The Kinetic\nPathways for the Formation of Complex\nCoacervate Micelles of Antimicrobial Peptides and Block Copolymers

The conceptual application of complex coacervate core\nmicelles\n(C3Ms) as drug delivery vehicles has obtained significant interest\nover the past several decades. Although C3Ms show promise in targeting\nspecific body areas and improving drug circulation times, their low\nreproducibility and limited stability have hindered their progression\nto clinical trials and commercial viability. One of the major obstacles\nis the lack of understanding of the formation and growth kinetics\nof C3Ms. Since their structure is often controlled kinetically rather\nthan thermodynamically, this insight is essential for achieving good\nreproducibility and stability of C3M drug delivery vehicles. Time-resolved\nsmall-angle X-ray scattering (TR-SAXS) offers excellent insights by\nresolving spatiotemporal kinetic processes on nanometer/millisecond\nscales. Here, we investigate the formation kinetics of C3Ms comprised\nof the antimicrobial peptide, colistin, and poly(ethylene oxide)-<i>b</i>-poly(methacrylic acid) (PEO-<i>b</i>-PMAA).\nUsing TR-SAXS coupled to stopped-flow mixing, we have identified three\ndistinct steps in the mechanism of this complex coacervation system:\nnucleation, fusion, and single chain/ion-pair insertion. Nucleation\ncannot be experimentally resolved as it is completed during the mixing\nprocess. The fusion kinetics proceed as a first-order reaction with\nrelaxation times of approximately 50–85 ms. The driving force\nfor fusion is larger at increased concentrations. Chain/pair insertion\noccurs at relaxation times of 15–25 s, where larger remaining\nsize mismatches (higher dispersity) drive the final equilibration\nprocess. As expected, the structural formation is concentration-dependent,\nproviding a handle to acquire control over the size and dispersity\nof these kinetically trapped C3Ms. These findings contribute to a\ndeeper understanding of C3M formation and stability, potentially advancing\nthe development of C3M-based drug delivery systems.