Name: anionic polymerization of an amphiphilic copolymer for preparation of block copolymer micelles stabilized by &#960;-&#960; stacking interactions
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Description: Watch this Scientific Journal Video about Anionic Polymerization of an Amphiphilic Copolymer for Preparation of Block Copolymer Micelles Stabilized by Ï€-Ï€ Stacking Interactions at JoVE.com

CA acknowledges a Discovery grant from the Natural Sciences and Engineering Research Council of Canada. CA acknowledges a Chair in Pharmaceutics and Drug Delivery from GSK. The authors declare no competing financial interest.

<img alt="Figure 1" src="/files/ftp_upload/54422/54422fig1.jpg" /> 
Figure 1. Schematic showing the nine key steps in the preparation of the mPEG-b-PPheGE copolymer. <a href="http://cloudfront.jove.com/files/ftp_upload/54422/54422fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Preparation of the Reagents under Dry Conditions
<ol>
	<li>Preparation of the reagents.
	<ol>
		<li>Weigh 15 g of mPEG-5K...

<ol>
	<li>Dickerson, T. J., Reed, N. N., Janda, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+Polymers+as+Scaffolds+for+Recoverable+Catalysts+and+Reagents.">Soluble Polymers as Scaffolds for Recoverable Catalysts and Reagents.</a> Chemical Reviews. 102, 3325-3344 (2002).</li><li>van Heerbeek, R., Kamer, P. C. J., van Leeuwen, P. W. N. M., Reek, J. N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendrimers+as+Support+for+Recoverable+Catalysts+and+Reagents.">Dendrimers as Support for Recoverable Catalysts and Reagents.</a> Chemical Reviews. 102 (10), 3717-3756 (2002).</li><li>Knop, K., Hoogenboom, R., Fischer, D., Schubert, U. 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G., Chaibundit, C., Ricardo, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Block+copolymers+for+drug+solubilisation:+Relative+hydrophobicities+of+polyether+and+polyester+micelle-core-forming+blocks.">Block copolymers for drug solubilisation: Relative hydrophobicities of polyether and polyester micelle-core-forming blocks.</a> International Journal of Pharmaceutics. 345 (1-2), 35-41 (2007).</li><li>Matsumura, Y., Kataoka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+and+clinical+studies+of+anticancer+agent-incorporating+polymer+micelles.">Preclinical and clinical studies of anticancer agent-incorporating polymer micelles.</a> Cancer Science. 100 (4), 572-579 (2009).</li><li>Chan, A. S., Chen, C. H., Huang, C. M., Hsieh, M. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerization+of+ethylene+oxide+by+alkali+metal-naphthalene+complexes+in+tetrahydrofuran.">Polymerization of ethylene oxide by alkali metal-naphthalene complexes in tetrahydrofuran.</a> European Polymer Journal. 7 (10), 1421-1433 (1971).</li><li>Crothers, M., 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=Micellization+and+Gelation+of+Diblock+Copolymers+of+Ethylene+Oxide+and+Styrene+Oxide+in+Aqueous+Solution.">Micellization and Gelation of Diblock Copolymers of Ethylene Oxide and Styrene Oxide in Aqueous Solution.</a> Langmuir. 18 (22), 8685-8691 (2002).</li><li>Taboada, P., 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=Block+Copolymers+of+Ethylene+Oxide+and+Phenyl+Glycidyl+Ether:+Micellization,+Gelation,+and+Drug+Solubilization.">Block Copolymers of Ethylene Oxide and Phenyl Glycidyl Ether: Micellization, Gelation, and Drug Solubilization.</a> Langmuir. 21 (12), 5263-5271 (2005).</li><li>Taboada, P., 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=Micellization+and+Drug+Solubilization+in+Aqueous+Solutions+of+a+Diblock+Copolymer+of+Ethylene+Oxide+and+Phenyl+Glycidyl+Ether.">Micellization and Drug Solubilization in Aqueous Solutions of a Diblock Copolymer of Ethylene Oxide and Phenyl Glycidyl Ether.</a> Langmuir. 22 (18), 7465-7470 (2006).</li><li>Attwood, D., Booth, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Colloid Stability. , 61-78 (2010).</li><li>Le Devedec, F., 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=Postalkylation+of+a+Common+mPEG-b-PAGE+Precursor+to+Produce+Tunable+Morphologies+of+Spheres,+Filomicelles,+Disks,+and+Polymersomes.">Postalkylation of a Common mPEG-b-PAGE Precursor to Produce Tunable Morphologies of Spheres, Filomicelles, Disks, and Polymersomes.</a> ACS Macro Letters. 5 (1), 128-133 (2016).</li><li>Chtryt, V., Ulbrich, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugate+of+Doxorubicin+with+a+Thermosensitive+Polymer+Drug+Carrier.">Conjugate of Doxorubicin with a Thermosensitive Polymer Drug Carrier.</a> Journal of Bioactive and Compatible Polymers. 16 (6), 427-440 (2001).</li><li>Kataoka, K., 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=Doxorubicin-loaded+poly(ethylene+glycol)-poly(&#946;-benzyl-l-aspartate)+copolymer+micelles:+their+pharmaceutical+characteristics+and+biological+significance.">Doxorubicin-loaded poly(ethylene glycol)-poly(&#946;-benzyl-l-aspartate) copolymer micelles: their pharmaceutical characteristics and biological significance.</a> Journal of Controlled Release. 64 (1-3), 143-153 (2000).</li><li>Cammas, S., Matsumoto, T., Okano, T., Sakurai, Y., Kataoka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+functional+polymeric+micelles+as+site-specific+drug+vehicles+based+on+poly+(&#945;-hydroxy+ethylene+oxide-co-&#946;-benzyl+l-aspartate)+block+copolymers.">Design of functional polymeric micelles as site-specific drug vehicles based on poly (&#945;-hydroxy ethylene oxide-co-&#946;-benzyl l-aspartate) block copolymers.</a> Materials Science and Engineering: C. 4 (4), 241-247 (1997).</li><li>Lv, S., 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=Doxorubicin-loaded+amphiphilic+polypeptide-based+nanoparticles+as+an+efficient+drug+delivery+system+for+cancer+therapy.">Doxorubicin-loaded amphiphilic polypeptide-based nanoparticles as an efficient drug delivery system for cancer therapy.</a> Acta Biomaterialia. 9 (12), 9330-9342 (2013).</li><li>Kim, J. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-porating+polymersomes+of+PEG-PLA+and+PEG-PCL:+hydrolysis-triggered+controlled+release+vesicles.">Self-porating polymersomes of PEG-PLA and PEG-PCL: hydrolysis-triggered controlled release vesicles.</a> Journal of Controlled Release. 96 (1), 37-53 (2004).</li><li>Uhrig, D., Mays, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+techniques+in+high-vacuum+anionic+polymerization.">Experimental techniques in high-vacuum anionic polymerization.</a> Journal of Polymer Science Part A: Polymer Chemistry. 43 (24), 6179-6222 (2005).</li><li>Parker, A. 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Due to the good control that anionic polymerization provides over molecular weight it is one of the most applied processes in the industry for the preparation of polymers based on oxirane monomers (PEG and PPG). Optimal and stringent conditions must be used for successful polymerization to be achieved. Rigorous purification of all reagents and appropriate apparatus are essential for the living character of the synthesis. Limitations of the current setup are mostly associated with the transfer technique that relies on can...

In aqueous media, amphiphilic block copolymers assemble to form nano-sized block copolymer micelles (BCMs) that consist of a hydrophobic core surrounded by a hydrophilic shell or corona. The micelle core can serve as a reservoir for the incorporation of hydrophobic drugs; while, the hydrophilic corona provides an interface between the core and the external medium. Poly(ethylene glycol) (PEG) and its derivatives are one of the most important classes of polymers and one of the most widely used in drug formulation.1-3 BCMs have proven to be a worthy drug delivery platform with several formulations relying on this technology now in late stage clinical development.4 Most commonly, the hydrophobic block of the copolymer is comprised of polycaprolactone, poly(D,L-lactide), poly(propylene oxide) or poly(&#946;-benzyl-L-aspartate).5-9
Kataoka&#39;s group investigated spherical micelles formed from PEO-b-PBLA and poly(ethylene oxide)-b-(polyaspartic acid-conjugated doxorubicin) for delivery of doxorubicin (DOX).10,11 In their reports, they put forward that &#960;-&#960; interactions between the polymer-conjugated drug or PBLA and free DOX act to stabilize the micelle core resulting in increases in drug loading and retention. It is established that compatibility or interactions between a drug and the core-forming block are determinants of key performance related parameters.12 In addition to DOX, a number of cancer therapeutics include aromatic rings within their core structure (e.g., methotrexate, olaparib, SN-38).
As a result there is significant interest in synthesis of copolymers that include benzyl rings in their core-forming blocks. Anionic ring-opening polymerization of PEG and its derivatives enable control over molecular weight and result in materials of low polydispersity in good yield.13,14 Ethylene oxide with phenyl glycidyl ether (PheGE) or styrene oxide (SO) can be (co)polymerized to form block copolymers that form micelles for solubilization of hydrophobic drugs.15-18 The current report describes the necessary steps for living anionic polymerization of phenyl glycidyl ether monomer on mPEG-OH as macroinitiator (Figure 1). The resulting block copolymer and its aggregates are then characterized in terms of properties of relevance to use in drug delivery.

<table border="0" cellpadding="0" cellspacing="0" width="742">
	<colgroup>
		<col span="2" />
		<col />
		<col span="2" />
		<col />
		<col span="2" />
		<col />
	</colgroup>
	<tbody>
		<tr height="62">
			<td colspan="3" dir="LTR" height="62" style="height:62px;width:224px;">DMEM/HAMF12</td>
			<td colspan="3" style="width:227px;">Gibco, Life Technologies</td>
			<td colspan="2" style="width:125px;">12500</td>
			<td dir="LTR" style="width:171px;">Supplemented with 10%FBS. Warm in 37 &#176;C water bath 
			&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr height="20">
			<td colspan="3" height="20" style="height:20px;">Trypsin-EDTA(0.25%)</td>
			<td colspan="3">Sigma-Aldrich</td>
			<td colspan="2">T4049</td>
			<td dir="LTR">Warm in 37 &#176;C water bath&#160;</td>
		</tr>
		<tr height="20">
			<td colspan="3" dir="LTR" height="20" style="height:20px;">Fetal bovine serum (FBS)</td>
			<td colspan="3">Sigma-Aldrich</td>
			<td colspan="2">F1051</td>
			<td dir="LTR">Canada origin</td>
		</tr>
		<tr height="20">
			<td colspan="3" dir="LTR" height="20" style="height:20px;">MDA-MB-468 cell line</td>
			<td colspan="3">ATCC</td>
			<td colspan="2">HTB-132</td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="3" dir="LTR" height="20" style="height:20px;">MTS tetrazolium reagent</td>
			<td colspan="3">PROMEGA</td>
			<td colspan="2">G111B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="3" height="20" style="height:20px;">Phenazine ethosulfate (PES)</td>
			<td colspan="3">Sigma-Aldrich</td>
			<td colspan="2">P4544</td>
			<td dir="LTR">&#62;95%</td>
		</tr>
		<tr height="20">
			<td colspan="3" dir="LTR" height="20" style="height:20px;">mPEG5K (Mn 5400 g/mol)</td>
			<td colspan="3">Sigma-Aldrich</td>
			<td colspan="2">81323</td>
			<td>PDI=1.02</td>
		</tr>
		<tr height="20">
			<td colspan="3" dir="LTR" height="20" style="height:20px;">Dimethylsolfoxide (DMSO)</td>
			<td colspan="3">Sigma-Aldrich</td>
			<td colspan="2">D4540</td>
			<td dir="LTR">&#62;99.5%</td>
		</tr>
		<tr height="20">
			<td colspan="3" dir="LTR" height="20" style="height:20px;">Naphthalene</td>
			<td colspan="3">Sigma-Aldrich</td>
			<td colspan="2">147141</td>
			<td dir="LTR">&#62;99%</td>
		</tr>
		<tr height="20">
			<td colspan="3" dir="LTR" height="20" style="height:20px;">Phenyl glycidyl ether</td>
			<td colspan="3">Sigma-Aldrich</td>
			<td colspan="2">A32608</td>
			<td dir="LTR">&#62;85%</td>
		</tr>
		<tr height="20">
			<td colspan="3" dir="LTR" height="20" style="height:20px;">Benzophenone</td>
			<td colspan="3">Sigma-Aldrich</td>
			<td colspan="2">427551</td>
			<td dir="LTR">&#62;99%</td>
		</tr>
		<tr height="20">
			<td colspan="3" dir="LTR" height="20" style="height:20px;">Potassium</td>
			<td colspan="3">Sigma-Aldrich</td>
			<td colspan="2">451096</td>
			<td dir="LTR">&#62;98%</td>
		</tr>
		<tr height="20">
			<td colspan="3" dir="LTR" height="20" style="height:20px;">Tetrahydrofuran</td>
			<td colspan="3">Caledon Laboratory Chemicals</td>
			<td colspan="2">8900 1</td>
			<td dir="LTR">ACS</td>
		</tr>
		<tr height="20">
			<td colspan="3" dir="LTR" height="20" style="height:20px;">Hexane</td>
			<td colspan="3">Caledon Laboratory Chemicals</td>
			<td colspan="2">5500 1</td>
			<td dir="LTR">ACS</td>
		</tr>
		<tr height="20">
			<td colspan="3" dir="LTR" height="20" style="height:20px;">Calcium hydride (CaH2)</td>
			<td colspan="3">ACP</td>
			<td colspan="2">C-0460</td>
			<td dir="LTR">&#62;99.5%</td>
		</tr>
		<tr height="20">
			<td colspan="3" dir="LTR" height="20" style="height:20px;">Diethyl Ether</td>
			<td colspan="3">Caledon Laboratory Chemicals</td>
			<td colspan="2">1/10/4800</td>
			<td dir="LTR">ACS</td>
		</tr>
		<tr height="20">
			<td colspan="3" dir="LTR" height="20" style="height:20px;">Microplate reader</td>
			<td colspan="3">BioTek Instruments</td>
			<td colspan="2"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="3" dir="LTR" height="20" style="height:20px;">Differential scanning calorimetry (DSC)</td>
			<td colspan="3">TA Instruments Inc</td>
			<td colspan="2">DSC Q100</td>
			<td></td>
		</tr>
		<tr height="32">
			<td colspan="3" dir="LTR" height="32" style="height:32px;">Gel permeation chromatography (GPC)</td>
			<td colspan="3">Waters</td>
			<td colspan="2" style="width:125px;">2695 separation moldule / 2414 detector&#160;</td>
			<td dir="LTR" style="width:171px;">2 Columns: Agilent Plgel 5&#181;m Mixed-D</td>
		</tr>
		<tr height="20">
			<td colspan="3" dir="LTR" height="20" style="height:20px;">NMR spectroscopy</td>
			<td colspan="3">Varian Mercury 400MHz</td>
			<td colspan="2"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="3" dir="LTR" height="20" style="height:20px;">Chloroform-d</td>
			<td colspan="3">Sigma-Aldrich</td>
			<td colspan="2">151858</td>
			<td dir="LTR">99.96%</td>
		</tr>
		<tr height="20">
			<td colspan="3" dir="LTR" height="20" style="height:20px;">DMSO-d</td>
			<td colspan="3">Sigma-Aldrich</td>
			<td colspan="2">156914</td>
			<td dir="LTR">99.96%</td>
		</tr>
		<tr height="20">
			<td colspan="3" height="20" style="height:20px;">Vaccum pump&#160;</td>
			<td colspan="3">Gardner Denver Welch Vacuum Tech, Inc.</td>
			<td colspan="2"></td>
			<td>Ultimate&#160; pressure 1.10-4 torr</td>
		</tr>
		<tr height="20">
			<td colspan="3" height="20" style="height:20px;">Drierit with indicator, 8 mesh</td>
			<td colspan="3">Sigma-Aldrich</td>
			<td colspan="2">238988</td>
			<td>Regenerated at 230&#176;C for 2 hrs</td>
		</tr>
	</tbody>
</table>

anionic polymerization of an amphiphilic copolymer for preparation of block copolymer micelles stabilized by &#960;-&#960; stacking interactions

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE). Characterization of the copolymer revealed a narrow molecular distribution (PDI &#60; 1.03) and confirmed the degree of polymerization of mPEG122-b-(PheGE)15. The critical micelle concentration of the copolymer was evaluated using an established fluorescence method with the aggregation behavior evaluated by dynamic light scattering and transmission electronic microscopy. The potential of the copolymer for use in drug delivery applications was evaluated in a preliminary manner including in vitro biocompatibility, loading and release of the hydrophobic anti-cancer drug doxorubicin (DOX). A stable micelle formulation of DOX was prepared with drug loading levels up to 14% (wt%), drug loading efficiencies &#62; 60% (w/w) and sustained release of drug over 4 days under physiologically relevant conditions (acidic and neutral pH, presence of albumin). The high drug loading level and sustained release is attributed to stabilizing &#960;-&#960; interactions between DOX and the core-forming block of the micelles.

<img alt="Figure 3" src="/files/ftp_upload/54422/54422fig3.jpg" /> 
Figure 3. Illustration of the anionic polymerization of phenyl glycidyl ether on mPEG macroinitiator to produce mPEG-b-(PheGE)15 for preparation of block copolymer micelles for loading of doxorubicin. The schematic illustrates the deprotonation of the hydroxyl group of mPEG using naphthalene potassium as a radical-anion, followed...

Watch this Scientific Journal Video about Anionic Polymerization of an Amphiphilic Copolymer for Preparation of Block Copolymer Micelles Stabilized by Ï€-Ï€ Stacking Interactions at JoVE.com

Anionic Polymerization of an Amphiphilic Copolymer for Preparation of Block Copolymer Micelles Stabilized by &#960;-&#960; Stacking Interactions

The key steps of living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE) are described. The resulting block copolymer micelles (BCMs) were loaded with doxorubicin 14% (wt%) and sustained release of drug over 4 days under physiologically relevant conditions was obtained.

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl ...

The overall goal of this work is to demonstrate the critical steps needed for synthesis of block copolymer materials by ring opening anionic polymerization, and to show the necessary flow of studies for block copolymer micelles formulation of a hydrophobic drug. This protocol demonstrates the proper execution of necessary steps and procedures for the synthesis of block copolymer by anionic polymerization. The big advantage of ring opening an anionic polymerization is the synthesis of materials in good yield, and low pollute dispersity.As well as the scalability of the process. This is an exciting time for polymeric drug delivery. With a number of drugs relying on formulation and block copolymer micelles, now in late stage pre-clinical, or clinical development.Prepare reagents and the materials for the transfer as described in the text protocol. Weigh methoxypolyethylene glycol with a molecular weight of 5, 000. Add the mPEG into a dried flask containing a stir bar, and seal the flushing adapter with a septum at the top.Connect the flask to the manifold, and perch the flask for two to three minutes with Argon. Turn the valve to the vacuum position to perch the flask. Rotate the flask manually, and dry the reaction vessel homogeneously, with a blowdryer or heat gun, until the mPEG melts.After one minute, break the vacuum by turning the valve on the manifold towards the Argon position with several quick snaps to cool down the flask. Keep the polymeric macroinitiator under vacuum for approximately two hours, and then under Argon before the reaction begins. Next, mount two high vacuum distillation apparatuses which have been dried previously in an oven under the hood.One apparatus is for the distillation of DMSO, and one apparatus is for the distillation of the monomer, phenyl glycerol ether. Connect the separate flasks to the two apparatuses, and install each on a hemispherical heating mantel, or in an oil bath. Connect cold water to the top of each apparatus, and to the manifold.Ensure that each apparatus is secure and well sealed. Engage the vacuum via the valve. Set the heating via a temperature controller, and start stirring the solutions.After two hours of circulation distillation of DMSO, close the high vacuum valve to collect approximately 20 milliliters of solution. Then, release the fraction into the flask, and repeat the operation once more, to ensure the purity of the desired fraction that is collected later. Heat the flask containing the mPEG under vacuum with the heat gun until the polymer melts, and then perch again with Argon.After two hours, close the high vacuum valve, and collect the volume of solvent. Stop heating, and break the vacuum from the manifold. Release Argon into the chamber, as described previously.Under a positive pressure of Argon, connect one side of the canula to a graduated cylinder, or directly to the flask containing the mPEG, also under Argon, and immerse the other end carefully into the freshly distilled fraction. Connect an extra bubbler to the flask, close off the Argon flow, and use only the Argon pressure from the distillation apparatus. To avoid any accidents caused by Argon pressure, open the glass stopcock for one to two seconds, and reclose to continue the flow of DMSO until the full transfer is completed.Next, under Argon, connect one side of the canula from the dried graduated cylinder, sealed by a septum, to a stock solution of 0.3 molar naphthalene potassium. Then, add an extra bubbler to the graduated cylinder, close the Argon flow to the cylinder, and under positive Argon pressure, transfer five milliliters of 0.3 molar naphthalene potassium via canulation from the stock solution, to the graduated cylinder. Insert another needle from the manifold into the cylinder, and remove the extra bubbler.Remove the canula connected to the stock solution, carefully, insert it rapidly into the reaction flask, and connect an extra bubbler. Next, as described previously, add the base drop by drop until the solution becomes dark. Following the slow disappearance of color, add another portion until the dark color appears again, and repeat until the full transfer.As described previously, transfer the desired volume of monomer to the reaction to reach a degree of polymerization of polyphenol glycol ether of about 18 to 20 units. Leave the reaction for 48 hours at 80 degrees celsius, under Argon atmosphere with constant stirring, to ensure complete polymerization. Quench the reaction by the addition of drops of one normal hydrochloric acid and methanol, as observed by a color disappearance.Extract the naphthalene from the DMSO solution with hexane. Remove approximately 70 milliliters of the DMSO by distillation under vacuum. Cool down the slurry solution, and add 15 milliliters of THF.Remove the salt from the slurry solution by centrifugation at 5, 000 times g for ten minutes. Transfer the supernatant, and add a dropwise to 500 milliliters of cold diethyl ether. Collect the precipitate by filtration, or centrifugation.After repeating the precipitation twice, dry the precipitate at room temperature under the hood, overnight, and then under vacuum at 30 degrees celsius for 24 to 48 hours, for a yield of 85%The copolymer is now ready for characterization, as described in the text protocol. Dissolve 12 milligrams of doxorubicin in one milliliter of acetonitrile. Add 10 microliters of triethylamine, and let the solution stir in the dark for two hours.Dissolve 45 milligrams of copolymer in one milliliter of THF, and stir for the same period of time. Add the copolymer solution to the doxorubicin solution, and rinse the vile containing residual copolymer with an extra volume of THF. Next, add the copolymer-drug mixture dropwise to a 20 milliliter vile containing 15 milliliters of 0.9%saline with stirring.Transfer the solution to a dialysis bag, and dialyze against 500 milliliters of 0.9%saline. Transfer the dialysate to a 15 milliliter tube and centrifuge at 5, 000 times g, for 15 minutes. Then, transfer the supernatant to an ultrafiltration system that contains a dialysis membrane.Put the stirring adapter into the ultrafiltration system, close the lid, and open to a stream of nitrogen. Concentrate the block copolymer micelles solution to a volume of four milliliters, and add six milliliters of fresh saline. Then, concentrate the block copolymer micelles solution to 4 milliliters again, rinse the chamber with 0.5 milliliters of saline, and add it to the solution.Store the resulting solution in brown viles at room temperature in the dark, prior to further use. The ring opening anionic polymerization of phenyl glycerol ether on the mPEG macroinitiator was used to prepare the amphiphilic diblock copolymer by the deep protonation of the hydroxyl group of mPEG. Using naphthalene potassium as a radical anion, followed by the polymerization of the phenyl glycerol ether monomer.The characterization of the block copolymer by gel permeation chromatography, and proton NMR spectroscopy analysis confirmed a narrow molecular weight distribution with a degree of polymerization of phenyl glycerol ether of 15 units. In acquis media, the amphiphilic block copolymers assemble to form micelles that consist of a hydrophobic core surrounded by a hydrophilic shell. Transmission electron microscopy confirmed a spherical morphology and dynamic light scattering indicated a hydrodynamic diameter of 25 nano meters.Doxorubicin was successfully encapsulated in the block copolymer micelles. Compared to the free protonated drug in solution, self-quenching of the doxorubicin, observed by fluorescence spectroscopy, confirmed the incorporation of the drug in the polymeric micelles, with a drug loading capacity of up to 14%The release profile of doxorubicin, from the block copolymer micelles, indicated good stability for this formulation at neutral pH. And sustained release of the drug over four days under physiologically relevant conditions.Once mastered, this technique can help trainees in pharmaceutical sciences to prepare the synthesis diblock copolymers for application in drug delivery. This procedure described herein, can be applied for synthesis of a wide range of copolymers with PEG as the initiator. Here emphasis has been placed on assuring good laboratory practices, that provide anhydrous conditions, and result in high quality materials.After watching this video, I should have a good understanding of how to work under anhydrous conditions and verse should be able to apply these steps for the synthesis of PEG polyester or PEG polyester materials. In pursuing any of the described research, it is essential to remember that you are working under controlled conditions, with potential hazardous organic solvents, and other agents. Furthermore, all drugs are toxic, and must be handled using necessary precautions.Such as, wearing gloves, a lab coat, close-toed shoes, as well as wearing a mask when you want to weigh the drugs.

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Unit 1

Synthetic Polymers

Ethers, Epoxides, Sulfides

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Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

Gyroid Nickel Nanostructures from Diblock Copolymer Supramolecules

Nanoporous metal foams possess a unique combination of properties - they are catalytically active, thermally and electrically conductive, and furthermore, have high porosity, high surface-to-volume and strength-to-weight ratio. Unfortunately, common approaches for preparation of metallic nanostructures render materials with highly disordered architecture, which might have an adverse effect on their mechanical properties. Block copolymers have the ability to self-assemble into ordered nanostructures and can be applied as templates for the preparation of well-ordered metal nanofoams. Here we describe the application of a block copolymer-based supramolecular complex - polystyrene-block-poly(4-vinylpyridine)(pentadecylphenol) PS-b-P4VP(PDP) - as a precursor for well-ordered nickel nanofoam. The supramolecular complexes exhibit a phase behavior similar to conventional block copolymers and can self-assemble into the bicontinuous gyroid morphology with two PS networks placed in a P4VP(PDP) matrix. PDP can be dissolved in ethanol leading to the formation of a porous structure that can be backfilled with metal. Using electroless plating technique, nickel can be inserted into the template&#39;s channels. Finally, the remaining polymer can be removed via pyrolysis from the polymer/inorganic nanohybrid resulting in nanoporous nickel foam with inverse gyroid morphology.

This article describes the preparation of well-ordered nickel nanofoams via electroless metal deposition onto nanoporous templates obtained from self-assembled diblock copolymer based supramolecules.

Nanoporous metal foams possess a unique combination of properties - they are catalytically active, thermally and electrically conductive, and furthermore, ...

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.

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 ...

Using Polystyrene-block-poly(acrylic acid)-coated Metal Nanoparticles as Monomers for Their Homo- and Co-polymerization

We present a template-free method for &#8220;polymerizing&#8221; nanoparticles into long chains without side branches. A variety of nanoparticles are encapsulated in polystyrene-block-poly(acrylic acid) (PSPAA) shells and then used as monomers for their self-assembly. Spherical PSPAA micelles upon acid treatment are known to assemble into cylindrical micelles. Exploiting this tendency, the core-shell nanoparticles are induced to aggregate, coalesce, and then transform into long chains. When more than one type of nanoparticles are used, random and block &#8220;copolymers&#8221; of nanoparticles can be obtained. Detailed procedures are reported for the PSPAA encapsulation of nanoparticles, homo- and co-polymerization of the core-shell nanoparticles, separation and purification of the resulting nanoparticle chains. Transformations of single-line chains into double- and triple-line chains are also presented. The synergy between the polymer shell and the embedded nanoparticles leads to an unusual chain-growth polymerization mode, giving long nanoparticle chains that are distinct from the products of the traditional step-growth aggregation process.

Using Polystyrene-block-polyacrylic acid-coated Metal Nanoparticles as Monomers for Their Homo- and Co-polymerization

We report protocols for &#8220;polymerizing&#8221; various types of polymer-encapsulated metal nanoparticles into long chains of &#8220;homo-&#8220; and &#8220;co-polymers&#8221;.

We present a template-free method for &#8220;polymerizing&#8221; nanoparticles into long chains without side branches. A variety of nanoparticles are ...

Preparation and Characterization of Individual and Multi-drug Loaded Physically Entrapped Polymeric Micelles

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar concentration forming hydrophobic cores surrounded by hydrophilic shells in aqueous environments. The core of these micelles can be utilized to load hydrophobic, poorly water soluble drugs like docetaxel (DTX) and everolimus (EVR). Systematic characterization of the micelle structure and drug loading capabilities are important before in vitro and in vivo studies can be conducted. The goal of the protocol described herein is to provide the necessary characterization steps to achieve standardized micellar products. DTX and EVR have intrinsic solubilities of 1.9 and 9.6 &#181;g/ml respectively Preparation of these micelles can be achieved through solvent casting which increases the aqueous solubility of DTX and EVR to 1.86 and 1.85 mg/ml, respectively. Drug stability in micelles evaluated at room temperature over 48 hr indicates that 97% or more of the drugs are retained in solution. Micelle size was assessed using dynamic light scattering and indicated that the size of these micelles was below 50 nm and depended on the molecular weight of the polymer. Drug release from the micelles was assessed using dialysis under sink conditions at pH 7.4 at 37 oC over 48 hr. Curve fitting results indicate that drug release is driven by a first order process indicating that it is diffusion driven.

The goal of this protocol is to describe the preparation and characterization of physically entrapped, poorly water soluble drugs in micellar drug delivery systems composed of amphiphilic block copolymers.

Amphiphilic block copolymers like polyethyleneglycol-block-polylactic acid (PEG-b-PLA) can self-assemble into micelles above their critical micellar ...

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

Functionalization of Single-walled Carbon Nanotubes with Thermo-reversible Block Copolymers and Characterization by Small-angle Neutron Scattering

We demonstrate a protocol for single-walled carbon nanotube functionalization using thermo-sensitive PEO-PPO-PEO triblock copolymers in an aqueous solution. In a carbon nanotube/PEO105-PPO70-PEO105 (poloxamer 407) aqueous solution, the amphiphilic poloxamer 407 adsorbs onto the carbon nanotube surfaces and self-assembles into continuous layers, driven by intermolecular interactions between constituent molecules. The addition of 5-methylsalicylic acid changes the self-assembled structure from spherical-micellar to a cylindrical morphology. The fabricated poloxamer 407/carbon nanotube hybrid particles exhibit thermo-responsive structural features so that the density and thickness of poloxamer 407 layers are also reversibly controllable by varying temperature. The detailed structural properties of the poloxamer 407/carbon nanotube particles in suspension can be characterized by small-angle neutron scattering experiments and model fit analyses. The distinct curve shapes of the scattering intensities depending on temperature control or addition of aromatic additives are well described by a modified core-shell cylinder model consisting of a carbon nanotube core cylinder, a hydrophobic shell, and a hydrated polymer layer. This method can provide a simple but efficient way for the fabrication and in-situ characterization of carbon nanotube-based nano particles with a structure-tunable encapsulation.

A method for the functionalization of carbon nanotubes with structure-tunable polymeric encapsulation layers and structural characterization using small-angle neutron scattering is presented.

We demonstrate a protocol for single-walled carbon nanotube functionalization using thermo-sensitive PEO-PPO-PEO triblock copolymers in an aqueous ...

Synthesis of Ligand-free CdS Nanoparticles within a Sulfur Copolymer Matrix

Aliphatic ligands are typically used during the synthesis of nanoparticles to help mediate their growth in addition to operating as high-temperature solvents. These coordinating ligands help solubilize and stabilize the nanoparticles while in solution, and can influence the resulting size and reactivity of the nanoparticles during their formation. Despite the ubiquity of using ligands during synthesis, the presence of aliphatic ligands on the nanoparticle surface can result in a number of problems during the end use of the nanoparticles, necessitating further ligand stripping or ligand exchange procedures. We have developed a way to synthesize cadmium sulfide (CdS) nanoparticles using a unique sulfur copolymer. This sulfur copolymer is primarily composed of elemental sulfur, which is a cheap and abundant material. The sulfur copolymer has the advantages of operating both as a high temperature solvent and as a sulfur source, which can react with a cadmium precursor during nanoparticle synthesis, resulting in the generation of ligand free CdS. During the reaction, only some of the copolymer is consumed to produce CdS, while the rest remains in the polymeric state, thereby producing a nanocomposite material. Once the reaction is finished, the copolymer stabilizes the nanoparticles within a solid polymeric matrix. The copolymer can then be removed before the nanoparticles are used, which produces nanoparticles that do not have organic coordinating ligands. This nascent synthesis technique presents a method to produce metal-sulfide nanoparticles for a wide variety of applications where the presence of organic ligands is not desired.

Herein we present a method to synthesize ligand-free cadmium sulfide (CdS) nanoparticles based on a unique sulfur copolymer. The sulfur copolymer operates as a high temperature solvent and a sulfur source during the nanoparticle synthesis and stabilizes the nanoparticles after the reaction.

Aliphatic ligands are typically used during the synthesis of nanoparticles to help mediate their growth in addition to operating as high-temperature ...

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 Photoredox Ring-Opening Polymerization of O-Carboxyanhydrides Mediated by Ni/Zn Complexes

Here, we describe an effective protocol that combines photoredox Ni/Ir catalysis with the use of a Zn-alkoxide for efficient ring-opening polymerization, allowing for the synthesis of isotactic poly(&#945;-hydroxy acids) with expected molecular weights (&#62;140 kDa) and narrow molecular weight distributions (Mw/Mn &#60; 1.1). This ring-opening polymerization is mediated by Ni and Zn complexes in the presence of an alcohol initiator and a photoredox Ir catalyst, irradiated by a blue LED (400 - 500 nm). The polymerization is performed at a low temperature (-15 &#176;C) to avoid undesired side reactions. The complete monomer consumption can be achieved within 4 - 8 hours, providing a polymer close to the expected molecular weight with narrow molecular weight distribution. The resulted number-average molecular weight shows a linear correlation with the degree of polymerization up to 1000. The homodecoupling 1H NMR study confirms that the obtained polymer is isotactic without epimerization. This polymerization reported herein offers a strategy for achieving rapid, controlled O-carboxyanhydrides polymerization to prepare stereoregular poly(&#945;-hydroxy acids) and its copolymers bearing various functional side-chain groups.

Controlled Photoredox Ring-Opening Polymerization of O-Carboxyanhydrides Mediated by Ni/Zn Complexes

A protocol for the controlled photoredox ring-opening polymerization of O-carboxyanhydrides mediated by Ni/Zn complexes is presented.

Here, we describe an effective protocol that combines photoredox Ni/Ir catalysis with the use of a Zn-alkoxide for efficient ring-opening polymerization, ...