Unravelling the immunomodulatory effect of Poly (glycolic acid) particles using new techniques

Xie, Bowen

Abstract

Non-degradable and biodegradable implants inevitably generate debris or degradation products in the body, and these induce acute and chronic inflammation. Polyglycolic acid (PGA) is a simple linear aliphatic polyester with a wide set of medical applications. It is a highly crystalline polymer and is insoluble in common organic solvents, hence the mechanisms of PGA immunomodulation have not been fully investigated due to the experimental challenges in PGA specimen processing.
In this study, PGA specimen was fabricated by a supercritical carbon dioxide meltfoaming technique into micro-scale particles with a controllable crystallinity and degradation rate. Both human (THP-1-derived) and murine (RAW 264.7) macrophage models were used for investigating the relationship among PGA degradation rate, PGA particle physical properties, and macrophage inflammatory responses. A fluorescent Rhodamine dye was grafted onto the PGA to facilitate the visualization of the dynamics and intracellular location during the particles’ internalization. The responses of three macrophage phenotypes’ (M0, M1, and M2) internalizing/culturing PGA particles against different particle properties (number, crystallinity, degradation rate, etc) have been studied. The induced activation and polarization of macrophages have been identified via morphological alterations and the expression of surface/intracellular markers and cytokine secretions. Lysosome-specific dyes were flexibly introduced to monitor the phagolysosomal pH changes of macrophages during the PGA particle internalization and degradation. Furthermore, two label-free techniques, Raman and FTIR spectroscopies were utilized to verify PGA internalization and the modulation of macrophage activation and polarization. Uniquely, this study created and used two types of PGA specimens with extremely different degradation properties: 1) a nondegraded type (ND) PGA which has a low average crystallinity and higher molecular weight, and 2) a partially degraded type (D) PGA which has a high average crystallinity and lower molecular weight. The two types of PGA specimens constituted differential acid-releasing systems to represent conditions in acute inflammation (a fast change of acidic environment) and chronic inflammation (a slow change of acidic environment). It was found that the human macrophages responded similarly to murine cells against the PGA particle internalization, whereas the RAW 264.7 were much easier to use, and straightforward for polarization and dynamic studies. It is confirmed that the internalization of different particles clearly triggered different immunomodulation patterns in the M0/M1/M2 macrophage phenotypes. The outcomes of spectroscopic and biological assays were consistent with each other.
The M0 macrophage with engulfing ND PGA exhibited a relatively strong inflammatory response that manifested as an increase of NO and IL-1β secretion and CD80 expression, and a reduction of phagolysosomal pH value. The FTIR showed no PGA feature peaks due to the small intake quantity and fast intracellular degradation of ND particles within 48 hours. The M0 had a moderate intake of D PGA particles, and exhibited a combination of inflammatory and anti-inflammatory behaviours.
Aggressive M1 macrophages massively internalized both ND and D PGA particles, and both types of particles triggered an inflammatory response but in different manners. The ND particles were excessively internalized and led to the apoptosis of very aggressive M1 population, and the surviving M1 cells exhibited less aggressive characteristics. The D PGA particles directly suppressed the M1’s feature by constant intercellular pH modulation and V-ATPase inhibition, and also reduced the expression of M1-related cytokines and surface markers.
The M2 macrophage had a negligible ND PGA intake, and although the CD206 marker and IL-10 secretion were reduced in these cells, they did not appear a pro-inflammatory response. Similarly, the D PGA internalization had minimal impact on the M2 macrophage. Therefore, no visible FTIR PGA peaks in the ND group, and weaker peaks of the D PGA in M2 culture groups were observed in comparison to that of M1 groups. The presence of PGA particles increased the level of pro-inflammatory factors, but the degraded PGA particles could promote anti-inflammatory cytokine secretion and marker expression. The PGA particle system and macrophage cell models used in this project demonstrated the possibility to control macrophage polarization and inflammatory stages by using degradable biomaterials, and could guide the future investigation of immunomodulation and the design of better biomaterial implants.

Citation

Xie, B. (2023). Unravelling the immunomodulatory effect of Poly (glycolic acid) particles using new techniques. (Thesis). Keele University. Retrieved from https://keele-repository.worktribe.com/output/674529

• Thesis Type: Thesis
• Deposit Date: Dec 21, 2023
• Public URL: https://keele-repository.worktribe.com/output/674529
• Additional Information: Embargo on access until 1 June 2025 - The thesis is due for publication, or the author is actively seeking to publish this material.
• Award Date: 2023-12