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. Síntesis de DOX-PA <ol><li> Pesar 390 mg de doxorrubicina y 243 mg de hidrazida de ácido palmítico, y transferir a un matraz de fondo redondo. </li><li> Añadir 150 ml de metanol anhidro al matraz con una jeringa de vidrio. Añadir 39 l de ácido trifluoroacético (TFA) con una pipeta. El uso de un agitador magnético, se agita la mezcla de reacción durante 18 horas a RT en la oscuridad. NOTA: Las cantidades de materiales de reacción se pueden escalar hacia arriba o hacia abajo para obt...

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En este trabajo se describe un método película de dispersión sencilla, rápida para la preparación de las micelas. Este método utiliza las propiedades de autoensamblaje de un polímero anfifílico (por ejemplo, DSPE-PEG) para formar micelas de núcleo-envuelta estructurados en un entorno acuoso. Este método de preparación de micelas tiene varias ventajas. 1. Se trata de un proceso de formulación simple, que evita el uso de pasos de reducción de tamaño complicadas (tales como la extrusión u homogeneiz...

La quimioterapia se utiliza comúnmente para tratar varias formas de cáncer. La mayoría, si no todos, los medicamentos de quimioterapia tienen efectos secundarios tóxicos que pueden variar de leves condiciones manejables, tales como náuseas y diarrea, a las condiciones que amenazan la vida más. Debido a que la mayoría de los medicamentos contra el cáncer son tóxicos, la exposición no selectiva de estos fármacos para el tejido normal inevitablemente causa toxicidad. Por lo tanto, hay una gran necesidad de un enfoque terapéutico que puede ofrecer selectivamente fármacos en células cancerosas. Otro de los desafíos con la administración de medicamentos contra el cáncer es su escasa solubilidad en agua. Por lo general, se necesitan agentes solubilizantes para formular estos fármacos poco solubles. Sin embargo, la mayoría de agentes solubilizantes, tales como sulfóxido de dimetilo (DMSO), Cremophor EL, y polisorbato 80 (Tween 80) puede causar toxicidad hepática y renal, hemólisis, reacciones de hipersensibilidad agudas y neuropatías periféricas. 1 Por lo tanto, se necesitan formulaciones seguras y biocompatibles para el uso clínico de pobremedicamentos contra el cáncer Ly solubles. Nanoportadores son prometedores sistemas de administración de fármacos para hacer frente a los retos anteriores. Estos nanoportadores incluyen liposomas, nanopartículas 2, 3, 4-7 micelas conjugados polímero-fármaco, 8 y materiales inorgánicos. 9 Varios productos de nanomedicina (por ejemplo, Doxil, Abraxane, y Genexol) han sido aprobados por las agencias reguladoras para el tratamiento de pacientes con cáncer. 10 Las micelas poliméricas son prometedores portadores de administración de fármacos a nanoescala, que se han utilizado con éxito para el suministro de fármacos contra el cáncer. 4-7,11,12 micelas poliméricas típicos se preparan a partir de polímeros anfífilos a través de un proceso de autoensamblaje. Las micelas poliméricas estructurados de núcleo-envoltura incluyen una vaina hidrófila y un núcleo hidrofóbico. La envoltura hidrofílica puede estabilizar estéricamente micelas y prolongar su circulación en el torrente sanguíneo. El núcleo hidrofóbico puede resumir eficazmente d hidrófoboalfombras. Debido al pequeño tamaño de las micelas (normalmente menos de 200 nm) y las propiedades de circulación largo, se cree que las micelas poliméricas para lograr tumor de orientación a través de efectos de retención (EPR) (tumor pasiva focalización) aumento de la permeabilidad y. estabilidad carga de fármaco es crítica para el tumor capacidad de las micelas de orientación. Para lograr la orientación óptima del tumor, micelas deben tener fugas mínimas de drogas antes de llegar al sitio del tumor, sin embargo, la liberación rápida del fármaco después de entrar en las células cancerosas. Además, estabilidad de la formulación es también un requisito esencial para el desarrollo de productos, porque la estabilidad formulación determina la viabilidad de desarrollo de productos, así como la vida útil de los productos desarrollados. Recientemente, se han hecho muchos esfuerzos para mejorar la carga de fármacos en vehículos de entrega. El enfoque de profármaco lipófilo es una estrategia que ha sido explorado para mejorar la carga de fármaco en nanopartículas lipídicas y emulsiones. 13,14 El conjugation de lípidos con fármacos puede mejorar significativamente su lipofilia y mejorar la carga y la retención de los componentes lipófilos de nanoportadores. A continuación, se describe un protocolo para la preparación de doxorrubicina lipofílica micelas cargadas de profármaco. En primer lugar, se describe el procedimiento para la síntesis de doxorrubicina lipofílica profármaco. A continuación, se introduce un protocolo para generar micelas con un método de película de dispersión. Este método ha sido utilizado con éxito en nuestros estudios anteriores. 5 DSPE-PEG fue seleccionado como el material de soporte para la preparación de micelas, ya que ha sido utilizado con éxito para la administración de fármacos micela. 15,16 Finalmente, se describen varios ensayos in vitro utilizados para caracterizar micela formulaciones y para evaluar la actividad contra el cáncer.

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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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<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.

La Figura 1 muestra el esquema de síntesis de DOX-PA. DOX-PA fue sintetizado mediante conjugación de ácido palmítico con doxorubicina a través de un enlace hidrazona sensible al pH. Un ligero exceso de hidrazida de ácido palmítico se utilizó para facilitar la finalización de la reacción. Este método de reacción tiene una eficacia muy alta y sólo una pequeña cantidad de doxorrubicina se mantuvo después de una reacción de 18 h (Figura 2). E...

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

Un protocolo para la preparación y caracterización de doxorrubicina lipofílica profármaco cargado 1,2-sn distearoyl- glicero-3-phosphoethanolamine- N - [amino (polietilenglicol) -2000] se describe (DSPE-PEG) micelas.

Preparación y caracterización de lipofílico Doxorrubicina micelas profármaco

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

The overall goal of this protocol is to describe procedures for preparing and characterizing lipophilic doxorubicin prodrug loaded micell formulations. This method provides a simple and reliable approach for micell formulation. The synthesis of lipophilic prodrug of doxorubicin can enhance drug loading and stability, thus addressing a significant challenge in developing micell formulation.The micell formulation can enhance drug targeting into tumors and therefore can reduce toxicities of cancer treatment. In addition to the application described in this project, this technical can also be used to prepare the nanomedicine for the agents. To synthesize lipophilic doxorubicin prodrug DOX-PA, weigh 390 milligrams of doxorubicin and 243 milligrams of palmitic acid hydrozide, and transfer them to a round bottom flask.Add 150 milliliters of anhydrous methanol to the flask with a glass syringe. Then add 39 microliters of trifluoroacetic acid, or TFA, with a pipette. Using a magnetic stirrer, stir the reaction mixture for 18 hours at room temperature in the dark.To purify DOX-PA using a silica gel column, first remove the solvent in the reaction mixture with a rotary evaporator. Add three grams of silica gel after the volume of the mixture is reduced to around 20 milliliters. Continue rotary evaporation to yield dry powders and to allow the absorption of products onto the silica gel.Keep the sample under a vacuum for an additional 30 minutes after the dry powders are formed. Pack 50 grams of silica gel into a column using dichloromethane as a solvent. Carefully add the silica gel sample containing absorbed product to the column.Elute the column with a mixture of dichloromethane and methanol while gradually increasing the percentage of methanol, thereby increasing solvent polarity. Collect fractions of elluent in test tubes at 25 milliliters per tube, and monitor the progress by thin layer chromatography, or TLC. Combine all fractions containing pure DOX-PA and remove the solvent using a rotary evaporator until dry powder is formed.Further dry the product under a vacuum overnight. To prepare micelles, dissolve 40 milligrams of DSPE-PEG and four milligrams of DOX-PA with two milliliters of methanol in a 10 milliliter glass vial. Remove the organic solvent under a vacuum using a rotary evaporator, until a thin film forms in the vial.Next, transfer two milliliters of Dulbecco's phosphate buffered saline, pH 7.4 to the glass vial. Select an ultrasonic bath that can generate enough ultrasonic power to disperse the thin polymer drug film. The output power of the ultrasonic bath used in this protocol is 110 watts.Place the vial in an ultrasonic bath for three minutes at room temperate to generate micelles. Keep micelles at four degrees Celsius for short term storage, and minus 20 degrees celsius for long term storage. Once micelles are prepared, perform various characterizations to determine drug concentration in micelles, particle sizes, and in vitro anticancer activities.To determine the DOX-PA concentration and drug encapsulation efficiency, dilute 25 microliters of drug loaded micelles with 500 microliters of DMSO. Measure the absorption at 490 nanometers with a UV-VIS spectrometer, and calculate drug concentrations and encapsulation efficiency as described in the text protocol. To evaluate in vitro anticancer activity, add the diluted cell suspensions into a 96 well cell culture plate at 100 microliters per well.Incubate the cells in a cell culture incubator for 18 hours to allow cell attachment. Next, dilute a DOX DMSO solution and a DOX-PA DMSO solution with cell culture medium to obtain final drug concentrations. Keep the final DMSO concentration in all samples at 0.5%Then dilute DOX-PA micelles with cell culture medium to obtain final drug concentrations.Use the blank cell culture medium as a control. Remove the 96 well cell culture plate from the incubator and replace the cell culture medium with 100 microliters of medium containing different treatment agents. Incubate the cells in the cell culture incubator for an additional 72 hours.Next, aspirate the medium and add 100 microliters of medium containing 0.5 milligrams per milliliter of MTT. Incubate the cells in the cell culture incubator for an additional two hours. Carefully remove the medium and add 100 microliters of DMSO to dissolve formazan crystals.Measure absorbence with the microplate spectrophotometer at a wavelength of 570 nanometers and a reference wavelength of 670 nanometers. Finally, calculate the cell viability as described in the text protocol. The synthesis scheme of DOX-PA is shown here.Palmitic acid is conjugated to doxorubicin through a pH sensitive hydrozine linker. The structure of DOX-PA was further confirmed with a proton NMR spectrum. Characteristic NMR peaks from both doxorubicin and palmitic acid are observed.Letters indicate peaks, and their corresponding moiety within the structure. Blank DSPE-PEG micelles without drug appear as a transparent liquid, whereas DOX-PA DSPE-PEG micelles appear as a red liquid, which is due to the DOX-PA loaded in the micelles. The mean particle size for blank DSPE-PEG micelles is around 17.0 nanometers.The loading of DOX-PA increased micell particle size slightly, with a mean particle size for DOX-PA DSPE-PEG micelles of around 25.7 nanometers. A concentration dependent reduction in cell viability was achieved by treating DU145 cells with free doxorubicin, free DOX-PA, or DOX-PA micelles. Although free doxorubicin is more effective than free DOX-PA or DOX-PA micelles at lower concentrations, DOX-PA groups showed greater reduction in cell viability than free doxorubicin at higher concentrations.Also, there appear to be no significant difference between DOX-PA and DOX-PA micelles at both higher and lower concentrations. After watching this video, you should have a good understanding of how to prepare the micell formulations with a film dispersion method. It's important to remember that the method described can be used to prepare micell with different drugs and carrier materials.The design of carrier material is critical to improve the formulation performance. Following this procedure, the micell formulation can be further evaluated in vivo with animal tumor models to test the anticancer activity.

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