Name: preparation and characterization of lipophilic doxorubicin pro-drug micelles
Uploaded: 2016-08-02T15:00:00
Description: Watch this Scientific Journal Video about Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles at JoVE.com

This work was supported by the following grants: NIH-SC3 grant, NSF-PREM grant, Hampton University Faculty Research Grant. We would like to thank Mrs. Michele A. Cochran at Virginia Institute of Marine Science (VIMS) for the use of the particle size analyzer. We would also like to thank Mrs. Corinne R. Ramaley for reviewing the manuscript.

1. Syntese af DOX-PA <ol><li> Afvej 390 mg doxorubicin og 243 mg palmitinsyre hydrazid, og overføres til en rundbundet kolbe. </li><li> Tilføj 150 ml vandfrit methanol til kolben med et glas sprøjte. Tilføj 39 pi trifluoreddikesyre (TFA) med en pipette. Anvendelse af en magnetisk omrører, reaktionsblandingen omrøres i 18 timer ved stuetemperatur i mørke. BEMÆRK: Mængden af reaktions- materialer kan skaleres op eller ned for at opnå forskellige mængder af DOX-PA. Forholdet mellem reak...

<ol>
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I dette arbejde beskriver vi en ukompliceret, hurtig film-dispersion fremgangsmåde til fremstilling af miceller. Denne metode udnytter selvsamlende egenskaber af en amfifil polymer (f.eks DSPE-PEG) til dannelse af kerne-skal strukturerede miceller i et vandigt miljø. Denne micelle fremstillingsmetode har flere fordele. 1. Den omfatter en simpel formulering proces, som undgår anvendelsen af komplicerede trin størrelse-reduktion (såsom ekstrudering eller homogenisering), almindeligvis anvendes i fremstilling...

Kemoterapi er almindeligt anvendt til behandling af forskellige former for kræft. De fleste, hvis ikke alle, kemoterapi narkotika har toksiske bivirkninger, som kan variere fra håndterbare mindre forhold, såsom kvalme og diarré, til mere livstruende tilstande. Fordi de fleste anticancerlægemidler er giftige, ikke-selektiv eksponering af disse lægemidler til normale væv uvægerligt forårsager toksicitet. Der er derfor et stort behov for en terapeutisk tilgang, der selektivt kan tilføre lægemidler til kræftceller. En anden udfordring med indgivelsen af ​​lægemidler mod cancer er deres ringe vandopløselighed. Normalt er der behov solubiliseringsmidler at formulere disse tungtopløselige lægemidler. Fleste solubiliseringsmidler, såsom dimethylsulfoxid (DMSO), Cremophor EL, og polysorbat 80 (Tween 80), kan imidlertid forårsage lever og nyre toksicitet, hæmolyse, akutte overfølsomhedsreaktioner og perifere neuropatier. 1 Derfor er der behov for sikre og biokompatible formuleringer til den kliniske anvendelse af fattigely opløselige anticancer lægemidler. Nanocarriers er lovende drug delivery systemer til opfyldelse af ovennævnte udfordringer. Disse nanocarriers omfatter liposomer, 2 nanopartikler, 3 miceller, 4-7 polymer-lægemiddel-konjugater, 8 og uorganiske materialer. 9 Flere nanomedicin produkter (f.eks Doxil, Abraxane, og Genexol) er blevet godkendt af de regulerende instanser til behandling af kræftpatienter. 10 Polymere miceller er lovende nano-skala lægemiddelafgivelsessystemer luftfartsselskaber, der med succes er blevet anvendt til levering af lægemidler mod cancer. 4-7,11,12 Typiske polymere miceller er fremstillet af amfifile polymerer gennem en selvstændig samling proces. Kerne-skal strukturerede polymermiceller omfatter en hydrofil skal og en hydrofob kerne. Den hydrofile skal kan sterisk stabilisere miceller og forlænge deres omsætning i blodet. Den hydrofobe kerne effektivt kan indkapsle hydrofobe dtæpper. På grund af den lille størrelse af miceller (typisk mindre end 200 nm) og lange cirkulation egenskaber, er polymere miceller menes at opnå tumor målretning gennem øget permeabilitet og fastholdelse (EPR) effekter (passiv tumor målretning). Lægemiddelfyldning stabilitet er kritisk for tumortargeting evne af miceller. For at opnå optimal tumor målretning bør miceller har minimal lægemiddel lækage, før de når tumorstedet, men hurtigt frigive lægemidlet efter indtastning cancerceller. Desuden formuleringsstabilitet er også et væsentligt krav for produktudvikling, fordi formulering stabilitet bestemmer mulighederne for produktudvikling samt holdbarheden af ​​udviklede produkter. For nylig er der gjort en stor indsats for at forbedre læsningen af ​​lægemidler ind leveringstid bærere. Den lipofile pro-drug tilgang er en strategi, der er blevet undersøgt for at forbedre lægemiddelfyldning i lipide nanopartikler og emulsioner. 13,14 The conjugation af lipider med lægemidler kan forbedre betydeligt deres lipofilicitet og forbedre lastning og fastholdelse i de lipofile komponenter i nanocarriers. Her beskriver vi en protokol for at forberede lipofile doxorubicin pro-drug indlæst miceller. Først syntese fremgangsmåde for doxorubicin lipofile pro-drug beskrevet. Derefter en protokol til at generere miceller med en film-spredning metode indført. Denne fremgangsmåde er med succes blevet anvendt i tidligere undersøgelser. 5 DSPE-PEG blev valgt som bæremateriale til fremstilling miceller, fordi det har været brugt med held til levering micelle lægemiddel. 15,16 Endelig beskriver vi flere in vitro assays anvendes til at karakterisere micelle formuleringer og evaluere anticanceraktivitet.

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	<tbody>
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			<td height="23" style="height:23px;width:216px;">DSPE-PEG2K</td>
			<td style="width:191px;">Cordenpharm</td>
			<td style="width:119px;">LP-R4-039</td>
			<td style="width:167px;">&#62;95%</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Doxorubicin</td>
			<td style="width:191px;">LC Laboratories</td>
			<td style="width:119px;">D-4000</td>
			<td>&#62;99%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Palmitic Acid Hydrazide</td>
			<td style="width:191px;">TCI AMERICA&#160;&#160;</td>
			<td style="width:119px;">P000425G</td>
			<td>&#62;98.0%</td>
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			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:191px;">ACROS Organics</td>
			<td style="width:119px;">610981000</td>
			<td>Anhydrous</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methylene chloride&#160;</td>
			<td style="width:191px;">FISHER&#160;</td>
			<td style="width:119px;">D151-4</td>
			<td>99.90%</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methyl sulfoxide-d6</td>
			<td style="width:191px;">ACROS Organics</td>
			<td style="width:119px;">AC320760075</td>
			<td>NMR solvent</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trifluoroacetic Acid&#160;</td>
			<td style="width:191px;">ACROS Organics</td>
			<td style="width:119px;">AC293811000</td>
			<td>99.50%</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Silica Gel</td>
			<td style="width:191px;">FISHER&#160;</td>
			<td style="width:119px;">L-7446</td>
			<td>230-400 mesh</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BAKER FLEX TLC PLATES&#160;</td>
			<td style="width:191px;">FISHER&#160;</td>
			<td style="width:119px;">NC9990129</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">DPBS</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DU 145&#160; Prostate Cancer Cells</td>
			<td style="width:191px;">ATCC</td>
			<td style="width:119px;">HTB-81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MTT</td>
			<td style="width:191px;">ACROS Organics</td>
			<td style="width:119px;">158990050</td>
			<td>98%</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640 Medium</td>
			<td style="width:191px;">MEDIATECH INC&#160;</td>
			<td style="width:119px;">10041CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antibiotic-Antimycotic&#160;</td>
			<td style="width:191px;">LIFE TECHNOLOGIES&#160;</td>
			<td style="width:119px;">15240062</td>
			<td>100x stock solution</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:191px;">LIFE TECHNOLOGIES&#160;</td>
			<td style="width:119px;">10437077</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nuclear Magnetic Resonance Spectroscopy</td>
			<td style="width:191px;">Varian, Inc</td>
			<td style="width:119px;"></td>
			<td>300 NMR&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">B&#252;chi R-3 Rotavapor</td>
			<td style="width:191px;">Buchi</td>
			<td style="width:119px;">1103022V1&#160;</td>
			<td>Rotary evaporator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultrasonic Bath</td>
			<td style="width:191px;">BRANSON ULTRASONICS CORPORATION&#160;</td>
			<td style="width:119px;">CPX952318R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UV-VIS spectrometer Biomate 3</td>
			<td style="width:191px;">Thermo Spectronic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Zetasizer Nano ZS90&#160;</td>
			<td style="width:191px;">Malvern Instruments</td>
			<td style="width:119px;"></td>
			<td>Particle Size Analyer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microplate Spectrophotometer&#160;</td>
			<td style="width:191px;">Rio-Rad</td>
			<td style="width:119px;">Benchmark Plus&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell Culture Incubator</td>
			<td style="width:191px;">Napco</td>
			<td style="width:119px;">CO2 6000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Biological Safety Cabinet</td>
			<td style="width:191px;">Nuaire</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SigmaPlot&#160;</td>
			<td style="width:191px;">Systat Software, Inc.</td>
			<td style="width:119px;"></td>
			<td>Analytical Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-Well Cell Culture Plate</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">353072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin&#160; 0.25%</td>
			<td style="width:191px;">Corning Cellgro</td>
			<td style="width:119px;">25-053-CI</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation and characterization of lipophilic doxorubicin pro-drug micelles

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous environment through self-assembly. The hydrophobic core of micelles functions as a drug reservoir and encapsulates hydrophobic drugs. The hydrophilic shell prevents the aggregation of micelles and also prolongs their systemic circulation in vivo. In this protocol, we describe a method to synthesize a doxorubicin lipophilic pro-drug, doxorubicin-palmitic acid (DOX-PA), which will enhance drug loading into micelles. A pH-sensitive hydrazone linker was used to conjugate doxorubicin with the lipid, which facilitates the release of free doxorubicin inside cancer cells. Synthesized DOX-PA was purified with a silica gel column using dichloromethane/methanol as the eluent. Purified DOX-PA was analyzed with thin layer chromatography (TLC) and 1H-Nuclear Magnetic Resonance Spectroscopy (1H-NMR). A film dispersion method was used to prepare DOX-PA loaded DSPE-PEG micelles. In addition, several methods for characterizing micelle formulations are described, including determination of DOX-PA concentration and encapsulation efficiency, measurement of particle size and distribution, and assessment of in vitro anticancer activities. This protocol provides useful information regarding the preparation and characterization of drug-loaded micelles and thus will facilitate the research and development of novel micelle-based cancer nanomedicines.

Figur 1 viser syntesen arrangement med DOX-PA. DOX-PA blev syntetiseret ved konjugering af palmitinsyre med doxorubicin gennem en pH-følsom hydrazonbinding. Et lille overskud af palmitinsyre hydrazid blev anvendt til at lette færdiggørelsen af ​​reaktionen. Denne reaktion fremgangsmåde har en meget høj effektivitet og kun en lille mængde af doxorubicin tilbage efter en 18 timers reaktion (figur 2). Udbyttet var ca. 88%. Ved slutningen af ​​...

Watch this Scientific Journal Video about Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles at JoVE.com

Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

En protokol til forberedelse og karakterisering af lipofile doxorubicin pro-drug indlæst 1,2-distearoyl- sn-3-phosphoethanolamine- N - [amino (polyethylenglycol) -2000] (DSPE-PEG) miceller beskrives.

Forberedelse og karakterisering af lipofile Doxorubicin Pro-drug Miceller

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous ...

Research

JoVE Journal

Bioengineering

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In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

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The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

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The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

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Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Påvisning af Endotoxin i Nano-formuleringer bruger Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

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Fokuseret Ionstrålithografi til etch Nano-arkitekturer i Mikroelektroderne

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Temperaturstyret samling og karakterisering af en dråbe interface Bilayer

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits ...