1Department of Chemical and Biological Engineering, Iowa State University, 2Department of Chemistry, Iowa State University
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Carrillo-Conde, B. R., Roychoudhury, R., Chavez-Santoscoy, A. V., Narasimhan, B., Pohl, N. L. B. High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles. J. Vis. Exp. (65), e3967, doi:10.3791/3967 (2012).
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).
1. High-throughput Carbohydrate Synthesis
2. High-throughput Nanoparticle Surface Functionalization
Notes: *Deposition volumes vary with the mass of nanoparticles contained in each tube.
**Reaction times for first and second reactions can be changed to adjust the final saccharide concentration.
***Each saccharide is deposited into test tubes depending on the desired group.
****For the specific reaction employed in this study for the attachment of carbohydrates, glycolic acid is used as a linker control since deprotected saccharides already have this molecule covalently linked, which allows for further attachment to nanoparticle surface.
3. Representative Results
The fully protected dimannoside shown in Figure 2 was synthesized using the automation platform. The synthesized compound was characterized by 1H NMR in a VXR 400 MHz spectrometer using CDCl3 as solvent. The NMR spectrum is shown in Figure 3.
Utilizing the high-throughput nanoparticle fabrication and functionalization of polyanhydride nanoparticles described herein, attachment of dimannose, lactose and galactose has been carried out successfully 10, 11. Using this set up, optimal reaction conditions (i.e., reaction temperature and time) were identified to achieve desired nanoparticle functionalization and morphology. When the reaction was carried out at 4 °C instead of room temperature, a reduction in nanoparticle aggregation was observed by SEM (data not shown). Table 1 shows representative results of the characterization of functionalized 50:50 CPTEG:CPH nanoparticles with either di-mannose or lactose, synthesized at 4 °C. The data indicate a small increase in the average nanoparticle diameter due to the functionalization. While the non-functionalized nanoparticles had a negative zeta potential of approx. -20 mV, the functionalized particles showed a positive zeta potential value, demonstrating successful functionalization of the nanoparticle surface. Lactose and di-mannose are both neutral sugars; however, free amine groups from the ethylene diamine linker utilized to attach the saccharides may be responsible of the positive zeta potential.
Reaction time is another variable that could affect both the final morphology of the nanoparticles and the degree of sugar attachment achieved. By adjusting the reaction time, the final sugar concentration attached to the nanoparticles surface can be controlled as shown in Figure 4A. As expected, the concentration of dimannose on the surface of 50:50 CPTEG:CPH nanoparticles increased with the total time of reaction and reached a maximum after 18 hr. Nanoparticles functionalized with the 24 hr total reaction time were used to evaluate their ability to target CLRs on mouse bone-marrow derived dendritic cells (DCs). Flow cytometry was used to evaluate the expression of two CL receptors (i.e., CIRE (CD209, DC-SIGN) and mannose receptor (CD206)) after stimulation with non-functionalized, and lactose and di-mannose functionalized nanoparticles (Figure 4B). A higher expression of both receptors, which is an indicative of effective targeting, was obtained when cells were stimulated with both lactose and di-mannose functionalized nanoparticles. However, di-mannose-functionalized particles showed a higher level of expression indicating a specificity of this ligand for the receptors that were studied.
|Nanoparticle type||Average Particle Diameter (nm)||Average Particle ζ-Potential (mV)|
|Non-functionalized||162 ± 43||-20 ± 0.6|
|Lactose||235 ± 34||26 ± 2.4|
|Di-mannose||243 ± 32||30 ± 4.2|
Table 1. Nanoparticle characterization. Non-functionalized and functionalized were characterized by quasi-elastic light scattering and zeta potential measurements. Particle size data represent the mean value ± standard deviation (SD) of dynamic light scattering data collected in three independent experiments. Zeta potential data represent the mean value ± SD of three independent readings. Change in the sign of the zeta potential demonstrates that sugar was efficiently conjugated to the 50:50 CPTEG:CPH nanoparticle surface.
Figure 1. (A) Graphical representation of the approach pursued with carbohydrate functionalization of polyanhydride nanoparticles and an example of the functionalized nanoparticle libraries that could be designed with the described high-throughput approach. (B) Schematic representation of the automated deposition apparatus utilized for particle functionalization, which consists of (i) three NE 1000 pumps; (ii) a robotic stage integrated by two actuators (Zaber): one for movement in the x direction and the other for movement in the y direction; (iii) a second robotic stage with two adjacent racks (appropriate for tubes and cuvettes) consisting of three actuators, one for each direction (x, y, and z). The pumps and a total of five actuators are connected in series. Actuators and pumps are operated by a computer using LabVIEW software. This diagram is not to scale. Click here to view larger figure.
Figure 2. Graphical representation of the automated iterative synthesis of carbohydrates using mannose as an example.
Figure 3. 1H NMR of the protected dimannoside.
Figure 4. (A) Effect of reaction time on nanoparticle surface concentration of saccharide. In the data shown, 50:50 CPTEG:CPH nanoparticles were functionalized with dimannose at different reaction times and the reaction was carried out at 4 °C. The average and standard error of two independent functionalization experiments is shown. (B) Lactose and di-mannose functionalized nanoparticles effectively target DC-SIGN (CIRE, CD209) and mannose receptor (CD206) on bone marrow-derived dendritic cells as demonstrated by the enhanced expression of these two markers after stimulation with functionalized 50:50 CPTEG:CPH nanoparticles when compared with the expression obtained with non-functionalized particles.
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—such as the polyanhydride chemistry, size, type of sugar or surface sugar density—need to be optimized and therefore increasing the throughput in the fabrication method to unearth the complexities associated with such a multi-parametric system will be of great value. In addition, the use of functionalized nanoparticles if of great value to other related areas of research, including biosensing, enzyme immobilization, and detection of foodborne pathogens.
By utilizing the described high-throughput synthesis of carbohydrates, the challenges in the reproducible synthesis of carbohydrate molecules can be mitigated. Automated parallel reactions of the same sugar can produce larger amounts of material as needed. The known roles of sugars and glycoconjugates are rapidly expanding. Nevertheless, an understanding of the molecular mechanisms of carbohydrates in many processes, e.g. signal transduction pathways or cellular recognition processes 21, relies on the easy and cheap availability of structurally well-defined saccharides. Protection/deprotection strategies to control the reactivity of various hydroxyl groups for precise chain extension are a prime requisite for sugar synthesis, but are tedious and time-consuming. Oligonucleotides and oligopeptides are regularly and efficiently synthesized by using automated synthesizers 22, 23. A solid phase synthesizer is available for oligosaccharide synthesis 24, but suffers from some serious disadvantages: e.g., large excesses of building blocks (5 to 20 equivalents per coupling step), the lack of facile monitoring of reaction progress, and the inherent variability of the solid-phase resins used. A new solution-phase automation platform, however, requires only 2 to 3 equivalents of these precious building blocks. In this platform a variety of fluorous tags, such as the alkenyl fluorous group, enables fluorous solid-phase extraction (FSPE) to purify the intermediate products easily from non-fluorous compounds 18, 25, 26. However, as shown here, these tags do not preclude solution-phase glycosylation and deprotection reactions in standard organic solvents. Also, unlike any solid-phase automated synthesizer, this new platform allows standard reaction monitoring strategies such as mass spectrometry (MS) and thin layer chromatography (TLC) at any stage.
As described in the results section, following the high-throughput functionalization of nanoparticles presented herein, reaction conditions (e.g., reaction time and temperature) to achieve optimal nanoparticle morphology after functionalization have been optimized. Optimal reaction temperature may need to be optimized depending on the polymer properties used to fabricate the nanoparticles (e.g., glass transition temperature (Tg), degradation rates). For example, when using polymers with low Tg (below room temperature), the functionalization reactions will need to carried out at low temperatures, which is the case for some of the polyanhydride chemistries utilized in our research group. Optimization of the total reaction time employed for particle functionalization is desired especially when particle chemistry with different degradation rates need to be functionalized. Shorter reaction time may be ideal to functionalize bulk-eroding materials especially when a targeting sugar needs to be attached to drug- or protein-loaded particles. Sugar concentration on particle surface may be an important variable to direct the biological performance of these carriers. The biological outcome of varying sugar concentration is a current area of study in our laboratories. The use of this high throughput set up to fabricate and functionalized polyanhydride nanoparticles allows for the testing of multiple variables faster than conventional fabrication and functionalization methods. The main limitation of the high throughput technique is the maximum batch size of particles that can be obtained since it is limited by the size of the containers that can fit in the apparatus holders: however, since the main use of this set up is for screening smaller size batch can be efficiently use for this purpose.
NLBP is cofounder and holds equity in the carbohydrate company LuCella Biosciences, Inc.
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.
|Motorized XYZ Stage: 3x T-LSM050A, 50 mm travel per axis||Zaber Technologies||T-XYZ-LSM050A-KT04|
|NE-1000 Single Syringe Pump||New Era Pump Systems||NE-1000|
|Pyrex* Vista* Rimless Reusable Glass Culture Tubes||Corning||07-250-125|
|ASW 1000||Chemspeed Technologies|
|SGE Gas Tight Syringes, Luer Loc||Sigma Aldrich||509507|
|Mini-tube rotator||Fisher Scientific||05-450-127|