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 (Mn=5,400 g/mol, PDI 1.03) and place at 50 &#176;C in an oven under vacuum for 48 hr prior to use.</li>
		<li>Dry 200 ml of dimethylsulfoxide (DMSO) over calcium hydride (CaH2) (~1 g), place under vacuum for 30 min, purge under argon and stir for 48 hr prior to use.</li>
		<li>Place 50 ml of the PheGE monomer in a dry and clean flask (100 ml), add 1 g of CaH2, seal under vacuum for 15 min on ice, purge under argon and leave to stir for 24 hr under argon prior to use.</li>
	</ol>
	</li>
</ol>
2. Preparation of the Potassium Naphthalene
<ol>
	<li>Carefully, cut small chunks of sodium (~ 1.5 g) dried with hexane to remove excess mineral oil and add to the round flask containing the tetrahydrofuran (THF) (v = 500 ml). 
	NOTE: The chunks of sodium must not be exposed to air for long due to risk of fire.</li>
	<li>Add benzophenone (~5 g), purge with argon and seal the round flask (2 necks) with glass stoppers.</li>
	<li>Following stirring under argon for 24 hr, connect the round-bottom flask to a distillation apparatus (Figure 2), distill the dark solution under argon while refluxing (i.e., reflux for about a 2 hr period after the solution turns blue). Begin to collect the desired volume ~ 150 ml of THF by closing the left valve (found in the middle of the distillation apparatus). 
	NOTE: If this solution does not turn blue, stop the distillation, cool down at room temperature (RT) and add more benzophenone or sodium and restart the distillation. This is an indication that THF still contains water.</li>
	<li>In a dry Erlenmeyer, add distilled THF (v=100 ml) and dissolve 3.9 g of naphthalene. 
	NOTE: Stop the distillation, cool down at RT and open the right valve to transfer the volume of THF.</li>
	<li>As described in point 2.1, cut small chunks of potassium (1.1 g) and add to the solution containing the naphthalene (final concentration ~0.3 mol/L). Seal the Erlenmeyer with a flushing adapter (T) (on/off) with a septum at the top and purge with argon.</li>
	<li>Following stirring under argon for 24 hr, observe the resulting solution of the potassium naphthalene base as a homogeneous dark green color.</li>
	<li>Under inert conditions, remove a 5 ml aliquot of the basic solution from the flask with a syringe and add to 10 ml of distilled water. Subsequently add 1-2 drops of phenolphthalein indicator to this solution, which turns the solution a fuchsia color.</li>
	<li>Use a burette to titrate the potassium naphthalene solution with a standard hydrochloric acid solution (0.1 N) until the solution turns colorless.</li>
</ol>
3. Materials and Necessary Precautions for Effective Living Anionic Polymerization
<ol>
	<li>System argon/vacuum manifold. 
	NOTE: As described in Figure 2, a double glass manifold with hollow glass stopcocks is used to switch between argon delivery and vacuum conditions in the glassware.
	<ol>
		<li>Connect the tank of argon (with manometer) to a dry desiccant column and to the manifold line using inert rubber tubing. At the other extremity of the argon line, connect a bubbler (containing mineral oil).</li>
		<li>To the glass stopcocks, connect flexible inert tubes and needles. To the other line of the manifold, connect a glass trap immersed in a cold Dewar flask (filled with ice/water or liquid nitrogen) to a high vacuum pump.</li>
	</ol>
	</li>
	<li>Apparatus for distillation of monomer and DMSO. 
	NOTE: A convenient (i.e., all in one) apparatus for high vacuum distillation is employed (Figure 2). The dry glassware is made with high vacuum valves, and built-in condensers with an inner refrigerated head.
	<ol>
		<li>Connect water flow through the inlet (A) and outlet (B) of the cooling unit (letter). Connect the other inlet/outlet (C) to the dual manifold for argon/vacuum. Add and seal a septum (metal wires) at the delivery/extraction port and connect a stainless steel cannula for the transfer of air sensitive liquids (D) (at the top/loop).</li>
		<li>Prior to polymerization, distill PheGE and DMSO on hemispherical heating mantles at 100 &#176;C and 70 &#176;C, respectively, for 2 hr under vacuum with stirring. The PheGE monomer&#39;s boiling point is 254 &#176;C whereas the boiling point for DMSO is 189 &#176;C at (1 atm).</li>
	</ol>
	</li>
	<li>Glassware for anionic polymerization.
	<ol>
		<li>In addition to the distillation system, use only high vacuum resistant glassware including round bottom flasks (certified by the manufacturer), graduated cylinders (for transfer of volumes of solvent, base and monomers), cannulas, septa and metal wires to seal the septa. 
		NOTE: For living polymerization, heat carefully (under vacuum) and cool down all glassware under argon flow prior to use. Keep the heat gun at a distance ~10 cm from the glassware.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/54422/54422fig2.jpg" /> 
Figure 2. Assembly and key distillation/transfer steps. <a href="http://cloudfront.jove.com/files/ftp_upload/54422/54422fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Description of the Key Steps of Living Anionic Polymerization: Distillation and Transfer
<ol>
	<li>Weigh mPEG-5K (2 mmol, 10 g) in a dry flask/Schlenk (oven) containing a stir bar and seal the flushing adapter (T) (on/off) with a septum at the top.</li>
	<li>Connect the flask to the manifold and purge the flask for 2-3 min with argon flushes. Turn the valve to the vacuum position to purge the flask.</li>
	<li>Rotate the flask manually and dry the reaction vessel homogeneously with a blow dryer (heat gun) until mPEG-5K melts. 
	NOTE: Keep the heat gun at a distance ~10 cm from the flask.</li>
	<li>After 1 min, break the vacuum by turning the valve on the manifold towards the argon position with several quick snaps. 
	NOTE: A continuous argon flow has to be observed in the bubbler. When the flow is continuous, the valve stays on the argon position. Repeat heating and cooling steps twice to remove all traces of moisture.</li>
	<li>Keep the polymeric macroinitiator under vacuum for ~ 2 hr and under argon before the reaction begins.</li>
	<li>Mount two high vacuum distillation apparatuses under the hood (Figure 2); one for the distillation of DMSO and one for the distillation of the monomer (PheGE).</li>
	<li>Connect the separate flasks containing the DMSO and monomer to the two apparatuses and install each on a hemispherical heating mantle (or in an oil bath). Connect cold water to the top of the apparatus (in/out) and to the manifold (argon/vacuum).</li>
	<li>Ensure that each apparatus is secure and well-sealed. Engage the vacuum via the valve. 
	NOTE: As described in step 3.3., repeat heating and cooling steps twice to remove all traces of moisture.</li>
	<li>Set the heating via a temperature controller and start stirring the solutions. After 2 hr of circulation/distillation of DMSO, close the high vacuum valve (found in the middle of the distillation apparatus) to collect approximately 20 ml of solution (to wash the inside of the apparatus). 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.</li>
	<li>Heat the flask containing the mPEG-5K (under vacuum) with the heat gun until the polymer (mPEG-5K) melts. Purge again with argon. 
	NOTE: The procedure will help to the dissolution after the transfer of the DMSO.</li>
	<li>After 2 hr, close the high vacuum valve and collect the volume of solvent (VDMSO = ~ 100 ml). Stop heating and break the vacuum from the manifold. Release argon (by snaps) into the chamber as described above.</li>
	<li>Under a positive pressure of argon, connect one side of the cannula (hold at the stopcock of the apparatus) to a graduated cylinder or directly to the flask containing the mPEG-5K (if the distillation apparatus has graduation) and, immerse the other end carefully into the freshly distilled fraction.</li>
	<li>Using argon pressure, drive the DMSO through the cannula into the reaction flask. Connect an extra bubbler to the flask (or cylinder if needed for measurement) and, close the glass stopcock connected to the bubbler on the opposite side of the manifold. 
	NOTE: When one side of a cannula is removed for the transfer, make sure that positive argon pressure is applied.</li>
	<li>To avoid any accidents caused by argon pressure, open the glass stopcock for 1-2 sec and reclose to continue the flow of DMSO (repeated once per 0.5 min) until the full transfer is completed. Reopen the stopcock when finished. 
	NOTE: The same procedure must now be followed for distillation and collection of the monomer. The solvent and monomer cannot be collected at the same time.</li>
	<li>Transfer 5 ml of 0.3 M naphthalene potassium via cannulation in a graduated cylinder sealed by a septum with cannula inserted (loop). 
	NOTE: Same precaution as described in NOTE 4.13. Positive argon pressure must be maintained first from the naphthalene potassium flask to the cylinder and then from the cylinder to the reaction flask to avoid air/water contamination.</li>
	<li>Insert another needle from the manifold into the cylinder (argon). Remove the cannula connected to the distillation system carefully and insert rapidly into the reaction flask. 
	NOTE: Use this technique for the transfer of base and monomer.</li>
	<li>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.</li>
	<li>Transfer the desired volume of monomer (VPheGE = 5 ml) to reach a degree of polymerization of PPheGE ~ n=18-20.</li>
	<li>Leave the reaction for 48 hr at 80 &#176;C under argon atmosphere with constant stirring to ensure complete polymerization.</li>
	<li>Quench the reaction by the addition of drops of HCl 1 N in methanol (measured using litmus paper (neutral pH)) and observed by a color disappearance.</li>
	<li>Extract the naphthalene from the DMSO solution with hexane (3&#215;50 ml). Remove the DMSO by distillation under vacuum ~ 70 ml (same apparatus). Cool down the slurry solution and add 50 ml of THF.</li>
	<li>Remove the salt from the slurry solution by centrifugation at 5,000 x g for 10 min. Transfer the supernatant, and add drop wise to 500 ml of cold diethyl ether.</li>
	<li>Collect the precipitate by filtration or centrifugation (repeat twice) and dry under vacuum at 30 &#176;C for 24-48 hr (yield 85%). 
	NOTE: The copolymer is now ready for characterization.</li>
</ol>
5. Characterization of the Copolymers
<ol>
	<li>Weigh 5-10 mg of copolymer (record the actual mass) in an aluminum sample pan and seal hermetically with the aluminum lid. Load sample pan and reference pan (empty) into the differential scanning calorimeter.</li>
	<li>Program a method (&#34;heat/cool/heat&#34;) cycle: 1) heat from 40 &#176;C to 100 &#176;C at 10 &#176;C/min, 2) cool to -70 &#176;C at 10 &#176;C/min, 3) heat to 100 &#176;C at 10 &#176;C/min. Repeat 2) and 3) twice. Determine melting point (Tm), crystallization (Tc) and glass transition temperatures (Tg), and heat of fusion (&#916;Hf) from the thermal traces from the third cycle (if applicable).</li>
	<li>Dissolve the polymers in THF (2 mg/ml) and filter through a 0.2-&#181;m PTFE filter. Inject the sample into a gel permeation chromatography system (50 &#181;l) and use the retention time for the sample and a calibration curve produced using a range of polystyrene standards to determine the molecular weight of the polymer.19</li>
	<li>Dissolve the (co)polymers (15 mg/ml) in d6 DMSO for 1H NMR spectroscopy analysis.19</li>
	<li>Determine the critical micelle concentration (CMC) of the copolymer using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a fluorescence probe.9
	<ol>
		<li>Prepare a DPH stock solution in THF (2.32 mg/L) in the dark and add 100 &#181;l of this stock solution to each of a series of vials.</li>
		<li>Prepare a copolymer stock solution in THF and add aliquots of equal volume (2 ml) to the series of vials (each containing an aliquot of the DPH stock solution) resulting in final copolymer concentrations that range from 0.01 to 1,000 &#181;g copolymer/ml.</li>
		<li>Subsequently, vortex the copolymer-DPH solutions and add dropwise to 10 ml of double-distilled water with stirring bar. The solutions must then be stirred vigorously in the dark for 48 hr under a stream of nitrogen to allow slow evaporation of THF. The final concentration of DPH in each solution is 0.232 mg/L.</li>
		<li>Measure the fluorescence emission of the samples at 430 nm (&#955;ex = 350 nm) using a dual-scanning microplate spectrofluorometer and plot fluorescence versus log[polymer]. The intercept between the two linear slopes provides the CMC value for the copolymer.</li>
	</ol>
	</li>
</ol>
6. Procedure for Loading Doxorubicin into BCMs
<ol>
	<li>Dissolve 12 mg of DOX in 1 ml of acetonitrile, add 10 &#181;l of triethylamine and let the solution stir in the dark for 2 hr.</li>
	<li>Dissolve the copolymer (45 mg) in 1 ml of THF and stir for the same period of time. Add the copolymer solution to the DOX solution and rinse the vial containing residual copolymer with an extra volume of THF (0.5 ml).</li>
	<li>Add the copolymer-drug mixture (2.5 ml) dropwise to a vial (20 ml) containing 15 ml of saline 0.9% (NaCl) with stirring.</li>
	<li>Transfer the solution to a dialysis bag (3.5 kDa cut off) and dialyze against saline 0.9% (500 ml). 
	NOTE: Change the external saline after 6 hr and let the dialysis continue for 24 hr with stirring in the dark at RT.</li>
	<li>Transfer the dialysate to a 50 ml tube and centrifuge at 5,000 x g for 15 min.</li>
	<li>Transfer the supernatant to an ultrafiltration system (with a 10 ml capacity) that contains a dialysis membrane (cut off 10 kDa). Put the stirring adapter into the ultrafiltration system, close the lid and open to a stream of nitrogen.</li>
	<li>Concentrate the BCM solution to a volume of 4 ml and add 6 ml of fresh saline and repeat the procedure twice.</li>
	<li>Concentrate the BCM solution to 4 ml, rinse the chamber with 0.5 ml of saline and add to the solution. Store in brown vials at RT in the dark prior to further use.</li>
</ol>
7. Evaluation of Doxorubicin Loading in DOX-BCMs
<ol>
	<li>Dissolve DOX-BCM in dimethylformamide (100 &#181;l in 400 &#181;l) to disrupt the micelles and dilute in HCl aqueous solution (0.1 N) prior to evaluation (100 &#181;l in 900 &#181;l HCl 0.1 N).</li>
	<li>Measure drug loading at 490 nm using a benchtop microplate spectrophotometric system. Use the following equations to determine the drug loading capacity (DLC) and drug loading efficiency (DLE): 
	DLC (wt%) = (weight of drug loaded / total weight of BCMs) x 100% 
	DLE (%) = (weight of drug loaded / weight of drug in feed) x 100%</li>
</ol>
8. Evaluation of In Vitro Release of DOX from DOX-BCMs
<ol>
	<li>Investigate the release of DOX from BCMs at 37 &#176;C in 0.1 M phosphate-buffered saline (PBS, pH 7.4) against PBS pH 7.4 containing 0.1% (w/v) Tween 80, BCMs + BSA (50 mg/ml) against PBS pH 7.4 and 0.1 M acetate-buffer at pH = 5.5.20,21</li>
	<li>Dilute the BCM-DOX formulation (700 &#181;l) in the selected buffer (2.3 ml) to result in a total amount of &#8776; 0.6-0.7 mg of DOX in the dialysis bag.</li>
	<li>Place the solution in the dialysis bag, seal with clips and immerse the bag into 200 ml of the respective external media.</li>
	<li>Remove 2 ml of the solution outside of the dialysis bag at predetermined time points and replace with the same volume of fresh buffer.</li>
	<li>Store the aliquots removed at -20 &#176;C prior to analysis by UV-Vis spectrophotometry (Abs490 nm). The cumulative percentage of drug released (Er) can be calculated using the following equation: 
	<img alt="Equation1" src="/files/ftp_upload/54422/54422eq1.jpg" /> 
	NOTE: Where mDOX represents the amount of DOX in the BCMs, V0 is the total volume of the release media (200 ml), Vt is the volume of the replaced media (Vt = 2 ml), Ci is the concentration before correction, and Cn represents the concentration of DOX in the sample.</li>
</ol>

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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. O., Oberoi, H. S., Desale, S., Kabanov, A. V., Bronich, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polypeptide+nanogels+with+hydrophobic+moieties+in+the+cross-linked+ionic+cores:+synthesis,+characterization+and+implications+for+anticancer+drug+delivery.">Polypeptide nanogels with hydrophobic moieties in the cross-linked ionic cores: synthesis, characterization and implications for anticancer drug delivery.</a> Journal of Drug Targeting. 21 (10), 981-993 (2013).</li><li>Zhao, C. L., Winnik, M. A., Riess, G., Croucher, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+probe+techniques+used+to+study+micelle+formation+in+water-soluble+block+copolymers.">Fluorescence probe techniques used to study micelle formation in water-soluble block copolymers.</a> Langmuir. 6 (2), 514-516 (1990).</li><li>Wilhelm, 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=Poly(styrene-ethylene+oxide)+block+copolymer+micelle+formation+in+water:+a+fluorescence+probe+study.">Poly(styrene-ethylene oxide) block copolymer micelle formation in water: a fluorescence probe study.</a> Macromolecules. 24 (5), 1033-1040 (1991).</li><li>Cammas, S., 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=Functional+poly[(ethylene+oxide)-co-(&#946;-benzyl-L-aspartate)]+polymeric+micelles:+block+copolymer+synthesis+and+micelles+formation.">Functional poly[(ethylene oxide)-co-(&#946;-benzyl-L-aspartate)] polymeric micelles: block copolymer synthesis and micelles formation.</a> Macromolecular Chemistry and Physics. 196 (6), 1899-1905 (1995).</li><li>Kwon, G., 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=Micelles+based+on+AB+block+copolymers+of+poly(ethylene+oxide)+and+poly(.beta.-benzyl+L-aspartate).">Micelles based on AB block copolymers of poly(ethylene oxide) and poly(.beta.-benzyl L-aspartate).</a> Langmuir. 9 (4), 945-949 (1993).</li><li>Ahmed, F., Discher, D. 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. 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+effects+of+solvation+on+the+properties+of+anions+in+dipolar+aprotic+solvents.">The effects of solvation on the properties of anions in dipolar aprotic solvents.</a> Quarterly Reviews, Chemical Society. 16 (2), 163-187 (1962).</li><li>Cram, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fundamentals o] Carbanion Chemistry. , (1965).</li><li>Szwarc, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ACS Symposium Series. 166, 1-15 (1981).</li><li>Cho, Y. W., Lee, J., Lee, S. C., Huh, K. 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The authors have nothing to disclose.

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 Ãâ‚¬-Ãâ‚¬ 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 ...

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13.9K Views.  University of Toronto. 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.

Video: Anionic Polymerization of an Amphiphilic Copolymer for Preparation of Block Copolymer Micelles Stabilized by π-π Stacking Interactions

Controlling the Size, Shape and Stability of Supramolecular Polymers in Water

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do so is highly important in view of a number of applications in functional soft matter including electronics, biomedical engineering, and sensors. In the past, successful strategies to control the size and shape of supramolecular polymers typically focused on the use of templates2,3, end cappers4 or selective solvent techniques5. 
Here we disclose a strategy based on self-assembling discotic amphiphiles that leads to the control over stack length and shape of ordered, chiral columnar aggregates. By balancing electrostatic repulsive interactions on the hydrophilic rim and attractive non-covalent forces within the hydrophobic core of the polymerizing building block, we manage to create small and discrete spherical objects6,7. Increasing the salt concentration to screen the charges induces a sphere-to-rod transition. Intriguingly, this transition is expressed in an increase of cooperativity in the temperature-dependent self-assembly mechanism, and more stable aggregates are obtained. 
For our study we select a benzene-1,3,5-tricarboxamide (BTA) core connected to a hydrophilic metal chelate via a hydrophobic, fluorinated L-phenylalanine based spacer (Scheme 1). The metal chelate selected is a Gd(III)-DTPA complex that contains two overall remaining charges per complex and necessarily two counter ions. The one-dimensional growth of the aggregate is directed by &pi;-&pi; stacking and intermolecular hydrogen bonding. However, the electrostatic, repulsive forces that arise from the charges on the Gd(III)-DTPA complex start limiting the one-dimensional growth of the BTA-based discotic once a certain size is reached. At millimolar concentrations the formed aggregate has a spherical shape and a diameter of around 5 nm as inferred from 1H-NMR spectroscopy, small angle X-ray scattering, and cryogenic transmission electron microscopy (cryo-TEM). The strength of the electrostatic repulsive interactions between molecules can be reduced by increasing the salt concentration of the buffered solutions. This screening of the charges induces a transition from spherical aggregates into elongated rods with a length &gt; 25 nm. Cryo-TEM allows to visualise the changes in shape and size. In addition, CD spectroscopy permits to derive the mechanistic details of the self-assembly processes before and after the addition of salt. Importantly, the cooperativity -a key feature that dictates the physical properties of the produced supramolecular polymers- increases dramatically upon screening the electrostatic interactions. This increase in cooperativity results in a significant increase in the molecular weight of the formed supramolecular polymers in water.

The goal of this experiment is to determine and control the size, shape and stability of self-assembled discotic amphiphiles in water. For aqueous based supramolecular polymers such level of control is very difficult. We apply a strategy using both repulsive and attractive interactions. The experimental techniques applied to characterize this system are broadly applicable.

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do ...

Engineering

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, ...

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

Bioengineering

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 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 ...

Self-assembling Morphologies Obtained from Helical Polycarbodiimide Copolymers and Their Triazole Derivatives

A facile method for the preparation of polycarbodiimide-based secondary structures (e.g., nano-rings, &#34;craters,&#34; fibers, looped fibers, fibrous networks, ribbons, worm-like aggregates, toroidal structures, and spherical particles) is described. These aggregates are morphologically influenced by extensive hydrophobic side chain-side chain interactions of the singular polycarbodiimide strands, as inferred by atomic force microscopy (AFM) and scanning electron microscopy (SEM) techniques. Polycarbodiimide-g-polystyrene copolymers (PS-PCDs) were prepared by a combination of synthetic methods, including coordination-insertion polymerization, copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) &#34;click&#34; chemistry, and atom transfer radical polymerization (ATRP). PS-PCDs were found to form specific toroidal architectures at low concentrations in CHCl3. To determine the influence of a more polar solvent medium (i.e., THF and THF/EtOH) on polymer aggregation behavior, a number of representative PS-PCD composites have been tested to show discrete concentration-dependent spherical particles. These fundamental studies are of practical interest to the development of experimental procedures for desirable architectures by directed self-assembly in thin film. These architectures may be exploited as drug carriers, whereas other morphological findings represent certain interest in the area of novel functional materials.

Here, we present a protocol to prepare and visualize secondary structures (e.g., fibers, toroidal architectures, and nano-spheres) derived from helical polycarbodiimides. The morphology characterized by both atomic force microscopy (AFM) and scanning electron microscopy (SEM) was shown to depend on molecular structure, concentration, and the solvent of choice.

A facile method for the preparation of polycarbodiimide-based secondary structures (e.g., nano-rings, &#34;craters,&#34; fibers, looped fibers, fibrous ...

Synthesis of Monodisperse Cylindrical Nanoparticles via Crystallization-driven Self-assembly of Biodegradable Block Copolymers

The production of monodisperse cylindrical micelles is a significant challenge in polymer chemistry. Most cylindrical constructs formed from diblock copolymers are produced by one of three techniques: thin film rehydration, solvent switching or polymerization-induced self-assembly, and produce only flexible, polydisperse cylinders. Crystallization-driven self-assembly (CDSA) is a method which can produce cylinders with these properties, by stabilizing structures of a lower curvature due to the formation of a crystalline core. However, the living polymerization techniques by which most core-forming blocks are formed are not trivial processes and the CDSA process may yield unsatisfactory results if carried out incorrectly. Here, the synthesis of cylindrical nanoparticles from simple reagents is shown. The drying and purification of reagents prior to a ring-opening polymerization of &#949;-caprolactone catalyzed by diphenyl phosphate is described. This polymer is then chain extended by methyl methacrylate (MMA) followed by N,N-dimethyl acrylamide (DMA) using reversible addition&#8722;fragmentation chain-transfer (RAFT) polymerization, affording a triblock copolymer that can undergo CDSA in ethanol. The living CDSA process is outlined, the results of which yield cylindrical nanoparticles up to 500 nm in length and a length dispersity as low as 1.05. It is anticipated that these protocols will allow others to produce cylindrical nanostructures and elevate the field of CDSA in the future.

Crystallization-driven self-assembly (CDSA) displays the unique ability to fabricate cylindrical nanostructures of narrow length distributions. The organocatalyzed ring-opening polymerization of &#949;-caprolactone and subsequent chain extensions of methyl methacrylate and N,N-dimethyl acrylamide are demonstrated. A living CDSA protocol that produces monodisperse cylinders up to 500 nm in length is outlined.

The production of monodisperse cylindrical micelles is a significant challenge in polymer chemistry. Most cylindrical constructs formed from diblock ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Assembly and Characterization of Polyelectrolyte Complex Micelles

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful platform for exploring the physics of polyelectrolyte interactions and also offer a promising solution to the pressing problem of delivering therapeutic oligonucleotides in vivo. Developing predictive structure-property relationships for PCMs has proven difficult, in part due to the presence of strong kinetic traps during nanoparticle self-assembly. This article discusses criteria for choosing polymers for PCM construction and provides protocols based on salt annealing that enable assembly of repeatable, low-polydispersity nanoparticles. We also discuss PCM characterization using light scattering, small-angle X-ray scattering, and electron microscopy.

We provide protocols and representative data for designing, assembling, and characterizing polyelectrolyte complex micelles, core-shell nanoparticles formed by polyelectrolytes and hydrophilic charged-uncharged block copolymers.

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful ...