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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53102/53102fig1large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a>

<table><tbody><tr><td>Nile Red (NiR)</td><td>Sigma</td><td>19123</td><td>BioReagent, suitable for fluorescence, &amp;ge;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 &amp;micro;m Supor Membrane Low Protein Binding</td><td>Life Sciences</td><td>PN4612</td><td>Non-Pyrogenic</td></tr><tr><td>Nanotrac Ultra&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>Sigma</td><td>P5493</td><td>For molecular biology</td></tr><tr><td>Recombinant murine GM-CSF&amp;nbsp;</td><td>Peprotech</td><td>315-03</td><td>&gt;97%, by SDS-PAGE under reducing conditions and visualized by silver stain.</td></tr><tr><td>Recombinant murine IL-4&amp;nbsp;</td><td>Peprotech</td><td>214-14</td><td>&gt;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

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

16.9K 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

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

Facile Herstellung von Intern Selbstorganisierte Lipidpartikel stabilisiert durch Kohlenstoff-Nanoröhren

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

Forschung

Chemie

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.

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 ...

Biotechnik

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

Gezielte Plasma Membrane Lieferung eines Hydrophobic Transport Encapsulated in einem Liquid Crystal Nanoparticle Träger

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Formulation and Characterization of Bioactive Agent Containing Nanodisks

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. Nanodisks are organized as a disk-shaped lipid bilayer whose perimeter is circumscribed by the scaffold protein, usually a member of the exchangeable apolipoprotein family. Numerous hydrophobic bioactive agents have been efficiently solubilized in nanodisks by their integration into the hydrophobic milieu of the particle&#39;s lipid bilayer, yielding a largely homogenous population of particles in the range of 10-20 nm in diameter. The formulation of nanodisks requires a precise ratio of individual components, an appropriate sequential addition of each component, followed by bath sonication of the formulation mixture. The amphipathic scaffold protein spontaneously contacts and reorganizes the dispersed bilayer forming lipid/bioactive agent mixture to form a discrete, homogeneous population of nanodisk&#160;particles. During this process, the reaction mixture transitions from an opaque, turbid appearance to a clarified sample that, when fully optimized, yields no precipitate upon centrifugation. Characterization studies involve the determination of bioactive agent solubilization efficiency, electron microscopy, gel filtration chromatography, ultraviolet visible (UV/Vis) absorbance spectroscopy, and/or fluorescence spectroscopy. This is normally followed by an investigation of biological activity using cultured cells or mice. In the case of nanodisks harboring an antibiotic (i.e., the macrolide polyene antibiotic amphotericin B), their ability to inhibit the growth of yeast or fungi as a function of concentration or time can be measured. The relative ease of formulation, versatility with respect to component parts, nanoscale particle size, inherent stability, and aqueous solubility permits myriad in vitro and in vivo applications of nanodisk technology. In the present article, we describe a general methodology to formulate and characterize nanodisks containing amphotericin&#160;B as the hydrophobic bioactive agent.

Here, we describe the production and characterization of bioactive agents containing nanodisks. Amphotericin B nanodisks are taken as an example to describe the protocol in a stepwise manner.

Formulierung und Charakterisierung bioaktiver Wirkstoffe, die Nanoscheiben enthalten

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. ...

Biochemie

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 ...

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 ...

Preparation of Neutrally-charged, pH-responsive Polymeric Nanoparticles for Cytosolic siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical success for treating previously &#8216;undruggable&#8217; hepatic disease targets with siRNA has been achieved. However, efficient tumor siRNA delivery necessitates additional pharmacokinetic design considerations, including long circulation time, evasion of clearance organs (e.g., liver and kidneys), and tumor penetration and retention. Here, we describe the preparation and in vitro physicochemical/biological characterization of polymeric nanoparticles designed for efficient siRNA delivery, particularly to non-hepatic tissues such as tumors. The siRNA nanoparticles are prepared by electrostatic complexation of siRNA and the diblock copolymer poly(ethylene glycol-b-[2-(dimethylamino)ethyl methacrylate-co-butyl methacrylate]) (PEG-DB) to form polyion complexes (polyplexes) where siRNA is sequestered within the polyplex core and PEG forms a hydrophilic, neutrally-charged corona. Moreover, the DB block becomes membrane-lytic as vesicles of the endolysosomal pathway acidify (&#60; pH 6.8), triggering endosomal escape and cytosolic delivery of siRNA. Methods to characterize the physicochemical characteristics of siRNA nanoparticles such as size, surface charge, particle morphology, and siRNA loading are described. Bioactivity of siRNA nanoparticles is measured using luciferase as a model gene in a rapid and high-throughput gene silencing assay. Designs which pass these initial tests (such as PEG-DB-based polyplexes) are considered appropriate for translation to preclinical animal studies assessing the delivery of siRNA to tumors or other sites of pathology.

Methods to prepare and characterize the physicochemical properties and bioactivity of neutrally-charged, pH-responsive siRNA nanoparticles are presented. Criteria for successful siRNA nanomedicines such as size, morphology, surface charge, siRNA loading, and gene silencing are discussed.

Vorbereitung von Neutral-aufgeladenen, pH-responsiven polymischen Nanopartikeln für zytosolische siRNA Delivery

The success of siRNA as a targeted molecular medicine is dependent upon its efficient cytosolic delivery to cells within the tissue of pathology. Clinical ...

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 ...

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 ...

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

Langfristige Silencing von Intersectin-1s in Mauslungen von wiederkehrenden Lieferung eines spezifischen siRNA durch kationische Liposomen. Auswertung der Knockdown Effects Electron Microscopy

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...