Funding for this project was through NSF CBET Award 1061834. The authors would like to acknowledge CIBA for a sample of photoinitiator.

1. Preparing Solutions for Use in Microgel Fabrication
<ol>
	<li>Before beginning the precipitation reaction, make the necessary solutions and warm them to 37 &#176;C. The required solutions include 0.5% photoinitiator, 1.5 M Na2SO4, buffer solution, 200 mg/ml PEG-diacrylate (PEG-DA) solution, and 1x phosphate buffered saline (PBS). Note: Acrylate all PEG precursors according to published methods and store at -20 &#176;C under argon until use12.</li>
	<li>Weigh out photoinitiator and dissolve in deionized (DI) H2O for a 0.5 (w/v)% solution. It is important to protect the solution from exposure to UV light.
	<ol>
		<li>After full dissolution, filter through a 0.22 &#956;m filter. This solution can be made in advance and stored at 4 &#176;C in the dark. Note: Decrease the stirring time to approximately 1 hr by heating the solution to 40 &#176;C while stirring.</li>
	</ol>
	</li>
	<li>For 1x PBS, add 1 tablet PBS to 1 L DI H2O and filter through a 0.22 &#956;m filter. PBS can be made in advance and stored at 4 &#176;C.</li>
	<li>Measure 39.4 ml&#160;of phosphate buffered saline (PBS, pH=7.4) and add 612 &#956;l triethanolamine (TEOA), and 260 &#956;l&#160;of 6 M hydrochloric acid (HCl). Note: TEOA is very viscous; preheat TEOA to 37 &#176;C prior to pipetting.
	<ol>
		<li>Adjust the pH of the buffer so that it is approximately 7.7 and filter through a 0.22 &#956;m filter (final pH ~7.8). The buffer solution can be scaled up, made in advance, and stored at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Make the PEG-DA solution prior to beginning the reaction each time; decreased reactivity has been observed if the solution is stored. Weigh out PEG-DA and add PBS/TEOA/HCl buffer to final concentration of 200 mg/ml. Note: Add buffer slowly to carefully control PEG concentration because PEG swells in solution. 
	Note: This solution is highly customizable. Options for customizing microgels include: changing the starting molecular weight of PEG to alter microgel properties, using a degradable PEG precursor to fabricate degradable microgels, sterile filtering all of the solutions for biologically sterile microgels, completing the process of formation in a laminar flow hood, and finally adding functional groups to the surface by incorporating them in the PEG solution.</li>
	<li>Weigh out Na2SO4 and dissolve in DI H2O for a 1.5 M solution. For best results, the sodium sulfate solution must also be made just prior to beginning the reaction.
	<ol>
		<li>Heat and vortex the solution until Na2SO4 is completely dissolved. Filter through 0.22 &#956;m filter as necessary.</li>
	</ol>
	</li>
</ol>
2. Microgel Fabrication
<ol>
	<li>Place 127.5 &#956;l&#160;of PBS/TEOA/HCl buffer, 10 &#956;l&#160;of 0.5% photoinitiator, and 25 &#956;l&#160;of 200 mg/ml&#160;PEG solution into the tubes where the microgels will be formed; typically 2 ml&#160;microcentrifuge tubes.
	<ol>
		<li>Heat these tubes along with the 1.5 M Na2SO4 solution to 37 &#176;C.</li>
		<li>Mix the tubes while heating. Note: This microgel protocol&#160;can be scaled up.</li>
	</ol>
	</li>
	<li>To encapsulate molecules into the microgels, add the desired molecule (e.g.&#160;ovalbumin) to the PBS/TEOA/HCl buffer, photoinitiator, and PEG solution. After crosslinking the molecule will be trapped in the microgel network.</li>
	<li>Complete each microgel reaction one at a time. Take the first tube and add 87.5 &#956;l&#160;of 1.5 M Na2SO4.</li>
	<li>Mix tube by pipetting 3-5x; alternatively wait 30 sec. After mixing, the salt concentration will be evenly distributed and the solution should be clear.</li>
	<li>Place the tube under the UV light and crosslink for 30 sec. Following crosslinking there should be a cloudy layer on top of the solution, this layer contains the microgels. See Figure 1 for reaction scheme.</li>
	<li>Add 750 &#956;l&#160;PBS to form a pellet upon centrifugation and mix well.</li>
	<li>Wash the microgels 5 x by centrifuging for 2 min at 4,000 x&#160;g to form a pellet and exchanging the supernatant with PBS. Note: Do not disturb the microgel pellet while buffer exchanging, it may not be possible to remove all the supernatant.</li>
</ol>
3. Microgel Size and Polydispersity
<ol>
	<li>Pipette 30 &#956;l&#160;of microgels from the pellet formed from centrifuging in 1 ml&#160;of DI H2O. For best results, sonicate the microgel solution in a water bath sonicator for 1 hr (50-60 Hz and 1.4 A) or mix vigorously to suspend the microgels.</li>
	<li>Pipette 15 &#956;l&#160;of the microgel solution onto a clean glass slide and put #1.5 cover slip on top. Flip the slide and cover slip over onto a laboratory tissue and push down on the slide to remove excess solution and ensure that the microgels are in a single layer.</li>
	<li>Capture several images of the microgels using a DIC 100X oil objective. Typically 5 images are taken per sample and 3 samples per batch are recommended for a total of 15 images. Note: no batch to batch variation has been observed to date.</li>
	<li>Measure the microgels to obtain a representative diameter.</li>
	<li>Calculate PDI using the following volume based equation in which N is the total number of microgels and Vi is the volume of microgel i.</li>
</ol>
4. Microgel Density
<ol>
	<li>Prepare dextran 7, 6, 5, 4, 3, and 1 (w/v)% solutions in DI H2O to determine microgel density. These densities were calculated to be 1.022, 1.020, 1.018, 1.016, 1.014, and 1.010 g/cm3, respectively.</li>
	<li>Add 1 ml&#160;of 7% dextran solution to a 15 ml&#160;centrifuge tube.
	<ol>
		<li>Slowly pipette 1 ml&#160;of 6% dextran solution to form a distinct layer atop the 7% dextran.</li>
		<li>Wait 5 min then add 1 ml&#160;of the 5% solution.</li>
		<li>Continue until all dextran solutions are layered in order of decreasing dextran concentration.</li>
	</ol>
	</li>
	<li>Carefully layer the microgels above the 1% dextran solution.</li>
	<li>Centrifuge the gradient for 10 min at 4,000 x&#160;g and 5 &#176;C. Microgel position after centrifugation allowed for simple calculation of density13.</li>
</ol>
5. Microgel Equilibrium Swelling
<ol>
	<li>To measure the swollen mass (Ms) of the microgels, defined as the mass after equilibrium swelling (48 hr), first wash the microgels 5x in DI H2O and swell for a minimum of 48 hr in preweighed microcentrifuge tubes.</li>
	<li>Centrifuge the microgels for 10 min at 4,000 x g.</li>
	<li>Remove all supernatant and record the wet mass. Use filtering methods to ensure all excess water is removed to obtain more accurate results.</li>
	<li>Freeze the microgels at -80 &#176;C then lyophilize to constant mass to obtain the dry mass (Md).</li>
	<li>Calculate the water content in the microgels by Water% = (Ms - Md) / Md x 100.</li>
	<li>Determine polymer content by Polymer% = 100 - Water%&#160;.</li>
	<li>Perform statistical analysis on all data using SAS with n-way ANOVA and Tukey post-hoc test. Differences are noted when p&#60;0.05.</li>
</ol>

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The authors declare that they have no competing financial interests.

Physical properties of PEG microgels were examined for changes in polymerization conditions. For this precipitation reaction, a buffer solution, 20% (w/v)&#160;PEG-diacrylate (MW 3,000, 4,600, or 6,000 Da) solution, and photoinitiator were mixed and warmed to 37 &#176;C. Addition of 1.5 M Na2SO4, a kosmotrophic salt that increases the interactions between water molecules, caused PEG to momentarily precipitate upon its addition. This effect is more prominent with higher molecular weight PEG8</su...

By definition, microgels are hydrogels of any shape with an equivalent diameter of approximately 0.1-100 &#956;m1. Because of their size and characteristics, polymeric microgels present a versatile tool for advancing drug delivery and tissue engineering systems. While bulk hydrogels are extensively utilized as tissue engineering scaffolds and drug delivery vehicles with great success2-4, a recent shift to microscale control of scaffolds provides a unique opportunity for microgels to be used as base materials for building scaffolds. In addition, microgels have a high surface area to volume ratio for cellular interactions and in solution have a low viscosity that makes them ideal for injections. Finally, microgels can be formed using numerous polymers by a variety of methods dependent on the desired microgel characteristics, making them highly customizable for a variety of applications.
Techniques to produce microgels include suspension, emulsion, dispersion, or precipitation polymerizations. Emulsion and suspension polymerizations typically require organic solvents and surfactants or stabilizers to form the microgels. The nature of these methods yield a highly disperse particle size distribution5. Dispersion and precipitation reactions render particles with a lower polydispersity6; however particles formed by dispersion polymerization still require the use of stabilizing agents6. Microgels formed by precipitation reactions are unique in that they form particles of uniform size and shape without the use of stabilizers or surfactants. Microgel formation is achieved when growing polymer chains phase separate from the continuous phase by enthalpic or entropic precipitation7. Precipitation polymerization is often at high temperatures that can be lowered by the use of kosmotrophic salts, which decrease the solubility of the polymer in the solvent8. This work focuses on microgels formed from poly(ethylene glycol) (PEG) by a photopolymerized precipitation reaction under biologically-compatible conditions, with variations to alter microgel properties and encapsulate molecules for drug delivery applications.
Previous studies with PEG hydrogels show that the polymerization conditions greatly influence the physical and mechanical properties of hydrogels, namely the hydrogel water content and compressive modulus3,9. These crosslinked materials are of interest because the relationship between structural and physical properties described by Flory10 can be utilized to tailor the crosslinked hydrogel for specific applications. These principles are similar for microgels. Precipitation-formed PEG microgels have been found to have potential for regenerative medicine11, however further investigation into the microgel properties was necessary to enhance their repertoire for future biomedical applications. This report describes the procedure to fabricate microgels by precipitation reaction and alter characteristics, such as microparticle diameter, polydispersity index (PDI), density, and swelling, that would be important to further develop these materials for drug delivery or regenerative medicine.

<table><tbody><tr><td>Phosphate Buffered Saline (PBS)</td><td>MP Biomedical</td><td>2810305</td><td></td></tr><tr><td>Triethanolamine (TEOA)</td><td>J.T. Baker</td><td>9468-01</td><td>Preheat to 37 &amp;deg;C prior to pipetting</td></tr><tr><td>Hydrochloric acid (HCl)</td><td>BDH Aristar</td><td>BDH3028</td><td></td></tr><tr><td>Sodium Sulfate</td><td>J.T. Baker</td><td>3891-01</td><td></td></tr><tr><td>Irgacure 2959</td><td>Ciba</td><td>029891301PS04</td><td></td></tr><tr><td>Ovalbumin (OVA)</td><td>Invitrogen</td><td>34782</td><td></td></tr><tr><td>PEG 1,500</td><td>Alfa Aesar</td><td>A16241</td><td></td></tr><tr><td>PEG 3,000</td><td>Fluka</td><td>03997-1KG</td><td></td></tr><tr><td>PEG 4,000</td><td>Alfa Aesar</td><td>A16151</td><td></td></tr><tr><td>PEG 4,600</td><td>Sigma</td><td>373001-250G</td><td></td></tr><tr><td>PEG 6,000</td><td>Fluka</td><td>03394-1KG</td><td></td></tr><tr><td>PEG 10,000</td><td>Alfa Aesar</td><td>B21955</td><td></td></tr><tr><td>Dextran 70</td><td>TCI</td><td>D1449</td><td></td></tr></tbody></table>

characteristics of precipitation-formed polyethylene glycol microgels are controlled by molecular weight of reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer several advantages over traditional microsphere fabrication techniques. Contrary to emulsion, suspension, and dispersion techniques, microgels formed by precipitation are of uniform shape and size, i.e.&#160;low polydispersity index, without the use of organic solvents or stabilizers. The mild conditions of the precipitation reaction, customizable properties of the microgels, and low viscosity for injections make them applicable for in vivo purposes. Unlike other fabrication techniques, microgel characteristics can be modified by changing the starting polymer molecular weight. Increasing the starting PEG molecular weight increased microgel diameter and swelling ratio. Further modifications are suggested such as encapsulating molecules during microgel crosslinking. Simple adaptations to the PEG microgel building blocks are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

Microgel size is dependent on polymerization conditions. Figure 2A illustrates how microgel diameter increases with increasing PEG starting molecular weight and crosslinking time. Representative images of microgels used for sizing for various molecular weights and crosslinked for 30 sec are shown in Figures 2C-G. For PEG with a molecular weight of 3,000 Da, the microgel average diameter increased from 1.65&#177;0.26 to 2.20&#177;0.54 &#956;m as UV exposure increased from 30-600 sec (<str...

Watch this Scientific Journal Video about Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants at JoVE.com

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Increasing the PEG molecular weight increased microgel diameter and swelling ratio. Simple adaptations to the PEG microgel precipitation reaction are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer ...

characteristics-precipitation-formed-polyethylene-glycol-microgels

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11.8K Views.  The University of Akron. This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Increasing the PEG molecular weight increased microgel diameter and swelling ratio. Simple adaptations to the PEG microgel precipitation reaction are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

Video: Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

Chemistry

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.

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

Facile Synthesis of Worm-like Micelles by Visible Light Mediated Dispersion Polymerization Using Photoredox Catalyst

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins with the synthesis of a hydrophilic poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) homopolymer using reversible addition-fragmentation chain-transfer (RAFT) polymerization. Under mild visible light irradiation (&#955; = 460 nm, 0.7 mW/cm2), this macro-chain transfer agent (macro-CTA) in the presence of a ruthenium based photoredox catalyst, Ru(bpy)3Cl2 can be chain extended with a second monomer to form a well-defined block copolymer in a process known as Photoinduced Electron Transfer RAFT (PET-RAFT). When PET-RAFT is used to chain extend POEGMA with benzyl methacrylate (BzMA) in ethanol (EtOH), polymeric nanoparticles with different morphologies are formed in situ according to a polymerization-induced self-assembly (PISA) mechanism. Self-assembly into nanoparticles presenting POEGMA chains at the corona and poly(benzyl methacrylate) (PBzMA) chains in the core occurs in situ due to the growing insolubility of the PBzMA block in ethanol. Interestingly, the formation of highly pure worm-like micelles can be readily monitored by observing the onset of a highly viscous gel in situ due to nanoparticle entanglements occurring during the polymerization. This process thereby allows for a more reproducible synthesis of worm-like micelles simply by monitoring the solution viscosity during the course of the polymerization. In addition, the light stimulus can be intermittently applied in an ON/OFF manner demonstrating temporal control over the nanoparticle morphology.

This article describes a process for producing polymeric self-assembled nanoparticles using visible light mediated dispersion polymerization. Using low energy visible light to control the polymerization allows for the reproducible formation of self-assembled worm-like micelles at high solids content.

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

Controlled Synthesis and Fluorescence Tracking of Highly Uniform Poly(N-isopropylacrylamide) Microgels

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this work, we use single particle tracking of fluorescently labeled PNIPAM microgels as a showcase for tuning microgel size by a rapid non-stirred precipitation polymerization procedure. This approach is well suited for prototyping new reaction compositions and conditions or for applications that do not require large amounts of product. Microgel synthesis, particle size and structure determination by dynamic and static light scattering are detailed in the protocol. It is shown that the addition of functional comonomers can have a large influence on the particle nucleation and structure. Single particle tracking by wide-field fluorescence microscopy allows for an investigation of the diffusion of labeled tracer microgels in a concentrated matrix of non-labeled microgels, a system not easily investigated by other methods such as dynamic light scattering.

Controlled Synthesis and Fluorescence Tracking of Highly Uniform PolyN-isopropylacrylamide Microgels

Non-stirred precipitation polymerization provides a rapid, reproducible prototyping approach to the synthesis of stimuli-sensitive poly(N-isopropylacrylamide) microgels of narrow size distribution. In this protocol synthesis, light scattering characterization and single particle fluorescence tracking of these microgels in a wide-field microscopy setup are demonstrated.

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this ...

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.

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

Generation of Size-controlled Poly (ethylene Glycol) Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices

Uniform and size-controllable poly (ethylene glycol) diacrylate (PEGDA) droplets could be produced via the flow focusing process in a microfluidic device. This paper proposes a semi-three-dimensional (semi-3D) flow-focusing microfluidic chip for droplet formation. The polydimethylsiloxane (PDMS) chip was fabricated using the multi-layer soft lithography method. Hexadecane containing surfactant was used as the continuous phase, and PEGDA with the ultraviolet (UV) photo-initiator was the dispersed phase. Surfactants allowed the local surface tension to drop and formed a more cusped tip which promoted breaking into tiny micro-droplets. As the pressure of dispersed phase was constant, the size of droplets became smaller with increasing continuous phase pressure before dispersed phase flow was broken off. As a result, droplets with size variation from 1 &#181;m to 80 &#181;m in diameter could be selectively achieved by changing the pressure ratio in two inlet channels, and the average coefficient of variation was estimated to be below 7%. Furthermore, droplets could turn into micro-beads by UV exposure for photo-polymerization. Conjugating biomolecules on such micro-beads surface have many potential applications in the fields of biology and chemistry.

Generation of Size-controlled Poly ethylene Glycol Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices

Here, we present a protocol to illustrate the fabrication processes and verifying experiments of a semi-three-dimensional (semi-3D) flow-focusing microfluidic chip for droplet formation.

Uniform and size-controllable poly (ethylene glycol) diacrylate (PEGDA) droplets could be produced via the flow focusing process in a microfluidic device. ...

Engineering

Forming Giant-sized Polymersomes Using Gel-assisted Rehydration

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier properties, chemical versatility and tunable physical characteristics. Typical methods used to prepare giant-sized (&gt; 4 &#181;m) vesicles, however, are both time and labor intensive, yielding low numbers of intact polymersomes. Here, we present for the first time the use of gel-assisted rehydration for the rapid and high-yielding formation of giant (&gt;4 &#181;m) polymer vesicles (polymersomes). Using this method, polymersomes can be formed from a wide array of rehydration solutions including several different physiologically-compatible buffers and full cell culture media, making them readily useful for biomimicry studies. This technique is also capable of reliably producing polymersomes from different polymer compositions with far better yields and much less difficulty than traditional methods. Polymersome size is readily tunable by altering temperature during rehydration or adding membrane fluidizers to the polymer membrane, generating giant-sized polymersomes (&gt;100 &#181;m).

We present a protocol to rapidly form giant polymer vesicles (pGVs). Briefly, polymer solutions are dehydrated on dried agarose films adhered to coverslips. Rehydration of the polymer films results in rapid formation of pGVs. This method greatly advances the preparation of synthetic giant vesicles for direct applications in biomimetic studies.

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier ...

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

Modulating Shape of Polyester Based Polymersomes using Osmotic Pressure

Polymersomes are membrane-bound, bilayer vesicles created from amphiphilic block copolymers that can encapsulate both hydrophobic and hydrophilic payloads for drug delivery applications. Despite their promise, polymersomes are limited in application due to their spherical shape, which is not readily taken up by cells, as demonstrated by solid nanoparticle scientists. This article describes a salt-based method for increasing the aspect ratios of spherical poly(ethylene glycol) (PEG)- based polymersomes. This method can elongate polymersomes and ultimately control their final shape by adding sodium chloride in post-formation dialysis. Salt concentration can be varied, as described in this method, based on the hydrophobicity of the block copolymer being used as the base for the polymersome and the target shape. Elongated nanoparticles have the potential to better target the endothelium in larger diameter blood vessels, like veins, where margination is observed. This protocol can expand therapeutic nanoparticle applications by utilizing elongation techniques in tandem with the dual-loading, long-circulating benefits of polymersomes.

Polymersomes are self-assembled polymeric vesicles that are formed in spherical shapes to minimize Gibb's Free Energy. In the case of drug delivery, more elongated structures are beneficial. This protocol establishes methods to create more rod-like polymersomes, with elongated aspect ratios, using salt to induce osmotic pressure and reduce internal vesicle volumes.

Polymersomes are membrane-bound, bilayer vesicles created from amphiphilic block copolymers that can encapsulate both hydrophobic and hydrophilic payloads ...

Interlinked Macroporous 3D Scaffolds from Microgel Rods

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions without further additives. Continuous photoinitiated on-chip gelation enables variation of the microgel aspect ratio, which determines the building block properties for the obtained constructs. Glycidyl methacrylate (GMA) or 2-aminoethyl methacrylate (AMA) monomers are copolymerized into the microgel network based on polyethylene glycol (PEG) star-polymers to achieve either epoxy or amine functionality. A focusing oil flow is introduced into the microfluidic outlet structure to ensure continuous collection of the functionalized microgel rods. Based on a recent publication, microgel rod-based constructs result in larger pores of several hundred micrometers and, at the same time, lead to overall higher scaffold stability in comparison to a spherical-based model. In this way, it is possible to produce higher-volume constructs with more free volume while reducing the amount of material required. The interlinked macroporous scaffolds can be picked up and transported without damage or disintegration. Amine and epoxy groups not involved in interlinking remain active and can be used independently for post-modification. This protocol describes an optimized method for the fabrication of microgel rods to form macroporous interlinked scaffolds that can be utilized for subsequent cell experiments.

Microgel rods with complementary reactive groups are produced via microfluidics with the ability to interlink in aqueous solution. The anisometric microgels jam and interlink into stable constructs with larger pores compared to spherical-based systems. Microgels modified with GRGDS-PC form macroporous 3D constructs that can be used for cell culture.

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions ...