Name: comprehensive evaluation of the effectiveness and safety of placenta-targeted drug delivery using three complementary methods
Uploaded: 2018-09-10T14:30:00
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Este trabalho foi financiado por doações da Fundação Nacional de ciências naturais (81771617) e a ciência Natural Fundação da província de Guangdong (2016A030313178) concedido a X.F.; uma concessão da Shenzhen básica fundo de investigação (JCYJ20170413165233512) atribuído a X.F; e a Eunice Kennedy Shriver National Institute de saúde infantil & desenvolvimento humano do institutos nacionais da saúde sob prêmio número R01HD088549 (o conteúdo é exclusivamente da responsabilidade dos autores e não representam, necessariamente, o funcionário vista para o National Institutes of Health) de N.N.

Todos os experimentos de rato rigorosamente os protocolos (SIAT-IRB-160520-YYS-FXJ-A0232), aprovados pelo cuidado Animal e uso Comissão de Shenzhen institutos de tecnologia avançada, da Academia Chinesa de Ciências.
1. síntese de nanopartículas de lipídios-polímero direcionados A placentária sulfato de condroitina
<ol><li>Sintetizar a nanopartículas de lipídios-polímero MTX e ICG-carregado (MNPs e INPs respectivamente) e plCSA-BP-conjugados de nanopartículas (plCSA-MNPs e plCSA-I...

<ol>
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Neste manuscrito, descrevem um sistema de três-método para determinar se plCSA BP-guiada por nanopartículas são uma ferramenta eficiente para o direcionamento a entrega de drogas para a placenta. O uso do na vivo de imagem para monitorar o sinal infravermelho de ICG fluorescente confirmou a especificidade de direcionamento da placenta de plCSA-BP usando HFUS e HPLC, demonstrámos que nanopartículas plCSA-BP-conjugados com eficiência podem entregar MTX apenas para o células da placenta, não para o feto.</...

Complicações da gravidez placenta-mediada, incluindo pré-eclâmpsia, perda de gravidez, descolamento prematuro da placenta e pequena idade gestacional (SGA), são comuns e levam a substancial morbidade fetal e materna e mortalidade1,2, 3e muito poucas drogas têm provado ser eficaz para tratar a gravidez distúrbios4,5. O desenvolvimento de estratégias para a entrega da droga placenta-alvo mais seletivo e segura durante a gravidez permanece desafiador em terapia com drogas modernas.
Nos últimos anos, vários relatos têm focado a alvo entrega de drogas aos tecidos uteroplacentária por nanopartículas de revestimento com peptídeos ou anticorpos como ferramentas de placenta-alvo. Estes incluem um anticorpo de6 do fator de crescimento epidérmico anti receptor (EGFR), tumor-homing peptídeos (CGKRK e iRGD)7, peptídeos de placenta-alvo8, peptídeos placentários vasculatura-alvo9 e anticorpos contra a receptor de ocitocina10.
Aqui, demonstramos que um peptídeo sintético placentária sulfato de condroitina A ligação (plCSA-BP) pode ser usado para a entrega de alvo de nanopartículas e suas cargas de drogas para a placenta11. As nanopartículas plCSA-BP-guiadas são complementares a uteroplacentária relatada visando métodos porque alvo o trofoblasto da placenta.
Como um método não-invasivo, na vivo a imagem tem sido usada para monitorar a expressão gênica de placenta específicas em ratos12e indocianina verde (ICG) tem sido amplamente utilizada para rastrear nanopartículas usando fluorescência de imagem sistemas13, 14,15. Assim, por via intravenosa injetamos nanopartículas plCSA-BP-conjugados, carregadas com o ICG (plCSA-INPs) para visualizar a distribuição de plCSA-INP em ratos grávidas com um gerador de imagens de fluorescência. Nós, em seguida, por via intravenosa injetado metotrexato (MTX)-carregado plCSA-NPs em ratos grávidos. Ultra-som de alta frequência (HFUS), outro não-invasiva, em tempo real de imagens ferramenta16,17 foi usado para monitorar o desenvolvimento fetal e placentário nos ratos. Finalmente, usamos a cromatografia líquida de alto desempenho (HPLC) para quantificar a distribuição de MTX na placenta e fetos.
Neste protocolo, descrevemos em detalhe o sistema de três-método usado para avaliar a eficiência da entrega da droga placenta-alvo por nanocarriers plCSA-BP-guiada.

<table border="0" cellpadding="0" cellspacing="0" style="width:785px;" width="786">
	<tbody>
		<tr>
			<td>CD-1 mice</td>
			<td>Beijing Vital River</td>
			<td>201</td>
			<td>Female (8-12 week)</td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>BD</td>
			<td>328421</td>
			<td>for IV injection</td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Sinopharm Chemical</td>
			<td>10009218</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Soybean lecithin</td>
			<td>Avanti Polar Lipids</td>
			<td>441601</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>DSPE-PEG-COOH</td>
			<td>Avanti Polar Lipids</td>
			<td>880125</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>PLGA</td>
			<td>Sigma-Aldrich</td>
			<td>719897</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Ultrasonic processor</td>
			<td>Sonics</td>
			<td>VCX130</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Methotrexate (MTX)</td>
			<td>Sigma-Aldrich</td>
			<td>V900324</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Indocyanine green (ICG)</td>
			<td>Sigma-Aldrich</td>
			<td>1340009</td>
			<td>for in vivo imaging</td>
		</tr>
		<tr>
			<td>phosphate-buffered saline (PBS)</td>
			<td>Hyclone</td>
			<td>SH30028.01</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS spectrum instrument</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>for in vivo imaging</td>
		</tr>
		<tr>
			<td>Ultrasound transmission gel</td>
			<td>Guanggong</td>
			<td>ZC4252418</td>
			<td>for ultrasound imaging</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Lunan Pharmaceutical</td>
			<td>I0040</td>
			<td>for maintain the anesthesia</td>
		</tr>
		<tr>
			<td>Depilatory cream</td>
			<td>Nair</td>
			<td>TMG001</td>
			<td>for removing fur</td>
		</tr>
		<tr>
			<td>40 MHz transducer</td>
			<td>VisualSonics</td>
			<td>MS550S</td>
			<td>for ultrasound imaging</td>
		</tr>
		<tr>
			<td>High-frequency ultrasound imaging system</td>
			<td>VisualSonics</td>
			<td>Vevo2100</td>
			<td>for ultrasound imaging</td>
		</tr>
		<tr>
			<td>Avertin</td>
			<td>Sigma-Aldrich</td>
			<td>T48402</td>
			<td>for anesthesia</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Mindray</td>
			<td>SK-500III</td>
			<td>forcardiac perfusion</td>
		</tr>
		<tr>
			<td>0.9% saline solution</td>
			<td>Meilunbio</td>
			<td>MA0083</td>
			<td>forcardiac perfusion</td>
		</tr>
		<tr>
			<td>1.5 mL Polypropylene tubes</td>
			<td>AXYGEN</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>-80 &#176;C freezer</td>
			<td>Thermo Fisher Scientific</td>
			<td>88600V</td>
			<td></td>
		</tr>
		<tr>
			<td>Centriguge</td>
			<td>Cence</td>
			<td>H1650R</td>
			<td></td>
		</tr>
		<tr>
			<td>Perchloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>311421</td>
			<td>for precipitating protein</td>
		</tr>
		<tr>
			<td>Homogenizer</td>
			<td>SCIENTZ</td>
			<td>SCIENTZ-48</td>
			<td>for homogenizing tissue</td>
		</tr>
		<tr>
			<td>Syringe filter (0.45 &#956;m)</td>
			<td>Millipore</td>
			<td>SLHV033RS01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sinopharm Chemical</td>
			<td>10019763</td>
			<td>for solving MTX</td>
		</tr>
		<tr>
			<td>HPLC vials</td>
			<td>Waters</td>
			<td>670650620</td>
			<td>for HPLC</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Sinopharm Chemical</td>
			<td>20032117</td>
			<td>for HPLC</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>JKchemical</td>
			<td>932537</td>
			<td>for HPLC</td>
		</tr>
		<tr>
			<td>C18 column</td>
			<td>Waters</td>
			<td>186003966</td>
			<td>for HPLC</td>
		</tr>
		<tr>
			<td>HPLC system</td>
			<td>Shimadzu</td>
			<td></td>
			<td>for HPLC</td>
		</tr>
	</tbody>
</table>

comprehensive evaluation of the effectiveness and safety of placenta-targeted drug delivery using three complementary methods

Não há tratamentos eficazes existem, atualmente, por complicações de gravidez associada a placenta, e desenvolver estratégias para a entrega de alvo de drogas para a placenta, minimizando efeitos colaterais fetais e maternos continua a ser um desafio. Transportadoras de nanopartículas alvo proporcionam novas oportunidades para tratar anormalidades da placenta. Recentemente demonstramos que um peptídeo sintético placentária sulfato de condroitina A ligação (plCSA-BP) poderia ser usado para guiar as nanopartículas para entregar drogas para a placenta. Neste protocolo, descrevemos detalhadamente um sistema para avaliar a eficiência da entrega da droga para a placenta pela plCSA-BP que emprega três métodos distintos, usados em combinação: na vivo de imagem, ultra-som de alta frequência (HFUS) e alto desempenho cromatografia líquida (HPLC). Usando na vivo da imagem latente, plCSA BP-guiada por nanopartículas foram visualizadas nas placentas de animais vivos, enquanto HFUS e HPLC demonstraram que nanopartículas plCSA-BP-conjugados com eficiência e especificamente entregues metotrexato da placenta. Assim, uma combinação desses métodos pode ser usada como uma ferramenta eficaz para a entrega de alvo de drogas para a placenta e desenvolvimento de novas estratégias de tratamento para várias complicações na gravidez.

Neste manuscrito, carregado com MTX (plCSA-MNPs) ou ICG (plCSA-INPs) plCSA-BP-conjugados de nanopartículas foram injetadas por via intravenosa ratos grávidas. Imagem latente na vivo revelou sinais ICG fortes na região do útero 30 min após a injeção de plCSA-INP. O INPs foram localizado principalmente na região do fígado e baço (figura 1A). A 48 h após a injeção de plCSA-INP, ratos grávidas foram sacrificados, revelando sinais ICG soment...

Watch this Scientific Journal Video about Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods at JoVE.com

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

Descrevemos um sistema que utiliza três métodos para avaliar a segurança e a eficácia da entrega da droga placenta-alvo: no vivo de imagem para monitorar o acúmulo de nanopartículas, ultra-som de alta frequência para monitorar o desenvolvimento placentário e fetal e HPLC para quantificar a entrega da droga ao tecido.

Avaliação global da eficácia e segurança de entrega de droga Placenta-alvo usando três métodos complementares

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

This method can help answer key questions in the nanomedicine field about the effectiveness and the safety of drug loaded on nanoparticle delivered to the mother and the fetus. The main advantage of this technique is that it facilitates an accurate biochemical measurement of the amount of drug delivered to the placenta. The implications of this technique extend toward the therapy of placenta-mediated pregnancy complications because it's important to ensure precise targeting of the drug delivery to the placenta.Visual demonstration of this method is critical, as high-frequency ultrasound evaluation of embryonic development requires patience and experience to master. For intravenous nanoparticle injection, transfer an Embryonic Day 14.5 pregnant mouse with heat-dilated tail veins into a restrainer, and clean the tail with an alcohol swab. Then insert the needle tip of a 28-gauge insulin syringe into a dilated tail vein, and slowly depress the plunger over five to 10 seconds with even pressure to deliver the nanoparticles into the tail vein.30 minutes after the injection, place the anesthetized pregnant mouse into the imaging chamber of an in vivo fluorescence imaging system in the supine position. Select 2D fluorescence and the appropriate photographic parameters for imaging the indocyanine green fluorescence signals, and set the exposure to auto and the excitation and emission wavelengths to 710 and 820 nanometers, respectively. Then image the animal, and return the pregnant mouse to its cage.48 hours after the injection, use Graefe forceps, Graefe tissue forceps, and dissecting scissors to collect the fetuses and placentas, and image the harvested tissues as just demonstrated for the whole animal. For high-frequency ultrasound evaluation of embryonic development, 24 hours after nanoparticle injection, secure the anesthetized pregnant dam onto a 37 to 42-degree Celsius preheated ultrasound imaging platform in the supine position, and place the 40-megahertz transducer into the mechanical arm. Then adjust the transducer position to obtain longitudinal images of the fetus and placenta with the region of interest within the focal zone.For B-mode imaging and analysis, select the B-mode and lower the transducer over the abdomen until the fetus and placenta come into view. Click Scan/Freeze to initiate and stop the imaging, Cine Store to store the cine loop, and Frame Store to store the frame images. Then click Measure to analyze the gestational sac length, fetal crown rump length, biparietal diameter, abdominal circumference, placental diameter, and placental thickness.For pulse wave Doppler imaging and analysis, using the same scan projection, first click Color, then select Pulse Wave, and place the sampling volume box in the center of the umbilical artery. Click Scan/Freeze to initiate imaging and Cine Store to collect the umbilical artery images. Then click Measure to calculate the umbilical artery peak velocity.For color Doppler mode imaging and analysis, using the same scan projection, click Color, and adjust the transducer position to obtain images of the fetal heart as demonstrated. Then click Measure to calculate the fetal heart rate of the stored cine image loop. For high-pressure liquid chromatography, or HPLC, analysis, first inject an Embryonic Day 14.5 pregnant mouse with a single dose of nanoparticles as demonstrated.After 24 hours, perfuse the heart of the pregnant animal with 50 milliliters of ice-cold 0.9%saline for 10 minutes to remove any unbound nanoparticles, and collect the fetuses and placentas for minus 80-degree Celsius storage. On the day of the analysis, add 500 microliters of freshly prepared homogenization solution to each 200-milligram tissue sample, and homogenize the samples two times at full speed for 30 seconds per dissociation. After the second homogenization, centrifuge the samples and filter the supernatants through a 0.45-micrometer syringe filter into an HPLC vial.Place the sample vials into an autosampler tray for injection, and turn on the HPLC degasser to remove air from the system. Next, turn on the flow to equilibrate the column with the mobile phase for 30 minutes to reduce baseline noise, and set the temperature of the column to 25 degrees Celsius. Then inject 20-microliter sample volumes at a one milliliter per minute flow rate within 30 minutes, and click Run Method to begin the analysis.When the runs are complete, manually change the mobile phase to HPLC-grade acetonitrile, and run the acetonitrile for approximately 15 minutes to protect the system. In vivo imaging reveals strong indocyanine green signals in the uterine tissue 30 minutes after placental chondroitin sulfate-bound fluorescence-conjugated nanoparticle injection, while the unbound nanoparticles mainly localize to the liver and splenic regions. 48 hours after injection, fluorescent signal is observed in the placenta of placental chondroitin sulfate-bound nanoparticle injected animals, with no signals detected within the fetus.High-frequency ultrasound monitoring of placental chondroitin sulfate-bound fluorescence-conjugated nanoparticle injected animals reveals a significant decrease in key fetal and placental developmental landmarks. Interestingly, treatment with methotrexate-bound nanoparticles also slightly impairs fetal and placental development, indicating that the nanoparticles may improve the delivery of methotrexate to the placenta via the enhanced permeability and retention effect. The measurement of methotrexate concentrations by HPLC indicates a retention time of seven minutes within the placentas of methotrexate-bound nanoparticle-injected mice.24 hours after injection, the placental methotrexate levels in the methotrexate-bound nanoparticle-injected group are significantly lower than that measured in the placental chondroitin sulfate and methotrexate-bound nanoparticle group, with no methotrexate detected in the fetuses of the latter. Methotrexate can still be detected in the placenta 48 hours after placental chondroitin sulfate and methotrexate-bound nanoparticle injection, demonstrating that placental chondroitin sulfate and methotrexate-bound nanoparticles cannot cross the placenta, thus minimizing potential adverse effects on the fetus. While attempting this procedure, it's important to remember that the placenta begin to form around Embryonic Day 9 1/2, and therefore the in vivo imaging experiment is better performed on Embryonic Day 10 1/2 or later.Following this procedure, other methods like immunofluorescent staining and analysis can be performed to answer additional questions about localization of the nanoparticles within the placenta. After its development, this technique paved the way for researcher in the field of reproductive medicine to explore the possibility of placenta-targeting treatment in pregnancy complications.

Research

JoVE Journal

Bioengineering

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Andaimes auto-relato para cultura de células 3-Dimensional

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Bioengenharia

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Modelos e Métodos para avaliar Transporte de Drug Delivery Systems através das barreiras celulares

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

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

Adaptação do Processo Electrospinning para fornecer três ambientes únicos para um Tri-camadas<em&gt; In Vitro</em&gt; Modelo da parede das vias aéreas

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

Manufacture and Drug Delivery Applications of Silk Nanoparticles

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Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

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Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Uso do elevado-Throughput Microbioreactor sistema automatizado para produção de IgG1 modelo em células CHO

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Purificação e análise de um anticorpo monoclonal de células de ovário de hamster chinês usando um sistema de Microbioreator automatizado

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Avaliação da proliferação queratinócito em duas e três dimensões tipo I colágeno substratos

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

Preparando proteínas que produzem células sintéticas usando extratos bacterianos livres de células, liposomos e transferência de emulsão

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Esclarecimento Primário do Fluido de Cultura Celular Colhida cho usando um separador acústico

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...