Poster CC4 - Small molecule drug-loaded microparticles for macrophage cell therapy in regenerative medicine

Introduction: Macrophages, integral to the innate immune system, are involved in numerous disease and injury models due to their unique ability to interact with neighboring cells. Consequently, macrophage cell therapy has become a promising approach for various medical conditions, including inflammatory disorders, autoimmune diseases, tissue regeneration, and cancer immunotherapy. However, following their administration into the body, macrophages quickly alter their phenotype in response to local cues, often assuming an undesired state. Thus, maintaining their phenotype is critical in cell therapy. Previously we showed that drug-releasing poly(lactic-co-glycolic acid) (PLGA) microparticles could intracellularly control macrophage phenotype, even in the presence of pro-inflammatory external stimuli. In this work, we explored the effects of microparticle size on drug release kinetics and macrophage phenotype, and we screened small molecule drugs that have been reported to be reparative (or M2) phenotype-promoting for their potential to mimic IL-4 effects in macrophage cell therapy for fibrotic models. We determined the effect of particle size on drug release kinetics and subsequent effects on macrophage phenotype. Furthermore, we identified other drugs to be anti-inflammatory (i.e., inhibiting pro-inflammatory or M1-like behavior), but they did not exhibit as significant an effect as IL-4 in terms of its potential as a reparative (M2-like) phenotype modulator for cell therapy in regenerative medicine applications. By elucidating these interactions, we aim to develop specific and tailored strategies for enhanced macrophage-based cell therapies in fibrotic conditions.

Materials and

Methods: Synthesis, size, and release of PLGA particles

90 mg of PLGA (RG502H, Sigma) was reconstituted in 2000 uL of solvent (dichloromethane DCM) and added with 20 uL of 2% polyvinyl alcohol (PVA). The mixture was sonicated at a higher setting for one minute, and then another 10 mL PVA was added before being sonicated for another minute on a lower setting. After sonication, vials were left to stir in a chemical hood for 4 hours and allow DCM to evaporate. Particles were then washed and centrifuged at decreasing speeds to isolate particles of specific sizes, and then left to be lyophilized. The sizes of particles were characterized by dynamic light scattering using a Malvern Nano ZS. Particle release studies were carried out with a model drug and a therapeutic drug, Drug X, until 14 days later.

In vitro murine bone-marrow-derived macrophage (BMDM) culture

Bone marrow from the murine hind limb was extracted and cultured with macrophage colony-stimulating factor (M-CSF, Peprotech) to differentiate monocytes into macrophages. All BMDM cultures were seeded onto 6-well non-tissue culture treated (NTCT) plates at approximately 1 million cells for a day before any treatment was made and then cultivated for the time of the experiment, changing media every day. The purity of RNA samples was verified with a NanoDrop One instrument. Sample analysis was made on the protein level through flow cytometry (BD Fortessa) and gene level with qRT-PCR.

Results, Conclusions, and Discussions: Particles of controlled sizes were isolated by centrifugation speed, with diameter decreasing by approximately 50 nm every 340 G of centrifugation force. The release rate of drugs from these particles showed an inverse relationship with both particle size and Molecular Weight (MW). Larger particles exhibit faster drug release compared to smaller particles, and this release induced a slightly more inflammatory and less reparative phenotype compared to release from smaller particles.

Moreover, we observed that IL-4 administration significantly upregulated reparative markers and downregulated inflammatory markers' expression in macrophages for up to three days than the other drugs. Further analysis on the gene level indicated that IL-4 significantly upregulated reparative markers Arg1 and Cd301b, while downregulating inflammatory marker Tnf, along with phagocytic markers Apoe and Mertk, as well as the fibrotic marker Spp1.

In conclusion, we determined the effect of particle size on drug release kinetics and subsequent effects on macrophage phenotype. In addition, we identified IL-4 as the strongest macrophage modulator for cell therapy in fibrotic models, with limited similarities from other drugs reported to be reparative or “M2-promoting” from the literature.

Acknowledgements (Optional): This work was supported by NHLBI R01 HL130037. Flow data was collected at Sidney Kimmel Cancer Center.