Name: comprehensive evaluation of the effectiveness and safety of placenta-targeted drug delivery using three complementary methods
Uploaded: 2018-09-10T14:30:00
Description: 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

Questo lavoro è stato sostenuto da borse di studio dalla Fondazione nazionale di scienze naturali (81771617) e la scienza naturale Fondazione della provincia di Guangdong (2016A030313178) assegnato a X.F.; una sovvenzione dalla Shenzhen Basic Research Fund (JCYJ20170413165233512) assegnato a X.F; Eunice Kennedy Shriver National Institute of Child Health e lo sviluppo umano del National Institutes of Health, sotto Premio numero R01HD088549 (il contenuto è di esclusiva responsabilità degli autori e non rappresentano necessariamente il funzionario viste del National Institutes of Health) di N.N.

Tutti gli esperimenti del mouse seguito rigorosamente i protocolli (SIAT-IRB-160520-YYS-FXJ-A0232) approvati dalla cura degli animali e uso Comitato di Shenzhen Institutes of Advanced Technology, Accademia cinese delle scienze.
1. sintesi di nanoparticelle mirate A lipido-polimero placentare Condroitin solfato
<ol><li>Sintetizzare MTX - e ICG-caricati del lipido-polimero nanoparticelle (MNPs e INPs rispettivamente) e nanoparticelle plCSA-BP-coniugato (plCSA-MNPs e plCSA-INPs) come descritto ...

<ol>
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In questo manoscritto, delineiamo un sistema di tre-metodo per determinare se le nanoparticelle plCSA-BP-guida sono uno strumento efficace per la consegna di farmaci alla placenta di targeting. L'uso di in vivo imaging per monitorare il segnale a infrarossi fluorescente ICG confermato la specificità di targeting placenta di plCSA-BP. Using HFUS e HPLC, abbiamo dimostrato che nanoparticelle plCSA-BP-coniugato possono fornire in modo efficiente MTX solo per il cellule della placenta, non per il feto.
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Complicazioni di gravidanza placenta-mediata, tra cui pre-eclampsia, la perdita di gravidanza, distacco della placenta e piccola età gestazionale (SGA), sono comuni e portare a sostanziale morbilità materna e fetale e mortalità1,2, 3e molto pochi farmaci hanno dimostrato di essere efficace per il trattamento di gravidanza disturbi4,5. Lo sviluppo di strategie per la consegna di droga placenta-mirati più selettivi e più sicura durante la gravidanza rimane impegnativo nella terapia farmacologica moderna.
Negli ultimi anni, parecchi rapporti sono concentrati sulla somministrazione mirata di farmaci di uteroplacental tessuti di rivestimento di nanoparticelle con peptidi o anticorpi come strumenti di targeting per placenta. Questi includono un anticorpo di anti-fattore di crescita recettore (EGFR)6 , tumore-homing peptidi (CGKRK e iRGD)7, placenta-mirato peptidi8, peptidi di targeting per sistema vascolare placentare9 e gli anticorpi contro il L'ossitocina del ricevitore10.
Qui, dimostriamo che un peptide sintetico placentare Condroitin solfato A associazione (plCSA-BP) può essere utilizzato per la somministrazione mirata di nanoparticelle e loro carichi di droga di placenta11. Le nanoparticelle plCSA-BP-guidati sono complementari alla uteroplacental segnalati metodi di targeting, perché prendono di mira il trofoblasto placenta.
Come un metodo non invasivo, in vivo imaging è stato utilizzato per monitorare l'espressione genica placenta-specifica in topi12e verde di indocianina (ICG) è stato ampiamente utilizzato per tenere traccia di nanoparticelle utilizzando sistemi13, di formazione immagine di fluorescenza 14,15. Così, abbiamo iniettato per via endovenosa plCSA-BP-coniugato nanoparticelle caricate con ICG (plCSA-INPs) per visualizzare la distribuzione di plCSA-INP in topi incinta con un imager di fluorescenza. Abbiamo quindi iniettato per via endovenosa di methotrexate (MTX)-caricato plCSA-NPs in topi incinti. L'ultrasuono ad alta frequenza (HFUS), un altro non-invasiva, in tempo reale strumento16,di imaging17 è stato utilizzato per monitorare lo sviluppo fetale e placenta nei topi. Infine, abbiamo usato ad alte prestazioni cromatografia liquida (HPLC) per quantificare la distribuzione di MTX in placente e feti.
In questo protocollo, descriviamo dettagliatamente il sistema di tre-metodo utilizzato per valutare l'efficienza di consegna della droga placenta-mirati da nanovettori plCSA-BP-guidata.

<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

Nessun trattamenti efficaci attualmente esistenti per le complicazioni di gravidanza placenta-collegata, e lo sviluppo di strategie per la somministrazione mirata di farmaci alla placenta, riducendo al minimo gli effetti collaterali materni e fetali rimane impegnativo. I vettori delle nanoparticelle mirate fornire nuove opportunità per trattare i disordini placentari. Recentemente abbiamo dimostrato che un peptide sintetico placentare Condroitin solfato A associazione (plCSA-BP) potrebbe essere usato per guidare le nanoparticelle per consegnare i farmaci alla placenta. In questo protocollo, descriviamo dettagliatamente un sistema per valutare l'efficienza di consegna della droga alla placenta di plCSA-BP che impiega tre metodi distinti utilizzati in combinazione: in vivo imaging, ultrasuono ad alta frequenza (HFUS) e ad alte prestazioni cromatografia liquida (HPLC). Utilizzo in vivo imaging, plCSA-BP-guidata nanoparticelle sono state visualizzate in placente di animali vivi, mentre HFUS e HPLC ha dimostrato che nanoparticelle plCSA-BP-coniugato in modo efficiente e in particolare consegnato metotrexato alla placenta. Così, una combinazione di questi metodi può essere utilizzata come strumento efficace per la somministrazione mirata di farmaci alla placenta e lo sviluppo di nuove strategie di trattamento per diverse complicazioni di gravidanza.

In questo manoscritto, plCSA-BP-coniugato nanoparticelle caricato con MTX (plCSA-MNPs) o ICG (plCSA-INPs) sono state iniettate per via endovenosa in topi incinti. In vivo imaging ha rivelato segnali forti di ICG nella regione dell'utero 30 min dopo l'iniezione plCSA-INP. L'INPs sono stata localizzata principalmente nella regione di fegato e milza (Figura 1A). A 48 h dopo l'iniezione plCSA-INP, incinto topi sono stati sacrificati, rivelando ICG segnal...

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

Descriviamo un sistema che utilizza tre metodi per valutare la sicurezza e l'efficacia della somministrazione di farmaci mirati placenta: in vivo imaging per monitorare l'accumulo delle nanoparticelle, ultrasuono ad alta frequenza per monitorare lo sviluppo placenta e fetale e HPLC per quantificare la consegna della droga al tessuto.

Valutazione globale dell'efficacia e della sicurezza di Placenta-Targeted Drug Delivery utilizzando tre metodi complementari

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

Research

JoVE Journal

Bioengineering

Self-reporting Scaffolds for 3-Dimensional Cell Culture

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

Ricerca

Bioingegneria

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

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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

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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

Automated Analysis Contrazione di ingegneria dei tessuti umani Cuore per lo screening cardiaco Drug Safety

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

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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

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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

Valutazione della proliferazione dei cheratinociti su bi - e tri - dimensionale substrati di collagene di tipo I

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

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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

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