Name: preparation of hollow polystyrene particles and microcapsules by radical polymerization of janus droplets consisting of hydrocarbon and fluorocarbon oils
Uploaded: 2018-01-25T13:30:00.000+00:00
Duration: 7 min 1 s
Description: Watch this Scientific Journal Video about Preparation of Hollow Polystyrene Particles and Microcapsules by Radical Polymerization of Janus Droplets Consisting of Hydrocarbon and Fluorocarbon Oils at J

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Attenzione: Si prega di indossare un camice da laboratorio, guanti e occhiali di sicurezza e leggere le schede di sicurezza (MSDS) prima dell'uso. Tutti i materiali acquistati sono stati utilizzati senza ulteriore purificazione.
1. preparazione di emulsioni costituite da miscele di SDS stirene/PFO/acquoso soluzione
<ol><li>Per preparare 5 mM di soluzione acquosa di SDS, sciogliere 14,5 mg di SDS in 10 mL di elevata purezza H2O. Nota: È stato utilizzato ad alta purezza H2O (resistività (R) = 18 cm MΩ, tensione superficiale (γ) = 72,0 mN m-1 a 25 ° C).</li>
	<li>Aggiungere 1,5 g di stirene, 0,6 g di PFO e 0,9 g di 5 mM di soluzione acquosa di SDS per un flaconcino di vetro da 10 mL con un'ancoretta. Nota: Nella scansione osservazioni di microscopia di fluorescenza confocale, aggiungere 2,6 mg di cumarina 102 e 0,062 mg di calceina alla miscela.</li>
	<li>Mescolare l'impasto per 60 min a 1.150 giri sotto temperatura ambiente e quindi aumentare la temperatura a 80 ° C.</li>
	<li>Mescolare l'impasto per 60 min a 1.150 giri e 80 ° C.</li>
</ol>2. fabbricazione di particelle di polimero usando emulsioni consistenti di stirene/PFO/acquoso SDS soluzione miscele
<ol><li>Aggiungere un flaconcino di vetro da 10 mL con un'ancoretta 3,9 mg di potassio Perossidisolfato, 2 mg di pirene, 1,5 g di stirene, 0,6 g di PFO e 0,9 g di 5 mM di soluzione acquosa di SDS e sigillare con un setto di gomma.</li>
	<li>Deoxygenate la miscela di gas di azoto bubbling attraverso un ago di siringa per 30 min. Attenzione: Lentamente il flusso del gas di azoto per non generare una grande quantità di schiuma.</li>
	<li>Per preparare l'emulsione, mescolare l'impasto per 60 min a 1.150 giri e temperatura ambiente e quindi aumentare la temperatura a 80 ° C.</li>
	<li>Mescolare il composto per 30 min a 1.150 giri e 80 ° C.</li>
	<li>Trasferire la parte nuvolosa (1,8 mL) della soluzione risultante (3ml) alla provetta e quindi aggiungere la soluzione acquosa di etanolo al 30% per terminare completamente la reazione di polimerizzazione.</li>
	<li>Sottoporre ad ultrasuoni (potenza: 130 W, frequenza: 4,2 kHz) per 10 min a lavare le particelle di polimero risultante e poi Centrifugare per 10 min a 2300 x g.</li>
	<li>Per ottenere le particelle di polimero, è necessario rimuovere il supernatante dalla provetta.</li>
	<li>Aggiungere 3 mL di acqua per i solidi risultanti nella provetta. Sottoporre ad ultrasuoni (potenza: 130 W, frequenza: 4,2 kHz) per 10 min e centrifugare per 10 min 2300 g. x rimuovere il supernatante dalla provetta.</li>
	<li>Ripetere la procedura di lavaggio (passo 2,8) fino a quando la schiuma non viene generato dalla soluzione surnatante. Evaporare l'acqua per ottenere il solido di particelle di polimero hollow.</li>
</ol>3. preparazione di microcapsule usando le particelle di polimero cava
<ol><li>Aggiungere 1 mg di particelle di polistirene hollow (punto 2.9) e 4 mL di acqua per un flaconcino di vetro da 10 mL con un'ancoretta.</li>
	<li>Sottoporre ad ultrasuoni (potenza: 130 W, frequenza: 4,2 kHz) per 10 min disperdere le particelle vuote in acqua.</li>
	<li>Aggiungere 0,1 mL di toluene all'acqua disperse le particelle e poi mescolare per 1 h a 100 giri/min a temperatura ambiente. Trasferire il liquido in una provetta. Attenzione: Se la rotazione al minuto aumenta, la morfologia delle particelle si deformerà.</li>
	<li>Centrifugare il liquido per 10 min a 2300 x g per isolare le microcapsule si formano. Rimuovere il supernatante dalla provetta.</li>
</ol>

<ol>
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Non abbiamo nulla di divulgare.

Le emulsioni contenenti goccioline di Janus di stirene e PFO sono state ottenute sopra 10 wt % della frazione peso di concentrazione acquosa di SDS e qualsiasi frazione di peso di stirene e PFO. Quando le varie frazioni di peso delle emulsioni contenenti le goccioline di Janus sono state polimerizzate per 30 min, le particelle di polistirene Cave sono ottenibili per ogni composizione. Questi risultati indicano che il protocollo ha dimostrato qui è semplice. Inoltre, il diametro, dimensione di foro e foro volume delle particelle polistirolo vuote può essere controllati con il tempo di polimerizzazione nel metodo descritto qui.
Suggeriamo il seguente meccanismo per la realizzazione della Cave particelle di polistirene. Gocce d'olio di Janus costituito di stirene e PFO formata in emulsioni O/W contenenti miscele di soluzione acquosa SDS, stirene e PFO a 80 ° C. Goccioline PFO si trovano sulla superficie delle goccioline di stirene. Poiché KPS, un iniziatore di polimerizzazione, è solubile nella fase acquosa, la polimerizzazione progredirà all'interfaccia della soluzione SDS stirene/acquosa. Stirene nelle emulsioni ternarie era polimerizzato a polistirolo a 80 ° C, mentre la PFO, non avendo nessun gruppi polimerizzabili, è rimasta in emulsioni a quella temperatura. Dopo la polimerizzazione è stata terminata, un foro sulla particella polistirolo fu creato dalla rimozione del PFO. Di conseguenza, le particelle di polistirene cava Coppa-tipo avendo un foro sulla superficie erano fabbricati Chiunquesi frazione di peso di stirene e PFO e tutti i tempi di polimerizzazione.
Aggiungendo una piccola quantità di toluene per le particelle di polistirene Cave fabbricate tramite polimerizzazione radicale per 90 min porta i fori sulle particelle di polistirolo vuote di tenuta. Questo fenomeno è coerente con quello riportato da Hyuk et al.9. Il metodo lipooligosaccharide può contenere un materiale acquoso nelle particelle.
In questo articolo, abbiamo dimostrato un metodo di fabbricazione delle particelle di polimero hollow utilizzando le goccioline di olio di Janus costituito di stirene e PFO, che sono mutuamente immiscibili. Il metodo di preparazione di particelle di polimero non-sferico è già stato studiato a causa del loro potenziale uso in varie applicazioni. Questa strategia utilizzando goccioline di olio di idrocarburi e fluorocarbon per Janus si applica alla fabbricazione di particelle di polimero non sferica da vari tipi di monomero e drug delivery system.

Mentre le particelle sferiche polimero sono stati ampiamente utilizzate in diverse applicazioni industriali, è ben noto che le particelle fossetta, emisferi, dischi ed Elissoidi, sono state preparate da seminato polimerizzazioni1,2, 3, foto-polimerizzazione delle goccioline di monomero non sferica utilizzando microreattore con geometrie specifiche4,5, auto-organizzazione utilizzando riprecipitazione di polimeri6e deformazione dei polimeri sferici le particelle da esterno meccanico forza7,8. In particolare, particelle di polimero cava con micrometro-dimensione sono state fabbricate da evaporazione di un buon solvente da particelle di polimero sferica gonfiata dal solvente9,10 e polimerizzazione utilizzando emulsioni multiple 11 , 12.
In questo lavoro, ci concentriamo sull'uso del reciproco immiscibilità di idrocarburi e fluorocarbon oli nella fabbricazione delle particelle di polimero. Tensioattivi di ibridi hanno una catena di idrocarburi e una catena di fluorocarburo nella molecola. Precedentemente, abbiamo segnalato le proprietà uniche dei tensioattivi ibrido che non si osservano in tensioattivi convenzionali13,14,15. Inoltre abbiamo studiato emulsioni utilizzando olio di idrocarburi, olio fluorocarburo e soluzione acquosa tensioattivo, che sono mutuamente immiscibili16. Tuttavia, ci sono pochissimi studi delle emulsioni17. Questi studi hanno descritto la morfologia delle goccioline di olio composto da olio di idrocarburi e olio di fluorocarbonio in soluzioni acquose tensioattivo.
Qui, indichiamo un protocollo dettagliato della fabbricazione delle particelle di polimero cava tramite polimerizzazione radicale usando gocce d'olio nelle emulsioni consistenti di idrocarburo, olio fluorocarburo e acquosa sodio dodecilsolfato (SDS) soluzioni. Suggeriamo una nuova strategia, che è diversa dai metodi convenzionali, per la preparazione di particelle di polimero non-sferico. Questo metodo può semplicemente fabbricare le particelle di polimero cava in breve tempo. Inoltre, il protocollo della preparazione di microcapsule attraverso le particelle di polimero hollow è indicato.

<table><tbody><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sodium dodecylsulfate, 95.0%</td><td>Wako Pure Chemical Industries, Ltd.</td><td>192-08672</td><td></td></tr><tr><td>Styrene, 99.0%</td><td>Tokyo Chemical Industry Co. Ltd.</td><td>S0095</td><td></td></tr><tr><td>Perfluorooctane, 99%</td><td>Fluorochem Ltd.</td><td>8706</td><td></td></tr><tr><td>Coumarin 102, 97.0%</td><td>Tokyo Chemical Industry Co. Ltd.</td><td>C2267</td><td></td></tr><tr><td>Calcein</td><td>Dojindo Molecular Technologies, Inc.</td><td>C001</td><td></td></tr><tr><td>Potassium peroxodisulfate, 98.0%</td><td>Kanto Chemical Co., Inc.</td><td>32375-30</td><td></td></tr><tr><td>Pyrene, 97.0%</td><td>Wako Pure Chemical Industries, Ltd.</td><td>167-05302</td><td></td></tr><tr><td>Ethanol, 99.5%</td><td>Kanto Chemical Co., Inc.</td><td>14033-00</td><td></td></tr><tr><td>Toluene, 99.5%</td><td>Kanto Chemical Co., Inc.</td><td>40180-00</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Scanning Electron Microscope</td><td>Hitachi High-Technologies Corporation</td><td>S-4800</td><td></td></tr><tr><td>Scanning Transmission Electron Microscope</td><td>Hitachi High-Technologies Corporation</td><td>S-4800</td><td></td></tr><tr><td>Ultrasonic cleaner</td><td>Branson Ultrasonics, Emerson Japan, Ltd.</td><td>Model 3510</td><td></td></tr><tr><td>Centrifuge</td><td>AS ONE Corporation</td><td>CN-1050</td><td></td></tr></tbody></table>

preparation of hollow polystyrene particles and microcapsules by radical polymerization of janus droplets consisting of hydrocarbon and fluorocarbon oils

In questo articolo, abbiamo dimostrato un metodo per produrre particelle vuote e microcapsule usando le goccioline di olio composto da olio di idrocarburi (stirene) e fluorocarburi (perfluoro -n-ottano, PFO) in acquoso tensioattivo (sodio dodecilsolfato, Soluzioni di SDS). Poiché fluorocarburo oli sono immiscibili con oli di idrocarburi, i due oli sono separati. Emulsioni sono preparati da agitazione stirene/PFO/acquosa miscele di soluzione SDS a 80 ° C. Il tipo delle emulsioni e la morfologia delle goccioline nelle emulsioni sono osservati da microscopio ottico e microscopio a fluorescenza confocale. È trovato che le goccioline di olio con morfologie di Janus-tipo costituito di stirene reciprocamente immiscibili e PFO si formano in soluzione acquosa di SDS. Particelle di polistirene sono fabbricate tramite polimerizzazione radicale di mischie ternarie di stirene/PFO/acquoso soluzione di SDS a 80 ° C. Le morfologie del polistirolo sono confermate da microscopia elettronica a scansione e scansione osservazioni di microscopia elettronica di trasmissione. Queste osservazioni mostrano la preparazione di particelle di polistirene Cave con un unico foro sulla superficie. A nostra conoscenza, questo metodo è una strategia novella utilizzando l'immiscibilità di oli idrocarburi e fluorocarbon. Le particelle di Cave possono essere applicate anche alla preparazione delle microcapsule.

Luce e osservazioni di microscopia di fluorescenza confocale di scansione sono stati effettuati per determinare la morfologia e la composizione delle goccioline nelle emulsioni costituito di stirene, PFO e soluzione di SDS acquosa 5 mM (Figura 1). Analisi in microscopia elettronica (SEM) e analisi osservazioni di microscopia elettronica (STEM) trasmissione ha mostrato la formazione di particelle vuote e microcapsule (Figura 2).
Il DIC e immagini di microscopia di fluorescenza confocale di emulsioni costituite da miscele ternarie di stirene/PFO/acquoso soluzione SDS indicano che la fase continua è la soluzione acquosa di SDS, perché fluorescenza verde corrispondente alla calceina è stata osservata in la fase continua, e di conseguenza, si formano emulsioni olio-in-acqua tipo (Figura 1a e 1b). Queste immagini mostrano anche che le goccioline di olio consistono di stirene e PFO, che sono mutuamente immiscibili. Il DIC e immagini di microscopia di fluorescenza confocale di emulsioni contenenti cumarina 102 rivelano che una gocciolina PFO si trova all'interfaccia tra acqua e una goccia di stirene. Le miscele ternarie di stirene, PFO e soluzione acquosa di SDS alle emulsioni di forma 5 mM contenenti Janus olio goccioline, che consistono di stirene e PFO.
Le immagini di SEM e staminali delle particelle di polimero fabbricate dalla polimerizzazione radicale delle emulsioni di soluzione acquosa di SDS stirene/PFO/5mm mostrano la preparazione di particelle di polistirene non sferica Cave con un foro sulla superficie (Figura 2a e 2b). il tasso di formazione, il diametro e la dimensione del foro della Cave particelle di polistirene vengono valutati osservando 200 particelle attraverso SEM e staminali. Il diametro medio stimato dalle osservazioni di SEM è 1.3 µm. La dimensione è coerenza con il diametro delle particelle di polimero stimato da misure di dispersione della luce dinamica. Il tasso di formazione è circa il 100%. La media di dimensione del foro e il volume del foro nelle particelle vuote sono 0,8 µm ± 0,4 e 0,9 ± 0,4 µm3, rispettivamente. Di conseguenza, le particelle di polistirene Cave munito di un foro di 0,8 µm furono fabbricate tramite polimerizzazione radicale di goccioline di Janus contenenti stirene nelle emulsioni.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56922/56922fig1.jpg"> Figura 1. Morfologie delle goccioline nelle emulsioni consistenti di soluzione acquosa di SDS stirene, PFO e 5 mM. (a) contrasto differenziale di interferenza (DIC) e (b) immagini di microscopia di fluorescenza confocale delle emulsioni contenenti calceina fluorescente acquosa. (c) DIC e (d) immagini di microscopia confocal di fluorescenza delle emulsioni contenenti olio solubile fluorescente cumarina 102. <a href="//cloudflare.jove.com/files/ftp_upload/56922/56922fig1large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56922/56922fig2.jpg"> Figura 2. Morfologie di Cave particelle di polistirene e microcapsule fabbricati con emulsioni consistenti di soluzione acquosa di SDS stirene, PFO e 5 mM. (a) immagini di staminali SEM e (b) di particelle di polistirene hollow. (c) SEM e (d) immagini di staminali di microcapsule. <a href="//cloudflare.jove.com/files/ftp_upload/56922/56922fig2large.jpg" target="_blank">Clicca qui per visualizzare una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Preparation of Hollow Polystyrene Particles and Microcapsules by Radical Polymerization of Janus Droplets Consisting of Hydrocarbon and Fluorocarbon Oils at J

Preparation of Hollow Polystyrene Particles and Microcapsules by Radical Polymerization of Janus Droplets Consisting of Hydrocarbon and Fluorocarbon Oils

Preparazione di Cave particelle di polistirene e microcapsule tramite polimerizzazione radicale di goccioline di Janus composto da idrocarburi e oli Fluorocarbon

Un protocollo per la fabbricazione di particelle di polimero hollow e microcapsule di polimerizzazione radicalica utilizzando emulsioni consistenti di stirene, perfluoro -n-ottano e acquosa SDS (sodio dodecilsolfato) soluzione è presentata.

In this article, we have demonstrated a method for producing hollow particles and microcapsules using oil droplets consisting of hydrocarbon oil (styrene) ...

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9.9K Views.  Tokyo University of Science. Un protocollo per la fabbricazione di particelle di polimero hollow e microcapsule di polimerizzazione radicalica utilizzando emulsioni consistenti di stirene, perfluoro -n-ottano e acquosa SDS (sodio dodecilsolfato) soluzione è presentata.

Video: Preparation of Hollow Polystyrene Particles and Microcapsules by Radical Polymerization of Janus Droplets Consisting of Hydrocarbon and Fluorocarbon Oils

Synthesis of Poly(N-isopropylacrylamide) Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability

Janus microparticles are compartmentalized particles with differing molecular structures and/or functionality on each of their two sides. Because of this unique property, Janus microparticles have been recognized as a new class of materials, thereby attracting a great deal of attention from various research fields. The versatility of these microparticles has been exemplified through their uses as building blocks for self-assembly, electrically responsive actuators, emulsifiers for painting and cosmetics, and carriers for drug delivery. This study introduces a detailed protocol that explicitly describes a synthetic method for designing novel Janus microhydrogels composed of a single base material, poly(N-isopropylacrylamide) (PNIPAAm). Janus microdroplets are firstly generated via a hydrodynamic focusing microfluidic device (HFMD) based on the separation of a supersaturated aqueous NIPAAm monomer solution and subsequently polymerized through exposure to UV irradiation. The resulting Janus microhydrogels were found to be entirely composed of the same base material, featured an easily identifiable compartmentalized morphology, and exhibited anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability. We believe that the proposed method introduces a novel hydrogel platform with the potential for advanced synthesis of multi-functional Janus microhydrogels.

Synthesis of PolyN-isopropylacrylamide Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability

We present a protocol to synthesize Janus microhydrogels composed entirely of the same base material, poly(N-isopropylacrylamide) (PNIPAAm), with a clearly compartmentalized structure base on the phase separation of a supersaturated NIPAAm monomer solution. The synthesized Janus microhydrogels show unique properties such as anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability.

Sintesi di Poly (<em&gt; N</em&gt; -isopropylacrylamide) Janus Microhydrogels per anisotropico Thermo-reattività e organofile / idrofilo Caricamento Capability

Janus microparticles are compartmentalized particles with differing molecular structures and/or functionality on each of their two sides. Because of this ...

Ricerca

Chimica

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;.

Utilizzando Polystyrene-<em&gt; Blocco</em&gt; -poly (Acido acrilico) Rivestiti metallo Nanoparticelle come monomeri per loro omo- e co-polimerizzazione

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

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

High-throughput sintesi dei carboidrati e funzionalizzazione di nanoparticelle polianidride

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

Bioingegneria

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

High Throughput singola cellula e cellula Multiple-Micro-incapsulamento

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

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.

Sintesi facile di micelle vermiforme dalla luce visibile mediata polimerizzazione in dispersione Utilizzando Photoredox Catalyst

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

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.

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.

Anionico polimerizzazione di un copolimero Amphiphilic per la preparazione di copolimero a blocchi micelle stabilizzata da π-¸ Interazioni Stacking

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

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.

Fabbricazione di microsfere di polimeri per risonatore ottico e applicazioni laser

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

Ingegneria

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.

Preparazione di microfluidica di liquido cristallino elastomero attuatori

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.

Sintesi di nanoparticelle cilindriche monodisperse tramite l'autoassemblaggio di copoliomeri a blocchi biodegradabili

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

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and morphology of the obtained microsphere. A microsphere copolymerized with an acrydite-DNA probe was successfully fabricated. Different methods such as asymmetric PCR, exonuclease digestion, and isolation on streptavidin-coated magnetic beads can be used to synthesize single-stranded DNA (ssDNA). However, these methods cannot efficiently use large amounts of highly purified ssDNA. Here, we describe a microsphere-PCR protocol detailing how ssDNA can be efficiently amplified and separated from dsDNA simply by pipetting from a PCR reaction tube. The amplification of ssDNA can be applied as potential reagents for the DNA microarray and DNA-SELEX (Systematic evolution of ligands by exponential enrichment) processes.

This work provides a method for the fabrication of droplet-based microfluidic platforms and the application of polyacrylamide microspheres for microsphere-PCR amplification. The microsphere-PCR method makes it possible to obtain single-stranded DNA amplicons without separating double-stranded DNA.

Preparazione di Cave particelle di polistirene e microcapsule tramite polimerizzazione radicale di goccioline di Janus composto da idrocarburi e oli Fluorocarbon

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Sintesi di Poly (

Utilizzando Polystyrene-