Research reported in this publication was supported by The Johns Hopkins Applied Physics Laboratory&#8217;s Research and Exploratory Development Department, Office of Technology Transfer, and Stuart S. Janney Fellowship Program, in addition to the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number R21HL127355.

1. Die Verarbeitung von SLNs <ol><li> Synthese von SLNs Hinweis: Mit der PIT-Methode 1,17,20, um die Unter 20 nm SLNs 1 vorzubereiten. <ol><li> Verwenden Sie aseptisch, Bioreagenzien oder Zellkultur-Reagenzien und sterilen Materialien (dh, Pipetten, Spatel, Fläschchen, etc.). </li><li> Verwenden Sie ein Biosicherheitswerkbank für die Synthese von SLN (Abbildung 2). </li><li> Kombinieren 0,6 mg Fluoreszenzfarbstoff (Nile Red (NIR) ist NiR Konzentration etwa 0,3 mg / ml) und 0,10 g Heneicosan (Lipid, 5 w / w% Lipid-Konzentration) in einem Glasfläschchen (15 ml), Co-Schmelze mit 90 ° C und umrühren. </li><li> Hinzufügen 0,11 g Polyoxyethylen (10) oleylether (Tensid, 5,5 Gewicht / Gewicht% Tensidkonzentration). Co-Schmelzen der resultierenden Mischung bei 90 ° C und rühren. </li><li> Hinzufügen 1,79 g steriles Wasser zu der Mischung, Erhitzen auf 90ºC und Rühren, bis eine transparente Nanoemulsion gebildet. Hinweis: Unter fortgesetztem Rühren und kontrollierte Kühlung, SLNs erzeugt. Diese Bedingungen verursachen eine Inversion der Wasser-in-Öl-Emulsion zu einer Öl-in-Wasser-Emulsion. Lipophile Moleküle wie NiR Partition auf die Lipidtröpfchen. </li><li> Bereiten Sie eine Nanoemulsion parallel ohne Zugabe des Fluoreszenzfarbstoffs, um als Kontrollprobe zu dienen. Kombinieren 0,10 g Heneicosan und 0,11 g Polyoxyethylen (10) oleylether in ein Fläschchen (15 ml) Co-Schmelze bei 90 ° C und umrühren. <ol><li> Hinzufügen 1,79 g steriles Wasser zu der Mischung, Erhitzen auf 90ºC und Rühren, bis eine transparente Nanoemulsion gebildet. </li></ol></li><li> Weitere Sterilisierung jedes Nanoemulsion mit einer sterilen 0,2 um Filter. </li></ol><img alt="Figur 2" src="/files/ftp_upload/53102/53102fig2.jpg" /> Abbildung 2. Schematische Darstellung der Synthese von SLN. <a href="https://www.jove.com/files/ftp_upload/53102/53102fig2large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> </li><li> Teilchengröße und Polydispersity <ol><li> Verwenden Sie dynamische Lichtstreuung (DLS), um die Teilchengröße und Polydispersität der SLN 1 unter Verwendung einer Glasküvette und ein Gerät zur Partikelgröße zu messen messen. </li><li> Vor den Messungen der Proben zu messen, die Teilchengrößen der beiden bekannten Normen nach Herstellerprotokoll für die Standards Vorbereitung und Messung. </li><li> Füllen Sie das Glas Küvetten mit 1,5 ml Standardlösung und messen Sie die Standardlösung mit den gleichen Messbedingungen wie die Proben. Hinweis: Vorheriger Schritt wurde für jeden Standard wiederholt. </li><li> Messen Sie die Proben vorbereitet. Für jeden, die folgenden Versuchsparameter: eine Laufzeit von 100 sec, Brechungsindex von Wasser (1,33), die Viskosität von Wasser bei 20 ° C (1.002 mPa · s). Anmerkung: Diese Technik verwendet frequenzverschobenen Lichts, um die Größe der Nanopartikel zu messen. </li><li> Berichten über den mittleren Teilchendurchmesser und Polydispersität particle Größenverteilung. </li></ol></li><li> Schmelzpunkt und latente Schmelzwärme <ol><li> Verwenden eines Differential-Scanning-Kalorimeter (DSC) mit einem Auto-Sampler und Flüssigstickstoffkühlquelle ausgestattet, um das thermische Verhalten von SLNs 1 zu untersuchen. </li><li> Pipettieren Sie den bei der Herstellung SLNs in einen 40 ul Aluminiumschale mit einer Masse von etwa 25 mg und hermetisch abzudichten die Pfanne mit einer Universal crimper drücken, um den Feuchtigkeitsverlust während der DSC-Scan zu minimieren. </li><li> Messen jedes Nanoemulsion 5 bis 80 ° C. </li><li> Bericht der Schmelzpunkt und die latente Schmelzwärme aus dem DSC-Diagramm, wo das Tal stellt den Schmelzpunkt und das Integral der Fläche unter der Kurve in der DSC-Diagramm, geteilt durch die Menge des Materials stellt die latente Schmelzwärme. </li></ol></li></ol> 2. SLNs Wechselwirkungen mit Fibroblasten und dendritischen Zellen <ol><li> Kultur von primären humanen Fibroblasten <ol><li> Kultur primären humanen fiFibroblasten gemäß den Anweisungen des Herstellers in einem flüssigen Medium für die Kultur von humanen dermalen Fibroblasten (Medium 106) mit der niedrigen Serum-Wachstumsergänzung (LSGS) -Kit ergänzt. Aufrechtzuerhalten Zellen in einer befeuchteten Atmosphäre bei 37 ° C, 5% CO 2 und Subkultur bei Erreichen 80% Konfluenz. </li><li> Für beide MTT und Bildanalyse, Samenzellen in einem 96-Well-Platte, bei einer Dichte von 2 × 10 4 Zellen pro Vertiefung, und lassen Sie sie bei 37 ° CO / N vor der SLN Belichtung ausgleichen lassen. </li><li> Zum Zeitpunkt Null, verdünnter SLNs in Komplettmedium ab (Abbildung 4), in Bezug auf Lipidkonzentration und Zugabe von 10 ul pro Vertiefung an jedem Replikat auch in der 96-Well-Platte. </li><li> Aufrechtzuerhalten Zellen bei 37 ° C, 5% CO 2 während der Inkubationszeit. </li></ol></li><li> MTT Toxizitätstest <ol><li> Nach 24 h Inkubation mit SLNs bestimmen zelluläre Lebensfähigkeit unter Verwendung des MTT-Assay nach dem Protokoll des Herstellers. &lt;/ li&gt; <li> Waschen Sie die Zellen einmal und fügen Sie frisches Medium in jede Vertiefung (100 ul / Vertiefung). Getötet Kontrolle Wells durch Inkubation in einer 70% igen Methanol-Lösung für 10 min vor dem Ersetzen mit frischem Medium. </li><li> Hinzufügen MTT-Reagens (10 ul pro Vertiefung) zu jeder Vertiefung der Mikrotiterplatte und Inkubieren O / N bei 37 ° C. </li><li> Nach O / N Inkubation solubilisieren intrazellulärem Formazin Kristalle mit dem vorgesehenen Detergenslösung (100 ul pro Vertiefung) gemäß dem Protokoll des Herstellers. </li><li> Nach 3 h Inkubation bei RT erhalten Absorptionswerte bei einer Meßwellenlänge von 570 nm mit einem Mikrotiterplatten-Lesegerät. Anmerkung: MTT-Reagenz verwendet wird, sollte weniger als 1% (w / v) 3- (4,5-dimethylthiazolyl-2) -2, 5-Diphenyl-Tetrazolium-Bromid. </li></ol></li><li> Fluoreszenzmikroskopie <ol><li> Zu bestimmten Zeitpunkten nach der Dosierung von Fibroblasten mit SLN, wäscht die Fibroblasten zweimal mit steriler phosphatgepufferter Salzlösung (PBS) und fixieren 10 min mit kaltem 70% igem Methanol. </li><li> Nach 10 min, Ersetzen Sie das Methanol mit PBS und die Aufnahmen mit einem inversen Mikroskop, mit einem Bandpass (BP) 690/50-nm-Filter-Set. </li></ol></li><li> Kultur von Knochenmark der Maus Dendritic Cells <ol><li> Induzieren Maus-Knochenmark gewonnene dendritische Zellen (BMDC) wie zuvor beschrieben 23,24. Kurz gesagt, Platte Einzelzellsuspensionen von C57BL / 10 Knochenmark in 100 mm Petrischalen bei 2 × 10 6 / Platte mit rekombinantem murinen Granulozyten-Makrophagen-Kolonie-stimulierender Faktor (GM-CSF, 300 U / ml) und rekombinantem murinem IL-4 (B-Zell-stimulierender Faktor, 200 U / ml). Ändern Medien alle 3 Tage. </li><li> Am 7. Tag hinzuzufügen NiR belastet SLN auf die Schalen in einer Endkonzentration von 0,5 und 5,0 ug / ml Lipid und Aufrechterhaltung der Zellen in einem 37 ° C, 5% CO 2 -Inkubator für die gewünschte Dauer der Exposition. </li><li> Sammeln Zellen von den Schalen durch Ansaugen alle Medien in der Platte gesammelt wird in ein 50 ml Röhrchen, gefolgt von gründlichem Waschen der Platte (lose ad verdrängenrente Zellen) mit 5 ml phosphatgepufferter Salzlösung (PBS), enthaltend 4 mM Ethylendiamintetraessigsäure (EDTA). Sammeln Sie die erhaltene Zellsuspension in der gleichen 50-ml-Tube. </li></ol></li><li> Beurteilung der SLNs Incorporation über Durchflusszytometrie <ol><li> Bestimmen Sie die Konzentration des in Punkt 2.4.3 erhaltene Zellsuspension und vertreiben 3x10 5 Zellen in eine 12 mm x 75 mm Rund FACS-Röhrchen. </li><li> Die Zellen werden durch Zugabe von 1 ml PBS und anschließendem Zentrifugieren der Röhrchen für 5 min bei 400 · g, bei einer Temperatur von 4 ° C. </li><li> Der Überstand verworfen und das Pellet mit 100 ul PBS / 2 mM EDTA / 0,1% Rinderserumalbumin (BSA) (Färbepuffer) mit anti-CD11c-FITC monoklonalen Antikörper (0,25 ug / ml final). Inkubieren für 30 min auf Eis im Dunkeln. </li><li> Die Zellen werden mit 1 ml PBS und dann zentrifugieren Röhrchen 5 min bei 400 · g, bei einer Temperatur von 4 ° C. </li><li> Überstand verwerfen und resuspendieren jedesPellet mit 400 &amp; mgr; l Färbepuffer. Die Zellen sind nun bereit für die Übernahme der Proben mit einem Durchflusszytometer (möglicherweise mit blauen und roten Lasern ausgestattet ist). </li><li> Mit Hilfe der Durchflusszytometrie-Analyse-Software, zu bewerten den Grad der Einbeziehung der SLN von DC durch Gating auf die Bevölkerung von CD11c + Zellen (FITC positiv) und Vergleichen der Intensität der SLN-assoziierte Fluoreszenz (in der PerCP-Cy5.5 oder APC-Kanälen gemessen). </li></ol></li></ol>

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The authors have nothing to disclose.

In dieser Studie wurden die Synthese SLNs und ihre Anwendbarkeit für eine intrazelluläre Targeting-Anwendungen erforscht. Diese biokompatible Nanopartikel versprechen als Lieferfahrzeuge für mehrere Anwendungen einschließlich der Arzneimittelabgabe, Gen-Silencing, und Impfstoff-Technologien 25-30 gezeigt. Ultrakleine SLNs wurden unter Verwendung eines einfachen Prozesses hergestellt, und ihre Wechselwirkungen mit primären Hautzellen und primären Immunzellen wurden untersucht. SLNs wurden zur Verkapselun...

Nanopartikel-basierten Lieferfahrzeuge haben ein großes Versprechen für die intrazelluläre Targeting-Anwendungen gezeigt, die Bereitstellung eines Mechanismus, um gezielt zu verändern zellulären Signaltransduktion und Genexpression. Diese Fahrzeuge können mit Medikamenten, Proteinen und Nukleinsäuren, einen Aufprall zellulären Reaktionen und erreichen eine gewünschte Wirkung auf Zielgewebe beladen werden. Viele Arten von Nano zu therapeutischen und diagnostischen Nutzen, einschließlich Lipide, Polymere, Silizium und magnetischen Materialien untersucht worden. Diese Systeme sind attraktiv aufgrund ihres Potentials zur lokalisierten Arzneimittelabgabe, erhöhten therapeutischen Konzentration im Zielgewebe, und eine Verringerung der systemischen Toxizität. Feste Lipidnanopartikel (SLN) sind ein gut untersuchtes Beispiel einer Nanopartikel-Abgabesystem, das als vielversprechende Arzneimittelabgabevehikel in den letzten Jahren abzeichnet. SLNs kann leicht für mehrere Anwendungen einschließlich Bio-Sensing 1, Kosmetika 2 und t formulierenherapeutic Lieferung 3-7. Ihr Nutzen ergibt sich aus der Tatsache, dass sie vollständig aus resorbierbaren, ungiftig Lipiden, was zu einer verbesserten Biokompatibilität. Während der Synthese können lipophile Medikamente in SLN Fahrzeuge aufgenommen werden, wodurch Arzneimittellöslichkeit und Eignung Erhöhung für die parenterale Verabreichung. SLN Fahrzeugen auch dazu beitragen, eingekapselt Therapeutika zu stabilisieren, wodurch deren Abbau und Freiraum, und die Maximierung der therapeutischen Wirkung. Diese Fahrzeuge sind vor allem für Langzeitwirkung, kontrollierte Trennöle gut geeignet aufgrund ihrer Stabilität bei Körpertemperatur 3,4,8,9. Wichtiger ist, Verkapselung von Wirkstoffen in Lipidnanopartikel ändert die intrinsische die pharmakokinetischen Profile der Arzneimittelmoleküle. Dies bietet einen Vorteil, indem sie die kontrollierte Freisetzung von Wirkstoffen mit einem engen therapeutischen Index. Die Freisetzungsrate des SLN-einge Therapeutika kann abgestimmt basieren auf der Lipidabbaurate oder das Medikament Diffusionsgeschwindigkeit in dieLipidmatrix. SLNs werden oft entwickelt, um in spezifischen Zielgeweben ansammeln. Beispielsweise ihre Größe (typischerweise größer als 10 nm) potenziert Retention im Blutkreislauf, wobei der Leaky-Vaskulatur Tumorgewebe erleichtert Abscheidung. Darüber hinaus hat der Weg der Verabreichung Partikel wurde gezeigt, daß die biologische Verteilung, die das Potenzial zu verändern, wie Lymphknoten 10,11 um spezifische physiologische Zielstrukturen. Bei der Abscheidung in Zielgeweben zu erzielen geeigneten zellulären Wechselwirkungen und eventuelle Internalisierung von Nanopartikeln ist schwierig aufgrund der Fähigkeit der Zellmembranen, um den Fluss von Ionen und Molekülen in die und aus der Zelle 12 selektiv zu steuern. Um die zelluläre Aufnahme zu erleichtern, ist es möglich, Nano mit spezifischen Liganden einschließlich Peptiden, kleine Moleküle, als auch monoklonale Antikörper 13,14 modifizieren. Verschiedene Mechanismen, die sowohl passive Penetration und aktiven Transport von Nanopartikelnüber die Zellmembran sind zuvor beschrieben worden 3,12,15. Im allgemeinen hat es sich gezeigt, daß zell Nanopartikel Wechselwirkungen werden durch die physikalisch-chemischen Eigenschaften der Nanopartikel einschließlich der Größe, Form, Oberflächenladung und Oberflächenchemie beeinflusst neben zellspezifischen Parameter wie Zelltyp oder Zellzyklus-Phase 12. Eine frühere Untersuchung zeigte die Synthese von Sub-10 nm SLNs zur topischen 16 und Biomarker-Erkennungsanwendungen 1 unter Verwendung der Phaseninversionstemperatur (PIT) Verfahren 17. Dies ist eine schonende Syntheseverfahren, wobei 2 die Zusammensetzung konstant bleibt, während die Temperatur allmählich verändert wird. Kontinuierliches Rühren der erhitzten Lösung beim Abkühlen auf Raumtemperatur führt zu einer Nanoemulsion. Dieses Verfahren resultiert in der Synthese von SLNs mit kleinerer Teilchengrße 1 als die zuvor unter Verwendung verschiedener Verfahren für die Synthese von Lipid nan gemeldetoparticles 17-22. Die resultierende Größenskala von weniger als 20 nm beträgt, stellt einen Vorteil für die intrazelluläre Targeting-Anwendungen aufgrund der erhöhten Oberfläche und dem Potential für erhöhte zelluläre Interaktionen. Eine schematische Darstellung SLNs designt einen fluoreszierenden Farbstoff oder therapeutischen liefern, ist in Abbildung 1 dargestellt. Die SLN bestehen aus einer Lipid Innenraum (zB lineares Alkan), die die Einarbeitung von lipophilen Verbindungen (beispielsweise Farbstoffe oder Therapeutika) und einem Tensid Außen (zB lineares nichtionisches Tensid) von Wasser umgeben ist. In dieser Studie wurden SLNs mit einem Fluoreszenzfarbstoff beladen, und als ein Modell verwendet, um Partikel-Zell-Wechselwirkungen zu untersuchen. Primäre humane dermale Fibroblasten und Maus dendritischen Zellen wurden farbstoffbeladenen SLN über die Zeit ausgesetzt, um Wechselwirkungen im Hinblick auf Toxizität und Partikelaufnahme charakterisieren. A 3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenylphenyltetrazolium (MTT) -Assay wurde utilium geeignete Dosierung Ebenen zu etablieren zed. Fluoreszenzmikroskopie und Durchflusszytometrie zwei eingesetzt werden, um Partikelaufnahme in vitro zu untersuchen Methoden. <img alt="Abbildung 1" src="/files/ftp_upload/53102/53102fig1.jpg" /> Abbildung 1. Schematische Darstellung des SLN, die die Hauptbestandteile. <a href="https://www.jove.com/files/ftp_upload/53102/53102fig1large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Nile Red (NiR)</td>
			<td>Sigma</td>
			<td>19123</td>
			<td>BioReagent, suitable for fluorescence, &#8805;98.0%</td>
		</tr>
		<tr>
			<td>Heneicosane</td>
			<td>Aldrich</td>
			<td>286052</td>
			<td>98%</td>
		</tr>
		<tr>
			<td>Brij O10</td>
			<td>Sigma</td>
			<td>P6136</td>
			<td>Brij 97, C18-1E10, Polyoxyethylene (10) oleyl ether</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma</td>
			<td>W3500</td>
			<td>Sterile-filtered, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Syringe Filter 0.2 &#181;m Supor Membrane Low Protein Binding</td>
			<td>Life Sciences</td>
			<td>PN4612</td>
			<td>Non-Pyrogenic</td>
		</tr>
		<tr>
			<td>Nanotrac Ultra&#160;</td>
			<td>Microtrac</td>
			<td>serial number U1985IS</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>Differential Scanning Calorimeter</td>
			<td>Mettlet-Toledo</td>
			<td>----</td>
			<td>Instument</td>
		</tr>
		<tr>
			<td>Primary human fibroblasts&#160;</td>
			<td>Life Technologies</td>
			<td>C-004-5C</td>
			<td>Neonatal (HDFn)</td>
		</tr>
		<tr>
			<td>Medium 106</td>
			<td>Life Technologies</td>
			<td>M-106-500</td>
			<td>A sterile, liquid medium for the culture of human dermal fibroblasts.</td>
		</tr>
		<tr>
			<td>Low Serum Growth Supplement Kit (LSGS Kit)</td>
			<td>Life Technologies</td>
			<td>S-003-K</td>
			<td>All the components of complete LSGS</td>
		</tr>
		<tr>
			<td>MTT Cell Proliferation Assay Kit</td>
			<td>Trevigen</td>
			<td>4890-025-K</td>
			<td>Sensitive kit for the measurement of cell proliferation based upon the reduction of the tetrazolium salt, 3,[4,5-dimethylthiazol-2- yl]-2,5-diphenyl-tetrazolium bromide (MTT)</td>
		</tr>
		<tr>
			<td>Safire2 microplate reader</td>
			<td>Tecan</td>
			<td>----</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline&#160;</td>
			<td>Sigma</td>
			<td>P5493</td>
			<td>For molecular biology</td>
		</tr>
		<tr>
			<td>Recombinant murine GM-CSF&#160;</td>
			<td>R&#38;D Systems</td>
			<td>415</td>
			<td>&#62;97%, by SDS-PAGE under reducing conditions and visualized by silver stain.</td>
		</tr>
		<tr>
			<td>Recombinant murine IL-4&#160;</td>
			<td>R&#38;D Systems</td>
			<td>404</td>
			<td>&#62;97%, by SDS-PAGE under reducing conditions and visualized by silver stain.</td>
		</tr>
	</tbody>
</table>

solid lipid nanoparticles (slns) for intracellular targeting applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

Die PIT-Verfahren wurde verwendet, um die SLN zu synthetisieren und die Phaseninversionstemperatur wurde bestimmt unter Verwendung eines Wasserbades. Die Proben wurden langsam erwärmt und vorsichtig gerührt, bis die Lösung klar erschien. Der Phaseninversionstemperatur für die SLNs unter Verwendung Heneicosan Lipid 45 ° C. Tabelle 1 faßt die Teilchengröße, Polydispersität, Schmelzpunkt und die latente Schmelzwärme für den SLN. Die synthetisierten Verwendung der oben beschriebenen Verarbeitungs...

Watch this Scientific Journal Video about Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications at JoVE.com

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Feste Lipidnanopartikel (SLN) für die intrazelluläre Targeting-Anwendungen

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

solid-lipid-nanoparticles-slns-for-intracellular-targeting

Research

JoVE Journal

Bioengineering

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

16.8K Aufrufe.  The Johns Hopkins Applied Physics Laboratory. In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions. 

Serie: Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

Viral Nanopartikel für<em&gt; In vivo</em&gt; Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Forschung

Biotechnik

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Zellzusammendrücken als Robust, Mikrofluidik Intrazelluläre Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

Harmonic Nanopartikel für Regenerative Forschung

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

Produktion und Targeting von Monovalenter Quantenpunkte

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Biofunktionalisierten Prussian Blue Nanopartikel für Molekulare Bildgebung Multimodale Anwendungen

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

Zellmarkierung und Targeting mit superparamagnetische Eisenoxid-Nanopartikel

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.

Herstellung und Drug-Delivery-Anwendungen von Silk Nanopartikel

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Aufnahme von neuen Lipid-beschichteten Nanopartikeln mit Falcarindiol von humanen mesenchymalen Stammzellen

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

Gleichzeitige Quantifizierung ausgewählter Kynurenine, analysiert durch Flüssigchromatographie-Massenspektrometrie in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

Formulierung und Charakterisierung von Lipid-Nanopartikeln für die Genabgabe mit einer mikrofluidischen Mischplattform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...