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>

<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 border="0" cellpadding="0" cellspacing="0" width="1293">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
		<col />
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:195px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:99px;">MP Biomedical</td>
			<td style="width:136px;">2810305</td>
			<td style="width:108px;"></td>
			<td style="width:63px;"></td>
			<td style="width:315px;">ThermoStat plus</td>
			<td style="width:123px;">Eppendorf</td>
			<td style="width:155px;">5352YL404573</td>
			<td style="width:104px;">Tube Warmer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:195px;">Triethanolamine (TEOA)</td>
			<td style="width:99px;">J.T. Baker</td>
			<td style="width:136px;">9468-01</td>
			<td style="width:108px;">Preheat to 37 &#176;C prior to pipetting</td>
			<td></td>
			<td>LSE Vortex Mixer</td>
			<td>Corning</td>
			<td>S004164</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Hydrochloric acid (HCl)</td>
			<td style="width:99px;">BDH Aristar</td>
			<td style="width:136px;">BDH3028</td>
			<td style="width:108px;"></td>
			<td></td>
			<td>XP205 Analytical Balance</td>
			<td>Mettler Toledo</td>
			<td>11106024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Sodium Sulfate</td>
			<td style="width:99px;">J.T. Baker</td>
			<td style="width:136px;">3891-01</td>
			<td style="width:108px;"></td>
			<td></td>
			<td>CureSpot 50</td>
			<td>ADAC Systems</td>
			<td>A121-031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Irgacure 2959</td>
			<td style="width:99px;">Ciba</td>
			<td style="width:136px;">029891301PS04</td>
			<td style="width:108px;"></td>
			<td></td>
			<td style="width:315px;">SevenMulti pH Meter&#160;</td>
			<td style="width:123px;">Mettler Toledo</td>
			<td>51302813</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Ovalbumin (OVA)</td>
			<td style="width:99px;">Invitrogen</td>
			<td style="width:136px;">34782</td>
			<td style="width:108px;"></td>
			<td></td>
			<td style="width:315px;">Class II, Type A2 Biological Safety Cabinet</td>
			<td style="width:123px;">Nuaire</td>
			<td align="right">1.39284E+11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">PEG 1,500</td>
			<td style="width:99px;">Alfa Aesar</td>
			<td style="width:136px;">A16241</td>
			<td style="width:108px;"></td>
			<td></td>
			<td style="width:315px;">Centrifuge 5430 R</td>
			<td>Eppendorf</td>
			<td>5428AH010419</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">PEG 3,000</td>
			<td style="width:99px;">Fluka</td>
			<td style="width:136px;">03997-1KG</td>
			<td style="width:108px;"></td>
			<td></td>
			<td style="width:315px;">Sonicator 8890</td>
			<td>Cole Parmer</td>
			<td>8890R-DTH</td>
			<td>SN QAC039907293D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">PEG 4,000</td>
			<td style="width:99px;">Alfa Aesar</td>
			<td style="width:136px;">A16151</td>
			<td style="width:108px;"></td>
			<td></td>
			<td>Coverslip 22 mm x 30 mm, 1.5 thickness</td>
			<td style="width:123px;">Fisherbrand</td>
			<td>12-544-A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">PEG 4,600</td>
			<td style="width:99px;">Sigma</td>
			<td style="width:136px;">373001-250G</td>
			<td style="width:108px;"></td>
			<td></td>
			<td style="width:315px;">Inverted Microscope with camera</td>
			<td style="width:123px;">Zeiss</td>
			<td>1022923629</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">PEG 6,000</td>
			<td style="width:99px;">Fluka</td>
			<td style="width:136px;">03394-1KG</td>
			<td style="width:108px;"></td>
			<td></td>
			<td style="width:315px;"></td>
			<td style="width:123px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">PEG 10,000</td>
			<td style="width:99px;">Alfa Aesar</td>
			<td style="width:136px;">B21955</td>
			<td style="width:108px;"></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Dextran 70</td>
			<td style="width:99px;">TCI</td>
			<td>D1449</td>
			<td style="width:108px;"></td>
			<td></td>
			<td></td>
			<td></td>
			<td></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.

Yağış oluşturulmuş polietilen glikol mikrojellerinin özellikleri reaktiflerin molekül ağırlığı ile kontrol edilir

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

Research

JoVE Journal

Bioengineering

11.8K Görüntüleme.  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.