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. Synthese von DOX-PA <ol><li> Wiegen 390 mg Doxorubicin und 243 mg Palmitinsäure Hydrazid, und in einen Rundkolben gegeben. </li><li> Zugabe von 150 ml wasserfreiem Methanol in den Kolben mit einer Glasspritze. In 39 ul Trifluoressigsäure (TFA) mit einer Pipette. Verwendung eines magnetischen Rührers, rühre das Reaktionsgemisch 18 h bei RT im Dunkeln. HINWEIS: Die Mengen an Reaktionsmaterialien skaliert werden können oder nach unten unterschiedlichen Mengen von DOX-PA zu erhalten. Das Verhältnis der Reaktanten sollte in den gleichen Proportionen eingehalten werden. Reaktionen unter Verwendung von DOX Mengen im Bereich von 78 mg bis 1.170 mg in einem regelmäßigen Chemielabor durchgeführt werden. </li><li> Reinigung von DOX-PA eine Kieselgelsäule mit. 17 <ol><li> Entferne das Lösungsmittel in dem Reaktionsgemisch mit einem Rotationsverdampfer. Werden 3 g Kieselgel, nachdem das Volumen des Gemisches auf etwa 20 ml reduziert. Weiter Rotationsverdampfen trockene Pulver zu ergeben, und t zu ermöglichener Adsorption von Produkten auf das Silicagel. Halten der Probe unter einem Vakuum für weitere 30 min, nachdem die Trockenpulver gebildet werden. </li><li> Pack 50 g Kieselgel in einer Kolonne von Dichlormethan als Lösungsmittel verwendet wird. Vorsichtig die Kieselgel-Probe adsorbierte Produkt an die Säule enthält. </li><li> Eluieren der Säule mit einem Gemisch aus Dichlormethan und Methanol, wobei nach und nach den Anteil an Methanol zu, wodurch Lösungsmittelpolarität zunehmende (Tabelle 1). </li><li> Sammle Fraktionen des Eluenten in Teströhrchen (25 ml / Röhrchen) und den Fortschritt durch Dünnschichtchromatographie (TLC). </li><li> Kombinieren Sie alle Fraktionen, die reine DOX-PA und entferne das Lösungsmittel mit einem Rotationsverdampfer bis trockenes Pulver gebildet wird. Trocknen das Produkt weiter unter Vakuum O / N. </li></ol></li><li> Analyse von DOX-PA durch TLC. <ol><li> Schneiden Sie ein 4 cm x 8 cm langen Abschnitt der DC-Platte. Spot-Probenlösungen 0,5 cm von der Unterseite der Platte mit TLC Spek Kapillaren unter Verwendung erfülltHANOL als Lösungsmittel. </li><li> Platzieren Sie die DC-Platte in eine Entwicklungskammer mit einer Mischung aus Dichlormethan und Methanol, das (3/1, v / v). Die Tiefe der Lösungsmittel sollte als 0,5 cm nur weniger. </li><li> Entfernen Sie die Platte aus der Entwicklerkammer, wenn die Lösungsmittelfront die Oberseite der Platte erreicht. Markieren Sie die Stelle des Lösungsmittelfront mit einem Bleistift und lassen Sie die Platte zu trocknen. Legen Sie die DC-Platte in eine Färbungskammer gesättigt Joddampfes enthält, um Proben zu visualisieren. </li></ol></li><li> Analyse von DOX-PA mit 1 H-Kernresonanzspektroskopie (1 H-NMR). 18 <ol><li> Man löst 15 mg des DOX-PA in 1 ml Methylsulfoxid-d 6 (DMSO) und Übertragung der Probe in ein NMR-Röhrchen. </li><li> Legen Sie die NMR-Röhrchen in den Magneten des NMR-Instrument. Messung der Protonenspektrum, Auswählen DMSO als Lösungsmittel. Entfernen Sie das NMR-Röhrchen von dem Magneten. Analysieren Sie den NMR - Ergebnis 18. </li></ol></li></ol> 2. Herstellung von DOX-PA Mizellen durch Film-Dispersionsverfahren <ol><li> Auflösen DSPE-PEG (40 mg) und DOX-PA (4 mg) mit 2 ml Methanol in einem 10 ml Glasfläschchen. </li><li> Entfernen des organischen Lösungsmittels im Vakuum am Rotationsverdampfer, bis sich ein dünner Film bildet sich in der Phiole verwendet. HINWEIS: Alternativ verdampfen die organischen Lösungsmittel unter Inertgas (zB Argon oder Stickstoffgas) einen Film zu bilden und das Fläschchen in einem Vakuumexsikkator halten , um weitere Lösungsmittelreste zu entfernen. </li><li> Transfer 2 ml Dulbeccos phosphatgepufferter Salzlösung (pH 7,4, DPBS) auf dem Glasfläschchen. </li><li> Legen Sie das Fläschchen in einem Ultraschallbad für 3 min bei RT zu Mizellen erzeugen. HINWEIS: Die Ultraschall-Leistung variiert zwischen den verschiedenen Modellen von Ultraschallbädern. Wählen Sie eine Einheit, die genügend Ultraschallleistung erzeugen kann, die dünne Polymer / Arzneimittel-Film zu zerstreuen. Die Ausgangsleistung des Ultraschallbades in diesem Protokoll verwendet ist 110 W. </li><li> Halten Mizellen bei 4 ° C für die kurzzeitige Lagerung und -20 ° C für lange-term Lagerung. HINWEIS: Alternativ können Mizellen auch gefriergetrocknet und vor Gebrauch mit Wasser rekonstituiert. Normalerweise kein Gefrierschutzmittel oder Lyoschutzmittel wird für diese Formulierung erforderlich. </li></ol> 3. Charakterisierung der DOX-PA Mizellen <ol><li> Bestimmung der DOX-PA - Konzentration in Mizellen und Drogen Verkapselungswirksamkeit <ol><li> Auflösen DOX-PA in vorherigen Schritten in DMSO synthetisiert DOX-PA-Lösungen von fünf verschiedenen Konzentrationen herzustellen: 1 &amp; mgr; g / ml, 5 ug / ml, 20 ug / ml, 50 ug / ml und 100 &amp; mgr; g / ml. Messen der Absorption von DOX-PA-Lösungen mit einem UV-VIS-Spektrometer bei 490 nm. Generieren Sie eine Standardkurve auf der Basis der DOX-PA Wirkstoffkonzentrationen und deren bei 490 nm entsprechenden Absorption. </li><li> Verdünne 25 &amp; mgr; l von mit Wirkstoff beladenen Mizelle mit 500 ul DMSO. Messen Sie die Absorption bei 490 nm mit einem UV-VIS-Spektrometer. Berechnen Wirkstoffkonzentrationen mit der Standardkurve in 3.1.1 erzeugt. </li><li> Berechnen Verkapselungseffizienz Verwendung der folgenden Gleichung: Drug Umhüllungs-Effektivität (%) = (Menge an Arzneimittel in Mizellen) / (Menge an zugesetztem drug) × 100% </li></ol></li><li> Charakterisierung von Partikelgröße mit dynamischer Lichtstreuung (DLS) <ol><li> Verdünnte Mizellen mit DPBS (pH 7,4) auf eine endgültige DSPE-PEG-Konzentration von 1 mg / ml. Analysieren eine 2 ml Probe mit einem Teilchengrßenanalysator das Z-gemittelte Größe und Polydispersitätsindex (PDI) zu erhalten. </li></ol></li><li> Bewertung der in - vitro - Aktivität gegen Krebs HINWEIS: Verwenden Sie geeignete sterile Technik und in einem Biosicherheitsschrank bedienen. <ol><li> Entfernen des Zellkulturmediums aus einer Zellkulturflasche (beispielsweise T25) enthält , DU-145 menschlichen Prostatakrebszellen und spülen Sie die Zellen mit 2 ml DPBS (pH 7,4). </li><li> Aspirat DPBS, 1 ml Trypsin-Lösung (0,25%) und bei 37 ° C für 2 min inkubiert, um die Zellen abzulösen. </li><li> In 10 ml Zellkulturmedium (RPMI 1640 + 10% fötales Rinderserum + 1% Antibiotic-Antimykotische), wenn die meisten der Zellen aus dem Kolben abgelöst werden. Transfer-Zellen in ein 15-ml-Zentrifugenröhrchen und zentrifugiere die Zellen bei 1.000 × g für 5 min. </li><li> Resuspendieren des Zellpellets mit 5 ml Kulturmedium Zelle und Entfernen mit einem Hämozytometer eine Probe Zellzahlen zum Zählen. Verdünne die Zellen mit Zellkulturmedium bis zu einer Dichte von 50.000 Zellen / ml. Fügen Sie die verdünnten Zellsuspensionen in einer 96-Well-Zellkulturplatte (100 &amp; mgr; l / Well). Inkubieren der Zellen in einem Zellkulturbrutschrank (37 ° C, 5% CO 2) für 18 h Zellanheftung zu ermöglichen. </li><li> Verdünnte DOX Dimethylsulfoxid (DMSO) -Lösung und DOX-PA DMSO Lösung mit Zellkulturmedium bis zur endgültigen Arzneimittelkonzentrationen von 0,1 &amp; mgr; M zu erhalten, 0,5 uM, 2 uM, 5 uM und 10 uM, respectively. Halten DMSO-Endkonzentration in allen obigen Proben auf 0,5%. Verdünnte DOX-PA Mizellen mit Zellkulturmedium endgültige Arzneimittel co zu erhaltenncentrations von 0,1 uM, 0,5 uM, 2 uM, 5 uM und 10 uM, respectively. Verwenden Sie die leere Zellkulturmedium, das eine Kontrolle. </li><li> Entfernen Sie die 96-Well-Zellkulturplatte aus dem Inkubator und ersetzen Sie die Zellkulturmedium mit 100 ul Medium unterschiedliche Behandlungsmittel enthalten, hergestellt in Schritt 3.3.5 (n = 4 für jede Gruppe). Inkubieren der Zellen in der Zellkulturbrutschrank (37 ° C, 5% CO 2) für eine weitere 72 hr. </li><li> Anzusaugen, das Medium und füge 100 ul Medium, enthaltend 0,5 mg / ml 3- (4,5-dimethyl-thiazol-2-yl) -2,5-diphenyl Tetrazolium Bromid (MTT). </li><li> Inkubieren der Zellen in der Zellkultur-Inkubator für weitere 2 Stunden. Sie vorsichtig das Medium zu entfernen und 100 ul DMSO hinzufügen Formazankristallen aufzulösen. </li><li> Messung der Extinktion mit dem Mikroplatten-Spektralphotometer bei einer Wellenlänge von 570 nm und einer Referenzwellenlänge von 670 nm. </li><li> Berechnen Sie die Lebensfähigkeit der Zellen unter Verwendung der folgenden Gleichung: <strong&gt; Zell Viability (%) = (A Test / A - Steuerung) × 100% HINWEIS: Vergleichen Sie die Lebensfähigkeit der Zellen zwischen den verschiedenen Gruppen unter Verwendung einer Einwegvarianzanalyse (ANOVA) statistischer Test. Berechnen Sie die IC 50 basierend auf die Lebensfähigkeit der Zellen gegen die Arzneimittelkonzentration Daten. </li></ol></li></ol>

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The authors have nothing to disclose.

In dieser Arbeit beschreiben wir eine unkomplizierte, schnelle Filmdispersionsverfahren zur Herstellung von Mizellen. Dieses Verfahren nutzt die Selbstorganisation eines amphiphilen Polymers (zB DSPE-PEG) Kern-Schale - strukturierten Mizellen in einer wässrigen Umgebung zu bilden. Diese Mizellen Herstellungsverfahren hat mehrere Vorteile. 1. Es handelt sich eine einfache Formulierungsverfahren, die die Verwendung von komplizierten Zerkleinerungsschritten (wie Extrusion oder Homogenisierung) vermeidet üblicher...

Chemotherapie wird häufig verwendet, um verschiedene Formen von Krebs zu behandeln. Die meisten, wenn nicht alle, chemotherapeutische Medikamente haben toxische Nebenwirkungen, die von handhabbaren minor Bedingungen, wie Übelkeit und Diarrhö, um mehr lebensbedrohlichen Zuständen variieren kann. Da die meisten Krebsmedikamente toxisch, nicht-selektive Belichtung dieser Medikamente zu normalem Gewebe sind verursacht unweigerlich Toxizität. Daher besteht ein großer Bedarf für einen therapeutischen Ansatz, der selektiv Drogen in Krebszellen liefern kann. Eine weitere Herausforderung bei der Verabreichung von Anti-Krebs-Medikamente ist ihre schlechte Wasserlöslichkeit. Üblicherweise werden Solubilisierungsmittel notwendig, um diese schlecht löslichen Arzneimitteln zu formulieren. Allerdings sind die meisten Lösungsvermittlern, wie Dimethylsulfoxid (DMSO), Cremophor EL, und Polysorbat 80 (Tween 80) kann zu Leber- und Nierentoxizität, Hämolyse und akute Überempfindlichkeitsreaktionen und periphere Neuropathien. 1 daher sicher und biokompatibel Formulierungen erforderlich sind für die klinische Verwendung von schlechtenly löslichen Krebsmedikamente. Nanocarriers sind vielversprechende Wirkstoffabgabesysteme für die oben genannten Herausforderungen. Diese Nanocarriern umfassen Liposomen, 2 Nanopartikel, 3 Mizellen, 4-7 Polymer-Wirkstoff - Konjugate, 8 und anorganischen Materialien. 9 Mehrere Nanoprodukte (zB Caelyx, Abraxane und Genexol) wurden von den Aufsichtsbehörden genehmigt worden , um Krebspatienten zu behandeln. 10 Polymere Mizellen sind vielversprechende nanoskalige Wirkstoffabgabeträger, die für die Abgabe von Antikrebsmitteln erfolgreich verwendet wurden. 4-7,11,12 Typische polymere Mizellen werden aus amphiphilen Polymeren durch eine Selbstorganisation. Die Kern-Schale-strukturierte polymere Mizellen umfassen eine hydrophile Schale und einen hydrophoben Kern. Die hydrophile Schale kann sterisch Mizellen stabilisieren und ihre Zirkulation im Blutstrom zu verlängern. Der hydrophobe Kern kann wirksam hydrophob d einzukapselnTeppiche. Aufgrund der geringen Größe der Mizellen (typischerweise weniger als 200 nm) und Langzirkulationseigenschaften werden polymere Mizellen angenommen Tumor-Targeting durch erhöhte Permeabilität und Retention (EPR) Effekte (passive Tumor Targeting) zu erreichen. Wirkstoffbeladung Stabilität ist für den Tumor kritische Fähigkeit von Mizellen Targeting. Um eine optimale Tumor-Targeting zu erreichen, sollte Mizellen minimale Medikamenten Leckage haben, bevor die Tumorstelle zu erreichen, noch kurz die Droge nach Krebszellen gelangt. Zusätzlich ist Formulierungsstabilität auch eine wesentliche Voraussetzung für die Produktentwicklung, weil Formulierungsstabilität, die Durchführbarkeit der Produktentwicklung bestimmt, sowie die Haltbarkeit der entwickelten Produkte. Kürzlich wurde viel Anstrengung unternommen, die Belastung von Medikamenten in Lieferträger zu verbessern. Die lipophile Prodrug - Ansatz ist eine Strategie , die erforscht wurde Wirkstoffbeladung in Lipid - Nanopartikel und Emulsionen zu verbessern. 13,14 Die Konjugation von Lipiden mit Medikamenten deutlich ihre Lipophilie verbessern und in den lipophilen Komponenten von Nanocarriern Laden und Retention zu verbessern. Hier beschreiben wir ein Protokoll zur Herstellung von lipophilen Doxorubicin Prodrug geladenen Mizellen. Zunächst wird die Syntheseprozedur lipophilen Prodrug für Doxorubicin beschrieben. Dann wird ein Protokoll für die Mizellen mit einem filmDispersionsVerfahren Erzeugen eingeführt. Diese Methode wurde in unseren früheren Studien. 5 DSPE-PEG wurde ausgewählt als Trägermaterial zur Herstellung von Micellen , weil es wurde für Micelle Arzneimittelabgabe erfolgreich eingesetzt erfolgreich eingesetzt. 15,16 Schließlich wir Micelle mehrere in vitro - Assays zur Charakterisierung beschreiben Formulierungen und Anti-Krebs-Aktivität zu bewerten.

<table border="0" cellpadding="0" cellspacing="0" width="693">
	<tbody>
		<tr height="23">
			<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>
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		<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.

Abbildung 1 zeigt das Syntheseschema von DOX-PA. DOX-PA wurde durch Konjugation von Palmitinsäure synthetisiert mit Doxorubicin über eine pH-sensitive Hydrazonbindung. Ein geringer Überschuß an Palmitinsäure Hydrazid wurde verwendet, um die Vervollständigung der Reaktion zu erleichtern. Dieses Reaktionsverfahren hat einen sehr hohen Wirkungsgrad und nur eine geringe Menge von Doxorubicin blieb nach 18 h Reaktion (Abbildung 2). Die Ausbeute betrug 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

Ein Protokoll für die Herstellung und Charakterisierung von lipophilen Doxorubicin Prodrug geladen 1,2-Distearoyl- sn -glycero-3-phosphoethanolamine- N - [Amino (Polyethylenglykol) -2000] (DSPE-PEG) Micellen beschrieben.

Herstellung und Charakterisierung von Lipophile Doxorubicin Pro-drug Mizellen

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

preparation-characterization-lipophilic-doxorubicin-pro-drug

Research

JoVE Journal

Bioengineering

14.8K Aufrufe.  Hampton University.  Ein Protokoll für die Herstellung und Charakterisierung von lipophilen Doxorubicin Prodrug geladen 1,2-Distearoyl- sn -glycero-3-phosphoethanolamine- N - [Amino (Polyethylenglykol) -2000] (DSPE-PEG) Micellen beschrieben. 

Serie: Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

Konstruktion und Charakterisierung eines neuartigen Bioreaktor Stimmlippen

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

Forschung

Biotechnik

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

Vorbereitung und Charakterisierung von neuartigen HDL-imitierenden Nanopartikeln für die Nervenwachstumsfaktorverkapselung

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

Synthesis of Monocyte-targeting Peptide Amphiphile Micelles for Imaging of Atherosclerosis

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. Atherosclerosis is also the leading cause of sudden death and myocardial infarction, instigated by unstable plaques that rupture and occlude the blood vessel without warning. Current imaging modalities cannot differentiate between stable and unstable plaques that rupture. Peptide amphiphiles micelles (PAMs) can overcome this drawback as they can be modified with a variety of targeting moieties that bind specifically to diseased tissue. Monocytes have been shown to be early markers of atherosclerosis, while large accumulation of monocytes is associated with plaques prone to rupture. Hence, nanoparticles that can target monocytes can be used to discriminate different stages of atherosclerosis. To that end, here, we describe a protocol for the preparation of monocyte-targeting PAMs (monocyte chemoattractant protein-1 (MCP-1) PAMs). MCP-1 PAMs are self-assembled through synthesis under mild conditions to form nanoparticles of 15 nm in diameter with near neutral surface charge. In vitro, PAMs were found to be biocompatible and had a high binding affinity for monocytes. The methods described herein show promise for a wide range of applications in atherosclerosis as well as other inflammatory diseases.

This paper presents a method involving the synthesis and characterization of monocyte-targeting peptide amphiphile micelles and the corresponding assays to test for biocompatibility and ability of the micelle to bind to monocytes.

Synthese von Monocyte-targeting Peptid Amphiphile Micellen für die Bildgebung der Atherosklerose

Atherosclerosis is a major contributor to cardiovascular disease, the leading cause of death worldwide, which claims 17.3 million lives annually. ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

Herstellung und Charakterisierung von optischen Gewebe Phantome, die Makrostruktur enthalten

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

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Vorbereitung, Reinigung und Anwendung von Fettsäure-haltigen Liposomen

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

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

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

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

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Nachweis von Endotoxin im Nano-Rezepturen mit Limulus Amoebocyte Lysate (LAL) Assays

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

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

Wachstum und Charakterisierung der bestrahlten Organoide aus den Brustdrüsen

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and ability to entrap both hydrophilic and hydrophobic compounds. The classical hydration of thin lipid films in an organic solvent is applied herein as a technique to encapsulate tarin, a plant lectin, in nanoliposomes. Nanoliposome size, stability, entrapment efficiency, and morphological characterization are described in detail. The nanoliposomes are prepared using 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt; DSPE-MPEG 2000), and cholesterylhemisuccinate (CHEMS) as the main constituents. Lipids are first dissolved in chloroform to obtain a thin lipid film that is subsequently rehydrated in ammonium sulfate solution containing the protein to be entrapped and incubated overnight. Then, sonication and extrusion techniques are applied to generate nanosized unilamellar vesicles. The size and polydispersity index of the nanovesicles are determined by dynamic light scattering, while nanovesicle morphology is assessed by scanning electron microscopy. Entrapment efficiency is determined by the ratio of the amount of unencapsulated protein to original amount of initially loaded protein. Homogeneous liposomes are obtained with an average size of 155 nm and polydispersity index value of 0.168. A high entrapment efficiency of 83% is achieved.

This study describes classical hydration using the thin lipid film method for nanoliposome preparation followed by nanoparticle characterization. A 47 kDa-hydrophilic and globular protein, tarin, is successfully encapsulated as a strategy to improve stability, avoid fast clearance, and promote controlled release. The method can be adapted to hydrophobic molecules encapsulation.

Vorbereitung und Charakterisierung von Nanoliposomen zur Entrapment ierung bioaktiver hydrophiler Globularproteine

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their ...

Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of sophisticated microelectrodes with greater resolution and expanded capabilities. The progress in fabrication technology has supported the development of biomimetic electrodes, which aim to seamlessly integrate into the brain parenchyma, reduce the neuroinflammatory response observed after electrode insertion and improve the quality and longevity of electrophysiological recordings. Here we describe a protocol to employ a biomimetic approach recently classified as nano-architecture. The use of focused ion beam lithography (FIB) was utilized in this protocol to etch specific nano-architecture features into the surface of non-functional and functional single shank intracortical microelectrodes. Etching nano-architectures into the electrode surface indicated possible improvements of biocompatibility and functionality of the implanted device. One of the benefits of using FIB is the ability to etch on manufactured devices, as opposed to during the fabrication of the device, facilitating boundless possibilities to modify numerous medical devices post-manufacturing. The protocol presented herein can be optimized for various material types, nano-architecture features, and types of devices. Augmenting the surface of implanted medical devices can improve the device performance and integration into the tissue.

We have shown that the etching of nano-architecture into intracortical microelectrode devices may reduce the inflammatory response and has the potential to improve electrophysiological recordings. The methods described herein outline an approach to etch nano-architectures into the surface of non-functional and functional single shank silicon intracortical microelectrodes.

Fokussierte Ionenstrahl-Lithographie zu Etch-Nano-Architekturen in Mikroelektroden

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of ...

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the context of liver diseases, 3-dimensional (3D) models based on cholangiocytes have emerged such as cysts, spheroids, or tube-like structures to mimic tissue topology for organogenesis, disease modeling, and drug screening studies. These structures have been mainly obtained by embedding cholangiocytes in a hydrogel. The main purpose was to study self-organization by addressing epithelial polarity, functional, and morphological properties. However, very few studies focus on cyst formation efficiency. When this is the case, the efficiency is often quantified from images of a single plane. Functional assays and structural analysis are performed without representing the potential heterogeneity of cyst distribution arising from hydrogel polymerization heterogeneities and side effects. Therefore, the quantitative analysis, when done, cannot be used for comparison from one article to another. Moreover, this methodology does not allow comparisons of 3D growth potential of different matrices and cell types. Additionally, there is no mention of the experimental troubleshooting for immunostaining cysts. In this article, we provide a reliable and universal method to show that the initial cell distribution is related to the heterogeneous vertical distribution of cyst formation. Cholangiocyte cells embedded in hydrogel are followed with Z-stacks analysis along the hydrogel depth over the time course of 10 days. With this method, a robust kinetics of cyst formation efficiency and growth is obtained. We also present methods to evaluate cyst polarity and secretory function. Finally, additional tips for optimizing immunostaining protocols are provided in order to limit cyst collapse for imaging. This approach can be applied to other 3D cell culture studies, thus opening the possibilities to compare one system to another.

Three-dimensional (3D) cellular systems are relevant models for investigating organogenesis. A hydrogel-based method for biliary cysts production and their characterization is proposed. This protocol unravels the barriers of 3D characterization, with a straightforward and reliable method to assess cyst formation efficiency, sizes, and to test their functionality.

Generierung und quantitative Charakterisierung funktioneller und polarisierter Gallenepithelzysten

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the ...