Poster K7 - Using Co-Flow Microfluidics to Control PLGA and PNHAN Microsphere Diameter

Saturday, October 26, 2024

10:00 AM - 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Caroline E. Bixby, N/A

Undergraduate student
Washington University in St. Louis
Dallas, Texas, United States

Primary Investigator(s)

Jianjun Guan, PhD

Earl E. & Myrtle E. Walker Professor of Engineering
Department of Mechanical Engineering & Materials Science, Washington University in St. Louis, United States

Co-Author(s)

CG

Changxin Guo

Graduate student
Washington University in St. Louis, United States

Introduction: Myocardial infarction (MI) is a heart disease that deprives cardiac cells of O2, causing ischemia. If the ischemic tissue is not treated immediately after acute MI, permanent cell death will rapidly occur. Therefore, MI treatments intend to supply O2 supplementation at the earliest possible stage of MI in order to promote the regeneration of ischemic tissue and restore cardiac function. However, the efficacy of current oxygen therapies is inconsistent, as they are often unsafe, unsustained, or otherwise unimpactful.

To address the shortcomings of current oxygen therapies, Fan et al. developed a delivery system based on oxygen-releasing microspheres. Microspheres allow for targeted and sustained drug delivery, making them suitable for the continuous supplementation of oxygen to infarcted tissue. The release system involves core-shell structured microspheres encapsulated by a thermosensitive hydrogel. A commonly used polymer to comprise the microsphere shell is poly(lactic-co-glycolic) acid (PLGA), a biodegradable, non-toxic, and functional polymer with long-acting sustained release capabilities. A second, newer polymer is poly(N-isopropylacrylamide-co-hydroxyethyl methacrylate-co-acrylate polylactide-co-N-acryloxysuccinimide) (PNHAN ), a synthetic copolymer developed by Guan et al., ideal for environmentally-triggered release due to its thermosensitive properties. The size of the microspheres is proportional to the oxygen release rate and its resulting distribution, so obtaining precise control over microsphere diameter allows for the regulation of its behavior.

Here, a microfluidic device was used to fabricate both PLGA and PNHAN based microspheres and explore the ways in which microsphere diameter can be controlled, allowing the oxygen release rate to be regulated when treating MI.

Materials and

Methods: A microfluidic device was used to fabricate single emulsion microspheres of equal volume, and an optic microscope was used to view and measure them in the droplet phase. The microfluidic device uses the co-flow microfluidic method, where a polymer solution (the dispersed phase) is injected through a cylindrical glass capillary into another capillary with a second liquid phase (the continuous phase), forming monodisperse droplets where the co-flowing streams meet. The diameter of the microspheres can be controlled through the operating parameters, such as the flow rates of the two solutions, and the properties of the solutions themselves, such as their concentrations.

Here, the polymer solution concentrations and the flow rates of the two solutions were manipulated, and the resulting microsphere diameters were measured. The continuous phase consisted of 1% poly(vinyl alcohol), and the dispersed (polymer) phase consisted of either PLGA or PNHAN dissolved in ethyl acetate. Five different concentrations of both PLGA and PNHAN were prepared (2%, 1.5%, 1%, .50%, and .25%). These 10 polymer solutions were run individually through the microfluidic device (as the dispersed phase), and 1% PVA was used as the continuous phase. Seven different flow rate ratios were used (PVA flow rate / polymer flow rate), including 95/.07, 75/.90, 65/1.10, 55/1.10, 45/1.30, 35/1.30, and 35/1.50 ("µL/min" /"µL/min" ). This way, the effects of both the polymer concentration and the ratio of flow rates could be observed. The diameters of 10 spheres were measured for each combination of conditions, and the averages and standard deviations were calculated.

Results, Conclusions, and Discussions: As shown in Fig.1-2, the data shows a negative correlation between flow rate ratio and diameter, where the 95/.70 flow rate ratio (135.71) was the largest ratio that produced spheres. The lower polymer concentrations consistently resulted in smaller microspheres. For a given concentration, the range of diameters was approximately 6.0 ±0.8 µm for PNHAN and 6.1±0.7 µm for PLGA. Overall, the diameters ranged from 6.11µm to 25.45µm for PNHAN and 13.57µm to 27.476µm for PLGA.

The PLGA microspheres tended to have higher diameters and less variation between the concentration conditions than the PNHAN microspheres. However, the range of diameters within each concentration condition was consistent for both polymers. Though the .25% and .50% conditions produced the smallest PNHAN microspheres, there was more irregularity in the shapes of the spheres compared to other concentrations. As shown in Fig.1, the standard deviations for the .25% condition are the greatest, so this concentration of PNHAN might be unsuitable for more complex applications. As shown in Fig.2, the PLGA microsphere diameters tended to have lower standard deviations than the PNHAN microspheres, suggesting that PLGA might be a more consistent choice for more advanced applications.

These results demonstrate that a higher continuous to dispersed phase flow rate ratio and a lower polymer solution concentration yield lower microsphere diameters. This proves that microsphere diameter can be accurately controlled by manipulating the properties of the dispersed phase and the operating parameters. The ability to fabricate microspheres of uniform and controlled size allows for the formation of more complex microspheres capable of encapsulating drugs, including double emulsion O2-carrying microspheres. Next steps include fabricating the thermosensitive hydrogel carrier to complete the safe, sustained, and effective delivery system to treat ischemic tissue.

Acknowledgements (Optional): We would like to acknowledge the support of the American Heart Association (AHA Award #906519) and the WashU Cardiovascular Summer Research (CardS) program.

References
1. Guan Y, Niu H, Wen J, Dang Y, Zayed M, Guan J. Rescuing Cardiac Cells and Improving Cardiac Function by Targeted Delivery of Oxygen-Releasing Nanoparticles after or Even before Acute Myocardial Infarction. ACS Nano. 2022;16(11):19551-19566. doi:10.1021/acsnano.2c10043
2. Fan Z, Xu Z, Niu H, et al. An Injectable Oxygen Release System to Augment Cell Survival and Promote Cardiac Repair Following Myocardial Infarction. Sci Rep. 2018;8(1):1371. doi:10.1038/s41598-018-19906-w
3. Guan J, Hong Y, Ma Z, Wagner WR. Protein-Reactive, Thermoresponsive Copolymers with High Flexibility and Biodegradability. Biomacromolecules. 2008;9(4):1283-1292. doi:10.1021/bm701265j
4. Ruan L, Su M, Qin X, et al. Progress in the application of sustained-release drug microspheres in tissue engineering. Mater Today Bio. 2022;16:100394. doi:10.1016/j.mtbio.2022.100394
5. Duncanson WJ, Lin T, Abate AR, Seiffert S, Shah RK, Weitz DA. Microfluidic synthesis of advanced microparticles for encapsulation and controlled release. Lab Chip. 2012;12(12):2135. doi:10.1039/c2lc21164e
6. Zhao CX. Multiphase flow microfluidics for the production of single or multiple emulsions for drug delivery. Adv Drug Deliv Rev. 2013;65(11-12):1420-1446. doi:10.1016/j.addr.2013.05.009