Name: synthesis and characterization of placental chondroitin sulfate a (plcsa)-targeting lipid-polymer nanoparticles
Uploaded: 2018-09-18T14:00:00
Description: Watch this Scientific Journal Video about Synthesis and Characterization of Placental Chondroitin Sulfate A plCSA-Targeting Lipid-Polymer Nanoparticles at JoVE.com

Dette arbejde blev støttet af tilskud fra den nationale nøgle-forskning og udvikling Program i Kina (2016YFC1000402), National Natural Sciences Foundation (81571445 og 81771617) og Natural Science Foundation of Guangdong provins (2016A030313178) til X.F. og Shenzhen Basic Research Fund (JCYJ20170413165233512) til X.F.

1. fremstilling af stamopløsninger
<ol><li>Forberede en vandig opløsning af 4% ethanol ved fortynding af 4 mL af absolut ethanol med 100 mL i ultrarent vand. Butik løsning ved 4 ° C. Bemærk: Ultrarent vand er defineret som vand uden forurenende stoffer såsom bakterier, partikler, ioner eller nukleaser. Ultrarent vand blev indhentet fra et vandrensningssystem med en target resistivitet af op til 18.2 mΩ·cm, hvilket betyder lav anioniske forurening.</li>
	<li>Forberede en 1 mg/mL soja lecithin stamopløsning v...

<ol>
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Denne protokol giver en effektiv og reproducerbar metode til syntese af plCSA-BP-konjugeret lipid-polymer nanopartikler. Trinvis sonikering metode at forberede lipid-polymer nanopartikler er hurtig, reproducerbare og adskiller sig fra typiske nanoprecipitation metoder, der involverer varme, vortexing eller fordampning. Derfor reducerer udviklede metoden syntese tid. Derudover er EDC/NHS bioconjugate anvendes i denne protokol et almindeligt anvendte og praktisk teknik til konjugat peptider og antistoffer for nanopartikler...

En effektiv kræft terapeutiske metode reducerer og eliminerer tumorer med minimal systemisk toksicitet. Dermed er selektiv tumor målretning nøglen til at udforske vellykket terapeutiske metoder. Nanopartikler giver en lovende mulighed for kræftbehandling, og molekylære enheder med forskellige funktionelle grupper vil forbedre drug effektivitet og reducere forbundet bivirkninger1,2. Derudover udnytter nanopartikel systemer hovedsagelig passive og aktive målretning til at nå målet tumorer3.
Passiv målretning udnytter de medfødte egenskaber af nanopartikler og øget permeabilitet og opbevaring (EPR) effekter at nå tumorceller. Kationiske Liposomer har held været anvendt til at levere forskellige anticancermedicin til tumorer i kliniske anvendelser4,5,6. Trods den potentielle effektive kræft terapeutiske effekt, en lav drug koncentration i regionen tumor og manglende evne til at skelne mellem tumorceller fra normalt væv er to store begrænsninger i passiv-targeting nanopartikler7.
Aktive målretning strategier drage fordel af antigen-antistof, ligand-receptor og andre Molekylær anerkendelse interaktioner specifikt levere lægemidler til tumorer8. Glykosaminoglykan placenta Chondroitinsulfat en (plCSA) udtrykkes bredt på de fleste kræftceller og placenta trophoblasts. Derudover malaria proteinet VAR2CSA kan specifikt binder til plCSA9,10. Derfor kan VAR2CSA være et redskab til at målrette menneskelige kræftceller. Men når VAR2CSA er konjugeret med nanopartikler, fuldlængde protein kan begrænse indtrængen af nanopartikler i tumorceller. For nylig opdagede vi en plCSA bindende peptid (plCSA-BP), afledt af malaria proteinet VAR2CSA. plCSA-BP-konjugeret lipid-polymer nanopartikler hurtigt bundet til choriocarcinoma celler og signifikant øget doxorubicin (DOX) anticancer aktivitet i vivo11; disse partikler også specifikt bundet til placenta trophoblasts og kunne tjene som et redskab til målrettet levering af lægemidler til moderkagen12.
Lipid-polymer nanopartikler består af en lipid éncellelag shell og en hydrofobe polymere kerne og repræsenterer en ny transportør for drug delivery. Disse nanopartikler kombinere fordelene ved Liposomer og polymere nanocarriers, kontrollerbar nanopartikel størrelse, høj biokompatibilitet, vedvarende drug frigivelse, høj drug lastning effektivitet (LE) og fremragende stabilitet13. I dette arbejde brugte vi en trinvis sonikering metode til at syntetisere lipid-polymer nanopartikler. Denne metode er hurtig, bekvem og egnet til opskalering og har været meget anvendt til at forberede lipid-polymer nanopartikler af vores gruppe11,14 og andre15,16,17,18 .
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochlorid (EDC) er en populær carbodiimide anvendes som en crosslinking agent for de tråddannende biomolekyler indeholdende aminer og carboxylates19. Ud over EDC er N-hydroxysulfosuccinimide (NHS) den mest almindelige konjugation reagens i overfladen og nanopartikel konjugation reaktioner20,21. NHS kan reducere antallet af side reaktioner og øge stabiliteten og udbytte af ester mellemprodukter22,23.
Her, beskriver vi en protokol for syntese af plCSA-målrettet lipid-polymer nanopartikler. Først, trinvis sonikering syntesen af DOX-loaded lipid-polymer nanopartikler (DNPs) er beskrevet. Derefter, en EDC/NHS bioconjugate teknik til at generere plCSA-BP-konjugeret lipid-polymer nanopartikler er indført. Denne bioconjugate teknik kan også bruges til konjugat andre antistoffer og peptider for nanopartikler. Endelig vil beskrive vi fysisk-kemiske egenskaber og in vitro- analysen bruges til at karakterisere plCSA-målrettet lipid-polymer nanopartikler. Vi mener, at disse plCSA-målrettet lipid-polymer nanopartikler kunne udgøre et effektivt system for målrettet levering af lægemidler til de fleste menneskelige kræftformer og målrettet levering af lasterne til moderkagen til behandling af placenta lidelser.

<table>
	<tbody>
		<tr>
			<td>plCSA peptide</td>
			<td>Shanghai GL Biochem</td>
			<td>573518</td>
			<td>for peptide synthesis</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>Doxorubicin</td>
			<td>JKChemical</td>
			<td>113424</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Shanghai Lingfeng</td>
			<td>1008621</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>Centrifuge filter (MWCO 10 kDa)</td>
			<td>Millipore</td>
			<td>UFC801024</td>
			<td>for nanoparticles purification</td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>Sigma</td>
			<td>3-18KS</td>
			<td>for nanoparticles purification</td>
		</tr>
		<tr>
			<td>2-[morpholino]ethanesulfonic acid(MES)</td>
			<td>Sigma-Aldrich</td>
			<td>M3671</td>
			<td>for peptide conjugation</td>
		</tr>
		<tr>
			<td>1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)</td>
			<td>Sigma-Aldrich</td>
			<td>3450</td>
			<td>for peptide conjugation</td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide (NHS)</td>
			<td>Sigma-Aldrich</td>
			<td>56480</td>
			<td>for peptide conjugation</td>
		</tr>
		<tr>
			<td>Dialysis bags</td>
			<td>Spectrum</td>
			<td>132592T</td>
			<td>for nanoparticles purification</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Hyclone</td>
			<td>SH30028.01</td>
			<td>for cell culture</td>
		</tr>
		<tr>
			<td>10 mL centrifuge tubes, polypropylene</td>
			<td>Aladdin</td>
			<td>S-025</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes, polypropylene</td>
			<td>Corning</td>
			<td>430791</td>
			<td>for various applications</td>
		</tr>
		<tr>
			<td>0.22 &#956;m sterile syringe filter</td>
			<td>Millipore</td>
			<td>SLGV033RB</td>
			<td>for nanoparticles purification</td>
		</tr>
		<tr>
			<td>1 ml syringe, polypropylene</td>
			<td>BD</td>
			<td>328421</td>
			<td>for nanoparticles synthesis</td>
		</tr>
		<tr>
			<td>Malvern Zetasizer</td>
			<td>Malvern</td>
			<td>Nano ZS</td>
			<td>for particle size analyer</td>
		</tr>
		<tr>
			<td>Phosphotungstic acid</td>
			<td></td>
			<td></td>
			<td>for TEM</td>
		</tr>
		<tr>
			<td>TEM grid</td>
			<td>EMCN</td>
			<td>BZ10024a</td>
			<td>for TEM</td>
		</tr>
		<tr>
			<td>UV-VIS spectrometer</td>
			<td>Leagene</td>
			<td>DZ0035</td>
			<td>for TEM</td>
		</tr>
		<tr>
			<td>Transmission 
			electron microscope</td>
			<td>JEOL</td>
			<td>JEM-100CXII</td>
			<td>for particle size analyer</td>
		</tr>
		<tr>
			<td>BCA reagent A</td>
			<td>Thermo Fisher Scientific</td>
			<td>23228</td>
			<td>for BCA assay</td>
		</tr>
		<tr>
			<td>BCA reagent B</td>
			<td>Thermo Fisher Scientific</td>
			<td>23224</td>
			<td>for BCA assay</td>
		</tr>
		<tr>
			<td>96-Well Plates</td>
			<td>Corning</td>
			<td>3599</td>
			<td>for BCA assay</td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>Thermo Fisher Scientific</td>
			<td>Multiskan&#8482; GO</td>
			<td>for BCA assay</td>
		</tr>
		<tr>
			<td>12-well plates</td>
			<td>Corning</td>
			<td>3513</td>
			<td>for cell culture</td>
		</tr>
		<tr>
			<td>JEG3 cell</td>
			<td>Cell Bank of the Chinese Academy of Sciences</td>
			<td>TCHu195</td>
			<td>Human placenta</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Hyclone</td>
			<td>SH30272.01</td>
			<td>phenol red-free</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>GIBCO</td>
			<td>10100</td>
			<td>for cell culture</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>GIBCO</td>
			<td>15070063</td>
			<td>for cell culture</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>OLYMPUS</td>
			<td>CKK53</td>
			<td>for celluar uptake</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Shanghai Lingfeng</td>
			<td>1372021</td>
			<td>for celluar uptake</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sangon Biotech</td>
			<td>A606584</td>
			<td>for celluar uptake</td>
		</tr>
		<tr>
			<td>Mounting medium</td>
			<td>Life</td>
			<td>P36961</td>
			<td>for celluar uptake</td>
		</tr>
	</tbody>
</table>

synthesis and characterization of placental chondroitin sulfate a (plcsa)-targeting lipid-polymer nanoparticles

En effektiv kræft terapeutiske metode reducerer og eliminerer tumorer med minimal systemisk toksicitet. Aktivt målretning nanopartikler tilbyder en lovende tilgang til kræftbehandling. Glykosaminoglykan placenta Chondroitinsulfat en (plCSA) er udtrykt på en bred vifte af kræftceller og placenta trophoblasts og malaria protein VAR2CSA kan specifikt binder sig til plCSA. En rapporteret placenta chondroitin sulfat A bindende peptid (plCSA-BP), afledt af malaria protein VAR2CSA, kan også specifikt binder sig til plCSA på kræftceller og placenta trophoblasts. Derfor kunne plCSA-BP-konjugeret nanopartikler bruges som et redskab til målrettet medicinafgivelse menneskelige kræftformer og placenta trophoblasts. I denne protokol beskriver vi en metode til at syntetisere plCSA-BP-konjugeret lipid-polymer nanopartikler fyldt med doxorubicin (plCSA-DNPs); Metoden består af en enkelt sonikering trin og bioconjugate teknikker. Derudover er flere metoder til at karakterisere plCSA-DNPs, herunder fastsættelsen af fysisk-kemiske egenskaber og cellulære optagelse af placenta choriocarcinoma (JEG3) celler, beskrevet.

I denne protokol, PLGA, DSPE-PIND-COOH og soja lecithin er et repræsentativt polymer, lipid-PIND-COOH konjugat og lipid, henholdsvis. Syntesen af plCSA-målrettet lipid-polymer nanopartikler via en enkelt-trins sonikering metode og EDC/NHS-teknik er illustreret i figur 1. Først under sonikering betingelser, soja lecithin, PLGA og DSPE-PIND-COOH selv samle til form core-shell struktureret DNPs. Kernen består af PLGA og indkapslet DOX og skallen bes...

Watch this Scientific Journal Video about Synthesis and Characterization of Placental Chondroitin Sulfate A plCSA-Targeting Lipid-Polymer Nanoparticles at JoVE.com

Synthesis and Characterization of Placental Chondroitin Sulfate A plCSA-Targeting Lipid-Polymer Nanoparticles

Vi præsenterer her, en protokol til syntese af placenta chondroitin sulfat A bindende peptid (plCSA-BP)-konjugeret lipid-polymer nanopartikler via trinvis ultralydbehandling og bioconjugate teknikker. Disse partikler udgør en roman redskab til målrettet levering af therapeutics til mest humane tumorer og placenta trophoblasts til behandling af kræft og placenta lidelser.

Syntese og karakterisering af placenta Chondroitin sulfat en (plCSA) - målretning Lipid - Polymer nanopartikler

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising ...

Denne metode kan hjælpe med at besvare centrale spørgsmål i narkotika levering område, såsom målrettet levering af terapeutiske til humane kræftformer og moderkagen cystoblast. Den største fordel ved denne teknik er, at det er en enkel og reproducerbar metode til at syntetisere peptider konjugeret i andre partikler. Til at begynde, tilføje tre millimeter af fire procent ethanol til en steril 10 milliliter centrifuge rør.Derefter tilsættes 90 mikrogram sojabønnebestandsopløsning, 210 mikrogram DSPE-PEG-carboxylsyreopløsning og 750 mikrogram DOX-lageropløsning. Brug en en milliliter sprøjte til pipette to milligram PLGA lager opløsning. Placer centrifugerøret i et isbad på en ultralydsprocessor og soniker i to minutter.Derefter tilsættes PLGA lageropløsning drop-wise i centrifugerøret. Hvis du vil rense DNP'erne, skal du bruge et centrifugefilter til at vaske opløsningen i 0,1 molar MES-buffer. Derefter centrifuge opløsningen ved 1000 G i tre minutter ved fire grader Celsius.Til at begynde ester aktivering, tilføje 0.4 milligram EDC til en milliliter DNPs. Derefter, tilføje 0.24 milligram NHS til reaktionen. Lad derefter reaktionen finde sted i op til en time ved stuetemperatur i mørke.Derefter blandes reaktionskomponenterne grundigt, og reaktionen på en shaker sættes på. For at starte aminreaktionen skal du bruge PBS til at øge bufferpH-zonen fra 7,2 til 7,5. Derefter, tilføje 0.5 milligram pICSA målretning peptid til reaktionsopløsningen.Bland opløsningen godt og læg den på en shaker. Lad derefter reaktionen fortsætte ved fire grader Celsius natten over i mørket. Den næste dag fyldes dialyseposer med konjugatopløsningen.Rens pICSA DNP'erne med PBS-buffer ved stuetemperatur i 24 timer i mørke. Efter dyrkning celler i henhold til tekstprotokollen, fjerne medierne og tilføje en milliliter friske, kolde medier med fem procent FBS og pICSA DNPs eller DNPs. Derefter inkuberes celle- og nanopartikelblandingen i en time ved fire grader Celsius.Derefter skal du fjerne mediet og vaske cellerne tre gange med PBS. Derefter tilsættes en milliliter friske medier og inkubere cellerne i 30 minutter ved 37 grader Celsius. Derefter skal du fjerne mediet og vaske cellerne tre gange med PBS.Derefter tilsættes to milliliter kulde, fire procent PFA og inkubere blandingen ved stuetemperatur i 10 minutter. Fjern PFA og vask cellerne med to milliliter PBS. Derefter tilsættes en milliliter PBS med DAPI for kernefarvning.Lad blandingen inkubere ved stuetemperatur i 10 minutter. Endelig tilføje montering medium og billede fluorescens med en fluorescens mikroskop. Ved hjælp af denne protokol blev DNP'er forberedt og konjugeret til pICSA BP. Efter bøjningen steg diameteren af de primære DNP'er til ca. 109 nanometer.TEM-billederne af DNP'erne og pICSA-DNP'erne viste, at partiklerne var godt spredte og generelt viste sfærisk morfologi. Nanopartiklernes zetapotentiale var negativt 20,1 og 29,9 millivolt, hvilket indikerer, at nanopartiklerne var meget stabile. DEN I3 cellulære optagelsesanalyse indikerede pICSA DNPs bundet til JEG3 celler inden for 30 minutter.Derfor blev pICSA BP effektivt konjugeret til overfladen. Mens du forsøger denne procedure, er det vigtigt at huske at sonikere blandingen i et til to minutter, før du tilføjer PLGA. Efter denne procedure, vores metoder som HPLC analyse kan udføres for at bestemme bøjning effektivitet mere præcist.Efter sin udvikling, denne teknik banede vejen for forskere inden for reproduktiv biologi til at undersøge muligheden for moderkagen-målretning behandling i graviditeten komplikationer.

Research

JoVE Journal

Cancer Research

Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles

Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NF&#954;B Activity and Breast Cancer Stem Cells

Synthesis and Characterization of an Aspirin-fumarate Prodrug that Inhibits NFÃƒÆ’Ã…Â½Ãƒâ€šÃ‚ÂºB Activity and Breast Cancer Stem Cells

Syntese og karakterisering af en Aspirin-fumarat prodrug, Hæmmer NFKB Aktivitet og brystkræft Stamceller

Inflammation is a cancer hallmark that underlies cancer incidence and promotion, and eventually progression to metastasis. Therefore, adding an ...

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Anticancer Effekten af ​​Fotodynamisk terapi med lungekræft-Målrettet Nanopartikler

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Target Cell pre berigelse og hele genom forstærkning for enkelt celle Downstream karakterisering

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

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Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

Isolering og karakterisering af tumor initierende celler fra sarkom patient afledte Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

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The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

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Isolation and Characterization of Patient-derived Pancreatic Ductal Adenocarcinoma Organoid Models

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Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies. Recently, next-generation organoid culture methods enabling the ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

Generation af Ortotopiske tumorer i bugspytkirtlen og ex vivo karakterisering af tumor Infiltrerende T-celle cytotoksicitet

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...