Name: a protocol for the production of gliadin-cyanoacrylate nanoparticles for hydrophilic coating
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1. Entfettung Gewerbe Gliadin <ol><li> Messen Sie 150 ml Aceton mit einem Messzylinder abgemessen und in 250 ml-Erlenmeyerkolben. <ol><li> Während mit einem Spin-Bar auf einem Magnetrührer Rühren bei RT 30 g handelsübliches Gliadin Pulver hinzufügen. Verschließen Sie die Öffnung der Flasche mit Aluminiumfolie und halten auf O / N in der Haube gerührt wurde. </li></ol></li><li> Filtrieren der Lösung mit einem Filterpapier. <ol><li> Das Filtrat wird mit frischem Aceton (ca. 50 ml). Lassen Sie stehen für 10 Minuten das Aceton ablaufen zu lassen. </li><li> Das Filtrat zusammen mit dem Filterpapier unter einer großen Schale wie beispielsweise eine Zellkultur quadratische Schale. Decken Sie die ganze Schüssel mit einem großen Filterpapier zu verlangsamen das Verdampfen von Rest Aceton nach unten. </li><li> Lassen Sie das Filtrat vollständig in die Haube O / N zu trocknen. Bewahren Sie die entfettet Gliadin in einem luftdichten Behälter bei RT. </li></ol></li></ol> 2. Herstellung von gereinigtem Gliadin <ol><li> Messen Sie 150 ml water mit einem Messzylinder abgemessen und in 1.000 ml Erlenmeyerkolben. <ol><li> Messen Sie 350 ml absolutem Ethanol mit einem Durcheinander Zylinder und strömen in die gleiche 1.000 ml Erlenmeyerkolben. Rühre kräftig (800 - 1000 rpm) mit einem Rührstab, bis keine Luftblase aus dem Lösungsgemisch erzeugt wird. </li></ol></li><li> Unter Rühren werden 20 g entfettetes kommerziellen Gliadin Pulver. Hinzufügen Gliadin in kleinen Mengen zu einer Zeit, um die Bildung von Klumpen zu vermeiden, dass Luft in der Mitte enthält. Halten Sie sich auf dem Rühren O / N. </li><li> Übertragen Sie die gesamte Lösung auf einen 1000 ml Chaos Zylinder. Lassen Sie zwei Tage lang stehen. Während dieser Zeit Verunreinigungen werden am Boden des Zylinders ausgefällt werden. </li><li> Den Überstand in einen Rotationsverdampfer mit einer Pipette, und Entfernen von Ethanol aus dem Überstand, so viel wie möglich. Da der Anteil von Ethanol reduziert wird, wird Gliadin in der Lösung als Aggregat erscheinen. </li><li> Frieren Sie die Lösung, die Gliadin-Aggregate durch Tauchen / Drehen in Methanol/ Trockeneis-Mischung und gefrier trocken bei -70 ° C im Vakuum. Vor dem Gefriertrocknen, stellen Sie sicher, dass die gesamte Lösung ohne Flüssigkeit gefroren ist. HINWEIS: Das gefriergetrocknete Gliadin einen porösen Feststoff zu bilden. </li><li> Crush das gefriergetrocknete Gliadin mit Mörser und Stößel, und schleifen mit einer Kaffeemühle eine feine Leistung (&lt;0,5 mm) zu erhalten. Das Produkt in einem luftdichten Behälter und Speicher bei RT. </li></ol> 3. Polymerisation Reaktion von ECA mit Gliadin <ol><li> Legen Sie ein Szintillationsphiole (Volumen etwa 20 ml) in einer Waage und tarieren. In 3,2 g destilliertes Wasser und 6,8 ​​g absolutem Ethanol in der scitillation Fläschchen. </li><li> Bewegen Sie den Szintillationsfläschchen auf einem Magnetrührer, legte einen magnetischen Spin bar (20 x 3 mm) in das Fläschchen und kräftig rühren (800 - 1000 Umdrehungen pro Minute), bis keine Luftblase aus der wäßrigen Ethanol-Gemisch erzeugt wird. </li><li> Werden 40 ul 4 N HCl in das Fläschchen unter Rühren. In 20 mg Gliadin in das Fläschchen während sti<html> Rring, und halten Sie auf dem Rühren bis Gliadin Pulver vollständig in der wässrigen Ethanol-Gemisch gelöst ist. </li><li> Nachdem die Gliadin-Lösung ist klar auf die bloßen Augen sicher, fügen Sie langsam 80-100 ul ECA während Rührgeschwindigkeit noch 800 - 1000 Umdrehungen pro Minute. <ol><li> Senken Sie die Rührgeschwindigkeit auf 500 Umdrehungen pro Minute, und weiterhin für 1 Stunde gerührt wurde. Da die Reaktion fortschreitet, beobachten Trübung, die die Polymerisationsreaktion zeigt an, dass im Gange ist. </li></ol></li><li> Wenn die Reaktion beendet ist, wird die Lösung in Zentrifugenröhrchen und das Gewicht der Rohre auszugleichen. Zentrifugieren Sie das Reaktionsprodukt bei 10.000 × g für 20 min. Während dieser Zeit bleiben Nebenprodukt fällt aus und Nanopartikel in der überstehende. Die Hauptkomponente von Nebenprodukt ist PACA-Homopolymer. </li><li> Nach der Zentrifuge, übertragen die erzeugte Nanopartikel-Suspension (Überstand) mit einer Pipette in ein Szintillationsphiole (oder einem luftdichten Behälter), und speichern Sie es bei RT. </li></ol>title &quot;&gt; 4. Charakterisierung des Produkts <ol><li> Bereiten Sie 10 g von 68 Gew% igen wässrigen Ethanol durch Mischen von 3,2 g Wasser und 6,8 ​​g absolutem Ethanol in 20 ml Szintillationsphiole und rühren, bis keine Luftblase aus dem Lösungsgemisch erzeugt wird. <ol><li> Werden 40 ul 4 N HCl-Lösung zu der Mischung unter Rühren. Zugabe von 50 &amp; mgr; l Nanopartikel-Suspension auf die vorbereitete Ethanollösung unter Rühren. </li></ol></li><li> Messen Sie die Größe der Nanopartikel mit Dynamic Light Scattering (DLS) von der Probenlösung unter Verwendung von oben und folgen Sie den Anweisungen des Herstellers. HINWEIS: Die Größe des Endprodukts hat kleiner als 200 nm sein. Wenn sie größer als 200 nm ist, der das Produkt nicht über einen längeren Zeitraum stabil sein. Größere Nanopartikel werden erzeugt, wenn die verwendete ECA nicht frisch ist. </li></ol> 5. Prüfung des Produkts <ol><li> Bereiten Sie eine Glasplatte wie einen Handspiegel. Waschen Sie die Oberfläche der Glasplatte mit Seifenwasser. Rinse der Oberfläche der Glasplatte mit fließendem Wasser. Trocknen der gereinigten Oberfläche oder verwenden, ohne für den nächsten Schritt zu trocknen. </li><li> Sprühen Sie die Nanopartikel-Suspension in 3.6) auf einem Teil der Glasplatte erhalten, und spülen Sie sofort die Oberfläche mit Wasser abfließt. </li><li> Spray reines Wasser auf der Oberfläche der Glasplatte. Achten Sie auf die beschichtete (versprüht mit Nanopartikel-Suspension) Oberfläche halten Wasserschicht, während die unbeschichteten Oberfläche ist klar. </li></ol>

<ol>
	<li>Vauthier, C., Dubernet, C., Fattal, E., Pinto-Alphandary, H., Couvreur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(alkylcyanoacrylates)+as+biodegradable+materials+for+biomedical+applications.">Poly(alkylcyanoacrylates) as biodegradable materials for biomedical applications.</a> Adv. Drug Deliver. Rev. 55, 519-548 (2003).</li><li>Wieser, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemistry+of+gluten+proteins.">Chemistry of gluten proteins.</a> Food Microbiol. 24, 115-119 (2007).</li><li>Bietz, J. A., Wall, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identity+of+high+molecular+weight+gliadin+and+ethanol+soluble+glutenin+subunits+of+wheat:+Relation+to+gluten+structure.">Identity of high molecular weight gliadin and ethanol soluble glutenin subunits of wheat: Relation to gluten structure.</a> Cereal Chem. 57, 415-421 (1980).</li><li>Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+composites+by+using+gliadin+as+a+bonding+material.">Production of composites by using gliadin as a bonding material.</a> J. Cereal Sci. 54, 168-172 (2011).</li><li>Kim, S., Kim, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+gliadin-poly(ethyl+cyanoacrylate)+nanoparticles+for+hydrophilic+coating.">Production of gliadin-poly(ethyl cyanoacrylate) nanoparticles for hydrophilic coating.</a> J. Nanopart. Res. 16, 1-10 (2014).</li><li>Behan, N., Birkinshaw, C., Clarke, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly+n-butyl+cyanoacrylate+nanoparticles:+a+mechanistic+study+of+polymerisation+and+particle+formation.">Poly n-butyl cyanoacrylate nanoparticles: a mechanistic study of polymerisation and particle formation.</a> Biomaterials. 22, 1335-1344 (2001).</li><li>Nicolas, J., Couvreur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+poly(alkyl+cyanoacrylate)-based+colloidal+nanomedicines.">Synthesis of poly(alkyl cyanoacrylate)-based colloidal nanomedicines.</a> Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 1, 111-127 (2009).</li><li>Kim, S., Evans, K., Biswas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+BSA-poly(ethyl+cyanoacrylate)+nanoparticles+as+a+coating+material+that+improves+wetting+property.">Production of BSA-poly(ethyl cyanoacrylate) nanoparticles as a coating material that improves wetting property.</a> Colloid Surface. B. 107, 68-75 (2013).</li><li>Lander, L. M., Siewierski, L. M., Brittain, W. J., Vogler, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+comparison+of+contact+angle+methods.">A systematic comparison of contact angle methods.</a> Langmuir. 9, 2237-2239 (1993).</li><li>Davies, J., Nunnerley, C. S., Brisley, A. C., Edwards, J. C., Finlayson, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Dynamic+Contact+Angle+Profile+Analysis+in+Studying+the+Kinetics+of+Protein+Removal+from+Steel+Glass,+Polytetrafluoroethylene,+Polypropylene,+Ethylenepropylene+Rubber,+and+Silicone+Surfaces.">Use of Dynamic Contact Angle Profile Analysis in Studying the Kinetics of Protein Removal from Steel Glass, Polytetrafluoroethylene, Polypropylene, Ethylenepropylene Rubber, and Silicone Surfaces.</a> J. Colloid Interf. Sci. 182, 437-443 (1996).</li><li>Giolando, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-crystals+of+titanium+dioxide+in+aluminum+oxide:+A+transparent+self-cleaning+coating+applicable+to+solar+energy.">Nano-crystals of titanium dioxide in aluminum oxide: A transparent self-cleaning coating applicable to solar energy.</a> Sol. Energy. 97, 195-199 (2013).</li></ol>

Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

There are several critical steps in the production of the nanoparticle suspension. If the purified gliadin contains impurities, the reaction with ECA will produce side products. Although these unwanted products can be removed from the reaction medium during the centrifugation stage, it lowers the yield of the major product. If the gliadin solution prepared during experimental step 2.3) does not show clear separation between supernatant and precipitate after two days, the solution needs to stand for longer time. Using fre...

The goal of this article is to show the protocol for the preparation of nanoparticle suspension that modifies the wetting property of materials by a simple spray. The presented nanoparticle suspension is made from alkyl cyanoacrylate1 and a cereal protein, gliadin2,3. During the manufacturing process, protein aggregates are formed in aqueous ethanol4. Subsequent reaction with monomer (alkyl cyanoacrylate) produces the nanoparticle that is comprised of a protein core surrounded by linear polymer chains [poly(alkyl cyanoacrylate)]5.
Poly(alkyl cyanoacrylate)s are biodegradable and have been used for the production of nanoparticles via emulsion polymerization6. This reaction is spontaneously initiated by the hydroxyl groups dissociated from water or by other nucleophilic groups in the reaction medium7. In the case of the reaction presented in this article, the amine groups on the surface of protein aggregates initiate the polymerization reaction of alkyl cyanoacrylate monomers5,8. As a result of this reaction, nanoparticles are formed in the reaction medium. The core of the nanoparticle is protein aggregates and the outer layer is poly(alkyl cyanoacrylate) (PACA) chains. The prepared nanoparticle has a strong affinity on most materials (more precisely, any material which PACA can adsorb to) and adheres onto their surface to form a thin coating on a nanometer scale. A simple spray coating instantly turns the surface of the materials hydrophilic.
Gliadin is one of the main fractions of gluten, which is in the endosperms of wheat. Gliadins are mainly monomeric proteins with molecular weights around 28,000 - 55,000. Non-covalent bonds such as hydrogen bonds, ionic bonds and hydrophobic bonds are responsible for the aggregation of gliadins2. Although gliadin is chosen as a reactant in this article, many other proteins can also be used for the same purpose. However, the reaction condition needs to be modified accordingly because the condition for inducing aggregation is dependent on the type of protein to be employed8. Compared with other proteins, gliadin is more readily available, purification is simple, and production cost is low. Although ethyl cyanoacrylate (ECA) is chosen as a monomer for the presented reaction, other alkyl cyanoacrylates can also be used for the same reaction. The reason for choosing ECA is that it is readily available at low cost.

<table><tbody><tr><td>Ethyl cyanoacrylate (ECA) monomer</td><td>K&amp;amp;R International (Laguna Niguel, CA)</td><td>I-1605</td><td>Any pure ECA can be used.</td></tr><tr><td>Gliadin</td><td>MGP Ingredients, Inc (Atchison, KS)</td><td>Gift from the company</td><td>Gliadin can be purchased from Sigma-Aldrich (cat #: G3375-25G). Instead of gliadin, any commercial&amp;nbsp; gluten can be used.</td></tr><tr><td>HCl</td><td>Any</td><td></td><td>Any reagent grade chemical can be used.</td></tr><tr><td>Acetone</td><td>Any</td><td></td><td>Any reagent grade chemical can be used.</td></tr><tr><td>Methanol</td><td>Any</td><td></td><td>Any reagent grade chemical can be used.</td></tr><tr><td>Ethanol (100%)</td><td>Any</td><td></td><td>Any reagent grade chemical can be used.</td></tr><tr><td>Filter paper</td><td>Any</td><td></td><td>Any grade filter paper larger than 10 cm can be used.</td></tr><tr><td>Cell culture square dish</td><td>Any</td><td></td><td>Any dish larger than 20 x 20 cm can be used.</td></tr><tr><td>Coffee grinder</td><td>Any</td><td></td><td>Any coffee grinder can be used.</td></tr><tr><td>Rotary evaporator</td><td>Any</td><td></td><td>Any rotary evaporator can be used.</td></tr><tr><td>Freeze Dryer</td><td>Any</td><td></td><td>Any freeze dryer that can reach -70 &amp;deg;C can be used.</td></tr><tr><td>Centrifuge</td><td>Any</td><td></td><td>Any centrifuge that can apply 1,000 x g can be used.</td></tr><tr><td>Magnetic stirrer</td><td>Any</td><td></td><td>Any magnetic stirrer that can turn spin bar to 1,000 rpm can be used.</td></tr><tr><td>Dynamic Light Scattering (DLS)</td><td>Brookhaven Instruments Corporation</td><td>NanoBrook Omni Zeta Potential Analyzer</td><td>DLS from any company can be used.</td></tr><tr><td>Scanning Electron Microscope (SEM)</td><td>Carl Zeiss Inc.</td><td></td><td>Any SEM can be used.</td></tr><tr><td>Dynamic Contact Angle (DCA)</td><td>Thermo Cahn Instruments</td><td></td><td>Any DCA can be used.</td></tr></tbody></table>

a protocol for the production of gliadin-cyanoacrylate nanoparticles for hydrophilic coating

Dieser Artikel stellt ein Protokoll für die Herstellung von Protein-basierten Nanopartikel, die die hydrophobe Oberfläche hydrophil durch eine einfache Spritzbelages ändert. Diese Nanopartikel werden durch die Polymerisationsreaktion von Alkylcyanoacrylat auf der Oberfläche der Getreideprotein (Gliadin) Moleküle hergestellt. Alkylcyanoacrylatzusammensetzung ist ein Monomer, das sofort bei RT polymerisiert, wenn es auf die Oberfläche von Materialien aufgebracht wird. Seine Polymerisationsreaktion wird durch die Spurenmengen von schwach basischen oder nukleophilen Spezies auf der Oberfläche, einschließlich der Feuchtigkeit initiiert. Einmal polymerisiert, zeigen die polymerisierten Alkylcyanoacrylaten eine starke Affinität zu den Objektmaterialien, weil Nitrilgruppen im Rückgrat aus Poly (Alkylcyanoacrylat) sind. Proteine ​​arbeiten auch als Initiator für diese Polymerisation, weil sie Amingruppen enthalten, die die Polymerisation von Cyanoacrylat initiieren kann. Wenn aggregiertem Protein als ein Initiator verwendet wird, Proteinaggregat wird durch die hydrophoben umgebenPoly (alkyl Cyanacrylat) Ketten nach der Polymerisationsreaktion von Alkylcyanoacrylat. Durch die Versuchsbedingungen zu steuern, Partikel im Nanometerbereich erzeugt werden. Die hergestellten Nanopartikel adsorbieren leicht an der Oberfläche der meisten Materialien einschließlich Glas, Metallen, Kunststoffen, Holz, Leder und Textilien. Wenn die Oberfläche eines Materials mit der hergestellten Nanopartikel-Suspension und wäscht mit Wasser besprüht wird, werden die micellare Struktur der Nanoteilchen ändert seine Konformation und die hydrophilen Proteine ​​der Luft ausgesetzt. Als Ergebnis ändert sich die Nanopartikel-beschichteten Oberfläche hydrophil.

Nanopartikel können in verschiedenen Reaktionsbedingungen hergestellt werden. Gliadin bildet aggregieren in breite Palette von Ethanolgehalt 5. Allerdings muss die Größe der Aggregate so klein wie möglich sein , weil eine zusätzliche Schicht (dh., Polymerisiertes ECA) wird zu diesem Aggregat zugesetzt werden , und dieser Prozess wird die Endgröße größer. Wenn die endgültige Größe der Teilchen zu groß ist, wird das Teilchen instabil und wird leicht ausgef?...

Watch this Scientific Journal Video about A Protocol for the Production of Gliadin-cyanoacrylate Nanoparticles for Hydrophilic Coating at JoVE.com

A Protocol for the Production of Gliadin-cyanoacrylate Nanoparticles for Hydrophilic Coating

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic. The produced nanoparticle is an assembly of gliadin-cyanoacrylate diblock copolymers. Spray coating with the produced nanoparticle changes the surface of target material to a hydrophilic surface.

Ein Protokoll zur Herstellung von Gliadin-cyanacrylat Nanopartikel für die hydrophile Beschichtung

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic by a simple spray ...

a-protocol-for-production-gliadin-cyanoacrylate-nanoparticles-for

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

6.9K Aufrufe.  USDA/ARS/NCAUR. This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic. The produced nanoparticle is an assembly of gliadin-cyanoacrylate diblock copolymers. Spray coating with the produced nanoparticle changes the surface of target material to a hydrophilic surface.

Serie: A Protocol for the Production of Gliadin-cyanoacrylate Nanoparticles for Hydrophilic Coating

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

Formulierung von Diblock Polymernanopartikel durch Nanopräzipitation Technik

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

Forschung

Biotechnik

Preparation and Characterization of SDF-1&#945;-Chitosan-Dextran Sulfate Nanoparticles

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under appropriate conditions. The glycan nanoparticles are useful carriers for protein factors, which facilitate the in vivo delivery of the proteins and sustain their retention in the targeted tissue. The glycan polyelectrolyte complexes are also ideal for protein delivery, as the incorporation is carried out in aqueous solution, which reduces the likelihood of inactivation of the proteins. Proteins with a heparin-binding site adhere to dextran sulfate readily, and are, in turn, stabilized by the binding. These particles are also less inflammatory and toxic when delivered in vivo. In the protocol described below, SDF-1&#945; (Stromal cell-derived factor-1&#945;), a stem cell homing factor, is first mixed and incubated with dextran sulfate. Chitosan is added to the mixture to form polyelectrolyte complexes, followed by zinc sulfate to stabilize the complexes with zinc bridges. The resultant SDF-1&#945;-DS-CS particles are measured for size (diameter) and surface charge (zeta potential). The amount of the incorporated SDF-1&#945; is determined, followed by measurements of its in vitro release rate and its chemotactic activity in a particle-bound form.

The objective of this protocol is to incorporate SDF-1&#945;, a stem cell homing factor, into dextran sulfate-chitosan nanoparticles. The resultant particles are measured for their size and zeta potential, as well as the content, activity, and in vitro release rate of SDF-1&#945; from the nanoparticles.

Herstellung und Charakterisierung von SDF-1α-Chitosan-Nanopartikel Dextransulfat

Chitosan (CS) and dextran sulfate (DS) are charged polysaccharides (glycans), which form polyelectrolyte complex-based nanoparticles when mixed under ...

Chemie

Using Polystyrene-block-poly(acrylic acid)-coated Metal Nanoparticles as Monomers for Their Homo- and Co-polymerization

We present a template-free method for &#8220;polymerizing&#8221; nanoparticles into long chains without side branches. A variety of nanoparticles are encapsulated in polystyrene-block-poly(acrylic acid) (PSPAA) shells and then used as monomers for their self-assembly. Spherical PSPAA micelles upon acid treatment are known to assemble into cylindrical micelles. Exploiting this tendency, the core-shell nanoparticles are induced to aggregate, coalesce, and then transform into long chains. When more than one type of nanoparticles are used, random and block &#8220;copolymers&#8221; of nanoparticles can be obtained. Detailed procedures are reported for the PSPAA encapsulation of nanoparticles, homo- and co-polymerization of the core-shell nanoparticles, separation and purification of the resulting nanoparticle chains. Transformations of single-line chains into double- and triple-line chains are also presented. The synergy between the polymer shell and the embedded nanoparticles leads to an unusual chain-growth polymerization mode, giving long nanoparticle chains that are distinct from the products of the traditional step-growth aggregation process.

Using Polystyrene-block-polyacrylic acid-coated Metal Nanoparticles as Monomers for Their Homo- and Co-polymerization

We report protocols for &#8220;polymerizing&#8221; various types of polymer-encapsulated metal nanoparticles into long chains of &#8220;homo-&#8220; and &#8220;co-polymers&#8221;.

Verwendung Polystyrol-<em&gt; Block</em&gt; -Poly (Acrylsäure) beschichteten Metallnanopartikel als Monomere für ihre Homo- und Polymerisation

We present a template-free method for &#8220;polymerizing&#8221; nanoparticles into long chains without side branches. A variety of nanoparticles are ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

Hochdurchsatz-Synthese von Kohlenhydraten und Funktionalisierung von Nanopartikeln Polyanhydrid

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their extracellular matrix (ECM) environment. Eukaryotic cells constantly sense their local microenvironment through surface mechanosensors to transduce physical changes of ECM into biochemical signals, and integrate these signals to achieve specific changes in gene expression. Interestingly, physicochemical and mechanical parameters of the ECM can couple with each other to regulate cell fate. Therefore, a key to understanding mechanotransduction is to decouple the relative contribution of ECM cues on cellular functions.
Here we present a detailed experimental protocol to rapidly and easily generate biologically relevant hydrogels for the independent tuning of mechanotransduction cues in vitro. We chemically modified polyacrylamide hydrogels (PAAm) to surmount their intrinsically non-adhesive properties by incorporating hydroxyl-functionalized acrylamide monomers during the polymerization. We obtained a novel PAAm hydrogel, called hydroxy-PAAm, which permits immobilization of any desired nature of ECM proteins. The combination of hydroxy-PAAm hydrogels with microcontact printing allows to independently control the morphology of single-cells, the matrix stiffness, the nature and the density of ECM proteins. We provide a simple and rapid method that can be set up in every biology lab to study in vitro cell mechanotransduction processes. We validate this novel two-dimensional platform by conducting experiments on endothelial cells that demonstrate a mechanical coupling between ECM stiffness and the nucleus.

We present a new polyacrylamide hydrogel, called hydroxy-PAAm, that allows a direct binding of ECM proteins with minimal cost or expertise. The combination of hydroxy-PAAm hydrogels with microcontact printing facilitates independent control of many cues of the natural cell microenvironment for studying cellular mechanostransduction.

Herstellung von Hydroxy-PAAm Hydrogele zur Entkopplung der Auswirkungen von Mechanotransduction Cues

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their ...

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

Synthese von thermogelierenden Poly (N-isopropylacrylamid) -Pfropf-Chondroitinsulfat Composites mit Alginate Mikroteilchen für Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

Herstellung von abbaubaren Thermoresponsive Hydrogele auf mehreren Längenskalen über reaktive Extrusion, Mikrofluidik, Selbstmontage und Elektrospinnen

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

Synthesis of Stimuli-responsive Nanogels using Aqueous One-step Crosslinking and Co-nanopolymerization

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to their versatile bottom-up synthesis and biocompatibility. While various methods have been employed for nanogel synthesis to date, very few have achieved it without the use of harsh organic solvents or high temperatures that can damage the integrity of the biological payload. In contrast, the methodology presented here accomplishes the synthesis of sub-100 nm sized, protein-loaded nanogels using mild reaction conditions. Here, we present a method for the non-covalent encapsulation of protein-based payloads within nano-gels that were synthesized using an aqueous-based, single-step, crosslinking copolymerization technique. In this technique, we initially electrostatically bind a protein-based payload to a cationic quaternary ammonium monomer and simultaneously cross-link and co-polymerize it using ammonium persulfate and N,N,N&#39;,N&#39;-tetramethylethylenediamine to form nanogels that entrap the protein payload. The size and polydispersity index of the nanogels is determined using dynamic light scattering (DLS), while the surface morphology is assessed by transmission electron microscopy (TEM). The mass of protein entrapped within nanogels is determined by calculating the encapsulation efficiency. Furthermore, the controlled-release ability of the nanogels via the gradual degradation of redox-responsive structural elements is also assessed in bioreduction assays. We provide examples of nanoparticle optimization data to demonstrate all caveats of nanogel synthesis and characterization using this technique. In general, uniformly sized nanogels were obtained with an average size of 57 nm and a polydispersity index value of 0.093. A high encapsulation efficiency of 76% was achieved. Furthermore, the nanogels exhibited controlled release of up to 86% of the encapsulated protein by gradual degradation of novel redox-responsive components in the presence of glutathione over 48 h.

Nanogels are an excellent and versatile nanoparticle platform for the delivery of biologics. Stimuli-responsive poly(ethylene) glycol-based polymeric nano-gels, capable of encapsulating protein-based payloads, were synthesized using a one-step cross-linking co-nanopolymerization strategy in aqueous conditions. The optimal fabrication and characterization of these novel nanoparticles are presented here.

Synthese von Stimuli-responsiven Nanogelen mittels wässriger Ein-Schritt-Vernetzung und Co-Nanopolymerisation

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to ...

Flash NanoPrecipitation for the Encapsulation of Hydrophobic and Hydrophilic Compounds in Polymeric Nanoparticles

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can improve bioavailability and modify drug distribution within the body. For hydrophilic drugs like peptides or proteins, encapsulation within NPs can also provide protection from natural clearance mechanisms. There are few techniques for the production of polymeric NPs that are scalable. Flash NanoPrecipitation (FNP) is a process that uses engineered mixing geometries to produce NPs with narrow size distributions and tunable sizes between 30 and 400 nm. This protocol provides instructions on the laboratory-scale production of core-shell polymeric nanoparticles of a target size using FNP. The protocol can be implemented to encapsulate either hydrophilic or hydrophobic compounds with only minor modifications. The technique can be readily employed in the laboratory at milligram scale to screen formulations. Lead hits can directly be scaled up to gram- and kilogram-scale. As a continuous process, scale-up involves longer mixing process run time rather than translation to new process vessels. NPs produced by FNP are highly loaded with therapeutic, feature a dense stabilizing polymer brush, and have a size reproducibility of &#177; 6%.

Flash NanoPrecipitation (FNP) is a scalable approach to produce polymeric core-shell nanoparticles. Lab-scale formulations for the encapsulation of hydrophobic or hydrophilic therapeutics are described.

Flash-NanoPrecipitation für die Kapselung der hydrophoben und hydrophilen Verbindungen in Polymeren Nanopartikel

The formulation of a therapeutic compound into nanoparticles (NPs) can impart unique properties. For poorly water-soluble drugs, NP formulations can ...

Injectable Supramolecular Polymer-Nanoparticle Hydrogels for Cell and Drug Delivery Applications

These methods describe how to formulate injectable, supramolecular polymer-nanoparticle (PNP) hydrogels for&#160;use as biomaterials. PNP hydrogels are composed of two components: hydrophobically modified cellulose as the network polymer and self-assembled core-shell nanoparticles that act as non-covalent cross linkers through dynamic, multivalent interactions. These methods describe both the formation of these self-assembled nanoparticles through nanoprecipitation as well as the formulation and mixing of the two components to form hydrogels with tunable mechanical properties. The use of dynamic light scattering (DLS) and rheology to characterize the quality of the synthesized materials is also detailed. Finally, the utility of these hydrogels for drug delivery, biopharmaceutical stabilization, and cell encapsulation and delivery is demonstrated through in vitro experiments to characterize&#160;drug release, thermal stability, and cell settling and viability. Due to its biocompatibility, injectability, and mild gel formation conditions, this hydrogel system is a readily tunable platform suitable for a range of biomedical applications.

This protocol describes the synthesis and formulation of injectable, supramolecular polymer-nanoparticle (PNP) hydrogel biomaterials. Applications of these materials for drug delivery, biopharmaceutical stabilization, and cell encapsulation and delivery are demonstrated.

Injizierbare supramolekulare Polymer-Nanopartikel-Hydrogele für Zell- und Arzneimittelverabreichungsanwendungen

These methods describe how to formulate injectable, supramolecular polymer-nanoparticle (PNP) hydrogels for&#160;use as biomaterials. PNP hydrogels are ...