use of a Rat Model to Study Ventral Abdominal Hernia Repair

복 부 복 부 헤 르 니 아 수리 공부 쥐 모델의 사용

Ventral abdominal hernia is a relatively common clinical condition that sometimes requires herniorraphy (surgical repair). The repair of ventral abdominal ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

<em&gt; 체외</em쥐의 추간판의&gt; 장기 문화 모델

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

매우 작은 공간에 팁 성장 하는 식물 세포의 신장 기능 연구를 미세 장치 개발

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...

Lucifer Yellow - A Robust Paracellular Permeability Marker in a Cell Model of the Human Blood-brain Barrier

루시퍼 옐로우 - 인간 혈액-뇌 장벽의 세포 모델에서 강력한 파라셀룰러 투과성 마커

The blood-brain barrier BBB consists of endothelial cells that form a barrier between the systemic circulation and the brain to prevent the exchange of ...