(M1230-06-31) Manipulating the PLGA Terminal Group Chemistry Alters Electrostatic Controlled Release from Polymeric Microparticles

Purpose: Protein drugs are a rapidly advancing arm of pharmaceuticals. Many of these protein drugs are limited in their delivery and application due to limited oral bioavailability and short half-lives [1]. Encapsulation of therapeutics within PLGA MP can significantly improve bioavailability of peptides and proteins while facilitating predictable and controllable release kinetics through tunable degradation kinetics [2]. However, simple degradation of PLGA fails to fully predict release in some cases, due in part to electrostatic interactions between the polymer and encapsulated compound [3]. Unaddressed electrostatic interactions have the potential to delay or totally halt the release of proteins and peptides [4]. These interactions principally occur between positively charged amino acid side chains and the negatively charged PLGA terminal carboxylic acid group [5]. Yet it is unknown if altering the PLGA terminal group chemistry has potential to reduce these electrostatic interactions. This work aims to determine how PLGA polymers with variable terminal group chemistry affects polymer properties, microparticle morphology, and the release of encapsulated protein drugs.

Methods: The physical properties of PLGA polymers terminated with hydroxyl (PLGA-OH), amine (PLGA-NH2), or carboxylic acid (PLGA-COOH) were assessed including hydrophobicity, viscosity, and glass transition temperature. Microparticles (MP) were fabricated from these polymers and their morphology and degradation rates were quantified via scanning electron microscopy (SEM) and size exclusion chromatography (SEC), respectively. Additionally, the pH change associated with PLGA MP degradation was quantified. MP were co-cultured with bone marrow derived macrophages to assess potential to alter macrophage phenotype and quantified using flow cytometry. Release rates of encapsulated model peptides and proteins were determined via spectrophotometry and enzyme-linked immunosorbent assay.

Results: For all microparticles, the morphology was consistent and the average diameter was within 15-40 µm. The polymer physical properties, pH evolution, and SEC indicates that PLGA-NH2 degrades faster than PLGA-COOH. Release rate of positively charged model peptide from PLGA-NH2 MP was significantly improved relative to neutral peptide release. This improvement in release rate was consistently observed for a larger and more complex protein with positive charge, CCL22. Macrophage co-culture data shows that M1 or M2 phenotypes are not induced, but there is an increase in phagocytosis markers (Glut1, CD36) and Ly6C activation marker.

Conclusion: These data reveal that a small alteration in the PLGA chemical composition, specifically adjusting the terminal group chemistry, can impart significant changes to the release rate of both charged and neutral biologic compounds. This change does not significantly affect microparticle morphology or interactions with the host immune system. Together, this innovation provides helpful insight and a novel approach when encapsulating peptides or proteins in polymeric microparticles.

References: 1. Werle, M.; Bernkop-Schnurch, A., Strategies to improve plasma half life time of peptide and protein drugs. Amino Acids 2006, 30 (4), 351-67.
2. Hines, D. J.; Kaplan, D. L., Poly(lactic-co-glycolic) acid-controlled-release systems: experimental and modeling insights. Crit Rev Ther Drug Carrier Syst 2013, 30 (3), 257-76.
3. Blanco, M. D.; Alonso, M. J., Development and characterization of protein-loaded poly(lactide-co-glycolide) nanospheres. European Journal of Pharmaceutics and Biopharmaceutics 1997, 43 (3), 287-294.
4. Houchin, M. L.; Topp, E. M., Chemical degradation of peptides and proteins in PLGA: a review of reactions and mechanisms. J Pharm Sci 2008, 97 (7), 2395-404.
5. Sophocleous, A. M.; Zhang, Y.; Schwendeman, S. P., A new class of inhibitors of peptide sorption and acylation in PLGA. J Control Release 2009, 137 (3), 179-84

Acknowledgements: M. Borrelli is supported by the National Institutes of Health [grant number 5T32HL076124-13] and the American Heart Association (AHA) Predoctoral Fellowship [https://doi.org/10.58275/AHA.23PRE1026713.pc.gr.161025]

Figure 1 – Material Properties of Variable End-Cap Group PLGA Microparticles. Main point of this figure (A) Wetted Contact Angle (WCA) measurements of variable end-cap group PLGA Polymer. (B) Microparticle size distribution for unloaded microparticles (MP). (C) Representative SEM images of MP interior cross section (Left, scale bar 10µm) and morphology (Right, scale bar 30µm). (D) Number weighted molecular weight (Mn) of PLGA MP normalized to the measured Mn measured on Day 3 of a degradation assay. Mn data was measured via size exclusion chromatography (SEC) calibrated to a polystyrene Mn standard curve. (E) pH evolution in the supernatant and inside the MP, data represents mean ± SD from n = 3 replicates.

Figure 2 – Bone Marrow Derived Macrophage Co-Culture with Variable End-Cap PLGA Microparticles. Main point of this figure (Co-culture show that MP do not polarize macrophages). (A) Gating strategy for macrophages. (B) Mean fluorescent intensity (MFI) of macrophage phenotype markers. (C) Representative gating for macrophage populations. (D) Quantification of macrophage population frequency. Data shown represents the mean from n = 2 replicates.

Figure 4 – Peptide MP Size and Release Rate. Main point of this figure Size distribution for positive charge peptide (A) and neutral charge peptide (B). Cumulative release rate (% of encapsulated peptide) of positive charge peptide (C) and neutral charge peptide (D).