Name: particles without a box: brush-first synthesis of photodegradable peg star polymers under ambient conditions
Uploaded: 2013-10-10T21:30:00
Description: Watch this Scientific Journal Video about Particles without a Box: Brush-first Synthesis of Photodegradable PEG Star Polymers under Ambient Conditions at JoVE.com

We thank the MIT Department of Chemistry and the MIT Lincoln Labs Advanced Concepts Committee for support of this work.

We first describe the synthesis and purification of PEG-MM B from 3 kDa O-(2-aminoethyl)polyethylene glycol (PEG-NH2) and norbornene-N-hydroxysuccinimidyl (NHS) ester. The former compound can be purchased from Sigma Aldrich Inc., or prepared via anionic polymerization according to literature procedures22,23. The latter compound can be prepared in two steps according to a published procedure21. Next we describe a synthesis of catalyst A</...

<ol>
	<li>Bielawski, C. W., Grubbs, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Living+ring-opening+metathesis+polymerization.">Living ring-opening metathesis polymerization.</a> Prog. Polym. Sci. 32, 1-29 (2007).</li><li>Hawker, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Convergence+of+Synthetic+Organic+and+Polymer+Chemistries.">The Convergence of Synthetic Organic and Polymer Chemistries.</a> Science. 309, 1200-1205 (2005).</li><li>Peer, D., Karp, J. M., Hong, S., Farokhzad, O. C., Margalit, R., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanocarriers+as+an+emerging+platform+for+cancer+therapy.">Nanocarriers as an emerging platform for cancer therapy.</a> Nat. Nano. 2, 751-760 (2007).</li><li>Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscience,+Nanotechnology,+and+Chemistry.">Nanoscience, Nanotechnology, and Chemistry.</a> Small. 1, 172-179 (2005).</li><li>Leitgeb, A., Wappel, J., Slugovc, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ROMP+toolbox+upgraded.">The ROMP toolbox upgraded.</a> Polymer. 51, 2927-2946 (2010).</li><li>Johnson, J. A., Lu, Y. Y., Burts, A. O., Lim, Y. -. H., Finn, M. G., Koberstein, J. T., Turro, N. J., Tirrell, D. A., Grubbs, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Core-Clickable+PEG-Branch-Azide+Bivalent-Bottle-Brush+Polymers+by+ROMP:+Grafting-Through+and+Clicking-To.">Core-Clickable PEG-Branch-Azide Bivalent-Bottle-Brush Polymers by ROMP: Grafting-Through and Clicking-To.</a> J. Am. Chem. Soc. 133, 559-566 (2010).</li><li>Bielawski, C. W., Grubbs, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Efficient+Ring-Opening+Metathesis+Polymerization+(ROMP)+Using+New+Ruthenium+Catalysts+Containing+N-Heterocyclic+Carbene+Ligands.">Highly Efficient Ring-Opening Metathesis Polymerization (ROMP) Using New Ruthenium Catalysts Containing N-Heterocyclic Carbene Ligands.</a> Angew. Chem. Int. Ed. 39, 2903-2906 (2000).</li><li>Love, J. A., Morgan, J. P., Trnka, T. M., Grubbs, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Practical+and+Highly+Active+Ruthenium-Based+Catalyst+that+Effects+the+Cross+Metathesis+of+Acrylonitrile.">A Practical and Highly Active Ruthenium-Based Catalyst that Effects the Cross Metathesis of Acrylonitrile.</a> Angew. Chem. Int. Ed. 41, 4035-4037 (2002).</li><li>Lu, J. A., Johnson, Y. Y., Burts, A. O., Xia, Y., Durrell, A. C., Tirrell, D. A., Grubbs, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-Loaded,+Bivalent-Bottle-Brush+Polymers+by+Graft-through+ROMP.">Drug-Loaded, Bivalent-Bottle-Brush Polymers by Graft-through ROMP.</a> Macromolecules. 43, 10326-10335 (2010).</li><li>Burts, A. O., Li, Y. J., Zhukhovitskiy, A. V., Patel, P. R., Grubbs, R. H., Ottaviani, M. F., Turro, N. J., Johnson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+EPR+To+Compare+PEG-branch-nitroxide+"Bivalent-Brush+Polymers"+and+Traditional+PEG+Bottle-Brush+Polymers:+Branching+Makes+a+Difference.">Using EPR To Compare PEG-branch-nitroxide "Bivalent-Brush Polymers" and Traditional PEG Bottle-Brush Polymers: Branching Makes a Difference.</a> Macromolecules. 45, 8310-8318 (2012).</li><li>Bazan, G. C., Schrock, R. R. <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+star+block+copolymers+by+controlled+ring-opening+metathesis+polymerization.">Synthesis of star block copolymers by controlled ring-opening metathesis polymerization.</a> Macromolecules. 24, 817-823 (1991).</li><li>Saunders, R. S., Cohen, R. E., Wong, S. J., Schrock, R. R. <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+amphiphilic+star+block+copolymers+using+ring-opening+metathesis+polymerization.">Synthesis of amphiphilic star block copolymers using ring-opening metathesis polymerization.</a> Macromolecules. 25, 2055-2057 (1992).</li><li>Buchmeiser, M. R., Wurst, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Access+to+Well-Defined+Heterogeneous+Catalytic+Systems+via+Ring-Opening+Metathesis+Polymerization+(ROMP):+Applications+in+Palladium(II)-Mediated+Coupling+Reactions.">Access to Well-Defined Heterogeneous Catalytic Systems via Ring-Opening Metathesis Polymerization (ROMP): Applications in Palladium(II)-Mediated Coupling Reactions.</a> J. Am. Chem. Soc. 121, 11101-11107 (1999).</li><li>Weichelt, F., Frerich, B., Lenz, S., Tiede, S., Buchmeiser, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ring-Opening+Metathesis+Polymerization-Based+Synthesis+of+CaCO3+Nanoparticle-Reinforced+Polymeric+Monoliths+for+Tissue+Engineering.">Ring-Opening Metathesis Polymerization-Based Synthesis of CaCO3 Nanoparticle-Reinforced Polymeric Monoliths for Tissue Engineering.</a> Macromol. Rapid Comm. 31, 1540-1545 (2010).</li><li>Weichelt, F., Lenz, S., Tiede, S., Reinhardt, I., Frerich, B., Buchmeiser, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROMP-Derived+cyclooctene-based+monolithic+polymeric+materials+reinforced+with+inorganic+nanoparticles+for+applications+in+tissue+engineering.">ROMP-Derived cyclooctene-based monolithic polymeric materials reinforced with inorganic nanoparticles for applications in tissue engineering.</a> Beilstein J. Org. Chem. 6, 1199-1205 (2010).</li><li>Mayr, M., Mayr, B., Buchmeiser, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monolithic+Materials:+New+High-Performance+Supports+for+Permanently+Immobilized+Metathesis+Catalysts.">Monolithic Materials: New High-Performance Supports for Permanently Immobilized Metathesis Catalysts.</a> Angew. Chem. Int. Ed. 40, 3839-3842 (2001).</li><li>Mayr, B. H., &#246;lzl, G., Eder, K., Buchmeiser, M. R., Huber, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophobic,+Pellicular,+Monolithic+Capillary+Columns+Based+on+Cross-Linked+Polynorbornene+for+Biopolymer+Separations.">Hydrophobic, Pellicular, Monolithic Capillary Columns Based on Cross-Linked Polynorbornene for Biopolymer Separations.</a> Anal. Chem. 74, 6080-6087 (2002).</li><li>Otani, H., Fujita, S., Watanabe, Y., Fujiki, M., Nomura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Facile,+Controlled+Synthesis+of+Soluble+Star+Polymers+Containing+a+Sugar+Residue+by+Ring-Opening+Metathesis+Polymerization+(ROMP).">A Facile, Controlled Synthesis of Soluble Star Polymers Containing a Sugar Residue by Ring-Opening Metathesis Polymerization (ROMP).</a> Macromol. Symp. 293, 53-57 (2010).</li><li>Nomura, K., Watanabe, Y., Fujita, S., Fujiki, M., Otani, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+Controlled+Synthesis+of+Soluble+Star+Shape+Polymers+by+Ring-Opening+Metathesis+Polymerization+(ROMP).">Facile Controlled Synthesis of Soluble Star Shape Polymers by Ring-Opening Metathesis Polymerization (ROMP).</a> Macromolecules. 42, 899-901 (2009).</li><li>Gao, H., Ohno, S., Matyjaszewski, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+Polydispersity+Star+Polymers+via+Cross-Linking+Macromonomers+by+ATRP.">Low Polydispersity Star Polymers via Cross-Linking Macromonomers by ATRP.</a> J. Am. Chem. Soc. 128, 15111-15113 (2006).</li><li>Liu, J., Burts, A. O., Li, Y., Zhukhovitskiy, A. V., Ottaviani, M. F., Turro, N. J., Johnson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Brush-First"+Method+for+the+Parallel+Synthesis+of+Photocleavable,+Nitroxide-Labeled+Poly(ethylene+glycol)+Star+Polymers.">"Brush-First" Method for the Parallel Synthesis of Photocleavable, Nitroxide-Labeled Poly(ethylene glycol) Star Polymers.</a> J. Am. Chem. Soc. 134, 16337-16344 (2012).</li><li>Studer, P., Larras, V., Riess, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+end-functionalized+poly(ethylene+oxide)-block-poly(methylidene+malonate+2.1.2)+block+copolymers:+synthesis,+characterization,+and+chemical+modification+for+targeting+purposes.">Amino end-functionalized poly(ethylene oxide)-block-poly(methylidene malonate 2.1.2) block copolymers: synthesis, characterization, and chemical modification for targeting purposes.</a> Eur. Polym. J. 44, 1714-1721 (2008).</li><li>Mosquet, M., Chevalier, Y., Le Perchec, P., Guicquero, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+poly+(ethylene+oxide)+with+a+terminal+amino+group+by+anionic+polymerization+of+ethylene+oxide+initiated+by+aminoalcoholates.">Synthesis of poly (ethylene oxide) with a terminal amino group by anionic polymerization of ethylene oxide initiated by aminoalcoholates.</a> Macromol. Chem. Phys. 198, 2457-2474 (1997).</li><li>Burchard, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+properties+of+branched+macromolecules.">Solution properties of branched macromolecules.</a> Adv. Polym. Sci. 143, 113-194 (1999).</li><li>Gao, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Star+Polymers+as+Unimolecular+Containers+for+Nanomaterials.">Development of Star Polymers as Unimolecular Containers for Nanomaterials.</a> Macromol. Rapid Comm. , 722-734 (2012).</li><li>Blencowe, A., Tan, J. F., Goh, T. K., Qiao, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Core+cross-linked+star+polymers+via+controlled+radical+polymerisation.">Core cross-linked star polymers via controlled radical polymerisation.</a> Polymer. 50, 5-32 (2009).</li><li>Burts, A. O., Liao, L., Lu, Y. Y., Tirrell, D. A., Johnson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brush-first+and+Click:+Efficient+Synthesis+of+Nanoparticles+that+Degrade+and+Release+Doxorubicin+in+Response+to+Light.">Brush-first and Click: Efficient Synthesis of Nanoparticles that Degrade and Release Doxorubicin in Response to Light.</a> Photochem. Photobiol. , (2013).</li></ol>

The key advantage of brush-first BASP synthesis is the unique ability to rapidly synthesize nanostructures of diverse size and composition in parallel without need for specialized equipment. In this study, we demonstrate the brush-first synthetic method using a norbornene functionalized PEG macromonomer (B, Figure 1) and a bis-norbornene nitrobenzyl ester crosslinker (C, Figure 1). The PEG chains from B impart ...

Polymeric nanoparticles have been widely studied for their potential use as platforms for drug delivery, supported catalysis, biological imaging, and self-assembly1-3. Modern applications require that nanoparticle syntheses be facile, reproducible, compatible with chemical functionalities, and amenable to diversification4,5. Ring-opening metathesis polymerization (ROMP) of strained olefins is a powerful methodology for the synthesis of functional polymeric nanostructures with controlled sizes and narrow mass distributions1,6-8. For example, norbornene-functionalized poly(ethylene glycol) (PEG) macromonomers (MMs) can be efficiently polymerized via ROMP to generate water soluble bottle-brush polymers. Using this approach, nanostructures that carry multiple releasable drug molecules, fluorophores, and spin-contrast agents can be prepared rapidly and in parallel6,9,10.
ROMP has also been used for the &#34;arm-first&#34; synthesis of star polymers. In the arm-first method, linear polymers are crosslinked with a multi-functional crosslinker to give spherical nanostructures with polymeric arms. Schrock and co-workers reported the first arm-first ROMP synthesis of star polymers via crosslinking of norbornene, dicarbomethoxynorbornadiene, and trimethylsilyl protected dicarboxynorbornene linear polymers with a bifunctional norbornene crosslinker.11,12 Buchmeiser has extended this methodology for the synthesis of materials with a range of applications that include supported catalysis, tissue-engineering, and chromatography13-17. Otani and coworkers have made star polymer nanoparticles with functional surfaces via a related &#34;in-out&#34; polymerization strategy18,19.
Most arm-first polymerizations involve a complex interplay of monomer, polymer, and star coupling reactions. The latter proceeds via a step-growth mechanism that typically leads to broad molecular weight (MW) distributions. To overcome this limitation in related arm-first atom transfer radical polymerization reactions, Matyjaszewski and coworkers performed arm-first crosslinking of preformed polymeric MMs to provide star polymers with very narrow MW distributions20. In this case, the steric bulk of the MMs, and the increased ratio of star arms to initiation sites, inhibited poorly controlled star+star coupling processes, and led to a living, chain growth mechanism.
When we attempted the same strategy in the context of ROMP with a norbornene-terminated PEG-MM and a bis-norbornene crosslinker, star polymers with very broad, multi-modal MW distributions were obtained. This result suggested that in this system the MM alone was not sufficiently bulky to inhibit star+star coupling. To increase the steric bulk of the star arms, and potentially limit this uncontrolled coupling, we attempted to first polymerize the MM to form bottle-brush polymers in the absence of crosslinker and then add the crosslinker. We were pleased to find that under certain conditions, this &#34;brush-first&#34; method provided straightforward access to &#34;brush-arm star polymers&#34; (BASPs) with narrow MW distributions and tunable core and corona functionalities.
We recently reported the brush-first ROMP synthesis of PEG BASPs using Grubbs 3rd generation catalyst A (Figure 1)21. In this work, exposure of PEG-MM B to catalyst A generated a living brush macroinitiator with defined backbone length (B1, Figure 1). Transfer of aliquots of the B1&#160;to vials that contained different amounts of crosslinker C initiated BASP formation. The MW, and therefore the size, of the BASPs increased geometrically with the amount of C added. We provided a mechanistic hypothesis for this geometric growth process and demonstrated that functional, nitroxide core- and corona-labeled BASPs could be readily prepared without the need for post-polymerization modification steps or sequential monomer additions. However, in all of the reported examples, we were concerned about catalyst deactivation; we carried out all reactions under N2 atmosphere inside a glovebox.
Since our initial report, we have found that the brush-first method is very effective for the formation of BASPs from a wide range of norbornene-terminated MMs and functional crosslinkers. We have also discovered that the method can be performed on the benchtop with no special precautions to remove air or moisture.
Herein, a series of three BASPs of differing MWs will be synthesized by the brush-first method under ambient conditions. In brief, 10 equivalents of B will be exposed to 1.0 equivalents of catalyst A (Figure 1a) for 15 min to yield a BI with an average degree of polymerization (DP) of 10. Three aliquots of this batch of BI will be transferred to separate vials that contain 10, 15, and 20 equivalents (N, Figure 1b) of C. After 4 hr, the polymerizations will be quenched via addition of ethyl vinyl ether. The star polymer MWs and MW distributions will be characterized using a gel permeation chromatography instrument equipped with a multi-angle laser light scattering detector (GPC-MALLS).

<table border="0" cellpadding="0" cellspacing="0" width="592">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="84" rowspan="2" style="height:84px;width:216px;">Grubbs Second Generation Catalyst</td>
			<td rowspan="2" style="width:91px;">Materia (or Sigma Aldrich)</td>
			<td rowspan="2" style="width:119px;">C848 (Sigma Aldrich: 569747)</td>
			<td style="width:167px;">
			Used as purchased from manufacturer. 
 *Provided as a generous gift.
			</td>
		</tr>
		<tr height="42">
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pyridine</td>
			<td style="width:91px;">Sigma Aldrich</td>
			<td style="width:119px;">270970</td>
			<td style="width:167px;">Used as purchased from manufacturer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">O-(2-aminoethyl)polyethylene glycol 3000</td>
			<td style="width:91px;">Sigma Aldrich</td>
			<td style="width:119px;">07969</td>
			<td style="width:167px;">Used as purchased from manufacturer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PEG-MM</td>
			<td style="width:91px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;">Synthesized following reported procedures (Ref. 21, protocol 1)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">norbornene-N-hydroxysuccinimidyl (NHS) ester</td>
			<td style="width:91px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;">Synthesized following reported procedures (Ref. 21)</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Bis-norb-NBOC Crosslinker</td>
			<td style="width:91px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;">Synthesized following reported procedures (Ref. 21)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pentane</td>
			<td style="width:91px;">Sigma Aldrich</td>
			<td style="width:119px;">158941</td>
			<td style="width:167px;">Used as purchased from manufacturer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Tetrahydrofuran (HPLC grade)</td>
			<td style="width:91px;">Sigma Aldrich </td>
			<td style="width:119px;">34865</td>
			<td style="width:167px;">Dried and purified over a solvent purification columns </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dichloromethane</td>
			<td style="width:91px;">VWR </td>
			<td>BDH1113-4LG</td>
			<td style="width:167px;">Used as purchased from manufacturer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Acetonitrile (HPLC grade)</td>
			<td style="width:91px;">Sigma Aldrich</td>
			<td style="width:119px;">34998</td>
			<td style="width:167px;">Used as purchased from manufacturer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Acetic Acid </td>
			<td style="width:91px;">Sigma Aldrich</td>
			<td style="width:119px;">A6283</td>
			<td style="width:167px;">Used as purchased from manufacturer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium sulfate </td>
			<td style="width:91px;">Sigma Aldrich</td>
			<td>239313</td>
			<td style="width:167px;">Used as purchased from manufacturer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Diethyl ether</td>
			<td style="width:91px;">Sigma Aldrich</td>
			<td style="width:119px;">673811</td>
			<td style="width:167px;">Used as purchased from manufacturer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dimethylformamide (HPLC grade) </td>
			<td style="width:91px;">Sigma Aldrich</td>
			<td style="width:119px;">270547</td>
			<td style="width:167px;">Used as purchased from manufacturer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lithium Bromide</td>
			<td style="width:91px;">Sigma Aldrich</td>
			<td style="width:119px;">213225</td>
			<td style="width:167px;">Used as purchased from manufacturer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MillQ Biocel A10</td>
			<td style="width:91px;">Millipore</td>
			<td style="width:119px;"> </td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Beckmann Coulter HPLC (127p solvent module, 166p detector)</td>
			<td style="width:91px;">Beckmann Coulter</td>
			<td style="width:119px;"> </td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Zorbax 300SB-C18 PrepHT reverse phase column </td>
			<td style="width:91px;">Agilent</td>
			<td style="width:119px;"> </td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1260 Infinity Liquid Chromatography </td>
			<td style="width:91px;">Agilent</td>
			<td style="width:119px;"> </td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GPC KD-806M column</td>
			<td style="width:91px;">Shodex</td>
			<td style="width:119px;"> </td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dawn Heleos II Light Scatterer</td>
			<td style="width:91px;">Wyatt</td>
			<td style="width:119px;"> </td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Optilab T-rEX Refractive Index Detector</td>
			<td style="width:91px;">Wyatt</td>
			<td style="width:119px;"> </td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass Scintillation Vials - 40 ml</td>
			<td style="width:91px;">Chemglass</td>
			<td>CG-4909-05</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass Scintillation Vials - 4 ml </td>
			<td style="width:91px;">Chemglass</td>
			<td style="width:119px;">CG-4904-06</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass Scintillation Vials (PTFE-lined cap) - 2 ml </td>
			<td style="width:91px;">Agilent</td>
			<td>5183-4518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stir-bars</td>
			<td style="width:91px;">VWR</td>
			<td style="width:119px;">5894x</td>
			<td style="width:167px;">various sizes </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">13 mm 0.45 &#181;m Nylon Syringe filter</td>
			<td style="width:91px;">PerkinElmer</td>
			<td>02542903</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">13 mm 0.45 &#181;m polytetrafluoroethylene syringe filter</td>
			<td style="width:91px;">PerkinElmer</td>
			<td>02542909</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1 ml disposable syringes </td>
			<td style="width:91px;">VWR</td>
			<td style="width:119px;">53548-001</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Swing bucket centrifuge or similar</td>
			<td style="width:91px;"> </td>
			<td style="width:119px;"> </td>
			<td style="width:167px;">Should be able to reach approximately 4,000 rpm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Round bottom flask</td>
			<td style="width:91px;"> </td>
			<td style="width:119px;"> </td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fritted glass filter assembly </td>
			<td style="width:91px;"> </td>
			<td style="width:119px;"> </td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rotary Evaporator </td>
			<td style="width:91px;"> </td>
			<td style="width:119px;"> </td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Balance </td>
			<td style="width:91px;"> </td>
			<td style="width:119px;"> </td>
			<td style="width:167px;"> </td>
		</tr>
	</tbody>
</table>

particles without a box: brush-first synthesis of photodegradable peg star polymers under ambient conditions

Convenient methods for the rapid, parallel synthesis of diversely functionalized nanoparticles will enable discovery of novel formulations for drug delivery, biological imaging, and supported catalysis. In this report, we demonstrate parallel synthesis of brush-arm star polymer (BASP) nanoparticles by the &#34;brush-first&#34; method. In this method, a norbornene-terminated poly(ethylene glycol) (PEG) macromonomer (PEG-MM) is first polymerized via ring-opening metathesis polymerization (ROMP) to generate a living brush macroinitiator. Aliquots of this initiator stock solution are added to vials that contain varied amounts of a photodegradable bis-norbornene crosslinker. Exposure to crosslinker initiates a series of kinetically-controlled brush+brush and star+star coupling reactions that ultimately yields BASPs with cores comprised of the crosslinker and coronas comprised of PEG. The final BASP size depends on the amount of crosslinker added. We carry out the synthesis of three BASPs on the benchtop with no special precautions to remove air and moisture. The samples are characterized by gel permeation chromatography (GPC); results agreed closely with our previous report that utilized inert (glovebox) conditions. Key practical features, advantages, and potential disadvantages of the brush-first method are discussed.

Figure 2 shows GPC traces for a variety of BASPs prepared from B1, B2, and B3. In all cases, the data illustrate that increasing the equivalents of crosslinker (N) leads to an increase in the size of the BASP. As was observed in our previous report, 10 equivalents of crosslinker is not sufficient to achieve uniform BASPs; the N = 10 sample shows a clearly multi-modal GPC trace with a large amount of residual b...

Watch this Scientific Journal Video about Particles without a Box: Brush-first Synthesis of Photodegradable PEG Star Polymers under Ambient Conditions at JoVE.com

Particles without a Box: Brush-first Synthesis of Photodegradable PEG Star Polymers under Ambient Conditions

Poly(ethylene glycol) (PEG) brush-arm star polymers (BASPs) with narrow mass distributions and tunable nanoscopic sizes are synthesized in via ring opening metathesis polymerization (ROMP) of a PEG-norbornene macromonomer followed by transfer of portions of the resulting living brush initiator to vials containing varied amounts of a rigid, photo-cleavable bis-norbornene crosslinker.

Convenient methods for the rapid, parallel synthesis of diversely functionalized nanoparticles will enable discovery of novel formulations for drug ...

particles-without-box-brush-first-synthesis-photodegradable-peg-star

Research

JoVE Journal

Chemistry

A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like oligomeric clusters of these yellow nanoparticles of narrow size distribution are synthesized under ambient conditions via two methods. The delay-time method controls the number of subunits in the oligoclusters by varying the time between the addition of HAuCl4 to alkaline solution and the subsequent addition of reducing agent, NaSCN. The yellow oligoclusters produced range in size from ~3 to ~25 nm. This size range can be further extended by an add-on method utilizing hydroxylated gold chloride (Na+[Au(OH4-x)Clx]-) to auto-catalytically increase the number of subunits in the as-synthesized oligocluster nanoparticles, providing a total range of 3 nm to 70 nm. The crude oligocluster preparations display narrow size distributions and do not require further fractionation for most purposes. The oligoclusters formed can be concentrated &#62;300 fold without aggregation and the crude reaction mixtures remain stable for weeks without further processing. Because these oligomeric clusters can be concentrated before derivatization they allow expensive derivatizing agents to be used economically. In addition, we present two models by which predictions of particle size can be made with great accuracy.

We describe a simple method for producing highly stable oligomeric clusters of gold nanoparticles via the reduction of chloroauric acid (HAuCl4) with sodium thiocyanate (NaSCN). The oligoclusters have a narrow size distribution and can be produced with a wide range of sizes and surface coats.

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like ...

Visualization of Ambient Mass Spectrometry with the Use of Schlieren Photography

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass spectrometer, it is necessary to characterize and understand the physical principles of the source. Most commercial ambient ionization sources utilize jets of nitrogen, helium, or atmospheric air to facilitate the ionization of the analyte. As a consequence, schlieren photography can be used to visualize the gas streams by exploiting the differences in refractive index between the streams and ambient air for visualization in real time. The basic setup requires a camera, mirror, flashlight, and razor blade. When properly configured, a real time image of the source is observed by watching its reflection. This allows for insight into the mechanism of action in the source, and pathways to its optimization can be elucidated. Light is shed on an otherwise invisible situation.

This paper presents a protocol for the visualization of gaseous streams of an ambient ionization source using schlieren photography and mass spectrometry.

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass ...

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 ...

Facile Preparation of Ultrafine Aluminum Hydroxide Particles with or without Mesoporous MCM-41 in Ambient Environments

An aqueous suspension of nanogibbsite was synthesized via the titration of aluminum aqua acid [Al(H2O)6]3+ with L-arginine to pH 4.6. Since the hydrolysis of aqueous aluminum salts is known to produce a wide array of products with a wide range of size distributions, a variety of state-of-the-art instruments (i.e., 27Al/1H NMR, FTIR, ICP-OES, TEM-EDX, XPS, XRD, and BET) were used to characterize the synthesis products and identification of byproducts. The product, which was comprised of nanoparticles (10-30 nm), was isolated using gel permeation chromatography (GPC) column technique. Fourier transform infrared (FTIR) spectroscopy and powder X-ray diffraction (PXRD) identified the purified material as the gibbsite polymorph of aluminum hydroxide. The addition of inorganic salts (e.g., NaCl) induced electrostatic destabilization of the suspension, thereby agglomerating the nanoparticles to yield Al(OH)3 precipitate with large particle sizes. By utilizing the novel synthetic method described here, Al(OH)3 was partially loaded inside the highly ordered mesoporous framework of MCM-41, with average pore dimensions of 2.7 nm, producing an aluminosilicate material with both octahedral and tetrahedral Al (Oh/Td = 1.4). The total Al content, measured using energy-dispersive X-ray spectrometry (EDX), was 11% w/w with a Si/Al molar ratio of 2.9. A comparison of bulk EDX with surface X-ray photoelectron spectroscopy (XPS) elemental analysis provided insight into the distribution of Al within the aluminosilicate material. Furthermore, a higher ratio of Si/Al was observed on the external surface (3.6) as compared to the bulk (2.9). Approximations of O/Al ratios suggest a higher concentration of Al(O)3 and Al(O)4 groups near the core and external surface, respectively. The newly developed synthesis of Al-MCM-41 yields a relatively high Al content while maintaining the integrity of the ordered silica framework and can be used for applications where hydrated or anhydrous Al2O3 nanoparticles are advantageous.

An ultrafine aluminum hydroxide nanoparticle suspension was prepared via the controlled titration of [Al(H2O)]3+ with L-arginine to pH 4.6 with and without cage-effect confinement within mesoporous channels of MCM-41.

An aqueous suspension of nanogibbsite was synthesized via the titration of aluminum aqua acid [Al(H2O)6]3+ with L-arginine to pH 4.6. Since the hydrolysis ...

Facet-to-facet Linking of Shape-anisotropic Colloidal Cadmium Chalcogenide Nanostructures

Here, we describe a protocol that allows for shape-anisotropic cadmium chalcogenide nanocrystals (NCs), such as nanorods (NRs) and tetrapods (TPs), to be covalently and site-specifically linked via their end facets, resulting in polymer-like linear or branched chains. The linking procedure begins with a cation-exchange process in which the end facets of the cadmium chalcogenide NCs are first converted to silver chalcogenide. This is followed by the selective removal of ligands at their surface. This results in cadmium chalcogenide NCs with highly reactive silver chalcogenide end facets that spontaneously fuse upon contact with each other, thereby establishing an interparticle facet-to-facet attachment. Through the judicious choice of precursor concentrations, an extensive network of linked NCs can be produced. Structural characterization of the linked NCs is carried out via low- and high-resolution transmission electron microscopy (TEM), as well as energy-dispersive X-ray spectroscopy, which confirm the presence of silver chalcogenide domains between chains of cadmium chalcogenide NCs.

A protocol detailing how shape-anisotropic colloidal cadmium chalcogenide nanocrystals can be covalently linked via their end facets is presented here.

Here, we describe a protocol that allows for shape-anisotropic cadmium chalcogenide nanocrystals (NCs), such as nanorods (NRs) and tetrapods (TPs), to be ...

Phase Behavior of Charged Vesicles Under Symmetric and Asymmetric Solution Conditions Monitored with Fluorescence Microscopy

Phase-separated giant unilamellar vesicles (GUVs) exhibiting coexisting liquid-ordered and liquid-disordered domains are a common biophysical tool to investigate the lipid raft hypothesis. Numerous studies, however, neglect the impact of physiological solution conditions. On that account, the current work presents the effect of high-salinity buffer and trans-membrane solution asymmetry on liquid-liquid phase separation in charged GUVs grown from dioleylphosphatidylglycerol, egg sphingomyelin, and cholesterol. The effects were studied under isothermal and varying temperature conditions.
We describe equipment and experimental strategies applicable for monitoring the stability of coexisting liquid domains in charged vesicles under symmetric and asymmetric high-salinity solution conditions. This includes an approach to prepare charged multicomponent GUVs in high-salinity buffer at high temperatures. The protocol entails the option to perform a partial exchange of the external solution by a simple dilution step while minimizing the vesicle dilution. An alternative approach is presented utilizing a microfluidic device that allows for a complete external solution exchange. The solution effects on phase separation were also studied under varying temperatures. To this end, we present the basic design and utility of an in-house built temperature control chamber. Furthermore, we reflect on the assessment of the GUV phase state, pitfalls associated with it and how to circumvent them.

Experiments on phase separated giant unilamellar vesicles (GUVs) frequently neglect physiological solution conditions. This work presents approaches to study the effect of high-salinity buffer on liquid-liquid phase separation in charged multicomponent GUVs as a function of trans-membrane solution asymmetry and temperature.

Phase-separated giant unilamellar vesicles (GUVs) exhibiting coexisting liquid-ordered and liquid-disordered domains are a common biophysical tool to ...

Synthesis of Core-shell Lanthanide-doped Upconversion Nanocrystals for Cellular Applications

Lanthanide-doped upconversion nanocrystals (UCNs) have attracted much attention in recent years based on their promising and controllable optical properties, which allow for the absorption of near-infrared (NIR) light and can subsequently convert it into multiplexed emissions that span over a broad range of regions from the UV to the visible to the NIR. This article presents detailed experimental procedures for high-temperature co-precipitation synthesis of core-shell UCNs that incorporate different lanthanide ions into nanocrystals for efficiently converting deep-tissue penetrable NIR excitation (808 nm) into a strong blue emission at 480 nm. By controlling the surface modification with biocompatible polymer (polyacrylic acid, PAA), the as-prepared UCNs acquires great solubility in buffer solutions. The hydrophilic nanocrystals are further functionalized with specific ligands (dibenzyl cyclooctyne, DBCO) for localization on the cell membrane. Upon NIR light (808 nm) irradiation, the upconverted blue emission can effectively activate the light-gated channel protein on the cell membrane and specifically regulate the cation (e.g., Ca2+) influx in the cytoplasm. This protocol provides a feasible methodology for the synthesis of core-shell lanthanide-doped UCNs and subsequent biocompatible surface modification for further cellular applications.

A protocol is presented for the synthesis of core-shell lanthanide-doped upconversion nanocrystals (UCNs) and their cellular applications for channel protein regulation upon near-infrared (NIR) light illumination.

Lanthanide-doped upconversion nanocrystals (UCNs) have attracted much attention in recent years based on their promising and controllable optical ...

Cercosporin-Photocatalyzed [4+1]- and [4+2]-Annulations of Azoalkenes Under Mild Conditions

The interest on nitrogen-containing heterocycles has expanded rapidly in the synthetic community since they are important motifs for new drugs. Traditionally, they were synthesized through thermal cycloaddition reactions, whereas today, photocatalysis is preferred due to the mild and efficient conditions. With this focus, a new photocatalytic method for the synthesis of nitrogen-containing heterocycles is highly desired. Here, we report a protocol for the biosynthesis of cercosporin, which could function as a metal-free photocatalyst. We then illustrate cercosporin-photocatalyzed protocols for the synthesis of nitrogen-containing heterocycles 1,2,3-thiadiazoles through annulation of azoalkenes with KSCN, and synthesis of 1,4,5,6-tetrahydropyridazines [4+2] through cyclodimerization of azoalkenes under mild conditions, respectively. As a result, there is a new bridge between the microbial fermentation method and organic synthesis in a mild, cost-effective, environmentally friendly and sustainable manner.

New routes for the synthesis of nitrogen-containing heterocycles utilizing cercosporin as a metal-free photocatalyst were developed.

The interest on nitrogen-containing heterocycles has expanded rapidly in the synthetic community since they are important motifs for new drugs. ...

A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and chemical research. To meet the demand for high-performance microreactors, we developed a femtoliter droplet array (FemDA) device and exemplified its application in massively parallel cell-free protein synthesis (CFPS) reactions. Over one million uniform droplets were readily generated within a finger-sized area using a two-step oil-sealing protocol. Every droplet was anchored in a femtoliter microchamber composed of a hydrophilic bottom and a hydrophobic sidewall. The hybrid hydrophilic-in-hydrophobic structure and the dedicated sealing oils and surfactants are crucial for stably retaining the femtoliter aqueous solution in the femtoliter space without evaporation loss. The femtoliter configuration and the simple structure of the FemDA device allowed minimal reagent consumption. The uniform dimension of the droplet reactors made large-scale quantitative and time-course measurements convincing and reliable. The FemDA technology correlated the protein yield of the CFPS reaction with the number of DNA molecules in each droplet. We streamlined the procedures about the microfabrication of the device, the formation of the femtoliter droplets, and the acquisition and analysis of the microscopic image data. The detailed protocol with the optimized low running cost makes the FemDA technology accessible to everyone who has standard cleanroom facilities and a conventional fluorescence microscope in their own place.

The overall goal of the protocol is to prepare over one million ordered, uniform, stable, and biocompatible femtoliter droplets on a 1 cm2 planar substrate that can be used for cell-free protein synthesis.

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and ...

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.

In this protocol, doxorubicin-loaded AS1411-g-PEI-g-PEG modified gold nanoparticles are synthesized via three-step amide reactions. Then, doxorubicin is loaded and delivered to target cancer cells for cancer therapy.

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the ...