The authors gratefully acknowledge the financial support of a &#34;Thousand Young Global Talents&#34; award from the Chinese Central Government, a &#34;Shuang Chuang&#34; award from the Jiangsu Provincial Government, start-up fund from College of Engineering and Applied Sciences, Nanjing University, China, award from the &#34;Tian-Di&#34; Foundation, grant from the Priority Academic Program Development Fund of Jiangsu Higher Education Institutions (PAPD), grant from the Jiangsu Province Natural Science Fund.

Caution: Due to the use of organic solvents, all operations should be done in a chemical fume hood. Due to the use of high electric voltage, avoid body contact with the apparatus when the power supply is on. Use all appropriate safety practices such as using personal protective equipment (safety glasses, gloves, lab coat, full-length pants, and closed-toe shoes). Consult all relevant material safety data sheets (MSDS).
1. Setup of Materials
<ol>
	<li>To prepare QD solution, dissolve 10 mg hydrophobic QDs (fluorescent emission peak wavelength = 605 nm, used as the model nanocrystals here) in 20 mL organic solvent (chloroform for producing spherical micelle shape or THF for producing worm-like micelle shape) and vortex for 20 s.</li>
	<li>To prepare PS-PEG solution, dissolve 100 mg PS-PEG (amphiphilic block copolymer, with 9.5 kDA PS segment and 18.0 kDA PEG segment) in 10 mL organic solvent (chloroform for producing spherical micelle shape or THF for producing worm-like micelle shape). Mix the solution by vortexing for 1 min (chloroform) or bath sonicate for 2 min (THF).</li>
	<li>Mix 1 mL of QD solution and 1 mL PS-PEG solution and vortex for 1 min. Add the mixture to Syringe A. The syringe is made of PTFE.</li>
	<li>To prepare PVA solution, dissolve 400 mg PVA (13-23 kDa, 87-89% hydrolyzed) in 10 mL water in a heated water bath at 60&#8211;80 &#176;C for 4&#8211;5 h. Allow the PVA solution to cool down to room temperature before use.</li>
	<li>Add 5 mL of PVA solution to Syringe B. The syringe is made of PTFE.</li>
</ol>
2. Setup of Equipment
<ol>
	<li>Insert the inner capillary into the outer capillary assembly and gently screw into position. Do not over tighten. Figure 2 shows the overall setup of the coaxial electrospray system. The inner capillary needle is a 27 G (outer diameter 500 &#181;m; inner diameter 300 &#181;m) stainless-steel capillary, and the outer needle is a 20 G (outer diameter 1,000 &#181;m; inner diameter 500 &#181;m) stainless-steel three-way connector. The PTEE tubing used has an inner diameter of 1.8 mm.</li>
	<li>Load Syringe A on Syringe Pump A as shown in Figure 2. Connect Syringe A to the inner stainless-steel capillary of the electrospray coaxial nozzle using PTFE tubing.</li>
	<li>Load Syringe B on Syringe Pump B as shown in Figure 2. Connect Syringe B to the outer stainless-steel capillary of the electrospray coaxial nozzle using PTFE tubing.</li>
	<li>Position the electrospray coaxial nozzle tip approximately 0.8 cm above a grounded steel ring (diameter of 1.5 cm).</li>
	<li>Place a glass collection dish approximately 10 cm below the coaxial nozzle.</li>
	<li>With the power supply turned off, connect the ground wire (black wire in Figure 2) to the grounded steel ring.</li>
	<li>With the power supply turned off, connect the positive terminal (red wire in Figure 2) of the power supply to the inner needle of the coaxial nozzle using a metal alligator clip.</li>
</ol>
3. Production of Micellar Nanocrystals
<ol>
	<li>Set the speed of Syringe Pump A to 0.6 mL/h.</li>
	<li>Set the speed of Syringe Pump B to 1.5 mL/h.</li>
	<li>Start both syringe pumps and wait for their respective flow rates to stabilize. Drops forming at the nozzle at a steady rate indicate a stable flow rate. This usually occurs within 60 s after starting syringe pumps. 
	NOTE: There should be no bubbles in the tubing and droplets should form at the electrospray coaxial nozzle.</li>
	<li>Turn on the power supply to apply a positive high voltage to the electrospray coaxial nozzle. Adjust the applied voltage within the range of 5&#8211;9 kV, until a concave cone-jet (i.e., a convergent jet, commonly known as a &#39;Taylor cone&#39;) is observed at the tip of the coaxial nozzle (as shown in the inset of Figure 3a). 
	Caution: Be sure not to touch electrospray nozzle when high voltage is applied. Follow appropriate safety precautions. 
	NOTE: Insufficient applied voltage will result in droplets forming at the tip of the nozzle (as shown in the inset of Figure 3b), while too high of applied voltage will cause an electrical arc between the nozzle and the grounded steel ring.</li>
	<li>After a stable Taylor cone (Figure 3a) has been obtained, add 10 mL deionized water to a clean collection dish and replace the glass collection dish in the setup. The new dish will collect the micellar nanocrystal product.</li>
	<li>Run the micellar nanocrystal production process for a certain time duration (for approximately 40 min for producing spherical micelle shape or approximately 90 min for producing worm-like micelle shape). Then remove the collection dish from underneath the electrospray nozzle.</li>
	<li>Stop Syringe Pump A and B.</li>
	<li>Turn off the high voltage power supply.</li>
	<li>Allow the organic solvent to evaporate (in a fume hood) from the uncovered collection dish overnight. 
	NOTE: Judging from the characterization results of the micellar nanocrystal products, evaporation overnight is sufficient for removing the organic solvent to obtain products with good quality.</li>
	<li>Finally, transfer the micellar nanocrystal product to a 15 mL centrifuge tube for characterization (e.g., fluorescent spectroscopy, dynamic light scattering, transmission electron microscopy, and thermal analysis), application, or storage. Store the final micellar nanocrystal product in a refrigerator at 4 &#176;C. 
	NOTE: The product can remain stable under this storage condition for at least one month.</li>
</ol>

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<li>Heath, J. R., Davis, M. E. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3706454/">Nanotechnology and Cancer.</a> Annu. Rev. Medicine. 59, 251-265 (2008).</li>
<li>Pu, K., Chattopadhyay, N., Rao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+advances+of+semiconducting+polymer+nanoparticles+in+in+vivo+molecular+imaging%5BTitle%5D)+AND+%22J.+Control.+Release%22%5BJournal%5D)">Recent advances of semiconducting polymer nanoparticles in in vivo molecular imaging.</a> J. Control. Release. 240, 312-322 (2016).</li>
<li>Swierczewska, M., Han, H. S., Kim, K., Park, J. H., Lee, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polysaccharide-based+nanoparticles+for+theranostic+nanomedicine%5BTitle%5D)+AND+%22Adv.+Drug+Deliv.+Rev%22%5BJournal%5D)">Polysaccharide-based nanoparticles for theranostic nanomedicine.</a> Adv. Drug Deliv. Rev. 99, 70-84 (2016).</li>
<li>Gao, X. H., Yang, L. L., Petros, J. A., Marshal, F. F., Simons, J. W., Nie, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+molecular+and+cellular+imaging+with+quantum+dots%5BTitle%5D)+AND+%22Curr.+Opin.+Biotechnol%22%5BJournal%5D)">In vivo molecular and cellular imaging with quantum dots.</a> Curr. Opin. Biotechnol. 16 (1), 63-72 (2005).</li>
<li>Dubertret, B., Skourides, P., Norris, D. J., Noireaux, V., Brivanlou, A. H., Libchaber, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+imaging+of+quantum+dots+encapsulated+in+phospholipid+micelles%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">In vivo imaging of quantum dots encapsulated in phospholipid micelles.</a> Science. 298 (5599), 1759-1762 (2002).</li>
<li>Ruan, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+magnetic+manipulation+and+fluorescent+tracking+of+multiple+individual+hybrid+nanostructures%5BTitle%5D)+AND+%22Nano+Lett%22%5BJournal%5D)">Simultaneous magnetic manipulation and fluorescent tracking of multiple individual hybrid nanostructures.</a> Nano Lett. 10 (6), 2220-2224 (2010).</li>
<li>Ruan, G., Winter, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alternating-color+quantum+dot+nanocomposites+for+particle+tracking%5BTitle%5D)+AND+%22Nano+Lett%22%5BJournal%5D)">Alternating-color quantum dot nanocomposites for particle tracking.</a> Nano Lett. 11 (3), 941-945 (2011).</li>
<li>Park, J. H., von Maltzahn, G., Ruoslahti, E., Bhatia, S. N., Sailor, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Micellar+hybrid+nanoparticles+for+simultaneous+magnetofluorescent+imaging+and+drug+delivery%5BTitle%5D)+AND+%22Angewandte+Chemie-International+Edition%22%5BJournal%5D)">Micellar hybrid nanoparticles for simultaneous magnetofluorescent imaging and drug delivery.</a> Angewandte Chemie-International Edition. 47 (38), 7284-7288 (2008).</li>
<li>Torchilin, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PEG-based+micelles+as+carriers+of+contrast+agents+for+different+imaging+modalities%5BTitle%5D)+AND+%22Adv.+Drug+Deliv.+Rev%22%5BJournal%5D)">PEG-based micelles as carriers of contrast agents for different imaging modalities.</a> Adv. Drug Deliv. Rev. 54 (2), 235-252 (2002).</li>
<li>Sun, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Examining+the+roles+of+emulsion+droplet+size+and+surfactant+in+the+interfacial+instability-based+fabrication+process+of+micellar+nanocrystals%5BTitle%5D)+AND+%22Nanoscale+Research+Letters%22%5BJournal%5D)">Examining the roles of emulsion droplet size and surfactant in the interfacial instability-based fabrication process of micellar nanocrystals.</a> Nanoscale Research Letters. 12, 434(2017).</li>
<li>Ding, X. Y., Han, N., Wang, J., Sun, Y. X., Ruan, G. <a target="_blank" href="http://pubs.rsc.org/en/content/articlehtml/2017/ra/c6ra28741g">Effects of organic solvents on the structures of micellar nanocrystals.</a> RSC Advances. 7 (26), 16131-16138 (2017).</li>
<li>Sailor, M., Park, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hybrid+nanoparticles+for+detection+and+treatment+of+cancer%5BTitle%5D)+AND+%22Adv.+Materials%22%5BJournal%5D)">Hybrid nanoparticles for detection and treatment of cancer.</a> Adv. Materials. 24 (28), 3779-3802 (2012).</li>
<li>Jing, L. H., Ding, K., Kershaw, S. V., Kempson, T. M., Rogach, A. L., Gao, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Magnetically+engineered+semiconductor+quantum+dots+as+multimodal+imaging+probes%5BTitle%5D)+AND+%22Adv.+Materials%22%5BJournal%5D)">Magnetically engineered semiconductor quantum dots as multimodal imaging probes.</a> Adv. Materials. 26 (37), 6367-6386 (2014).</li>
<li>Bao, G., Mitragotri, S., Tong, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multifunctional+nanoparticles+for+drug+delivery+and+molecular+imaging%5BTitle%5D)+AND+%22Annu.+Rev.+Biomed.+Eng%22%5BJournal%5D)">Multifunctional nanoparticles for drug delivery and molecular imaging.</a> Annu. Rev. Biomed. Eng. 15, 253-282 (2013).</li>
<li>Mura, S., Couvreur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanotheranostics+for+personalized+medicine%5BTitle%5D)+AND+%22Adv.+Drug+Delivery+Rev%22%5BJournal%5D)">Nanotheranostics for personalized medicine.</a> Adv. Drug Delivery Rev. 64 (13), 1394-1416 (2012).</li>
<li>Louie, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multimodality+imaging+probes%3A+design+and+challenges%5BTitle%5D)+AND+%22Chem.+Rev%22%5BJournal%5D)">Multimodality imaging probes: design and challenges.</a> Chem. Rev. 110 (5), 3146-3195 (2010).</li>
</ol>

The authors have nothing to disclose.

The fabrication method of micellar nanocrystals described in the present work combines top-down electrospray, bottom-up self-assembly, and solvent-based structure control. An effective and convenient quality control method is to use the Taylor cone formed at the coaxial nozzle tip. This is because a properly formed Taylor cone indicates balance (or near balance) between electric force and surface tension, which in turn indicates successful formation of micro-reactors (uniform ultrafine droplets) for the self-assembly rea...

Nanocrystals such as semiconductor quantum dots (QDs) and superparamagnetic iron oxide nanoparticles (SPIONs) have demonstrated great potential for biological detection, imaging, manipulation, and therapy1,2,3,4,5,6. Encapsulating one or more nanocrystals into a micelle has been a widely used method to interface nanocrystals with biological environments3,6. The thus-formed micellar nanocrystals (micelles with nanocrystals encapsulated) have become an emerging class of nanobiomaterials7,8,9,10. Commonly used methods to fabricate micelles that encapsulate various materials (e.g., nanocrystals, small molecule drugs, and dyes) include film hydration, dialysis, and several others7,11.
The present work describes a method of fabricating micellar nanocrystals based on combining top-down electrospray, bottom-up self-assembly, and solvent-mediated structural control. Compared with other fabrication methods of micellar nanocrystals, our method offers several beneficial features: (1) It is a largely continuous production process. This feature is mainly due to the fact that electrospray is used in our method to form emulsion droplets. In contrast, some other methods use vortexing or sonication to form emulsion droplets, thereby making these methods batch processes in nature12. (2) It results in products with high water-dispersibility, excellent colloidal stability, and intact physical functions of the encapsulated nanocrystals. This process can often give products with superior quality compared with other micelle encapsulation methods, to a large extent because electrospray can form ultrafine and uniform emulsion droplets. (3) The structures of the products, including micelle shape and number of encapsulated nanocrystals, can be controlled by the solvent, which is much more inexpensive compared with other ways of control such as changing the amphiphilic polymers used, and can produce not only the commonly available spherical micelle shape but worm-like micelle shape via micelle fusion13. The thus-formed worm-shaped micellar nanocrystals are found to offer greatly reduced non-specific cellular uptake than the spherical counterparts13. On the other hand, it is worth pointing out that this method requires the setup of an electrospray device, which is somewhat more technically demanding (although far from prohibitive) than the need of instrumentation in the other methods.
The fabrication method involves first generating ultrafine liquid (often oil-in-water emulsion) droplets with uniform sizes by electrospray, followed by evaporation of organic solvent resulting in self-assembly to form micellar nanocrystals (Figure 1).The electrospray setup has a coaxial configuration using concentric needles: the oil phase, which contains amphiphilic block copolymers and hydrophobic nanocrystals dissolved in organic solvent, is delivered to the inner needle (27 G stainless-steel capillary) with a syringe pump; the water phase, which contains a surfactant dissolved in water, is delivered to the outer needle (20 G stainless-steel three-way connector) with a second syringe pump. A high voltage is applied to the coaxial nozzle. Ultrafine droplets with uniform sizes are generated due to electrodynamic force overcoming surface tension and inertial stress in the liquid. Each droplet essentially functions as a &#39;micro-reactor&#39;, in which, upon removal of the organic solvent by evaporation, the self-assembly &#39;reaction&#39; occurs spontaneously due to hydrophobic interactions. Using different organic solvents leads to different structures of micellar nanocrystals: a water-immiscible organic solvent chloroform leads to spherical micelle shape, while a water-miscible organic solvent THF with a long reaction time leads to worm-like micelle shape along with enhanced nanocrystal encapsulation.

<table><tbody><tr><td>Hydrophobic quantum dots</td><td>Ocean Nanotech</td><td>QSP</td><td>Solid hydrophobic CdSe/ZnS quantum dots. Peak fluorescence emission wavelength is 605 nm.</td></tr><tr><td>Poly(styrene)-b-poly(ethylene glycol) (PS-PEG)</td><td>Sigma-Aldrich</td><td>666476-500MG</td><td>Molecular weight of PS segment is 9.5 kDa and that of PEG segment is 18.0 kDa.</td></tr><tr><td>Poly(vinyl alcohol) (PVA)</td><td>Sigma-Aldrich</td><td>363170-500G</td><td>Molecular weight 13&amp;ndash;23 kDa, 87&amp;ndash;89% hydrolyzed.</td></tr><tr><td>Tetrahydrofuran (THF)</td><td>Sinopharma Chemical Reagent</td><td>80124418</td><td></td></tr><tr><td>Chloroform</td><td>Sinopharma Chemical Reagent</td><td>40007960</td><td></td></tr><tr><td>Syringe pumps</td><td>Bao Ding Shen Chen</td><td>SPLab01</td><td></td></tr><tr><td>Tubing</td><td>Shanghei Lai Xing</td><td></td><td>2 mm outer diameter and 1.8 mm inner diameter PTFE tubing.</td></tr><tr><td>Syringes</td><td>Yi Ming</td><td>5.CC</td><td>5 mL disposable syringe made of PTFE.</td></tr><tr><td>High voltage power supply</td><td>Dong Wen</td><td>DW Series</td><td>Direct current power supply (0&amp;ndash;50 kV range).</td></tr><tr><td>Electrospray coaxial nozzle</td><td>Hunan Chang Sha Na Yi</td><td></td><td>Stainless steel assembly. Inner capillary needle was a 27 gauge (outer diameter 500 &amp;mu;m; inner diameter 300 &amp;mu;m). Outer capillary was a 20 gauge (outer diameter 1,000 &amp;mu;m; inner diameter 500 &amp;mu;m).</td></tr><tr><td>Vortexer</td><td>Xi&#039;an HEB Biotechnology Co., Ltd. China</td><td>MX-S</td><td>MX-S with wide speed range of 0&amp;ndash;2,500 rpm, stepless speed regulation, touch and continuous operations.</td></tr><tr><td>Steel ring</td><td>Yiwu Wan Tu</td><td></td><td>Rings with a range of diameters (0.8&amp;ndash;1.8 cm) can be constructued. For example, a 1.3 cm diameter ring was constructed by curling an approximately 25 cm (length) of 0.5-mm diamter (24 gauge, AWG) steel wire.</td></tr><tr><td>Glass collecting dish</td><td>Grainger</td><td>1u5084</td><td>25-mm height and 120-mm diameter glass dish.</td></tr><tr><td>15 mL centrifuge tube</td><td>Jiangsu Xinkang Medical Instrument Co., Ltd.</td><td>X-407</td><td>Centrifuge tube is made of transparent polypropylene (PP).</td></tr></tbody></table>

fabrication of spherical and worm-shaped micellar nanocrystals by combining electrospray, self-assembly, and solvent-based structure control

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of fabricating micellar nanocrystals based on combining top-down electrospray, bottom-up self-assembly, and solvent-based structure control. This method involves first using electrospray to generate uniform ultrafine liquid droplets, each of which functions as a micro-reactor in which self-assembly reaction occurs forming micellar nanocrystals, with the structures (micelle shape and nanocrystal encapsulation) controlled by the organic solvent used. This method is largely continuous and produces high quality micellar nanocrystal products with an inexpensive structure control approach. By using a water-miscible organic solvent tetrahydrofuran (THF), worm-shaped micellar nanocrystals can be produced due to solvent-induced/facilitated micelle fusion. Compared with the common spherical micellar nanocrystals, worm-shaped micellar nanocrystals can offer minimized non-specific cellular uptake, thus enhancing biological targeting. By co-encapsulating multiple nanocrystals into each micelle, multifunctional or synergistic effects can be achieved. Current limitations of this fabrication method, which will be part of the future work, primarily include imperfect encapsulation in the micellar nanocrystal product and the incompletely continuous nature of the process.

Figure 1 shows a schematic summarizing the control of structures (shape and encapsulation) of micellar nanocrystals by the organic solvent used in the production process. Briefly, dichloromethane leads to spherical micelles with no encapsulation of nanocrystals; chloroform leads to spherical micelles with a low encapsulation number of nanocrystals; THF leads to spherical micelles with a high encapsulation number of nanocrystals at a short reaction time and wo...

Watch this Scientific Journal Video about Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control at JoVE.com

Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control

The present work describes a method to fabricate micellar nanocrystals, an emerging major class of nanobiomaterials. This method combines top-down electrospray, bottom-up self-assembly, and solvent-based structure control. The fabrication method is largely continuous, can produce high quality products, and possesses an inexpensive means of structure control.

Micellar nanocrystals (micelles with encapsulated nanocrystals) have become an emerging major class of nanobiomaterials. We describe a method of ...

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Chemistry

13.9K Views.  Nanjing University. Het huidige werk beschrijft een methode om te fabriceren micellaire nanokristallen, een opkomende grote klasse van nanobiomaterials. Deze methode combineert topdown electrospray, onderop zelf-assemblage, en op basis van oplosmiddel structuur controle. De fabricage methode is grotendeels continu kan produceren hoogwaardige producten en bezit een goedkoop middel structuur controle.

Video: Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control

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

A Technique to Functionalize and Self-assemble Macroscopic Nanoparticle-ligand Monolayer Films onto Template-free Substrates

This protocol describes a self-assembly technique to create macroscopic monolayer films composed of ligand-coated nanoparticles1,2. The simple, robust and scalable technique efficiently functionalizes metallic nanoparticles with thiol-ligands in a miscible water/organic solvent mixture allowing for rapid grafting of thiol groups onto the gold nanoparticle surface. The hydrophobic ligands on the nanoparticles then quickly phase separate the nanoparticles from the aqueous based suspension and confine them to the air-fluid interface. This drives the ligand-capped nanoparticles to form monolayer domains at the air-fluid interface.&#160; The use of water-miscible organic solvents is important as it enables the transport of the nanoparticles from the interface onto template-free substrates.&#160; The flow is mediated by a surface tension gradient3,4 and creates macroscopic, high-density, monolayer nanoparticle-ligand films.&#160; This self-assembly technique may be generalized to include the use of particles of different compositions, size, and shape and may lead to an efficient assembly method to produce low-cost, macroscopic, high-density, monolayer nanoparticle films for wide-spread applications.

A simple, robust and scalable technique to functionalize and self-assemble macroscopic nanoparticle-ligand monolayer films onto template-free substrates is described in this protocol.

This protocol describes a self-assembly technique to create macroscopic monolayer films composed of ligand-coated nanoparticles1,2. The simple, robust and ...

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 ...

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully understood. We have traced the trajectories of individual nanoparticles using liquid-cell transmission electron microscopy (TEM) to investigate the mechanism of the assembly process. Herein, we present the protocols used for liquid-cell TEM studies of the self-assembly mechanism. First, we introduce the detailed synthetic protocols used to produce uniformly sized platinum and lead selenide nanoparticles. Next, we present the microfabrication processes used to produce liquid cells with silicon nitride or silicon windows and then describe the loading and imaging procedures of the liquid-cell TEM technique. Several notes are included to provide helpful tips for the entire process, including how to manage the fragile cell windows. The individual motions of nanoparticles tracked by liquid-cell TEM revealed that changes in the solvent boundaries caused by evaporation affected the self-assembly process of nanoparticles. The solvent boundaries drove nanoparticles to primarily form amorphous aggregates, followed by flattening of the aggregates to produce a 2-dimensional (2D) self-assembled structure. These behaviors are also observed for different nanoparticle types and different liquid-cell compositions.

Here we introduce experimental protocols for the real-time observation of a self-assembly process using liquid-cell transmission electron microscopy.

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully ...

Fabrication of Polymer Microspheres for Optical Resonator and Laser Applications

This paper describes three methods of preparing fluorescent microspheres comprising &#960;-conjugated or non-conjugated polymers: vapor diffusion, interface precipitation, and mini-emulsion. In all methods, well-defined, micrometer-sized spheres are obtained from a self-assembling process in solution. The vapor diffusion method can result in spheres with the highest sphericity and surface smoothness, yet the types of the polymers able to form these spheres are limited. On the other hand, in the mini-emulsion method, microspheres can be made from various types of polymers, even from highly crystalline polymers with coplanar, &#960;-conjugated backbones. The photoluminescent (PL) properties from single isolated microspheres are unusual: the PL is confined inside the spheres, propagates at the circumference of the spheres via the total internal reflection at the polymer/air interface, and self-interferes to show sharp and periodic resonant PL lines. These resonating modes are so-called "whispering gallery modes" (WGMs). This work demonstrates how to measure WGM PL from single isolated spheres using the micro-photoluminescence (&#181;-PL) technique. In this technique, a focused laser beam irradiates a single microsphere, and the luminescence is detected by a spectrometer. A micromanipulation technique is then used to connect the microspheres one by one and to demonstrate the intersphere PL propagation and color conversion from coupled microspheres upon excitation at the perimeter of one sphere and detection of PL from the other microsphere. These techniques, &#181;-PL and micromanipulation, are useful for experiments on micro-optic application using polymer materials.

Protocols for the synthesis of microspheres from polymers, the manipulation of microspheres, and micro-photoluminescence measurements are presented.

This paper describes three methods of preparing fluorescent microspheres comprising &#960;-conjugated or non-conjugated polymers: vapor diffusion, ...

Microfluidic Preparation of Liquid Crystalline Elastomer Actuators

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation usually consists in the formation of droplets containing low molar mass liquid crystals at elevated temperatures. Subsequently, these particle precursors are oriented in the flow field of the capillary and solidified by a crosslinking polymerization, which produces the final actuating particles. The optimization of the process is necessary to obtain the actuating particles and the proper variation of the process parameters (temperature and flow rate) and allows variations of size and shape (from oblate to strongly prolate morphologies) as well as the magnitude of actuation. In addition, it is possible to vary the type of actuation from elongation to contraction depending on the director profile induced to the droplets during the flow in the capillary, which again depends on the microfluidic process and its parameters. Furthermore, particles of more complex shapes, like core-shell structures or Janus particles, can be prepared by adjusting the setup. By the variation of the chemical structure and the mode of crosslinking (solidification) of the liquid crystalline elastomer, it is also possible to prepare actuating particles triggered by heat or UV-vis irradiation.

This article describes the microfluidic process and parameters to prepare actuating particles from liquid crystalline elastomers. This process allows the preparation of actuating particles and the variation of their size and shape (from oblate to strongly prolate, core-shell, and Janus morphologies) as well as the magnitude of actuation.

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation ...

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 ...

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 ...

Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to nanomolar ranges, several orders of magnitude less than many physiological ligand concentrations. Optical nanostructures called zero mode waveguides (ZMWs), which are 100&#8722;200 nm in diameter apertures fabricated in a thin conducting metal such as aluminum or gold, allow imaging of individual molecules at micromolar concentrations of fluorophores by confining visible light excitation to zeptoliter effective volumes. However, the need for expensive and specialized nanofabrication equipment has precluded the widespread use of ZMWs. Typically, nanostructures such as ZMWs are obtained by direct writing using electron beam lithography, which is sequential and slow. Here, colloidal, or nanosphere, lithography is used as an alternative strategy to create nanometer-scale masks for waveguide fabrication. This report describes the approach in detail, with practical considerations for each phase. The method allows thousands of aluminum or gold ZMWs to be made in parallel, with final waveguide diameters and depths of 100&#8722;200 nm. Only common lab equipment and a thermal evaporator for metal deposition are required. By making ZMWs more accessible to the biochemical community, this method can facilitate the study of molecular processes at cellular concentrations and rates.

Described here is a nanosphere lithography method for parallel fabrication of zero mode waveguides, which are arrays of nanoapertures in a metal-clad glass microscopy coverslip for single molecule imaging at nano- to micromolar concentrations of fluorophores. The method takes advantage of colloidal crystal self-assembly to create a waveguide template.

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to ...

Bioengineering

Developing Sodium Alginate Hydrogel Microspheres for Biomedical Use

Preparation of Cross-Linked Sodium Alginate Microspheres with Different Metal Ions Using the Microfluidic Electrospray Technology

Microspheres are micrometer-sized particles that can load and gradually release drugs via physical encapsulation or adsorption onto the surface and within polymers. In the field of biomedicine, hydrogel microspheres have been extensively studied for their application as drug carriers owing to their ability to reduce the frequency of drug administration, minimize side effects, and improve patient compliance. Sodium alginate (ALG) is a naturally occurring linear polysaccharide with three backbone glycosidic linkages. There are two auxiliary hydroxyl groups present in each of the moieties of the polymer, which have the characteristics of an alcohol hydroxyl moiety. The synthetic ALG units can undergo chemical cross-linking reactions with metal ions, forming a cross-linked network structure of polymer stacks, ultimately forming a hydrogel. Hydrogel microspheres can be prepared using a simple process involving the ionic cross-linking properties of ALG. In this study, we prepared ALG-based hydrogel microspheres (ALGMS) using a microfluidic electrodeposition strategy. The prepared hydrogel microspheres were uniformly sized and well-dispersed, owing to accurate control of the microfluidic electrospray flow. ALGMS cross-linked with different metal ions were prepared using a microfluidic electrospray technique combining microfluidic and high electric field, and its antimicrobial properties, slow drug release ability, and biocompatibility were investigated. This technology holds promise for application in advanced drug development and production.

Author Spotlight: Advancing Therapeutics with Biocompatible Sodium Alginate Hydrogel Microspheres

The protocol describes the preparation of sodium alginate microspheres cross-linked with different metal ions using a microfluidic device for drug carrier design. The antimicrobial properties and slow drug release of these microspheres were also investigated.

Microspheres are micrometer-sized particles that can load and gradually release drugs via physical encapsulation or adsorption onto the surface and within ...

Fabrication of Spherical and Worm-shaped Micellar Nanocrystals by Combining Electrospray, Self-assembly, and Solvent-based Structure Control

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