We would like to acknowledge the following funding sources: the CSU Infectious Disease Translational Research Training Program (ID:TRTP) and the NIH research grant program (R01 NS075214-01A1). We would like to thank the Telling lab for the use of their monoclonal antibody PRC5. We would also like to thank the Dow lab for DOTAP liposomes, and for sharing their expertise in generating liposomes.

Alle Mäuse wurden in Labortier Ressourcen gezüchtet und gepflegt, von der Association akkreditiert für Evaluierung und Akkreditierung von Lab Animal Care International, in Übereinstimmung mit den Protokollen von der Institutional Animal Care und Use Committee an der Colorado State University genehmigt. 1. Herstellung von LSPCs <ol><li> Verwenden Sie ein 1: 1 DOTAP (1,2-Dioleoyl-3-Trimethylammonium-Propan): Cholesterin-Verhältnis für LSPCs. Für ein 4 nmol Liposom-Zubereitung, Mischung 2 nmol DOTAP und 2 nmol von Cholesterin in 10 ml eines 1: 1 Chloroform: Methanol-Lösung in einem Kolben. </li><li> Verdunsten 9 ml Chloroform: Methanol - Lösung unter Verwendung von N 2 -Gas in einer Abzugshaube. Dampft die letzten 1 ml Lösungsmittel in einem Abzug über Nacht ohne Gas. Beobachten eines dünnen Lipidfilm auf dem Boden des Kolbens. </li><li> Wärme 10 ml einer 10% Saccharose-Lösung auf 55 ° C. </li><li> Gießen Sie die erhitzte 10% Saccharose - Lösung auf die dünne Lipidfilm langsam, dh 1 ml / min, während gently Schwenken des Kolbens. Aufrechterhaltung der 10% Sucrose und Kolben mit Lipiden bei 55 ° C so, dass die Lipidmoleküle in dem Gel-Flüssigkeits-Phase verbleiben. Rehydrierung Ergebnisse in multilamellare Vesikel (MLV) Bildung. </li><li> Erlauben Lipide für mindestens eine Stunde bei 55 ° C rehydriert, bevor die Liposomen Größen. </li><li> Montieren eines Extruders den Anweisungen des Herstellers mit 1,0 &amp; mgr; m-Filter oder verwenden 1,0 um Spritzenfilter für die Dimensionierung. </li><li> 1 ml der erwärmten Liposomensuspension auf eine der Spritzen auf den Extruder, und passieren die Suspension zwischen den zwei Spritzen 11 mal. Stellen Sie sicher, dass die Suspension in die entgegengesetzte Spritze ist als die Ausgangs Spritze nach den 11 verläuft. </li><li> Größe die Liposomen wieder durch 0,45 um dann 0,2 um Filter große unilamellare Vesikel (LUV) zu erzeugen. Halten Sie die Liposomensuspension zur einfacheren Dimensionierung erwärmt. </li><li> Inkubiere 20 ul des 4 nmol Liposom-Suspension (200 &amp; mgr; M) mit 200 &amp; mgr; l einer 20 &amp; mgr; M (4nmol gesamt) Lager siRNA-Lösung für 10 Minuten bei Raumtemperatur. </li><li> Inkubiere 80 ul einer 500 uM (40 nmol Gesamt) stock RVG-9R-Lösung mit der siRNA / Liposom-Lösung für 10 min bei RT. LSPCs sollte so bald wie möglich verwendet werden, sondern kann bis zu mehreren Stunden nach der Herstellung verwendet werden. Store LSPCs bei 4 ° C für mehrere Stunden nach der Zubereitung. </li></ol> 2. Herstellung von PALETS <ol><li> Verwenden, um eine 55: 40: 5 Molverhältnis von entweder DSPE (1,2-Distearoyl-sn-glycero-3-phosphoethanolamin): Cholesterin: DSPE-PEG oder DOTAP: Cholesterin: DSPE-PEG für PALETS. Für ein 4 nmol Liposom-Zubereitung, Mischung 2,2 nmol entweder DSPE oder DOTAP, 1,6 nMol von Cholesterin und 0,2 nmol von DSPE-PEG in 10 ml eines 2: 1 Chloroform: Methanol-Lösung in einem Kolben. </li><li> Bereiten Sie Liposomensuspension genau wie in Schritt 1.1 und 1.2 oben beschrieben. Rehydrieren DSPE-Liposomen in 10 ml 1x PBS erhitzt auf 75ºC, Zugabe von 1 ml / min. Erlauben Lipiden bei 75 ° C rehydriert zuminmindestens 1 Stunde vor dem Kalibrieren. </li><li> Größe der Liposomensuspension genau wie in Schritt 1.6-1.8 beschrieben. </li><li> Pipettieren 20 ul jeder der 4 nmol (200 uM) Liposomensuspensionen in einzelne Aliquots nach DOTAP und DSPE Liposomen Größe gewesen, und für 30 min lyophilisieren ein Benchtop - Gefriertrockner (Abbildung 2). </li><li> Inkubieren 200 ul einer 20 uM (4 nmol Gesamt) stock siRNA-Lösung mit 1,4 ul einer 26,6 &amp; mgr; M Lösung von Protaminsulfat für 10 min bei RT für siRNA, die in DSPE PALETS Liposomen verkapselt werden soll. </li><li> Rehydratisieren die DOTAP PALETS Liposomen mit 200 ul einer 4 nmol Stammlösung von siRNA oder DSPE PALETS Liposomen mit 201,4 &amp; mgr; l der siRNA / Protamin Lösung rehydratisiert. Inkubieren Liposom / siRNA-Lösung für 10 min bei RT. </li><li> Werden 10 ul einer 60 mM 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimidhydrochlorid (EDC) Lösung der Liposomen / siRNA Lösung sowohl DOTAP und DSPE PALETS. </li><li> Werden 10 ul einer 150 mM N-Hydroxysulfosuccinimid (sulfo-NHS) Lösung der Liposomen / siRNA Lösung sowohl DOTAP und DSPE PALETS. </li><li> Inkubieren des EDC und sulfo-NHS mit der siRNA / Liposomen-Suspension für 2 h bei RT. </li><li> Inkubiere 80 ul einer 500 uM (40 nmol Gesamt) stock RVG-9R-Lösung mit der siRNA / Liposom-Vernetzungslösung für 10 min bei RT. HINWEIS: Die EDC / Sulfo-NHS die RVG-9R zu kovalent an die PEG-Lipide ermöglicht. Verwenden Sie PALETS innerhalb von mehreren Stunden nach der Vorbereitung. Store PALETS bei 4 ° C für mehrere Stunden nach der Zubereitung. </li><li> Um siRNA Verkapselungswirksamkeit von PALETS bestimmen: <ol><li> Messen der Konzentration von siRNA vor und nach der Zugabe von Protamin und Liposomen unter Verwendung eines Spektrophotometers bei 260 nm eingestellt. </li><li> Filtern Sie die nicht eingekapselten siRNA aus dem Liposom / verkapselt siRNA Suspension mit 50 kDa Zentrifugalfilter. Zentrifuge bei 1000 × g für 20 min. </li><li> Messen der Konzentration vonunverkapselte siRNA im Filtrat und der Konzentration an verkapseltem siRNA im Retentat eines Spektrophotometers bei 260 nm verwendet wird. </li></ol></li></ol> 3. Injizierender Mäuse mit LSPCs oder PALETS <ol><li> Expose Mäuse für 5-10 min einer Wärmelampe Gefäß aufzuweiten. </li><li> Anesthetize Maus mit einem 2-3% Isofluran / Sauerstoff fließen 5-10 min vor der Injektion und während der Injektion. Bestätigen anesthetization wenn Tier ist nicht mehr ambulanter und Respiration verlangsamt haben durch eine Zehe Prise zu tun. Platzieren Sie die Maus auf dem Rücken oder der Seite, um die Schwanzvene zugreifen. Verwenden Sie vet Salbe auf die Augen der Maus Austrocknen zu verhindern, während sie unter Narkose. </li><li> Wischen / Spray den Schwanz mit 70% EtOH und injizieren 300 ul LSPCs oder PALETS auf die Schwanzvene der Maus eine 26 G Insulin-Spritze. Beginnen Sie distal in die Schwanzvene Injektion und proximal zu bewegen, wenn die Vene oder Brüche zusammenbricht. </li><li> Bewegen Sie die Maus in einem sauberen Käfig, bis sie Brustlage beibehält. Stellen Sie keine moverwenden, um mit anderen Käfiggenossen, bis sie vollständig erholt hat. Die Mäuse sollten in 5 bis 15 min zu erholen. Sie können auf einem Heizkissen oder unter einer Heizlampe angeordnet werden Körpertemperatur zu halten, wenn Erholung länger braucht. Die Mäuse sollten genau überwacht werden, um Schutz vor Überhitzung. </li><li> Überwachen Sie die Tier mehrere Stunden nach der Injektion, um sicherzustellen, Anästhesie nachlässt, und es gibt keine negativen Auswirkungen der Behandlung. Lassen Sie die LSPCs oder PALETS für mindestens 24 Stunden zirkulieren zu lassen. </li></ol> 4. Analyse der Proteinexpression über Durchflusszytometrie <ol><li> Euthanize Mäuse mit einer 20% CO 2 Durchflussrate für 15 min. </li><li> Präparieren aus einem halben Hemisphäre des Gehirns durch Schneiden der Haut und Schädel der Maus mit einer Schere entfernt. Peel weg halben Hemisphäre des Gehirns aus dem Schädel entfernt Zangen oder das Ende einer Schere. </li><li> Press halben Hemisphäre des Gehirns von jeder Maus durch ein 40 &amp; mgr; m mesh Zellsieb 2,5 ml FACS Puffer (1x; PBS, 1% fötalem Rinderserum, 10 mM EDTA) in einer Petrischale mit dem Kolben einer Spritze. </li><li> Spülen Sie die Zelle Sieb und Petrischale mit einem zusätzlichen 2,5 ml FACS-Puffer. Legen Sie die Einzelzellsuspension in einem 15 ml konischen Röhrchen auf Eis. </li><li> Jeweils 100 ul der 5 ml Einzelzellsuspension in ein 1,5 ml Mikrozentrifugenröhrchen und zentrifugiere für 5 Minuten bei 350 x g. Überstand verwerfen und resuspendieren Zellpellet in 1 ml FACS-Puffer. Spin bei 350 xg für 5 min. Wiederholen Sie mit weiteren 1 ml FACS-Puffer. Halten Sie die Zellsuspension auf Eis. </li><li> Inkubieren der Zellen in 100 ul 1: 100-Verdünnung einer 0,5 mg / ml Ratten-anti-Maus-Fc-Block in FACS-Puffer für 30-60 min auf Eis. Pelletieren, und die Zellen wie in Schritt 4.5 waschen. Halten Sie die Zellsuspension auf Eis. </li><li> Inkubieren der Zellen in 100 ul einer 20 ug / ml Fluoreszenz - Antikörper gegen Maus - PrP C in 7% Mausserum in FACS - Puffer auf Eis für 30-60 min. Pelletieren, und die Zellen wie in Schritt 4.5 waschen. Halten Sie die Zellsuspensionauf Eis. </li><li> Pellet und die Zellen in 1 ml RBC - Lyse - Puffer (1x PBS, 155 mM NH 4 Cl, 12 mM NaHCO 3, 0,1 mM EDTA) für 1 min resuspendieren. Pellet wie in Schritt 4.5 und die Zellen in 1 ml FACS-Puffer resuspendieren. Halten Sie die Zellsuspension auf Eis. </li><li> Analysieren PrP C - Ausdruck ein Durchflusszytometer unter Verwendung von wie zuvor 10 beschrieben. Tor lebende Zellen aus Gesamtzellpopulation. Tor PrP C + Zellen von lebenden Zellpopulation MFI (mittlere Fluoreszenzintensität) zu bestimmen. </li></ol>

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The authors declare that they have no competing financial interests.

Dieser Bericht beschreibt ein Protokoll, das zwei Systeme zur gezielten Verabreichung zu schaffen, die effizient siRNA zum ZNS transportiert. Frühere Verfahren zur enthalten siRNA zum ZNS Liefern Injizieren siRNA / shRNA Vektoren direkt in das Gehirn, intravenöse Injektion von gezielten siRNA oder intravenöse Injektion von nicht-zielgerichteten Liposomen-siRNA-Komplexe. Injektion von siRNA / shRNA Vektoren in das ZNS verursacht eine Abnahme der Zielprotein-Expressionsniveaus. Jedoch die siRNA / shRNA diffundiert nich...

Prionen sind schwere neurodegenerative Erkrankungen, die das ZNS beeinflussen. Prionenkrankheiten ergeben sich aus der Fehlfaltung des normalen zellulären Prionproteins PrP C, durch einen infektiösen Isomer genannt PrP Res. Diese Krankheiten betreffen eine Vielzahl von Arten , einschließlich der bovinen spongiformen Enzephalopathie bei Rindern, Scrapie bei Schafen, Chronic Wasting Disease bei Hirschartigen, und der Creutzfeldt-Jakob - Krankheit beim Menschen 1-3. Prionen verursachen Neurodegeneration, die mit synaptischen Verlust beginnt, und zu Vakuolisation fortschreitet, Gliose, Neuronenverlust und Plaque-Ablagerungen. Schließlich, was zum Tod des Tieres / Einzel - 4. Seit Jahrzehnten haben Forscher untersuchten Verbindungen gemeint, zu verlangsamen oder das Fortschreiten der Prionenerkrankung stoppen. Allerdings haben die Forscher nicht entweder eine erfolgreiche Therapie oder eine wirksame systemische Abgabe Fahrzeug gefunden. Endogenes PrP C Expression für die Entwicklung von Prionenkrankheiten notwendigen 5 </sup&gt;. Daher Verringern oder Eliminieren PrP C - Expression in einer Verzögerung oder Linderung von Krankheiten führen kann. Mehrere Gruppen erstellt transgene Mäuse mit reduzierten Mengen an PrP C oder injiziert lentivectors shRNA direkt in murine Hirngewebe exprimiert die Rolle von PrP C - Expressionsniveaus in Prion - Krankheit zu untersuchen. Diese Forscher Verringerung der Menge an neuronaler PrP C gefunden führte die progressive neuropathology von Prion - Erkrankungen in Anhalten und verlängert die Lebensdauer der Tiere 6-9. Wir haben in der Clearance von PrP Res in Maus - Neuroblastomzellen 10 , dass PrP C siRNA Behandlungsergebnisse berichtet. Diese Studien legen nahe , dass die Verwendung von Therapien PrP C Expressionsniveaus, wie small interfering RNA (siRNA), die spaltet mRNA, ausreichend verzögert das Fortschreiten der Prion - Krankheiten können zu verringern. Allerdings sind die meisten Therapien für Prionenerkrankungen untersucht wurden in einer Weise geliefert, die nicht praktikabel wärein einer klinischen Umgebung. Daher muss ein siRNA-Therapie eine systemische Verabreichungssystem, das an das ZNS intravenös und gezielte geliefert wird. Die Ermittler haben die Verwendung von Liposomen als Abgabevehikel für die Gentherapie Produkte untersucht. Kationische und anionische Lipide zur Bildung von Liposomen verwendet beides. Kationische Lipide sind weiter verbreitet als anionische Lipide verwendet, weil die Ladungsdifferenz zwischen dem kationischen Lipid und DNA / RNA für eine effiziente Verpackung ermöglicht. Ein weiterer Vorteil von kationischen Lipiden ist , dass sie die Zellmembran leichter als andere Lipide 11-14 kreuzen. Jedoch kationische Lipide sind immunogener als anionische Lipide 13,14. Daher haben begonnen Forscher unter Verwendung von kationischen zu verschieben Lipiden in Liposomen anionische. Die Gentherapie Produkte effizient in anionische Liposomen verpackt werden , um die positiv geladenen Peptid Protaminsulfat verwendet, die DNA kondensiert / RNA - Moleküle 15-19. Da anionische lipids sind weniger immunogen als kationische Lipide sie Zirkulationszeiten zugenommen haben kann, und kann in Tiermodellen 13,14 toleriert werden. Liposome werden auf spezifische Gewebe gezielt Peptide unter Verwendung von Targeting, die an die Liposomen gebunden sind. Die RVG-9R Neuropeptid, das an nikotinische Acetylcholin - Rezeptoren bindet, wurde verwendet, siRNA und Liposomen an das ZNS 17-20 abzuzielen. Dieser Bericht beschreibt ein Protokoll drei siRNA Fahrzeuge zu produzieren und zu verpacken und die siRNA zu neuronalen Zellen (Abbildung 1) zu liefern. Liposom-siRNA-Peptid-Komplexe (LSPCs) bestehen aus Liposomen mit siRNA und die RVG-9R Peptid elektrostatisch an der äußeren Oberfläche des Liposoms Targeting. Peptid gerichtet Liposomen therapeutische siRNA (PALETS) eingekapselt sind, bestehend aus siRNA und Protamin im Liposom verkapselt, mit RVG-9R zu Lipid PEG-Gruppen kovalent gebunden sind. die folgenden Methoden verwenden LSPCs und PALET zu erzeugenS, PrP C siRNA verringert PrP C Ausdruck zu 90% in neuronalen Zellen auf, die enorme Versprechen hält das Auftreten von Prion - Krankheit Pathologie zu heilen oder erheblich verzögern.

<table border="0" cellpadding="0" cellspacing="0" width="606">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DOTAP lipid</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">890890</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cholesterol</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">700000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DSPE</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">850715</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DSPE-PEG</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">880125</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Chloroform</td>
			<td style="width:104px;">Fisher Scientific</td>
			<td style="width:119px;">AC268320010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:104px;">EMD Millipore</td>
			<td style="width:119px;">113351</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N2 Gas</td>
			<td style="width:104px;">AirGas</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sucrose</td>
			<td style="width:104px;">Fisher Scientific</td>
			<td style="width:119px;">S5-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Extruder</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">610023</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1.0, 0.4, and 0.2um filters</td>
			<td style="width:104px;">Avanti Lipids</td>
			<td style="width:119px;">610010, 610007, 610006</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PBS</td>
			<td style="width:104px;">Life Technologies</td>
			<td style="width:119px;">70011-044</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Protamine sulfate</td>
			<td style="width:104px;">Fisher Scientific</td>
			<td style="width:119px;">ICN10275205</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EDC</td>
			<td style="width:104px;">Thermo Scientific</td>
			<td style="width:119px;">22980</td>
			<td>Aliquoted for single use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sulfo-NHS</td>
			<td style="width:104px;">Thermo Scientific</td>
			<td style="width:119px;">24510</td>
			<td>Aliquoted for single use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">40um Cell Strainer</td>
			<td style="width:104px;">Fisher Scientific</td>
			<td style="width:119px;">08-771-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rat anti-mouse CD16/CD32 Fc block</td>
			<td style="width:104px;">BD Pharmigen</td>
			<td style="width:119px;">553141</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-PrP antibody (PRC5)</td>
			<td style="width:104px;">Proprietary - PRC</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

delivery of therapeutic sirna to the cns using cationic and anionic liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

Um die Effizienz der siRNA Die Verkapselung in anionischer PALETS erhöhen, wurde die siRNA mit Protamin gemischt. Um die beste Protamin Konzentration für die siRNA bestimmen, wurde die siRNA mit verschiedenen Konzentrationen von Protamin gemischt, von 1: 1 bis 2: 1 (3A). Es gab eine 60-65% siRNA Verkapselungseffizienz in anionischen Liposomen ohne Verwendung von Protamin. Proben mit Protamin: siRNA Molverhältnissen von 1: 1 bis 1,5: 1 (133-266 nM) hatten 80-90% siRNA ...

Watch this Scientific Journal Video about Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes at JoVE.com

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

Abgabe von therapeutischen siRNA an das ZNS mit kationischen und anionische Liposome

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

delivery-therapeutic-sirna-to-cns-using-cationic-anionic

Research

JoVE Journal

Bioengineering

10.4K Aufrufe.  Colorado State University. The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases. 

Serie: Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Entwurf eines zyklischen Druck Bioreaktor für die Ex Vivo Studium der Aorten-Herzklappen

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Forschung

Biotechnik

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

Gentransfer und Transfektion in humanen Pankreaskarzinom-Zellen mit Epidermal Growth Factor Gelatin-Based Engineered Nanovektoren Rezeptor-gezielte

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for silencing specific genes1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies13. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. 
Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies19, 24-26. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection27. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. 
Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (MEA).

Hohe Effizienz, Site-spezifische Transfektion adhärenter Zellen mit siRNA mit Mikroelektroden-Arrays (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

Schnelle und Low-Cost-Prototyping für Medizinische Geräte mit 3D Printed Formen für Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

Poröses Silizium Mikropartikel für die Lieferung von siRNA-Therapeutika

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Mehrfarbenfluoreszenzdetektion für Droplet Mikrofluidik Verwendung von Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

Intraduktale Lieferung an die Kaninchen Brustdrüse

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

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 ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

Umfassende Bewertung der Wirksamkeit und Sicherheit von Plazenta-gezielte Medikamentenverabreichung mit drei komplementäre Methoden

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive properties of in vitro transcribed (IVT) mRNA. With the help of IVT mRNA, the de novo synthesis of a desired protein can be induced without changing the physiological state of the target cell. Moreover, protein biosynthesis can be precisely controlled due to the transient effect of IVT mRNA.
For the efficient transfection of cells, nanoliposomes (NLps) may represent a safe and efficient delivery vehicle for therapeutic mRNA. This study describes a protocol to generate safe and efficient cationic NLps consisting of DC-cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a delivery vector for IVT mRNA. NLps having a defined size, a homogeneous distribution, and a high complexation capacity, and can be produced using the dry-film method. Moreover, we present different test systems to analyze their complexation and transfection efficacies using synthetic enhanced green fluorescent protein (eGFP) mRNA, as well as their effect on cell viability. Overall, the presented protocol provides an effective and safe approach for mRNA complexation, which may advance and improve the administration of therapeutic mRNA.

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.

Generation von kationischen Nanoliposomes für die effiziente Bereitstellung von In-vitro- transkribiert Boten-RNA

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive ...