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.

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 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

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically ...

Molecular Entanglement and Electrospinnability of Biopolymers

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular entanglement of the constituent polymers in the spinning dope was found to be an essential prerequisite for successful electrospinning. Rheology is a powerful tool to probe the molecular conformation and interaction of biopolymers. In this report, we demonstrate the protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan, from their dimethyl sulfoxide (DMSO)/water dispersions. Well-formed starch and pullulan fibers with average diameters in the submicron to micron range were obtained. Electrospinnability was evaluated by visual and microscopic observation of the fibers formed. By correlating the rheological properties of the dispersions to their electrospinnability, we demonstrate that molecular conformation, molecular entanglement, and shear viscosity all affect electrospinning. Rheology is not only useful in solvent system selection and process optimization, but also in understanding the mechanism of fiber formation on a molecular level.

Electrospinning is a fascinating technique used to fabricate micro- to nano-scale fibers from a wide variety of materials. Molecular entanglement of the constituent polymers in the spinning dope is essential for successful electrospinning. We present a protocol for utilizing rheology to evaluate the electrospinnability of two biopolymers, starch and pullulan.

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular ...

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white blood cells and platelets that are suspended in blood plasma. Due to the abundance of the RBCs (40% to 45% of the blood volume), their behavior dictates the rheological behavior of blood especially in the microcirculation. At very low shear rates, RBCs are seen to assemble and form entities called aggregates, which causes the non-Newtonian behavior of blood. It is important to understand the conditions of the aggregates formation to comprehend the blood rheology in microcirculation. The protocol described here details the experimental procedure to determine quantitatively the RBC aggregates in microcirculation under constant shear rate, based on image processing. For this purpose, RBC-suspensions are tested and analyzed in 120 x 60 &#181;m poly-dimethyl-siloxane (PDMS) microchannels. The RBC-suspensions are entrained using a second fluid in order to obtain a linear velocity profile within the blood layer and thus achieve a wide range of constant shear rates. The shear rate is determined using a micro Particle Image Velocimetry (&#181;PIV) system, while RBC aggregates are visualized using a high speed camera. The videos captured of the RBC aggregates are analyzed using image processing techniques in order to determine the aggregate sizes based on the images intensities.

The protocol described details an experimental procedure to quantify Red Blood Cell (RBC) aggregates under a controlled and constant shear rate, based on image processing techniques. The goal of this protocol is to relate RBC aggregate sizes to the corresponding shear rate in a controlled microfluidic environment.

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Magnetic and Thermal-sensitive Poly(N-isopropylacrylamide)-based Microgels for Magnetically Triggered Controlled Release

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were designed and fabricated for magnetically triggered release. PNIPAAm-based magnetic microgels with a spherical structure were produced via a temperature-induced emulsion followed with physical-crosslinking by mixing PNIPAAm, polyethylenimine (PEI), and Fe3O4-NH2 magnetic nanoparticles. Because of their dispersity, the Fe3O4-NH2 nanoparticles were embedded inside the polymer matrix. The amine groups exposed on the Fe3O4-NH2 and PEI surface supported the spherical structure by physically crosslinking with the amide groups of the PNIPAAm. The hydrophobic anti-cancer drug curcumin can be dispersed in water after encapsulation into the microgels. The microgels were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), and UV-Vis spectral analysis. Furthermore, magnetically triggered release was studied under an external high frequency magnetic field (HFMF). A significant &#34;burst release&#34; of curcumin was observed after applying the HFMF to the microgels due to the magnetic inductive heating (hyperthermia) effect. This manuscript describes the magnetically triggered controlled release of Cur-PNIPAAm/Fe3O4-NH2 encapsulated curcumin, which can be potentially applied for tumor therapy.

Magnetic and Thermal-sensitive PolyN-isopropylacrylamide-based Microgels for Magnetically Triggered Controlled Release

This manuscript describes the preparation of magnetic and thermal-sensitive microgels via a temperature-induced emulsion without chemical reaction. These sensitive microgels were synthesized by mixing poly(N-isopropylacrylamide) (PNIPAAm), polyethylenimine (PEI) and Fe3O4-NH2 nanoparticles for the potential use in magnetically and thermally triggered drug-release.

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits versus other methods: DIBs are stable and often long-lasting, bilayer area can be reversibly tuned, leaflet asymmetry is readily controlled via droplet compositions, and tissue-like networks of bilayers can be obtained by adjoining many droplets. Forming DIBs requires spontaneous assembly of lipids into high density lipid monolayers at the surfaces of the droplets. While this occurs readily at room temperature for common synthetic lipids, a sufficient monolayer or stable bilayer fails to form at similar conditions for lipids with melting points above room temperature, including some cellular lipid extracts. This behavior has likely limited the compositions&#8212;and perhaps the biological relevance&#8212;of DIBs in model membrane studies. To address this problem, an experimental protocol is presented to carefully heat the oil reservoir hosting DIB droplets and characterize the effects of temperature on the lipid membrane. Specifically, this protocol shows how to use a thermally conductive aluminum fixture and resistive heating elements controlled by a feedback loop to prescribe elevated temperatures, which improves monolayer assembly and bilayer formation for a wider set of lipid types. Structural characteristics of the membrane, as well as the thermotropic phase transitions of the lipids comprising the bilayer, are quantified by measuring the changes in electrical capacitance of the DIB. Together, this procedure can aid in evaluating biophysical phenomena in model membranes over various temperatures, including determining an effective melting temperature (TM) for multi-component lipid mixtures. This capability will thus allow for closer replication of natural phase transitions in model membranes and encourage the formation and use of model membranes from a wider swath of membrane constituents, including those that better capture the heterogeneity of their cellular counterparts.

This protocol details the use of a feedback temperature-controlled heating system to promote lipid monolayer assembly and droplet interface bilayer formation for lipids with elevated melting temperatures, and capacitance measurements to characterize temperature-driven changes in the membrane.

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits ...