Name: comprehensive evaluation of the effectiveness and safety of placenta-targeted drug delivery using three complementary methods
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Detta arbete stöds av anslag från National Natural Sciences Foundation (81771617) och naturvetenskap Foundation i Guangdongprovinsen (2016A030313178) tilldelas X.F.; ett bidrag från Shenzhen grundläggande forskningsfonden (JCYJ20170413165233512) tilldelas X.F; och Eunice Kennedy Shriver National Institute of Child Health & mänsklig utveckling av det nationella Institutes of Health under Award nummer R01HD088549 (innehållet ansvarar enbart för författarna och representerar inte nödvändigtvis officiella visningar av National Institutes of Health) att N.N.

Alla musen experiment följde strikt protokoll (SIAT-IRB-160520-YYS-FXJ-A0232) godkänd av djur vård och användning kommittén av Shenzhen institut av avancerad teknik, kinesiska vetenskapsakademin.
1. Sammanfattning av placenta Chondroitin sulfat A-riktade Lipid-polymera nanopartiklar
<ol><li>Syntetisera MTX - och ICG-loaded lipid-polymera nanopartiklar (MNPs och INPs respektive) och plCSA-BP-konjugerad nanopartiklar (plCSA-MNPs och plCSA-INPs) som beskrivs i detalj på annan plats<sup cl...

<ol>
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Vi Beskriv i detta manuskript, en tre-metoden system för att avgöra om plCSA-BP-guidad nanopartiklar är ett effektivt verktyg för att rikta leverans av läkemedel till moderkakan. Användning av in-vivo imaging för att övervaka den infraröda fluorescerande ICG signalen bekräftade placenta inriktning idrottens plCSA-BP. med HFUS och HPLC, vi visat att plCSA-BP-konjugerad nanopartiklar effektivt kan leverera MTX endast till den moderkakan celler, inte för fostret.
I vivo</e...

Placenta-medierad graviditetskomplikationer, inklusive havandeskapsförgiftning, missfall, moderkaksavlossning och små gestationsålder (SGA), är vanliga och leda till betydande fetal och maternell sjuklighet och dödlighet1,2, 3, och mycket få läkemedel har visat sig vara effektivt för behandling av graviditet störningar4,5. Utvecklingen av strategier för mer selektiv och säkrare placenta-targeted drug delivery under graviditet förblir utmanande i modern läkemedelsbehandling.
Under de senaste åren har flera rapporter fokuserat på riktade leverans av läkemedel till uteroplacentalt vävnader av beläggning nanopartiklar med peptider eller antikroppar som moderkakan riktade verktyg. Dessa inkluderar en anti-epidermal tillväxtfaktor receptor (EGFR)6 antikropp, tumör-homing peptider (CGKRK och iRGD)7, moderkakan riktade peptider8, placenta vaskulatur riktade peptider9 och antikroppar mot de oxytocin-receptorn10.
Här visar vi att en syntetisk placenta chondroitin sulfat A bindande peptid (plCSA-BP) kan användas för riktade leverans av nanopartiklar och deras drog nyttolaster till moderkakan11. De plCSA-BP-guidad nanopartiklarna är ett komplement till de rapporterade uteroplacentalt inriktningsmetoder eftersom de rikta moderkakan trofoblaster.
En icke-invasiv metod, i vivo imaging har använts för att övervaka placenta-specifika genuttryck i möss12samt indocyanine green (ICG) har använts i stor utsträckning att spåra nanopartiklar med fluorescens imaging system13, 14,15. Således injiceras vi intravenöst plCSA-BP-konjugerad nanopartiklar laddad med ICG (plCSA-INPs) att visualisera plCSA-INP distribution hos dräktiga möss med en fluorescens imager. Vi sedan injiceras intravenöst metotrexat (MTX)-laddade plCSA-NPs i dräktiga möss. Högfrekvent ultraljud (HFUS), en annan icke-invasiv, realtid imaging verktyg16,17 användes för att övervaka fostrets och placenta utveckling i möss. Slutligen, vi brukade högpresterande vätskekromatografi (HPLC) kvantifiera MTX distribution i moderkakor och foster.
I detta protokoll beskriver vi i detalj systemet tre-metoden används för att bedöma effektiviteten av placenta-targeted drug leverans av plCSA-BP-guidad nanocarriers.

<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

Det finns för närvarande inga effektiva behandlingar för placenta-associerade komplikationer, och utveckla strategier för riktade leverans av läkemedel till moderkakan samtidigt minimera fetal och maternell biverkningar förblir utmanande. Riktade nanopartiklar bärare ger nya möjligheter att behandla sjukdomar i placenta. Vi har nyligen visat att en syntetisk placenta chondroitin sulfat A bindande peptid (plCSA-BP) kan användas för att vägleda nanopartiklar för att leverera läkemedel till moderkakan. I detta protokoll, beskriver vi i detalj ett system för att bedöma effektiviteten av drogen leverans till moderkakan genom plCSA-BP som sysselsätter tre separata metoder som används i kombination: i vivo imaging, högfrekvent ultraljud (HFUS) och hög prestanda vätskekromatografi (HPLC). Hjälp i vivo var imaging, plCSA-BP-guidade nanopartiklar visualiseras i moderkakor av levande djur, medan HFUS och HPLC visat att plCSA-BP-konjugerad nanopartiklar effektivt och specifikt levereras metotrexat till moderkakan. Således kan en kombination av dessa metoder användas som ett effektivt verktyg för den riktade leveransen av läkemedel till moderkakan och utvecklingen av nya behandlingsstrategier för flera komplikationer under graviditeten.

I detta manuskript, skulle plCSA-BP-konjugerad nanopartiklar laddad med Metotrexat (plCSA-MNPs) eller ICG (plCSA-INPs) injiceras intravenöst i dräktiga möss. In vivo imaging visade stark ICG signaler i regionen i livmodern 30 min efter plCSA-INP injektion. INPs var huvudsakligen lokaliserade till regionen lever och mjälte (figur 1A). På 48 h efter plCSA-INP injektion offrades dräktiga möss, avslöjar ICG signaler endast i moderkakan, medan med...

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

Vi beskriver ett system som använder tre metoder för att utvärdera säkerheten och effektiviteten av placenta-targeted drug delivery: i vivo imaging för att övervaka nanopartiklar ackumulering, högfrekvent ultraljud för att övervaka placenta och fostrets utveckling , och HPLC att kvantifiera drogen leverans till vävnad.

Omfattande utvärdering av effektiviteten och säkerheten av Placenta-Targeted Drug Delivery med tre kompletterande metoder

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

Självrapporterings Ställningar för 3-dimensionell cellodling

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

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

Modeller och metoder för att utvärdera Transport av Drug Delivery System Across Cellular Hinder

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

Anpassning av Electro processen med att ge tre unika miljöer för en Tri-lager<em&gt; In Vitro</em&gt; Modell av Airway Wall

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

Tillverkning och Drug Delivery Tillämpningar av Silk Nanopartiklar

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

Automatiserad Contraction analys av Human Engineered hjärtvävnad för Cardiac Drug Safety Screening

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

Användning av High-Throughput automatiserat Microbioreactor System för produktion av modell IgG1 i CHO-celler

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

Rening och analys av en monoklonal antikropp från äggstocksceller från kinesisk hamster med hjälp av ett automatiserat Mikrobioreaktor system

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

Utvärdering av keratinocyter spridning på två - och tredimensionella typ I kollagen substrat

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

Förbereda proteinproducerande syntetiska celler med cellfria bakterieextrakt, liposomer och emulsion överföring

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

Primärt förtydligande av CHO skördad cellodlingsvätska med hjälp av en akustisk separator

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