This work was supported by the Experiment Station Chemical Laboratories of the University of Missouri, and by the Cystic Fibrosis Association of Missouri.

1. Synthesis of Glycomonomer 2-Lactobionamidoethyl Methacrylamide
<ol>
	<li>Dissolve 2 g of lactobionic acid in 3.0 ml of anhydrous methanol and slowly add absolute ethanol in a drop wise fashion until the solution just turns cloudy, then remove the solvents via rotoevaporation.</li>
	<li>Dissolve the residue, from step 1.1, in 3.0 ml anhydrous methanol and, once again, slowly add absolute ethanol until just cloudy, then evaporate the solvents via rotoevaporation. Repeat this step 3 times to obtain lactobiono-1,5-lactone (1.94 g, 98% yield). This product is of sufficient purity to be used in the following reactions.</li>
	<li>Add 1.0 g of lactobionolactone in 3.0 ml of methanol to N-(2-aminoethyl) methacrylamide (AEMA, 0.58 g) and hydroquinone monomethyl ether (MEHQ, 1.0 mg), an inhibitor of self-polymerization, in 2.0 ml of methanol, followed by 1.0 ml of triethylamine. Stir at RT for 48 hr.</li>
	<li>Add 20 ml of deionized H2O (dH2O) to the reaction flask, then remove the methanol and free triethylamine by evaporation to dryness via rotoevaporation.</li>
	<li>To remove any remaining lactobionic acid, add 20 ml of dH2O, then pass the aqueous solution through an anion exchange column (OH- form, 10 mm x 20 mm) into a receiving beaker containing 1.0 mg of MEHQ.</li>
	<li>Remove triethylamine, produced in Step 1.5, by evaporating to dryness via rotoevaporation.</li>
	<li>Add 20 ml of dH2O and remove unreacted AEMA by slowly adding 1 mg aliquots of cation exchange resin (H+ form) until no ninhydrin reactive materials are detectable. Monitor the removal by taking 1 &#181;l aliquots of the solution after each resin addition, applying it to a thin-layer chromatography plate, then spraying the plate with a 2% ninhydrin in ethanol solution. When no deep blue color is observed to develop when the plate is heated to 90 &#176;C for 1 min, the end point has been reached.</li>
	<li>Filter the solution through a fritted glass funnel, transfer the filtrate to a test tube, freeze the sample at -80 &#176;C, and then lyophilize.</li>
	<li>Remove MEHQ from the sample by dissolving the freeze-dried material in a minimum amount of methanol (~0.5 ml), then add cold anhydrous acetone (-20 &#176;C, 15 ml) to precipitate the product. Collect the precipitate by filtration using a fritted glass funnel, then dry the precipitate in a desiccator under vacuum to obtain 2-lactobionamidoethyl methacrylamide (LAEMA) as an off-white powder (0.94 g, 68% yield). This product is of sufficient purity to be used in the following reactions.</li>
</ol>
2. Synthesis of Monomer 2-Gluconamidoethyl Methacrylamide
Note: The preparation of 2-gluconamidoethyl methacrylamide (GAEMA), which does not possess a pendant sugar, was adapted from a published method15.
<ol>
	<li>Add 2.0 g of AEMA dissolved in 10 ml of methanol to a solution of D-gluconolactone (1.6 g) in 30 ml of methanol and, with stirring, slowly add 1.6 ml of triethylamine.</li>
	<li>Stir the reaction at RT for 24 hr.</li>
	<li>Filter the precipitated product using a fritted glass funnel and rinse the precipitate three times with 10 ml each of isopropanol, then wash with 10 ml of dry acetone. Dry the precipitated product in a desiccator under vacuum.</li>
</ol>
3. RAFT Glycopolymer Synthesis
<ol>
	<li>To remove the inhibitor MEHQ present in commercial N-(2-hydroxyethyl) acrylamide (HEAA), add 1 ml of HEAA to a 2 ml microcentrifuge tube, followed by adding 0.5 g of aluminum oxide nanoparticles. Centrifuge the tube at 300 x g for 30 sec, and use the top layer HEAA in the following reaction.</li>
	<li>Carefully add 32.8 mg of LAEMA (70.0 &#181;mol), 1.7 mg of AEMA (10.5 &#181;mol) and 27.5 &#181;l of HEAA (270 &#181;mol), all dissolved in 0.4 ml of dH2O, to a well-cleaned 1 ml Schlenk tube, thus having a monomer molar ratio of 20:3:77.</li>
	<li>In a parallel reaction, in order to produce control polymers that do not possess any pendant sugar, in lieu of using LAEMA in step 3.2, substitute 21.4 mg of GAEMA (70.0 &#181;mol) in the reaction.</li>
	<li>To the respective Schlenk tube (i.e., 3.2 or 3.3), sequentially add 50 &#181;l of DMF containing 0.53 mg of (4-cyanopentanoic acid)-4-dithiobenzoate (1.9 &#181;mol, RAFT agent), and 50 &#181;l of DMF containing 250 &#181;g of 4,4&#8242;-azobis-(4-cyanovaleric acid) (0.9 &#181;mol, initiator). Gently mix by finger tapping.</li>
	<li>Freeze the contents contained in the Schlenk tube employing a dry ice:ethanol bath (75 g dry ice in 100 ml ethanol), apply a vacuum to within 10-50 mTorr, then close the Schlenk valve and permit the solution to slowly thaw to RT. Repeat this freeze&#8211;evacuate&#8211;thaw cycle twice more. Ensure that all reagents are dissolved after the last thaw.</li>
	<li>Place the Schlenk tube into a sealable plastic bag, evacuate the bag&#8217;s air, and then seal it. Transfer the bag containing the Schlenk tube to a water bath preheated at 70 &#176;C and incubate for 24 hr.</li>
	<li>Carefully transfer the solution in the Schlenk tube to a prepared dialysis bag (MWCO = 3,500), and dialyze against dH2O (10 x 2 L) for 24 hr, changing the dH2O every hour for the first 8 hr. Following dialysis, transfer the sample from the dialysis tubing to a test tube, freeze the sample at -80 &#176;C, and then lyophilize it.</li>
</ol>
Note: The resultant statistical poly-methacrylamide/acrylamide (PMA) copolymers containing pendant 4-O-&#946;-D-galactopyranosyl-D-gluconamide (lactobionamide) (from Step 3.2) or D-gluconamide (from Step 3.3), respectively, are obtained. For convenience of discussion, these two glycopolymers are abbreviated as PMA-LAEMA and PMA-GAEMA, respectively.
4. Post-modification of Glycopolymers with Fluorophores
<ol>
	<li>Dissolve 5.0 mg of glycopolymer PMA-LAEMA or PMA-GAEMA containing ~0.9 &#181;mole of primary amine functional groups in 0.9 ml of phosphate buffered saline (PBS, 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.5), respectively.</li>
	<li>Slowly add 0.6 mg of carboxyfluorescein succinimidyl ester in 100 &#181;l of DMF to the solutions with rapid stirring. Gently stir the reactions for 16 hr in the dark at RT.</li>
	<li>While protected from light, load the sample into a prepared dialysis tubing (MWCO = 3,500) and dialyze against dH2O (2 L) for 16 hr, changing the dialysis solution every hour for the first 8 hr. Following dialysis, transfer the sample from the dialysis tubing to a test tube, freeze the sample at -80 &#176;C, and then lyophilize it.</li>
</ol>
Note: Following lyophilization, fluorescent glycopolymers PMA-LAEMA-Fluorescein and PMA-GAEMA-Fluorescein, respectively, are obtained.
5. Characterization of the Glycopolymers
<ol>
	<li>Determine the number average molecular weight&#160;(Mn), the weight average molecular weight (Mw) and the dispersity (Mw/Mn) of the glycopolymers on a commercial HPLC system equipped with gel permeation chromatography (GPC) software, a GPC column suitable for the molecular weight of interest, and a refractive index detector, using 0.1 M Tris/0.1 M sodium chloride buffer (pH 7) as eluent at a flow rate of 0.6 ml/min14. Use polyethylene glycol standards as molecular weight standards (MW: 200-1,200,000 g/mol).</li>
	<li>Quantify the actual concentrations of primary amine functional groups within the glycopolymers16. Analyze the total carbohydrate content of the synthesized glycopolymers according to a published method17.</li>
	<li>Perform tests of structural composition and purity of the glycomonomers LAEMA, GAEMA and glycopolymers PMA-LAEMA, PMA-GAEMA in D2O by NMR spectroscopy14.</li>
</ol>
6. Binding Tests of the Synthetic Glycopolymers with Lectin-coated Agarose Beads
<ol>
	<li>Add 1.5 ml of PBS to 50 &#181;l of suspension of Erythrina crista-galli lectin (ECL)-coated agarose beads, centrifuge at 300 x g for 1 min, and carefully remove and dispose of the supernatant. Repeat this step twice, and then resuspend the beads in 0.5 ml of PBS.</li>
	<li>Add 3 &#181;g of PMA-LAEMA-Fluorescein or PMA-GAEMA-Fluorescein (negative control) in 6 &#181;l of PBS to the beads suspension, and incubate the mixtures, in the dark, at RT for 1 hr.</li>
	<li>Wash the mixtures with 1.5 ml of PBS three times, and resuspend beads in 0.2 ml of PBS. Load an aliquot (4 &#181;l) into a well on an immunofluorescence microscope slide (Teflon-coated), cover with a cover slip, and observe by fluorescence microscopy using a FITC filter (excitation wavelength: 467-498 nm, emission wavelength: 513-566 nm) and a 10X objective to examine the binding of the fluorescent glycopolymers with the beads14.</li>
</ol>

<ol>
	<li>Scharfman, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+binds+to+neoglycoconjugates+bearing+mucin+carbohydrate+determinants+and+predominantly+to+sialyl-Lewis+x+conjugates.">Pseudomonas aeruginosa binds to neoglycoconjugates bearing mucin carbohydrate determinants and predominantly to sialyl-Lewis x conjugates.</a> Glycobiology. 9 (8), 757-764 (1999).</li><li>Song, E. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+targeting+of+alveolar+macrophages+via+RAFT-based+glycopolymers.">In vivo targeting of alveolar macrophages via RAFT-based glycopolymers.</a> Biomaterials. 33 (28), 6889-6897 (2012).</li><li>Wolfenden, M. L., Cloninger, M. J., Wang, B., Boons, G. .. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+14.+Multivalency+in+carbohydrate+binding.">Chapter 14. Multivalency in carbohydrate binding.</a> Carbohydrate Recognition: Biological Problems, Methods, and Applications. , 349-370 (2011).</li><li>Moad, G., Rizzardo, E., Thang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radical+addition-fragmentation+chemistry+in+polymer+synthesis.">Radical addition-fragmentation chemistry in polymer synthesis.</a> Polymer. 49 (5), 1079-1131 (2007).</li><li>Spain, S. G., Gibson, M. I., Cameron, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+the+synthesis+of+well-defined+glycopolymers.">Recent advances in the synthesis of well-defined glycopolymers.</a> J. Polym. Sci., Part A: Polym. Chem. 45 (11), 2059-2072 (2007).</li><li>Bernard, J., Hao, X., Davis, T. P., Barner-Kowollik, C., Stenzel, M. H. <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+various+glycopolymer+architectures+via+RAFT+polymerization:+From+block+copolymers+to+stars.">Synthesis of various glycopolymer architectures via RAFT polymerization: From block copolymers to stars.</a> Biomacromolecules. 7 (1), 232-238 (2006).</li><li>Bulmus, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAFT+polymerization+mediated+bioconjugation+strategies.">RAFT polymerization mediated bioconjugation strategies.</a> Polym. Chem. 2, 1463-1472 (2011).</li><li>Ting, S. R. S., Chen, G., Stenzel, M. H. <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+glycopolymers+and+their+multivalent+recognitions+with+lectins.">Synthesis of glycopolymers and their multivalent recognitions with lectins.</a> Polymer Chemistry. 1, 1392-1412 (2010).</li><li>Vazquez-Dorbatt, V., Lee, J., Lin, E. W., Maynard, H. D. <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+glycopolymers+by+controlled+radical+polymerization+techniques+and+their+applications.">Synthesis of glycopolymers by controlled radical polymerization techniques and their applications.</a> Chembiochem. 13, 2478-2487 (2012).</li><li>Jiang, X., Ahmed, M., Deng, Z., Narain, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotinylated+glyco-functionalized+quantum+dots:+Synthesis,+characterization,+and+cytotoxicity+studies.">Biotinylated glyco-functionalized quantum dots: Synthesis, characterization, and cytotoxicity studies.</a> Bioconjugate Chem. 20 (5), 994-1001 (2009).</li><li>Deng, Z., Li, S., Jiang, X., Narain, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Well-defined+galactose-containing+multi-functional+copolymers+and+glyconanoparticles+for+biomolecular+recognition+processes.">Well-defined galactose-containing multi-functional copolymers and glyconanoparticles for biomolecular recognition processes.</a> Macromolecules. 42 (17), 6393-6405 (2009).</li><li>Qin, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Galactosylated+N-2-hydroxypropyl+methacrylamide-b-N-3-guanidinopropyl+methacrylamide+block+copolymers+as+hepatocyte-targeting+gene+carriers.">Galactosylated N-2-hydroxypropyl methacrylamide-b-N-3-guanidinopropyl methacrylamide block copolymers as hepatocyte-targeting gene carriers.</a> Bioconjugate Chem. 22 (8), 1503-1512 (2011).</li><li>Albertin, L., Wolnik, A., Ghadban, A., Dubreuil, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aqueous+RAFT+polymerization+of+N-acryloylmorpholine,+synthesis+of+an+ABA+triblock+glycopolymer+and+study+of+its+self-association+behavior.">Aqueous RAFT polymerization of N-acryloylmorpholine, synthesis of an ABA triblock glycopolymer and study of its self-association behavior.</a> Macromol. Chem. Phys. 213 (17), 1768-1782 (2012).</li><li>Wang, W., Chance, D. L., Mossine, V. V., Mawhinney, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAFT-based+tri-component+fluorescent+glycopolymers:+synthesis,+characterization+and+application+in+lectin-mediated+bacterial+binding+study.">RAFT-based tri-component fluorescent glycopolymers: synthesis, characterization and application in lectin-mediated bacterial binding study.</a> Glycoconj. J. 31 (2), 133-143 (2014).</li><li>Deng, Z., Ahmed, M., Narain, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+well-defined+glycopolymers+synthesized+via+the+reversible+addition+fragmentation+chain+transfer+process+in+aqueous+media.">Novel well-defined glycopolymers synthesized via the reversible addition fragmentation chain transfer process in aqueous media.</a> J. Polymer Sci. Part A: Polym. Chem. 47 (2), 614-627 (2009).</li><li>Noel, S., Liberelle, B., Robitaille, L., De Crescenzo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+primary+amine+groups+available+for+subsequent+biofunctionalization+of+polymer+surfaces.">Quantification of primary amine groups available for subsequent biofunctionalization of polymer surfaces.</a> Bioconjugate Chem. 22 (8), 1690-1699 (2011).</li><li>Fox, A., Morgan, S. L., Gilbart, J., Biermann, C. J., McGinnis, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+alditol+acetates+and+their+analysis+by+gas+chromatography+(GC)+and+mass+spectrometry+(MS).">Preparation of alditol acetates and their analysis by gas chromatography (GC) and mass spectrometry (MS).</a> Analysis of Carbohydrates by GLC and MS. , 87-170 (1989).</li><li>Thomas, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+and+molecular+weight+control+of+the+polymerization+of+acrylamide+via+RAFT.">Kinetics and molecular weight control of the polymerization of acrylamide via RAFT.</a> Macromolecules. 37 (24), 8941-8950 (2004).</li></ol>

The authors have nothing to disclose.

A facile and efficient protocol for RAFT-based tri-component fluorescent glycopolymers, with and without a pendant carbohydrate, and their use in a lectin-binding test, is demonstrated in this report. The protocol starts with the preparation of glycomonomers LAEMA and GAEMA. Through a one-step RAFT-controlled copolymerization, glycopolymers with reproducible yield, predictable monomer composition and low dispersity, are obtained. Following post-modification of glycopolymers with carboxyfluorescein succinimidyl ester, the binding of the resulting respective fluorescent-labeled glycopolymer is readily testable for its lectin-binding specificity.
In the initial preparative steps of the glycomonomers that are to be employed in the subsequent glycopolymer syntheses, readily available lactobionic acid and gluconolactone were utilized. In theory, any carbohydrates of interest, from monosaccharides to complex oligosaccharides, can be converted to glycomonomers by conjugating the target sugar onto the primary hydroxyl group on C6 of glucose. Following oxidation of the reducing glucose residue, and its subsequent dehydration to a lactone, the product can then be readily reacted with the primary amine on AEMA to form the corresponding glycomonomer. Further examples of this route can be seen in a recent report14. It should be noted that before initiating any polymerization step, MEHQ, a potent polymerization inhibitor, must be removed from all monomer and glycomonomer preparations just prior to use. This is readily accomplished by using the minimum amount of methanol to dissolve the glycomonomer that possesses MEHQ then immediately treat it with acetone at -20 &#176;C to precipitate the inhibitor-free product in high yield.
Essential in any radical polymerization scheme, attention to detail and monomer purities are emphasized. As is typical of a RAFT polymerization system, it consists of a radical source, a RAFT reagent, a monomer and solvent. In this visualized presentation, a single-step RAFT polymerization system is described that focuses on the production of statistical copolymers generated from a reaction mixture possessing three different monomers in an aqueous solution. Two separate RAFT-mediated reactions are presented in which one utilizes a glycomonomer that possesses a pendant, non-reducing carbohydrate terminus (i.e.,&#160;&#946;-D-galactose), and the other, possessing a polyol with no bound carbohydrate residue. Common to both RAFT-mediated reactions were monomers possessing a singular hydroxyl group that serves as a spacer molecule, and another possessing a free amine for post-modification with an amino-reactive fluorophore.
Since the presence of oxygen in the reaction mixture and environment is detrimental to RAFT-mediated polymerization, its removal to trace levels is readily accomplished via several freeze&#8211;evacuate&#8211;thaw cycles while maintaining the Schlenk tube reaction vessel under high vacuum.
It should be noted that the molar ratio of different monomers in the reaction can be adjusted as needed. Also, by varying the amount of RAFT agent used, the length of the resulting polymers can be controlled18. However, the molar ratio of the RAFT agent to initiator should always be greater than two to assure the low dispersity of the product. Under these conditions, the evolution of the copolymerization is steady, and the reproducibility of the reaction is very high. That being said, it is unlikely that one obtains a totally uniform distribution of all participating monomers within a statistical copolymer, due to their different polymerization speeds. Characterizing the distribution of different monomers within the polymer is still very challenging.
The post-modification method, presented here, is both simpler and more amenable to the use of a wider selection of fluorescent labels, compared to other protocols applied to label glycopolymers2,11. These would include many of the water-soluble amine-reactive fluorophores, quantum dots, biotins, and others. The binding specificities of the synthesized, labeled glycopolymers are readily verifiable using lectins with known binding affinities. PMA-GAEMA possessing no pendant sugar is an appropriate negative control. Glycopolymers with different fluorescent labels prepared via this route have been successfully used in investigations of lectin-mediated bacterial binding14. As presented, this facile and efficient preparation of statistical fluorescent glycopolymers should provide great potential to a wide variety of glycobiological research.

In the past two decades, investigations with synthetic glycopolymers have undergone slow but continual development, demonstrating significant potential in examining infectious mechanisms that include research which focuses on lectin recognition processes1-3. Since synthetic glycopolymers possessing multivalent sugar moieties exhibit much higher lectin-binding efficacies, as compared to monovalent carbohydrates, they are of great demand in the glycobiology field3. Of particular interest in clinical research is the use of fluorescent glycopolymers to characterize the lectin-mediated bacterial binding with carbohydrates available on human respiratory cell surfaces and mucous glycoprotein. Early in vitro studies employed commercially available polyacrylamide-based glycopolymers in bacterial binding tests. Several of these probes showed promising results, but raised concerns regarding, obtainability, and lot-to-lot variances in both polymer molecular weight and glycoepitope content. An economical in-laboratory protocol was developed which would provide for a satisfactory control of structure content, size, and purity of synthetic glycopolymers targeting bacterial lectins.
In search for a suitable synthetic approach to glycopolymers, a relatively new polymerization technique was tested using a type of controlled radical polymerization that employed reversible addition-fragmentation chain-transfer (RAFT) agents4. Such RAFT reagents have recently been used in a few glycopolymer preparations5-7. Compared with other glycopolymer preparation protocols, RAFT-mediated polymerizations demonstrate several advantages, including the tolerance to a variety of monomer structures and reaction conditions, potential compatibility with aqueous solutions, and low size dispersity of the desired polymeric products8,9. Of notable interest are protocols for preparation of RAFT-based tri-component glycopolymers, allowing control of compositions of different monomers, each of which may have distinct functions10-13. However, most of the previous research endeavors either lacked anomeric pendant carbohydrates10, or employed stepped polymerizations resulting in tri-block copolymers, that consist of covalently linked homopolymers, which often serve different purposes than statistical polymers which are copolymers in which the sequence of monomer residues follow a statistical rule9-13.
Recently, employing the thiocarbonylthio RAFT compound (4-cyanopentanoic acid)-4-dithiobenzoate in an aqueous environment, the preparation of a group of RAFT-based linear tri-component statistical glycopolymers containing specific pendant sugars and their application in lectin-mediated bacterial binding tests was reported14. The overall goal of this method, presented in a visual manner, is to prepare tri-component statistical fluorescent glycopolymers via RAFT-controlled copolymerization. Because of the ease of the one-step polymerization protocol, the fine control over the polymer length and compositions, and the high reproducibility of the reaction, this protocol can be readily applied to other RAFT-based syntheses of glycopolymers with desired structures.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent&lt;/strong&gt;</td></tr><tr><td>Lactobionic acid</td><td>Sigma-Aldrich</td><td>153516</td><td></td></tr><tr><td>D-Gluconolactone&amp;nbsp;</td><td>Sigma-Aldrich</td><td>G2164</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-(2-hydroxyethyl) acrylamide (HEAA)</td><td>Sigma-Aldrich</td><td>697931</td><td></td></tr><tr><td>Orange II sodium salt</td><td>Sigma-Aldrich</td><td>O8126</td><td></td></tr><tr><td>Hydroquinone monomethyl ether (MEHQ)</td><td>Sigma-Aldrich</td><td>54050</td><td>Polymerization inhibitor</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-(2-aminoethyl) methacrylamide hydrochloride (AEMA)</td><td>Polysciences, Inc</td><td>24833-5</td><td></td></tr><tr><td>Triethylamine</td><td>Fisher Scientific</td><td>BP-616</td><td></td></tr><tr><td>Anion-exchange resin IRN-78 hydroxide-form, 80 mesh</td><td>Sigma-Aldrich</td><td>10343-U</td><td></td></tr><tr><td>Cation-exchange resin 50Wx8, 200 mesh</td><td>Sigma-Aldrich</td><td>217514</td><td></td></tr><tr><td>Aluminum oxide, ~150 mesh&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A1522</td><td>Type WN-6, Neutral, Activity Grade Super I</td></tr><tr><td>Ninhydrin</td><td>Sigma-Aldrich</td><td>N4876</td><td>An ethanol solution of 0.2% ninhydrin was used in the test</td></tr><tr><td>4-Cyano-4-(phenylcarbonothioylthio)pentanoic acid</td><td>Sigma-Aldrich</td><td>722995</td><td>RAFT agent</td></tr><tr><td>4,4&amp;prime;-Azobis(4-cyanovaleric acid)</td><td>Sigma-Aldrich</td><td>11588</td><td>Polymerization initiator</td></tr><tr><td>Carboxyfluorescein succinimidyl ester&amp;nbsp;</td><td>Life Technologies</td><td>C1157</td><td></td></tr><tr><td>Erythrina Cristagalli lectin coated agarose bead</td><td>Vector Laboratories</td><td>AL-1143&amp;nbsp;</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Solvent&lt;/strong&gt;</td></tr><tr><td>dH2O</td><td></td><td></td><td>Produced by Barnstead water purification system, 18 megOhm-cm</td></tr><tr><td>Isopropanol</td><td>Fisher Scientific</td><td>A461-4</td><td>ACS grade or better</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A454-4</td><td>ACS grade or better</td></tr><tr><td>Absolute ethanol</td><td>Fisher Scientific</td><td>BP2818-100</td><td>ACS grade or better</td></tr><tr><td>Dimethylformamide</td><td>Sigma-Aldrich</td><td>22705</td><td>ACS grade or better</td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>A929-4</td><td>ACS grade or better</td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Dialysis membrane (MWCO: 3,500)</td><td>Spectrum Labs</td><td>132720</td><td></td></tr><tr><td>Polyethylene glycol analytical standard standard</td><td>Sigma-Aldrich</td><td>O2393</td><td></td></tr><tr><td>Schlenk tube, 1 ml</td><td>Quark Glass</td><td></td><td>Customized</td></tr><tr><td>TSK-GEL G4000 PWxl&amp;nbsp;</td><td>Tosoh Bioscience&amp;nbsp;</td><td>8022</td><td>Used for GPC analysis of the glycopolymers</td></tr><tr><td>Empower 3 with GPC/SEC package</td><td>Waters Corporation</td><td></td><td></td></tr><tr><td>Waters Alliance HPLC system&amp;nbsp;</td><td>Waters Corporation</td><td></td><td>Equipped with refractive index detector (Waters 2414) and fluorescence detector (Waters 2475)</td></tr><tr><td>Avance III 800 MHz NMR Spectrometer</td><td>Bruker Corporation</td><td></td><td></td></tr><tr><td>BX43 fluorescence microscope</td><td>Olympus Corporation</td><td></td><td>Used with FITC filter in the glycopolymer binding test</td></tr><tr><td>Rotavap / Rotoevaporator</td><td>Heidolph</td><td></td><td></td></tr><tr><td>Fritted disc funnel</td><td>Fisher Scientific</td><td>10-310-109</td><td></td></tr><tr><td>Lyophilizer</td><td>Labconco</td><td></td><td></td></tr><tr><td>Immunofluorescence microscope slide</td><td>Polysciences</td><td>18357-1</td><td></td></tr><tr><td>Revco Ultima Plus -80&amp;nbsp;&amp;#176;C Freezer</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Plastic Vacuum Bag and Hand Pump</td><td>Ziploc</td><td></td><td></td></tr><tr><td>Vacuum Pump, Direct Drive, Maxima C Plus</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Vacuum Gauge</td><td>Sargent-Welch</td><td></td><td></td></tr></tbody></table>

facile and efficient preparation of tri-component fluorescent glycopolymers via raft-controlled polymerization

Synthetic glycopolymers are instrumental and versatile tools used in various biochemical and biomedical research fields. An example of a facile and efficient synthesis of well-controlled fluorescent statistical glycopolymers using reversible addition-fragmentation chain-transfer (RAFT)-based polymerization is demonstrated. The synthesis starts with the preparation of &#946;-galactose-containing glycomonomer 2-lactobionamidoethyl methacrylamide obtained by reaction of lactobionolactone and N-(2-aminoethyl) methacrylamide (AEMA). 2-Gluconamidoethyl methacrylamide (GAEMA) is used as a structural analog lacking a terminal &#946;-galactoside. The following RAFT-mediated copolymerization reaction involves three different monomers: N-(2-hydroxyethyl) acrylamide as spacer, AEMA as target for further fluorescence labeling, and the glycomonomers. Tolerant of aqueous systems, the RAFT agent used in the reaction is (4-cyanopentanoic acid)-4-dithiobenzoate. Low dispersities (&#8804;1.32), predictable copolymer compositions, and high reproducibility of the polymerizations were observed among the products. Fluorescent polymers are obtained by modifying the glycopolymers with carboxyfluorescein succinimidyl ester targeting the primary amine functional groups on AEMA. Lectin-binding specificities of the resulting glycopolymers are verified by testing with corresponding agarose beads coated with specific glycoepitope recognizing lectins. Because of the ease of the synthesis, the tight control of the product compositions and the good reproducibility of the reaction, this protocol can be translated towards preparation of other RAFT-based glycopolymers with specific structures and compositions, as desired.

Synthesis of glycomonomer
Lactobionic acid was used herein as an example for the preparation of glycomonomers. Using methods in the initial report on the synthesis of LAEMA11, varied yields in the preparation with unsatisfactory purity were observed. The modified purification method using cation and anion exchange resins to remove unreacted starting material offered stable product yield and high purity, which is confirmed by 1H and 13C NMR spectroscopy (Figure 1).
RAFT glycopolymer synthesis and post-modification of glycopolymers with fluorophores
In contrast to the block-glycopolymers prepared through stepped RAFT polymerizations, this one-step copolymerization protocol provides a uniform glycomonomer distribution throughout the polymer backbone. The glycopolymers shown here contain 20 mol% of glycomonomer, 77 mol% of HEAA as a spacer, and 3 mol% of AEMA as a target for post-modifications (see Figure 2). 1H- and 13C-NMR spectroscopy confirmed the structures of PMA-LAEMA and PMA-GAEMA (Figures 3 and 4). As shown in Figure 5, when plotted against the GPC elution profiles of the glycopolymer synthesized without RAFT, both PMA-LAEMA and PMA-GAEMA have low dispersities, proving the efficacy of the RAFT approach. As expected, PMA-GAEMA has a Mn smaller than that of PMA-LAEMA due to PMA-GAEMA&#8217;s lack of a pendant sugar. Analysis of the carbohydrates and primary amine functional groups content of the RAFT glycopolymers revealed that the ratio of monomers in the product glycopolymers is consistent with the stoichiometric ratio of starting monomers employed in the RAFT-mediated polymerization reaction (Table 1). This signifies a tight control of the monomer compositions in the synthesized glycopolymers, as designed.
Reaction of primary amine functional groups with activated fluorophores is a widely-used technique in protein labeling. This technique was employed here to label purified glycopolymers with carboxyfluorescein. Following post-modification, fluorescent polymers were obtained (Figure 6). No degradation of the fluorescein-labeled polymers in the reaction was detected by GPC analysis (data not shown).
Binding tests of the synthetic glycopolymers with lectin-coated agarose beads 
To assess the lectin-binding specificity of the synthesized glycopolymers, lectin-coated agarose beads with known carbohydrate binding specificity was used. Erythrina crista-galli lectin (ECL), employed in the experiments, has a binding specificity towards &#946;-D-galactoside. Figure 7A clearly demonstrates that PMA-LAEMA-Fluorescein, which contains &#946;-D-galactoside as a pendant carbohydrate, exhibited strong binding with the ECL lectin. In contrast, the negative binding to the ECL of the glycopolymer PMA-GAEMA-Fluorescein, which does not possess a pendant sugar, is shown in Figure 7B. This result exemplifies the binding effectiveness and affinity of the synthesized fluorescent glycopolymer.
<img alt="Figure 1" src="/files/ftp_upload/52922/52922fig1.jpg" /> 
Figure 1. Assigned 1H- (a) and 13C-NMR (b) spectra (D2O) for LAEMA. (This figure has been modified from Wang et al.14) <a href="https://www.jove.com/files/ftp_upload/52922/52922fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52922/52922fig2.jpg" /> 
Figure 2. Schematic illustration of the synthesis of fluorescent glycopolymer PMA-LAEMA containing &#946;-galactoside as the pendant sugar. <a href="https://www.jove.com/files/ftp_upload/52922/52922fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52922/52922fig3.jpg" /> 
Figure 3. Assigned 1H- (A) and 13C-NMR (B) spectra (D2O) for PMA-LAEMA glycopolymer. (This figure has been modified from Wang et al.14) <a href="https://www.jove.com/files/ftp_upload/52922/52922fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52922/52922fig4.jpg" /> 
Figure 4. Assigned 1H- (A) and 13C-NMR (B) spectra (D2O) for PMA-GAEMA. (This figure has been modified from Wang et al.14) <a href="https://www.jove.com/files/ftp_upload/52922/52922fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52922/52922fig5.jpg" /> 
Figure 5. Gel permeation chromatography traces of RAFT-based PMA-GAEMA and PMA-LAEMA prepared with and without using RAFT agent. In contrast to the PMA-LAEMA prepared without RAFT agent (blue), RAFT-based PMA-LAEMA (green) has a much lower dispersity (Mw/Mn). RAFT-based PMA-GAEMA (red) and PMA-LAEMA have similar GPC profiles, but the former has a smaller Mn due to the absence of any pendant sugars. <a href="https://www.jove.com/files/ftp_upload/52922/52922fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52922/52922fig6.jpg" /> 
Figure 6. PMA-LAEMA before and after post-modification with fluorophore. (A) Compared with white non-labeled glycopolymer (left tube), fluorescein-labeled PMA-LAEMA shows a strong yellow color (right tube). (B) Under UV, non-labeled PMA-LAEMA (left tube, 1 mg/ml in PBS) is dark and presents with no fluorescence, whereas fluorescein-labeled PMA-LAEMA (right tube, 1 mg/ml in PBS) shows strong green fluorescence. <a href="https://www.jove.com/files/ftp_upload/52922/52922fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/52922/52922fig7.jpg" /> 
Figure 7. Erythrina crista-galli lectin (ECL)-coated agarose beads bind &#946;-D-galactoside containing glycopolymers, and not those not possessing a pendant sugar. (A) PMA-LAEMA-Fluorescein (3 &#181;g) demonstrated strong binding with ECL, whereas in (B) PMA-GAEMA-Fluorescein, which possesses no pendant &#946;-D-galactoside residue, showed no binding with the lectin-coated beads. Scale bar = 100 &#181;m.
Table 1. Targeting valuesa of synthetic parameters and actual compositions of the glycopolymers. a) Targeting values, values that are desired of the products; b) DP, degree of polymerization; c) NA, not available. <a href="https://www.jove.com/files/ftp_upload/52922/52922fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody style="vertical-align:middle;text-align:center">
		<tr>
			<td style="width:120px"></td>
			<td style="width:40px">DPb&#160;</td>
			<td style="width:70px">Dispersity</td>
			<td style="width:120px">Actual content of glycomonomers 
			mol %</td>
			<td>Actual content of primary amine 
			mol %</td>
		</tr>
		<tr>
			<td>Targeting values</td>
			<td>100</td>
			<td>&#60; 1.3</td>
			<td>20</td>
			<td>3</td>
		</tr>
		<tr>
			<td>PMA-LAEMA</td>
			<td>99</td>
			<td>1.26</td>
			<td>19</td>
			<td>3.2</td>
		</tr>
		<tr>
			<td>PMA-GAEMA</td>
			<td>89</td>
			<td>1.32</td>
			<td>NAc</td>
			<td>2.7</td>
		</tr>
	</tbody>
</table>

Watch this Scientific Journal Video about Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization at JoVE.com

Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization

An efficient, three-step synthesis of RAFT-based fluorescent glycopolymers, consisting of glycomonomer preparation, copolymerization, and post-modification, is demonstrated. This protocol can be used to prepare RAFT-based statistical glycopolymers with desired structures.

Synthetic glycopolymers are instrumental and versatile tools used in various biochemical and biomedical research fields. An example of a facile and ...

facile-efficient-preparation-tri-component-fluorescent-glycopolymers

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Chemistry

9.7K Views.  University of Missouri. An efficient, three-step synthesis of RAFT-based fluorescent glycopolymers, consisting of glycomonomer preparation, copolymerization, and post-modification, is demonstrated. This protocol can be used to prepare RAFT-based statistical glycopolymers with desired structures.

Video: Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9. 
DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.
A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10.

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

Bioengineering

Facile Synthesis of Worm-like Micelles by Visible Light Mediated Dispersion Polymerization Using Photoredox Catalyst

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins with the synthesis of a hydrophilic poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) homopolymer using reversible addition-fragmentation chain-transfer (RAFT) polymerization. Under mild visible light irradiation (&#955; = 460 nm, 0.7 mW/cm2), this macro-chain transfer agent (macro-CTA) in the presence of a ruthenium based photoredox catalyst, Ru(bpy)3Cl2 can be chain extended with a second monomer to form a well-defined block copolymer in a process known as Photoinduced Electron Transfer RAFT (PET-RAFT). When PET-RAFT is used to chain extend POEGMA with benzyl methacrylate (BzMA) in ethanol (EtOH), polymeric nanoparticles with different morphologies are formed in situ according to a polymerization-induced self-assembly (PISA) mechanism. Self-assembly into nanoparticles presenting POEGMA chains at the corona and poly(benzyl methacrylate) (PBzMA) chains in the core occurs in situ due to the growing insolubility of the PBzMA block in ethanol. Interestingly, the formation of highly pure worm-like micelles can be readily monitored by observing the onset of a highly viscous gel in situ due to nanoparticle entanglements occurring during the polymerization. This process thereby allows for a more reproducible synthesis of worm-like micelles simply by monitoring the solution viscosity during the course of the polymerization. In addition, the light stimulus can be intermittently applied in an ON/OFF manner demonstrating temporal control over the nanoparticle morphology.

This article describes a process for producing polymeric self-assembled nanoparticles using visible light mediated dispersion polymerization. Using low energy visible light to control the polymerization allows for the reproducible formation of self-assembled worm-like micelles at high solids content.

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins ...

Controlled Synthesis and Fluorescence Tracking of Highly Uniform Poly(N-isopropylacrylamide) Microgels

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this work, we use single particle tracking of fluorescently labeled PNIPAM microgels as a showcase for tuning microgel size by a rapid non-stirred precipitation polymerization procedure. This approach is well suited for prototyping new reaction compositions and conditions or for applications that do not require large amounts of product. Microgel synthesis, particle size and structure determination by dynamic and static light scattering are detailed in the protocol. It is shown that the addition of functional comonomers can have a large influence on the particle nucleation and structure. Single particle tracking by wide-field fluorescence microscopy allows for an investigation of the diffusion of labeled tracer microgels in a concentrated matrix of non-labeled microgels, a system not easily investigated by other methods such as dynamic light scattering.

Controlled Synthesis and Fluorescence Tracking of Highly Uniform PolyN-isopropylacrylamide Microgels

Non-stirred precipitation polymerization provides a rapid, reproducible prototyping approach to the synthesis of stimuli-sensitive poly(N-isopropylacrylamide) microgels of narrow size distribution. In this protocol synthesis, light scattering characterization and single particle fluorescence tracking of these microgels in a wide-field microscopy setup are demonstrated.

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this ...

Synthesis of Cyclic Polymers and Characterization of Their Diffusive Motion in the Melt State at the Single Molecule Level

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single molecule level. An electrostatic self-assembly and covalent fixation (ESA-CF) process is used for the synthesis of the cyclic poly(tetrahydrofuran) (poly(THF)). The diffusive motion of individual cyclic polymer chains in a melt state is visualized using single molecule fluorescence imaging by incorporating a fluorophore unit in the cyclic chains. The diffusive motion of the chains is quantitatively characterized by means of a combination of mean-squared displacement (MSD) analysis and a cumulative distribution function (CDF) analysis. The cyclic polymer exhibits multiple-mode diffusion which is distinct from its linear counterpart. The results demonstrate that the diffusional heterogeneity of polymers that is often hidden behind ensemble averaging can be revealed by the efficient synthesis of the cyclic polymers using the ESA-CF process and the quantitative analysis of the diffusive motion at the single molecule level using the MSD and CDF analyses.

A protocol for the synthesis and characterization of diffusive motion of cyclic polymers at the single molecule level is presented.

We demonstrate a method for the synthesis of cyclic polymers and a protocol for characterizing their diffusive motion in a melt state at the single ...

Forming Giant-sized Polymersomes Using Gel-assisted Rehydration

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier properties, chemical versatility and tunable physical characteristics. Typical methods used to prepare giant-sized (&gt; 4 &#181;m) vesicles, however, are both time and labor intensive, yielding low numbers of intact polymersomes. Here, we present for the first time the use of gel-assisted rehydration for the rapid and high-yielding formation of giant (&gt;4 &#181;m) polymer vesicles (polymersomes). Using this method, polymersomes can be formed from a wide array of rehydration solutions including several different physiologically-compatible buffers and full cell culture media, making them readily useful for biomimicry studies. This technique is also capable of reliably producing polymersomes from different polymer compositions with far better yields and much less difficulty than traditional methods. Polymersome size is readily tunable by altering temperature during rehydration or adding membrane fluidizers to the polymer membrane, generating giant-sized polymersomes (&gt;100 &#181;m).

We present a protocol to rapidly form giant polymer vesicles (pGVs). Briefly, polymer solutions are dehydrated on dried agarose films adhered to coverslips. Rehydration of the polymer films results in rapid formation of pGVs. This method greatly advances the preparation of synthetic giant vesicles for direct applications in biomimetic studies.

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

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.

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.

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

Fabrication and Implementation of a Reference-Free Traction Force Microscopy Platform

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their microenvironment. While many approaches exist for measuring cell-induced material strain, here we provide a methodology for monitoring strain with sub-micron resolution in a reference-free manner. Using a two-photon activated photolithographic patterning process, we demonstrate how to generate mechanically and bio-actively tunable synthetic substrates containing embedded arrays of fluorescent fiducial markers to easily measure three-dimensional (3D) material deformation profiles in response to surface tractions. Using these substrates, cell tension profiles can be mapped using a single 3D image stack of a cell of interest. Our goal with this methodology is to make traction force microscopy a more accessible and easier to implement tool for researchers studying cellular mechanotransduction processes, especially newcomers to the field.

This protocol provides instructions for implementing multiphoton lithography to fabricate three-dimensional arrays of fluorescent fiducial markers embedded in poly(ethylene glycol)-based hydrogels for use as reference-free, traction force microscopy platforms. Using these instructions, measurement of 3D material strain and calculation of cellular tractions is simplified to promote high-throughput traction force measurements.

Quantifying cell-induced material deformation provides useful information concerning how cells sense and respond to the physical properties of their ...

Interlinked Macroporous 3D Scaffolds from Microgel Rods

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions without further additives. Continuous photoinitiated on-chip gelation enables variation of the microgel aspect ratio, which determines the building block properties for the obtained constructs. Glycidyl methacrylate (GMA) or 2-aminoethyl methacrylate (AMA) monomers are copolymerized into the microgel network based on polyethylene glycol (PEG) star-polymers to achieve either epoxy or amine functionality. A focusing oil flow is introduced into the microfluidic outlet structure to ensure continuous collection of the functionalized microgel rods. Based on a recent publication, microgel rod-based constructs result in larger pores of several hundred micrometers and, at the same time, lead to overall higher scaffold stability in comparison to a spherical-based model. In this way, it is possible to produce higher-volume constructs with more free volume while reducing the amount of material required. The interlinked macroporous scaffolds can be picked up and transported without damage or disintegration. Amine and epoxy groups not involved in interlinking remain active and can be used independently for post-modification. This protocol describes an optimized method for the fabrication of microgel rods to form macroporous interlinked scaffolds that can be utilized for subsequent cell experiments.

Microgel rods with complementary reactive groups are produced via microfluidics with the ability to interlink in aqueous solution. The anisometric microgels jam and interlink into stable constructs with larger pores compared to spherical-based systems. Microgels modified with GRGDS-PC form macroporous 3D constructs that can be used for cell culture.

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions ...

Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization

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