Antimicrobial Peptide-Poly(ethylene glycol) Conjugates: Connecting Molecular Architecture, Solution Properties, and Functional Performance

Abstract

Antimicrobial peptides (AMPs) are promising alternatives to conventional antibiotics for treating infections caused by drug-resistant bacteria; yet, many peptides are limited by toxicity to eukaryotic cells and instability in biological environments. Conjugation to linear polymers that reduce cytotoxicity and improve stability, however, often decreases antimicrobial activity. In this work, we combine the biocompatibility advantages of poly(ethylene glycol) (PEG) with the efficacy merits of nonlinear polymer architectures that accommodate multiple AMPs per molecule. By conjugating a chemokine-derived AMP, stapled Ac-P9, to linear and star-shaped PEG with various arm numbers and lengths, we investigated the role of molecular architecture in solution properties (i.e., ζ-potential, size, and morphology) and performance (i.e., antimicrobial activity, hemolysis, and protease resistance). Linear, 4-arm, and 8-arm conjugates with 2-2.5 kDa PEG arms were found to form nanoscale structures in solution with lower ζ-potentials relative to the unconjugated AMP, suggesting that the polymer partially shields the cationic AMP. Reducing the length of the PEG arms of the 8-arm conjugate to 1.25 kDa appeared to better reveal the peptide, seen by the increased ζ-potential, and promote assembly into particles with a larger size and defined spherical morphology. The antimicrobial effects exerted by the short 8-arm conjugate rivaled that of the unconjugated peptide, and the AMP constituents of the short 8-arm conjugate were protected from proteolytic degradation. All other conjugates examined also imparted a degree of protease resistance, but exhibited some reduced level of antimicrobial activity as compared to the AMP alone. None of the conjugates caused significant cytotoxic effects, which bodes well for their future potential to treat infections. While enhancing proteolytic stability often comes with the cost of lower antimicrobial activity, we have found that presenting AMPs at high density on a neutral nonlinear polymer strikes a favorable balance, exhibiting both enhanced stability and high antimicrobial activity.

Figure 1

Conjugation scheme, in which the thiol-terminated AMP stapled Ac-CGGP9 was appended to maleimide-functionalized linear and star-shaped PEG, using 1.1 or 1.25 equiv of peptides relative to the maleimide amount on linear and star-shaped polymers, respectively.

Figure 2

SEC traces of stapled Ac-CGGP9 (black), PEG maleimide with different architectures (light colors), and the corresponding conjugates (dark colors): (a) linear; (b) 4-arm star-shaped conjugate; (c) 8-arm star-shaped conjugate; and (d) short 8-arm star-shaped conjugate.

Figure 3

ζ-potential, size, and morphology of stapled Ac-P9 and its conjugates at a constant concentration of 200 μM peptide equivalent. (a) ζ-potential (circles) and size (bars) of stapled Ac-P9 alone and conjugates in 10 mM PBS. We report the Z-average hydrodynamic diameter from DLS (D_h, DLS) and the average diameter from TEM images (D_TEM). Error bars represent the standard deviation. (b) Representative TEM images of stapled Ac-P9 and conjugates in 10 mM PBS, showing more distinctly spherical assemblies of the linear and short 8-arm conjugate than from the other conjugates. Scale bar = 50 nm. (c) Proposed assembled structures. Stapled Ac-P9 is soluble or forms very small aggregates in solution. The low ζ-potential and larger size of the spherical particles formed from the linear conjugates suggest the formation of micelles with PEG coronas partially sequestering the peptide within the cores. The 4- and 8-arm star-shaped conjugates with 2.5 kDa PEG arms have lower peptide contents and form structures that likely still involve small-peptide aggregates connecting multiple conjugates into particles. As seen with the short 8-arm conjugate, shortening the PEG arms increases the peptide content and restores the spherical morphology, though the high ζ-potential is consistent with the presentation of peptides at the particle surface, where electrostatic repulsion may limit particle size.

Figure 4

AlamarBlue assay was used to test the antimicrobial activity of stapled Ac-P9 and its conjugates against K. pneumoniae. (a) Reduction of the alamarBlue reagent by metabolically active bacteria yields a pink, fluorescent product. (b) Photograph of a 96-well plate showing wells with pink, purple, and blue solutions indicative of high, medium, and low amounts, respectively, of viable bacteria. (c) Bacterial survival (%) after treatment with the indicated AMP or conjugate (peptide equiv. concentration = 100 μM in RPMI medium). Three independently prepared batches of materials (square, triangle, and circle) were tested in triplicate. Results are plotted for each batch and the bars indicate the average. Error bars represent the standard deviation. The illustrations are intended to indicate the different conjugate formulations rather than to imply their structures in solution.

Figure 5

Protease resistance of stapled Ac-P9 and conjugates against Proteinase K in RPMI medium. (a) The primary amino acid sequence of stapled peptide Ac-P9 with predicted cleavage sites indicated by red arrows (sites identified using Peptide Cutter). (b) Summary of intact peptide/conjugate fraction after incubation with Proteinase K for 0.5, 1, 2, and 24 h. Fractions were calculated from the peak area ratio of the HPLC traces at each time point relative to the untreated control (Table S2). The illustrations are intended to indicate the different conjugate formulations rather than to imply their structures in solution.