Rational multivalency construction enables bactericidal effect amplification and dynamic biomaterial design

Abstract

The multivalency of bioligands in living systems brings inspiration for not only the discovery of biological mechanisms but also the design of extracellular matrix (ECM)-mimicking biomaterials. However, designing controllable multivalency construction strategies is still challenging. Herein, we synthesized a series of well-defined multivalent antimicrobial peptide polymers (mAMPs) by clicking ligand molecules onto polymers prepared by reversible addition-fragmentation chain transfer polymerization. The multiple cationic ligands in the mAMPs could enhance the local disturbance of the anionic phospholipid layer of the bacterial membrane through multivalent binding, leading to amplification of the bactericidal effect. In addition to multivalency-enhanced antibacterial activity, mAMPs also enable multivalency-assisted hydrogel fabrication with an ECM-like dynamic structure. The resultant hydrogel with self-healing and injectable properties could be successfully employed as an antibacterial biomaterial scaffold to treat infected skin wounds. The multivalency construction strategy presented in this work provides new ideas for the biomimetic design of highly active and dynamic biomaterials for tissue repair and regeneration.

Graphical abstract

Figure 1

Schematic illustration of the preparation and application of mAMPs and the dynamic hydrogel (A) Synthesis of the mAMP polymers (left) and bactericidal amplification effect of an mAMP through locally enhanced disruption of bacterial membrane lipid bilayers (right). (B) Fabrication of the mAMP-derived dynamic hydrogel through multiple electrostatic interactions with nanoclays (upper) and its application for treating infected open skin wounds (lower).

Figure 2

Chemical characterization and antibacterial activities of mAMP_n (A and B) ¹H NMR spectra (A) and GPC analysis (B) of PHA-PPA_n. (C) ¹H NMR spectra of mAMP_n. (D and E) Bacterial colony images of E. coli (D) and S. aureus (E) incubated with various concentrations of AMP and mAMP_n for 8 h. (F and G) SEM (left) and TEM (right) images of E. coli (F) and S. aureus (G) after 8 h of treatment with AMP and mAMP₆ (40 μmol L⁻¹). Data are represented as mean ± SD (n = 4).

Figure 3

The enhanced antibacterial mechanism of mAMP_n (A) Schematic illustration of the bactericidal amplification effect provided by mAMP_n. (B and C) CLSM images (B) and quantitative analysis (C) of the binding of FITC-labeled AMP or mAMP₆ to bacteria. (D) Real-time QCM frequency changes during binding of AMP or mAMP₆ (40 μmol L⁻¹). (E) Quantitative results of frequency changes. (F) Efficiency of S. aureus biofilm eradication after treatment with AMP and mAMP₆ for 8 h. (G) CLSM images of S. aureus biofilms after treatment (640 μmol L⁻¹). (H) Schematic illustration of biofilm eradication using multivalent polymers. Data are represented as mean ± SD (n = 4); a statistically significant difference compared with the control group is indicated by ∗∗∗p < 0.001.

Figure 4

In vivo antibacterial activity of mAMP_n (A) Skin wound model for the in vivo anti-infection assay. (B) Wound healing processes in different groups of rats. (C and D) Images (C) and quantitative analysis (D) of the bacterial colonies obtained from the wound sites on day 3. (E) Quantitative analysis of wound sizes. (F and G) Immunofluorescence staining of TNF-α (F) and IL-1β (G) in the wound site on day 7. Data are represented as mean ± SD (n = 5); a statistically significant difference compared with the control group is indicated by ∗∗∗p < 0.001.

Figure 5

Characterization of the mAMP-Gel (A) ¹H NMR spectra. (B) Gelation of mAMP-Gel. (C) SEM image of mAMP-Gel. (D) EDS mapping image of mAMP-Gel (red, Mg; green, Si). (E) The self-healing ability of mAMP-Gel. Block sizes: 0.2 × 0.5 cm. (F) The injectability and remoldability of mAMP-Gel. Teflon molds: 2.0 × 2.0 cm. (G) Dynamic oscillatory frequency sweeps (strain = 1%) of mAMP-Gel. (H) Strain amplitude sweeps (γ = 66%) of mAMP-Gel. (I) Continuous step strain sweep (strain = 1 or 100%) of mAMP-Gel. Temperature: 25°C. (J) The swelling properties of mAMP-Gel. (K) Images of bacterial colonies treated with PEG-mAMP and mAMP-Gel for 12 h. (L and M) SEM images of bacteria seeded on mAMP-Gel for 6 h. (N and O) Hemolytic photos (N) and hemolysis rates (O) after 2 h of treatment with mAMP-Gel. (P and Q) Live/dead staining images (P) and proliferation profiles (Q) of L929 cells incubated with mAMP-Gel. Data are represented as mean ± SD (n = 4).

Figure 6

In vivo antibacterial activity of mAMP-Gel (A) Schematic illustration of the treatment and healing of infected skin wounds after injection of mAMP-Gel. (B and C) Wound healing processes (B) and changes in wound sizes (C) in different groups. (D) Quantitative analysis of the number of bacterial colonies obtained from the wound tissues of different groups on day 3. (E and F) H&E (E) and Masson’s trichrome (F) staining of the skin wound tissues harvested from different groups on days 7 and 14. Green arrows indicate the hair follicles. (G–I) Quantification of the number of hair follicles (G), granulation length (H), and collagen deposition (I) in different groups on day 14. (J) Immunofluorescence staining of TNF-α and IL-1β in different groups on day 7. Data are represented as mean ± SD (n = 5); a statistically significant difference compared with the control group is indicated by ∗∗∗p < 0.001.