From molecular bridge to macro-encapsulation: Unraveling the HEPES-porosity synergy for ultra-high peptide loading in PLGA microspheres

The clinical translation of long-acting peptide-loaded microspheres is often hampered by low and unpredictable encapsulation efficiency (EE), stemming from the complex interplay among formulation parameters. This study clarified these interactions by establishing a holistic, mechanism-guided framework that integrated polymer chemistry, matrix microstructure, and the incubation microenvironment. Using leuprolide acetate as a model peptide, we systematically deconstructed the key determinants of EE in a self-healing poly(lactic-co-glycolic acid) (PLGA) microsphere system. We found that the terminal carboxyl content of PLGA was a major determinant of peptide–polymer binding affinity, while the engineered pore architecture balanced peptide accessibility and retention and modulated loading-associated peptide–polymer interactions. Crucially, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) provided a more favorable loading environment than phosphate-buffered saline or Tris(hydroxymethyl)aminomethane hydrochloride and promoted peptide–PLGA association. Convergent experimental and simulation evidence supported a proposed HEPES-mediated non-covalent ternary association among leuprolide, HEPES, and PLGA. Optimization of the HEPES concentration and self-healing conditions further enhanced peptide–polymer association and encapsulation. Applying these findings, we fabricated optimized leuprolide-loaded microspheres with an EE of 96.44% and a drug loading of 9.64%, which were higher than the reported EE and drug-loading values for Lupron Depot®. The applicability of this formulation framework beyond the leuprolide acetate formulation was further supported by the high EEs achieved for two additional cationic peptides, triptorelin acetate (90.26%) and octreotide acetate (91.04%). This work provides a rational, multi-parameter formulation framework for improving the encapsulation of selected cationic peptides in self-healing PLGA microspheres by integrating mechanistic evidence with pharmaceutical formulation design.