(T1230-09-56) Encapsulation of Low Molecular Weight Chemokines via Single Emulsion Technique

Purpose: Over the past several decades, the FDA has approved numerous controlled drug delivery systems utilizing poly(lactic-co-glycolic) acid. These commercially available PLGA drug delivery systems typically encapsulate hydrophobic small molecules drugs or short peptides via a single emulsion solvent evaporation procedure. However, significant research is being done to develop drug delivery systems for biologics. Our group has focused on developing PLGA microparticles to deliver various chemokines or proteins in different inflammatory disease models to promote resolution of inflammation. We have demonstrated that local, sustained delivery of CCL22, a chemokine known to recruit regulatory T cells, promotes immune homeostasis within the context of periodontal disease, transplant, and dry eye disease[1–3]. However, these particles were fabricated using a more complex double emulsion technique, thus limiting the scalability of particle fabrication and potentially clinical translation. Here, we investigated the feasibility of using a single emulsion technique to encapsulate CCL22 chemokine, to develop a more streamlined process for large-scale manufacturing.

Methods: Using a single emulsion water in oil evaporation method, we investigated whether a small chemokine, CCL22, could be encapsulated into polymeric microparticles. To assess the ability to encapsulate CCL22, we used PLGA with different end caps and molecular weights. CCl22 was first dissolved in DMSO and mixed with a solution of PLGA in dichloromethane to form the discrete phase. Then, the discrete phase was added to an aqueous continuous phase with PVA as the surfactant and homogenized to form an emulsion. Particles were then left to stir for 3 hours to allow for solvent evaporation. Following solvent evaporation, microparticles were then washed 4 times with water, flash frozen in liquid nitrogen, and placed on a lyophilizer for 48 hours. The microparticles were then characterized by Coulter Counter volume impedance measurements, scanning electron microscopy (SEM), and in vitro release assays to assess their size, morphology, and release kinetics.

Results: Initially, CCL22 was encapsulated using acid terminated PLGA with a molecular weight of 5 kDa. Utilization of this low molecular weight polymer demonstrated that CCL22 could be encapsulated via a single emulsion method, however, there was significant lag phase (Figure 1). To minimize electrostatic interactions between cationic CCL22 and the anionic PLGA polymer matrix and improve release, CCL22 microparticles were formulated with hydroxyl terminated PLGA with molecular weights of 5 kDa or 10 kDa. Encapsulation of CCL22 in these different polymers demonstrated that both polymer molecular weight and end cap can significantly impact release kinetics. With both the 5kDa and 10kDa hydroxyl terminated PLGA, we observed linear release; however, release from the 10kDa hydroxyl terminated PLGA was significantly depressed in comparison to the 5kDa polymer (Figure 2). These different molecular weight and end-capped polymers did not show any significant differences in size or morphology.

Conclusion: These results suggest that at least some low molecular weight chemokines can be encapsulated utilizing a single emulsion technique, which is typically used to encapsulate hydrophobic drugs or small peptides. Altogether, we found that we could successfully encapsulate CCL22 in PLGA with different end groups and molecular weight PLGA. Future studies will investigate the biological activity of CCL22 post encapsulation, test production of CCL22 microparticles using a continuous microfluidic process and determine whether the single emulsion technique can be used to encapsulate other low molecular weight chemokine or therapeutic proteins.

References: 1. Fisher JD, Zhang W, Balmert SC, Aral AM, Acharya AP, Kulahci Y, et al. In situ recruitment of regulatory T cells promotes donor-specific tolerance in vascularized composite allotransplantation. Sci Adv. 2020;6:eaax8429.
2. Ratay ML, Glowacki AJ, Balmert SC, Acharya AP, Polat J, Andrews LP, et al. Treg-recruiting microspheres prevent inflammation in a murine model of dry eye disease. J Control Release. 2017;258:208–17.
3. Glowacki AJ, Yoshizawa S, Jhunjhunwala S, Vieira AE, Garlet GP, Sfeir C, et al. Prevention of inflammation-mediated bone loss in murine and canine periodontal disease via recruitment of regulatory lymphocytes. P Natl Acad Sci Usa. 2013;110:18525–30.

Acknowledgements:Julie Kobyra was supported by the National Institutes of Health [grant number T90 DE030853-01A1; NIH/NIDCR] during this project.

Figure 1: Characterization of rhCCL22 Microparticles using 5 kDa carboxylic acid terminated PLGA (A) Release curve of rhCCL22 from PLGA microparticles as measured via ELISA (B) Size distribution measured via volume impedance measurements on Coulter Counter (c) Scanning Electron Microscopy image rhCCL22 microparticles

Figure 1: Characterization of rhCCL22 Microparticles using 5 kDa hydroxyl terminated PLGA (A) Release curve of rhCCL22 from PLGA microparticles as measured via ELISA (B) Size distribution measured via volume impedance measurements on Coulter Counter (c) Scanning Electron Microscopy image rhCCL22 microparticles