The authors would like to thank the U.S. Army Medical Research and Materiel Command (Grant # W81XWH-10-1-0806) and the National Institutes of Health (Grant # U19 AI091031-01 and Grant # 1R01GM090280) for financial support. BN acknowledges the Balloun Professorship in Chemical and Biological Engineering and NLBP acknowledges the Wilkinson Professorship of Interdisciplinary Engineering. We thank Julia Vela for her assistance in performing the nanoparticle functionalization experiments.

1. High-throughput Carbohydrate Synthesis
<ol>
 <li>Prior to the automated synthesis of dimannoside, a suitably protected sugar donor, typically trichloroacetimidate, and acceptor, mainly an alkenyl fluorous alcohol, are synthesized on the bench-top.</li>
 <li>A program is written for the automated synthesis of dimannoside. A schematic representation of the basic automated procedure is presented in Figure 2. In the program, it is ensured that before the addition of the promoter, the mixture of donor an...

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NLBP is cofounder and holds equity in the carbohydrate company LuCella Biosciences, Inc.

The efficacy of carbohydrates as targeting agents to direct nanoparticle interactions to immune cells has been previously demonstrated 10, 11. Previous research in our laboratories have shown that specific sugars attached to polyanhydride nanoparticles are able to target different CLRs on antigen presenting cells (APCs), thereby enhancing the activation of immune cells which may be important for further T cell activation 10, 11. However, to achieve optimal targeting several parameters&mdash;su...

<table border="1">
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 <td>Name</td>
 <td>Company</td>
 <td>Catalog number</td>
 </tr>
 <tr>
 <td>Motorized XYZ Stage: 3x T-LSM050A, 50 mm travel per axis</td>
 <td>Zaber Technologies</td>
 <td>T-XYZ-LSM050A-KT04</td>
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 <td>NE-1000 Single Syringe Pump</td>
 <td>New Era Pump Systems</td>
 <td>NE-1000</td>
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 <td>Pyrex* Vista* Rimless Reusable Glass Culture Tubes</td>
 <td>Corning</td>
 <td>07-250-125</td>
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 <td>ASW 1000</td>
 <td>Chemspeed Technologies</td>
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 </tr>
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 <td>LabVIEW</td>
 <td>National Instruments</td>
 <td>776671-35</td>
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 <td>SGE Gas Tight Syringes, Luer Loc</td>
 <td>Sigma Aldrich</td>
 <td>509507</td>
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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).

Watch this Scientific Journal Video about High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles at JoVE.com

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

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.

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

high-throughput-synthesis-carbohydrates-functionalization

Research

JoVE Journal

Bioengineering

11.3K vues.  Iowa State University. Dans cet article, une méthode à haut débit est présenté pour la synthèse d'oligosaccharides et de leur attachement à la surface de nanoparticules polyanhydrides pour une utilisation ultérieure dans le ciblage des récepteurs spécifiques sur les cellules présentatrices d'antigènes.