Trio Act of Boronolectin with Antibiotic-Metal Complexed Macromolecules toward Broad-Spectrum Antimicrobial Efficacy

Abstract

Bacterial infections, particularly by Gram-negative pathogens, have become a serious threat to global healthcare due to the diminishing effectiveness of existing antibiotics. We report a nontraditional therapy to combine three components in one macromolecular system, in which boronic acid adheres to peptidoglycan or lipopolysaccharide via boron-polyol based boronolectin chemistry, cationic metal polymer frameworks interact with negatively charged cell membranes, and β-lactam antibiotics are reinstated with enhanced vitality to attack bacteria via evading the detrimental enzyme-catalyzed hydrolysis. These macromolecular systems exhibited high efficacy in combating pathogenic bacteria, especially Gram-negative strains, due to synergistic effects of multicomponents on interactions with bacterial cells. In vitro and in vivo cytotoxicity and hemolysis evaluation indicated that these multifunctional copolymers did not induce cell death by apoptosis, as well as did not alter the phenotypes of immune cells and did not show observable toxic effect on red blood cells.

Keywords: antibiotics; antimicrobial; boronic acid; boronolectin; cationic macromolecules; cobaltocenium.

Figure 1

FTIR spectra of (A) L-rhamnose, PCo-PPB copolymer, and PCo-PPB-Rhamnose conjugate; (B) D-mannose, PCo-PPB copolymer, and PCo-PPB-Mannose conjugate; (C) D-glucose, PCo-PPB copolymer, and PCo-PPB-Glucose conjugate; (D) peptidoglycan, PCo-PPB copolymer, and PCo-PPB-peptidoglycan conjugate. Note: the B–O–C is stretching vibration (1030–1050 cm⁻¹) of boronate ester. The PCo-PPB copolymer (PCo-PPB-1, 10.0 mg) and model sugars (10 mg) were mixed in water (2 mL, pH = 7.0) for 6 h at room temperature and then examined by FTIR after freeze-drying.

Figure 2

Agar diffusion tests: (A, B) against E. coli by PCo, PPB, PCo-PPB-1, PCo-PPB-2, and PCo-PPB-3; (C–F) against E. coli, P. aeruginosa, P. vulgaris, and K. pneumonia by penicillin-G, PCo-Peni, and PCo-PPB-Peni. All compounds at different amounts in 30 μL of water were added to disks, and the plates were incubated at 28 °C for 18 h.

Figure 3

CLSM and SEM images of control, PCo-PPB (11 μg/mL), penicillin-G (5 μg/mL), and PCo-PPB-Peni (16 μg/mL, with the concentration of penicillin-G at 5 μg/mL) against four strains of bacteria. All scale bars are 2.0 μm (using BacLight live/dead stain, green indicates live cells, red indicates dead cells). Bacteria concentrations were 1.0 × 106 CFU/mL.

Figure 4

UV–vis absorption of nitrocefin solution with PCo-PPB copolymers (PCo-PPB-1 as an example) after adding β-lactamase for 1 h. 50 μL of nitrocefin (DMSO solution 1.0 mg/mL) and PCo-PPB copolymers with different amounts (100, 200, and 400 μg) were added into 1 mL of H₂O and stirred for 12 h. Then, 1 μL of β-lactamase PBS buffer solution (0.1 mg/mL, determined by Biorad Protein Assay) was added to the above solution. A characteristic absorption peak appeared near 480 nm due to hydrolysis of the β-lactam ring in nitrocefin.

Figure 5

Cytotoxicity of copolymers on mammalian cells: (A) Flow cytometry of murine splenocytes and (B) the percentage of apoptotic cells after injecting with PBS or copolymers at the concentration of 10 mg/kg body weight in vivo. Splenocytes were collected after 48 h, cultured for 24 h, and followed by detection of apoptosis using flow cytometry after staining of Annexin V. (C) Splenocytes from in vivo-treated mice were stained with anti-CD3, -CD4, -CD8, and -CD19 Abs and analyzed by flow cytometry. (D) Hemolysis was detected by culturing RBCs with copolymers and determining hemolysis. PBS was used as the control.

Scheme 1

(A) Illustration of Synergistic Antimicrobial Mechanisms of Multifunctional Macromolecules against Gram-Negative Bacteria via a Trio Act of Boronolectin Chemistry on Lipopolysaccharide, Electrostatic Attraction between Cationic Metal and Negatively Charged Membrane, and Reinstated Antibiotic Targeting on Peptidoglycan; (B) Preparation of Multi-Component Macromolecules (PCo-PPB and PCo-PPB-Peni) Containing Boronic Acid, Cobaltocenium, and/or Penicillin via RAFT Polymerization and Ion-Exchange

Scheme 2

Boronolectin Chemistry Involving Phenylboronic Acid Polymers and Polyols and Representative Sugars Containing Cis-1,2 and 1,3-Diols from LPS and/or Peptidoglycan