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 and acceptor is stirred for at least 30 min.</li>
 <li>Solutions of the synthetic donor, acceptor and trimethylsilyltrifluoromethanesulfonate are made in dichloromethane. Toluene and dichloromethane are used most frequently for glycosylation reactions.</li>
 <li>Also, prepare solutions of reagents for the deprotection of temporary protecting groups in 80% methanol and 100% methanol.</li>
 <li>Prior to the start of the program, ensure that the relative humidity in the room is 30% or lower in the automation chamber. High humidity is detrimental for glycosylation reactions.</li>
 <li>Once the program is started, the robotic arm transfers the solutions of donor and acceptor into the reaction vial sequentially. Then the mixture is stirred for 30 min.</li>
 <li>Next the robotic arm transfers 0.2 to 0.3 equivalents of trimethylsilyltrifluoromethanesulfonate into the mixture, typically at room temperature although lower temperatures like -20 &deg;C can be attained. The reaction mixture is stirred for 30 min.</li>
 <li>After 30 min, the reaction is stopped and a small aliquot removed to monitor the reaction progress. If not complete, the reaction can be continued and ultimately the time required can be modified.</li>
 <li>Once the reaction is complete, the reaction mixture is transferred to the fluorous solid phase extraction (FSPE) cartridges containing C8F17-modified silica gel for purification.</li>
 <li>The cartridges are first washed with an 80% methanol-water mixture (8 mL) to get rid of the non-fluorous fraction.</li>
 <li>Then the cartridges are washed with 100% methanol to obtain the desired fluorous-tagged product. If additional purification is desired, the machine can be stopped and the reaction product(s) removed for purification by additional means.</li>
 <li>After the purification cycle, the robotic arm dispenses sodium methoxide into the reaction vial. The reaction is stirred for 2 hr. If not complete, the reaction can again be continued for a longer period and ultimately the programmed time required can be modified.</li>
 <li>After the completion of the reaction, the product is purified by FSPE and then subjected to dissolution in anhydrous toluene followed by evaporation to remove residual water.</li>
 <li>Then the cycle (from step 6 to 13) is repeated till the desired chain length is obtained for the target molecule.</li>
 <li>The protected product obtained from automation is then further purified and fully characterized by techniques such as nuclear magnetic resonance (NMR) spectroscopy. Complete deprotection (removal of all remaining protecting groups) of the final target molecule is then completed outside the automation platform as a rule because it usually involves explosive hydrogen gas and palladium. The final deprotection step was carried out on bench-top outside the automation platform. The first step was ozonolysis of the double bond in fluorous tag followed by oxidation of the produced aldehyde to a carboxylic acid. The product was purified by column chromatography. The final step was deprotection of benzyl ether groups by palladium-catalyzed hydrogenation. The product was passed through celite pad to get rid of palladium to get pure final product.</li>
</ol>
2. High-throughput Nanoparticle Surface Functionalization
<ol>
 <li>High-throughput polymer synthesis and nanoparticle fabrication is carried out following the same protocol and robotic set up described by Petersen et al19. The copolymer systems used for particle fabrication are based on sebacic acid (SA) and 1,6-bis(para-carboxyphenoxy)hexane (CPH), and 1,8-bis(para-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and CPH. A schematic representation of the robotic deposition apparatus utilized is presented in Figure 1b.</li>
 <li>Following nanoparticle fabrication, the holder containing tubes with the nanoparticle library is reattached to the linear actuator stage.</li>
 <li>For attachment of carbohydrates to the surface of polyanhydride particles, an amine-carboxylic acid coupling reaction 20 consisting of two consecutive reactions is performed.</li>
 <li>For the first reaction, the syringe in the first programmable syringe pump is filled with 10 equivalents (eq.) (equivalents of average molar carboxylic acid concentration on particle surface) of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and 10 eq. of ethylenediamine in an aqueous solution, while the syringe in the second programmable syringe pump is loaded with 12 eq. of N-hydroxysuccinimide (NHS) in aqueous solution.</li>
 <li>Using the LabVIEW program, reagent suspensions are deposited into the nanoparticle library*.</li>
 <li>Next, each sample is sonicated (30 s at 40 Hz) and the tube holder is detached from the robotic platform.</li>
 <li>Nanoparticle suspensions are incubated for 9 h** with constant rotation at 4 &deg;C.</li>
 <li>After reaction time is completed, tubes are centrifuged (12000 x g for 5 min) and returned to the robotic station to perform two washing steps.</li>
 <li>For washing, a syringe remains empty and loaded in the first programmable syringe pump while the syringe in the second syringe pump is filled with cold water. The supernatant in each tube is withdrawn into the empty syringe and the second pump deposits cold water.</li>
 <li>Homogenization of nanoparticle suspension is performed as described on step 2.6. Tubes are then centrifuged (12000 x g for 5 min) and a second wash step is performed as described on step 2.9.</li>
 <li>For the second reaction, two deposition steps are used. In the first deposition step, 12 eq. of EDC are loaded with one pump and 12 eq. of NHS are loaded with the second pump.</li>
 <li>The second deposition step includes 10 eq. of an specific saccharide on the first and second pumps (i.e., galactose, lactose or di-mannose)*** and a third pump with 10 eq. of glycolic acid (used as control****).</li>
 <li>Nanoparticle suspensions is homogenized as described on step 2.6 and incubated for 9 h with constant rotation at 4 &deg;C.</li>
 <li>After the time of reaction is completed, a wash step is performed as described in steps 2.8, 2.9 and 2.10.</li>
 <li>The functionalized nanoparticle library is then placed in a vacuum chamber to dry for at least 2 hr.</li>
 <li>The functionalized nanoparticles are then characterized by X-ray photoelectron spectroscopy and a high throughput phenol-sulfuric acid assay to determine the surface composition and concentration of the saccharide respectively. Scanning electron microscopy and dynamic light scattering are utilized to determine particle size, size distribution, and surface charge.</li>
</ol>
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 &deg;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 &deg;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.
<table border="1">
 <tbody>
 <tr>
 <td>Nanoparticle type</td>
 <td>Average Particle Diameter (nm)</td>
 <td>Average Particle &zeta;-Potential (mV)</td>
 </tr>
 <tr>
 <td>Non-functionalized</td>
 <td>162 &plusmn; 43</td>
 <td>-20 &plusmn; 0.6</td>
 </tr>
 <tr>
 <td>Lactose</td>
 <td>235 &plusmn; 34</td>
 <td>26 &plusmn; 2.4</td>
 </tr>
 <tr>
 <td>Di-mannose</td>
 <td>243 &plusmn; 32</td>
 <td>30 &plusmn; 4.2</td>
 </tr>
 </tbody>
</table>
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 &plusmn; standard deviation (SD) of dynamic light scattering data collected in three independent experiments. Zeta potential data represent the mean value &plusmn; 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.
<img alt="Figure 1" src="/files/ftp_upload/3967/3967fig1.jpg" /> 
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. <a href="https://www.jove.com/files/ftp_upload/3967/3967fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/3967/3967fig2.jpg" /> 
Figure 2. Graphical representation of the automated iterative synthesis of carbohydrates using mannose as an example.
<img alt="Figure 3" src="/files/ftp_upload/3967/3967fig3.jpg" /> 
Figure 3. 1H NMR of the protected dimannoside.
<img alt="Figure 4" src="/files/ftp_upload/3967/3967fig4.jpg" /> 
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 &deg;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.

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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">
	 <tbody>
 <tr>
 <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>
 </tr>
 <tr>
 <td>NE-1000 Single Syringe Pump</td>
 <td>New Era Pump Systems</td>
 <td>NE-1000</td>
 </tr>
 <tr>
 <td>Pyrex* Vista* Rimless Reusable Glass Culture Tubes</td>
 <td>Corning</td>
 <td>07-250-125</td>
 </tr>
 <tr>
 <td>ASW 1000</td>
 <td>Chemspeed Technologies</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>LabVIEW</td>
 <td>National Instruments</td>
 <td>776671-35</td>
 </tr>
 <tr>
 <td>SGE Gas Tight Syringes, Luer Loc</td>
 <td>Sigma Aldrich</td>
 <td>509507</td>
 </tr>
 <tr>
 <td>XL-2000 Sonicator</td>
 <td>Qsonica </td>
 <td>Q55</td>
 </tr>
 <tr>
 <td>Mini-tube rotator</td>
 <td>Fisher Scientific</td>
 <td>05-450-127</td>
 </tr>
	 </tbody>
 </table>

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

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JoVE Journal

Bioengineering

11.3K Views.  Iowa State University. 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.

Video: High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the synthesis of hydrophobic nanoparticles, our method is based on an adaptation from thermal decomposition method to microwave driven synthesis. The new methodology produces a decrease in the reaction times in comparison with traditional procedures. Moreover, the use of the microwave technology increases the reproducibility of the reactions, something important from the point of view of clinical applications. The novelty of this iron oxide nanoparticle is the attachment of Neridronate. The use of this molecule leads a bisphosphonate moiety towards the outside of the nanoparticle that provides Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. The protocol allows the synthesis and plaque detection in about 3 hr since the initial synthesis from organic precursors. Their accumulation in the atherosclerotic area in less than 1 hr provides a contrast agent particularly suitable for clinical applications.

Microwave technology enables extremely fast synthesis of iron oxide nanoparticles for atherosclerosis plaque characterization. The use of an aminobisphosphonate in the external side of the nanoparticle provides a fast accumulation in the atherosclerotic area.

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...