Poster C8 - With Water-In-Oil Emulsion of Hydrogels, Time Matters

Saturday, October 26, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Neda Rehan

Student
University of Virginia
Henrico, Virginia, United States

Co-Author(s)

Jules Claire Bates

PhD student
University of Virginia, United States

Primary Investigator(s)

Donald R. Griffin, PhD

Associate Professor
University of Virginia
Charlottesville, Virginia, United States

Introduction: One of the main variables to control the permeability of granular hydrogels is porosity . Increasing porosity increases the swelling ratio of the gel (De Masi et al.) and diffusion rate of biomolecules through the gel (Weber et al.). Porosity also affects cell viability (Pfaff et al.) and macrophage phenotype (Liu and Segura). The control of porosity, therefore, is an important consideration in biomaterial fabrication of high fidelity.
Hydrogel microparticles are the building blocks of granular hydrogels. When synthesizing hydrogel microparticles via water-in-oil emulsion, the viscosity of the aqueous solution has a significant impact on particle size (i.e., the greater the viscosity, the larger the particle)(Pennington et al.; Yao et al.). Relatedly, gelation of hydrogels is preceded by a chemical entanglement process between growing polymer macromers, and, thus, the viscosity of the hydrogel precursor solution increases prior to gelation. As a result, we hypothesize that reaction time prior to start of segmentation is a critical variable to control to avoid large batch-to-batch particle size variability in bulk emulsion droplet formation processes.
Current literature shows that bulk water-in-oil segmentation via emulsion produces a wide range of particle sizes, oftentimes with large coefficients of variation (CV). In the presented work we demonstrate the impact of controlling for reaction time and argue that it should be observed and recorded to allow for replication and to improve CV.

Materials and

Methods: A solution of 0.05% surfactant and 7300 Novec served as the oil phase. PEG-VS was dissolved in TEOA (pH of 8.0) and MMP was dissolved in deionized H2O; these solutions served as the aqueous phase. Fluorophore was added to the MMP solution. 50uL of PEG-VS solution and 50uL of MMP solution were vortexed for 3 seconds ; at this point, the timer began. At the designated time, the entire vortexed aqueous solution was pipetted into a glass vial containing the oil phase while a screw stirrer mixed the oil phase at 1200 rpm. Time intervals were 0, 75, 150, and 225 seconds after vertexing. The stirring continued for 5 minutes after all the aqueous solution was added. A time of 5 minutes was selected because at this point, the gelation has reached the exponential curve on the viscometer. At this point, the stir apparatus was removed, and the glass vial was capped and solution left to finish gelling for 30 additional minutes.
After gelation, the gel was purified by first straining the oil-gel solution through a 20µm filter and washing with 10mL of PBS. The gel was left to swell in 5mL of PBS overnight. The next day, particle sizes were determined by imaging in a confocal microscope with a FITC channel. Particle sizes were averaged for each replication and standard deviations were determined.
200µL of PEG-VS solution and 200µL of MMP solution were vortexed and immediately placed on a viscometer which analyzed viscosity over the course of one hour.

Results, Conclusions, and Discussions: Of the 24 experiments, the diameters of gel particles pipetted 0, 75, 150, and 225 seconds prior to water-in-oil segmentation were 159±31, 177±39, 264±52, and 294±63 microns, respectively. As time prior to water-in-oil segmentation increased, particle size increased. This association is strongly demonstrated with a correlation coefficient of 0.97.
The coefficient of variation for all four times ranged from 19-22%. This variation is lower than that of particles produced in multiple publications (summarized in Table 1) where time was not a controlled factor.
Variance component analysis found that 36±16% of variation in particle diameter was due to gelation time. Controlling for gelation time, therefore, can alleviate a sizeable amount of variation across batches of gel.
Discussion
We found that controlling for gelation time prior to water-in-oil segmentation via emulsion decreases variability in particle sizes. Time prior to water-in-oil emulsion is a simple metric to control and can thus be used as a tool to control hydrogel particle sizes. To maintain reproducibility, researchers may find it beneficial to standardize the time prior to emulsion.

Acknowledgements (Optional):