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. 2023 Jul 13;4(5):100483. doi: 10.1016/j.xinn.2023.100483

Rational multivalency construction enables bactericidal effect amplification and dynamic biomaterial design

Xu Chen 1, Xinrui Li 1, Wenbo He 1, Miao Wang 1, Ang Gao 2, Liping Tong 2,∗, Shun Guo 1, Huaiyu Wang 2,∗∗, Guoqing Pan 1,∗∗∗
PMCID: PMC10407542  PMID: 37560332

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

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Public summary

  • •

    The multivalent bioligands that are ubiquitous in biosystems can be rationally designed via precision chemistries.

  • •

    Multivalency construction based on antimicrobial peptides leads to enhanced membrane disturbance and bactericidal amplification effects.

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    The electrostatic interacting pairs in multivalent antimicrobial peptides enable the fabrication of hydrogels with dynamic structures.

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    The multivalent antimicrobial peptide presented here and its hydrogel both demonstrate great potential for the treatment of infected wounds.

Introduction

Multivalency is widely involved in the physiological and pathological processes in life systems.1,2 These multiple and reversible intermolecular interactions occurring in the microenvironment composed of cells and the extracellular matrix (ECM) are central to diverse biological responses.3,4 For example, defense against pathogens involves competitive recognition of multiple carbohydrate ligands between pathogenic and mammalian lectins,1,5 and cell signaling and intracellular cascades are more inclined to be triggered by efficient activation of multiple membrane receptors.2,6,7,8,9 In addition to immune defense and various cell responses, multivalency is also the intrinsic driving force that supports the structural integrity of living organisms.10 Multivalent interactions in the biomacromolecular network, including proteins, lipids, and polysaccharides, are crucial to the dynamic but stable three-dimensional structures of cells and ECM.11,12,13 In brief, multivalency participates in both biochemical and biophysical processes in biosystems. Therefore, the concept of multivalency deserves in-depth exploration. From the perspective of tissue repair and regeneration, multivalency brings inspiration for not only the discovery of certain biological mechanisms but also the design of advanced biomaterials, since multivalent bioactivities (i.e., biochemical cues) and multivalency-supported dynamic structures (i.e., biophysical cues) are both highly desired for ECM-mimicking biomaterials.11,14,15,16,17,18

In recent decades, multivalency has been a well-studied new type of molecular strategy that can enhance specific recognition events.19,20,21,22,23 Synthetic multivalent ligand molecules have been proposed as therapeutic modalities to enhance the neutralization of toxins,20,24,25 improve the prevention of pathogen infections, and even accelerate tissue regeneration.5,26,27,28,29,30 Despite their vast prospects, synthetic multivalent systems as bioactive factors or functional biomaterials for biomedical applications are still very rare.19,31 This could be ascribed to the complexity (i.e., high reactivity that can damage bioactivities) and low controllability (i.e., hard to control the valence number) of traditional chemistries.32,33,34 In this context, precise chemical strategies with mild reaction conditions, high selectivity, and controllability are highly desired for multivalency construction.

As the molecular entities of multivalency, multivalent ligand molecules are reminiscent of polymers with multiple or repeating units. Polymerization might be a feasible means to realize bioligand multivalency through side group grafting. Moreover, the past decades have witnessed the great success of controlled radical polymerization (CRP),35,36 which can easily obtain well-defined linear polymers with controlled repeating units. Thus, CRP may provide a viable solution to improve controllability during multivalency construction. As another kind of precision chemistry, the click reaction (typically based on azido-alkynyl cycloaddition) enables specific, rapid, and thorough molecular conjugation, thus exhibiting great promise in biomolecule-related molecular grafting, modification, and even synthesis.37,38 Therefore, we propose to construct multivalency by combining the above two precision chemistries, which may synergistically improve the controllability, operability, and reproducibility for the synthesis of multivalent bioligand molecules.

In this work, we reported a precise multivalency construction strategy by clicking ligand molecules onto well-defined polymers. As a proof of concept, an antimicrobial peptide (AMP) was employed as the model ligand for multivalency construction due to its ability to enhance antibacterial activity and improve therapeutic efficiency for tissue infections.39 Moreover, the charged molecular structures of polymeric AMPs might facilitate the formation of dynamic biomedical hydrogel materials through multiple electrostatic interactions.40 These two aspects exactly correspond to the biochemical (i.e., multivalent bioactivities) and biophysical (i.e., dynamic multivalent structures) functions of multivalency in biosystems. Herein, we first synthesized a series of well-defined linear polymers with different alkynyl side groups through reversible addition-fragmentation chain transfer (RAFT) polymerization of alkynyl-containing propargyl acrylamide (PA) and hydrophilic 2-hydroxyethyl acrylamide. Subsequently, the alkynyl-containing polymers were clicked with azido-capped AMPs, leading to precisely controlled multivalent AMPs (mAMPs) (Figure 1A, left). The bactericidal amplification effect of the mAMPs was demonstrated both in vitro and in vivo (Figure 1A, right). In addition, the optimal mAMP was further employed in the design of a dynamic hydrogel through multiple electrostatic interactions (Figure 1B, upper). As an ECM-mimicking dynamic biomaterial, the mAMP-derived hydrogel exhibited self-healing and injectable properties. Furthermore, the hydrogel was used for treating infected open skin wounds due to its inherent antibacterial activity (Figure 1B, lower). Such high adaptability to wound geometry, efficient resistance to stress-induced fragmentation, and enhanced bactericidal effects may facilitate efficient disinfection of injured sites and continuous support during skin wound repair. In summary, the precision strategy presented in this work may provide an efficient means for multivalency construction, and the multivalency-assisted fabrication of ECM-like dynamic biomaterials will also bring new insights for the development of biomimetic scaffolds for tissue engineering and regenerative medicine.

Figure 1.

Figure 1

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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).

Results

Multivalent polymer synthesis

The alkynyl-capped functional monomer PA was obtained by amidation of acryloyl chloride with propargylamine according to a reported method (Figures S1 and S2A).41 During RAFT polymerization, the number of PA repeating units was controlled by feeding different ratios of PA and 2-Hydroxyethyl acrylamide (HEAA). The resultant poly(HA-co-PA) polymers (named PHA-PPAn, where n is the number of PA repeating units) were then characterized by NMR. The characteristic peaks of alkynyl hydrogen (–C≡CH, 3.08 ppm), aromatic hydrogen (–C6H5, 7.6 ppm and 7.9 ppm), and methine and methylene hydrogens (–CH– and –CH2–, 1.34–1.93 ppm) in the 1H NMR spectra of the PHA-PPAn polymers (Figure 2A) were clearly observed. According to the yields and quantification detected from the 1H NMR spectra, the number of PA repeating units and molecular weights of the polymers were determined (Table S1). Three polymers, PHA-PPA3, PHA-PPA6 and PHA-PPA10, with increasing numbers of alkynyl repeating units but similar chain lengths (polymerization degree of approximately 30) were obtained. Gel permeation chromatography (GPC) confirmed that all three PHA-PPAn polymers had very similar molecular weights and low polydispersity indices (approximately 1.09–1.17) (Figure 2B), indicating the high controllability of RAFT polymerization for the preparation of multivalent alkynyl polymers.

Figure 2.

Figure 2

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Chemical characterization and antibacterial activities of mAMPn

(A and B) 1H NMR spectra (A) and GPC analysis (B) of PHA-PPAn.

(C) 1H NMR spectra of mAMPn.

(D and E) Bacterial colony images of E. coli (D) and S. aureus (E) incubated with various concentrations of AMP and mAMPn 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).

The three alkynyl polymers (PHA-PPAn) were then clicked by an azido-capped antimicrobial peptide (N3-RWRWRW) for multivalent molecular construction (Figures S2B–S2D).42,43 The multivalent AMP polymers (where n is the valence number or repetitive number of AMP) were also characterized by NMR. In comparison to those of PHA-PPAn, the 1H NMR spectra of the mAMPn samples displayed the characteristic AMP peaks (10.81 ppm and 6.83–7.88 ppm), confirming the successful synthesis of mAMPn (Figure 2C). The valence number of mAMPn was further determined (Table S2), and three multivalent AMP polymers, mAMP2, mAMP4 and mAMP6, were obtained, corresponding to the original PHA-PPA3, PHA-PPA6, and PHA-PPA10, respectively. Note that the ratio of alkynyl groups that reacted with azido-AMP was only approximately 60%, probably due to steric effects. The above results demonstrated that mAMPn with precisely controlled multivalent ligands could be efficiently synthesized through the combination of RAFT and click chemistry.

In vitro antibacterial activity

The influence of multivalency on the intrinsic antibacterial activity of the AMP was evaluated. The mAMPn polymers and free AMP molecules all exhibited concentration-dependent antibacterial activity against gram-negative Escherichia coli (Figure 2D). When the concentration AMP increased to 40 μmol L−1, the E. coli could be completely killed, indicating a decent bactericidal effect of the monovalent AMP that is consistent with previous studies.43 In contrast, all the mAMPn polymers exhibited significantly enhanced bactericidal effects, and the minimum bactericidal concentrations (MBCs) of the mAMPn polymers against E. coli were determined to be approximately 2.5–10 μmol L−1 (Figure S3; Table S3). In particular, the MBC of mAMP6 (2.5 μmol L−1) was less than one-tenth of that of the monovalent AMP (40 μmol L−1). Similar results were observed in the tests against gram-positive Staphylococcus aureus (Figure 2E). For example, the MBC of the monovalent AMP was 20 μmol L−1, which is about eight times higher than that of mAMP6 (2.5 μmol L−1). In addition, mAMPn showed faster antibacterial kinetics than the AMP (Figure S4), further indicating the significant effect of mAMPn polymers to amplify the antibacterial activity.

To more intuitively reveal the antibacterial mechanism,44 the membrane morphology of the bacteria after treatment was examined by scanning electron microscope (SEM) and transmission electron microscope (TEM) (Figures 2F and 2G). The SEM images showed that the number of bacteria after treatment was significantly reduced, and their morphology mostly showed collapsed and fused states, demonstrating highly efficient destruction of the bacterial cell wall. The TEM images further revealed that the clearly visible membranes on bacteria in the control groups disappeared after treatment with AMP and mAMP6, leaving an incomplete morphology and only a trace of leaked cytoplasmic matrix. We considered that stronger electrostatic binding forces and the ligand confinement of mAMPn might lead to the locally enhanced membrane distribution compared with that of monovalent AMP, which contributed greatly to the amplification of antibacterial activity.2

Multivalent antibacterial mechanism

We speculated that the bactericidal amplification effect of mAMPn was due to locally enhanced ligand binding of multivalent molecules (Figure 3A). To verify this hypothesis, the binding of AMP and mAMPn to bacteria was investigated by confocal laser scanning microscopy (CLSM). After incubation with fluorescein isothiocyanate (FITC)-labeled AMP and mAMPn (Tables S4 and S5) for 20 min, we found that the green fluorescence from FITC that appeared on the mAMP6-treated bacteria was significantly stronger than that on the AMP-treated bacteria (Figures 3B and S5). Quantitative analysis showed that the bacterial binding rates for the AMP and the mAMP6 polymers were 22.9% and 51.9%, respectively (Figure 3C). The quartz crystal microbalance (QCM) tests confirmed this result. The significantly decreased frequency (f) after solution injection more intuitively demonstrated the stronger binding action and confirmed the multivalency-enhanced ligand binding of the mAMP6 polymer on bacterial membranes (Figures 3D and 3E). Therefore, the local ligand concentration and the binding force of mAMPn on the bacterial membrane are much higher, which could significantly enhance the disturbance of the anionic phospholipid layer, thus exhibiting amplified bactericidal effects.

Figure 3.

Figure 3

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The enhanced antibacterial mechanism of mAMPn

(A) Schematic illustration of the bactericidal amplification effect provided by mAMPn.

(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.

Biofilm inhibition

In the clinic, bacterial biofilms in chronic wound infections are extremely difficult to cure.45,46 Thus, we attempted to apply the multivalent antibacterial polymer to eliminate biofilms. As shown in Figure 3F, the minimum eradication biofilm concentrations of AMP and mAMP6 were determined to be 1,280 and 640 μmol L−1, respectively. Live/dead staining showed that the original dense biofilm was significantly reduced after mAMP6 treatment for 8 h, and the surface became sparse and irregular (Figure 3G). Therefore, we have a preliminary inference that multivalent binding could accelerate the permeation and removal of the protective layer of the biofilm (e.g., extracellular polymeric substances), thus exposing the inner bacteria.47,48 After losing its barrier function, the damaged biofilm could be easily penetrated by mAMP6 polymers, leading to an improved biofilm eradication efficiency (Figure 3H).

Biocompatibility

Both AMP and mAMPn exhibited very low hemolytic toxicity, which was almost negligible at concentrations below 10 μmol L−1 (Figures S6A–S6C). Thus, the mAMP6 polymer could be considered to have no hemolytic toxicity in its effective antibacterial concentration ranges (2.5–10 μmol L−1). In addition to hemolytic toxicity, the cytotoxicity of mAMPn was also determined with live/dead staining. As expected, treated mouse fibroblast (L929) cells showed only very few red dead cells (Figure S6D). Moreover, the treated cells could be recovered and exhibited perfect proliferative ability (Figure S6E). The above results indicated the excellent biocompatibility of the mAMPn polymers.

In vivo anti-infective ability

We then established an S. aureus-infected skin wound model in rats to investigate the in vivo anti-infective potential of mAMPn (Figure 4A). The anti-infection and wound healing processes were then evaluated (Figure 4B). In the early stages, yellow pus was clearly found on the infected wound bed in the PBS and PH-PPA groups, indicating the severity of infection. However, such pyogenic infection was not observed in the AMP and mAMP6 groups, which was also confirmed by the agar plate count method of tissue exudate from the wound sites (Figures 4C and 4D). In addition to stronger anti-infection properties, the mAMP6-treated wounds exhibited the fastest wound closure rate over 14 days, probably due to the faster elimination of bacteria in vivo and the initiation of the tissue reconstruction process (Figure 4E). Hematoxylin and eosin (H&E) and Masson’s trichrome staining were also used to examine the anti-infection and wound healing efficacy. The skin defects in the mAMP6 group were completely closed with highly regular epithelium, numerous hair follicles (green arrows), and almost complete regeneration of the dermal tissue (improved granulation length and collagen deposition) on day 14 (Figure S7). The infection-induced inflammatory responses were also examined (Figures 4F and 4G). Two typical proinflammatory cytokines,49,50 tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), were highly expressed on day 7 in the other groups in contrast to their significantly reduced expression in the mAMP6 group. These results together demonstrated the promise of mAMP6 as an antibacterial therapeutic for treating in vivo bacterial infections and improving wound healing.

Figure 4.

Figure 4

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In vivo antibacterial activity of mAMPn

(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.

Dynamic hydrogel fabrication

We further used the multivalent mAMPn polymer to fabricate an ECM-like biomaterial with a dynamic structure. The mAMP6 polymer was successfully linked onto four-armed poly(ethylene glycol) (4-arm-PEG) through thiol reduction and Michael addition reactions (Figures 5A and S8). Due to the multiple guanidinium groups, the PEG-mAMP6 polymer could be rapidly crosslinked by clay nanosheets (CNSs) through multiple electrostatic interactions (Figures 5B, 5D, and S9).51,52 The porous structure of the hydrogel (mAMP-Gel) also implied its potential as a biomaterial to support cell growth in tissue engineering (Figure 5C).

Figure 5.

Figure 5

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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).

Due to the reversibility of multiple electrostatic interactions, we expect the mAMP-Gel to display typical dynamic properties, such as self-healing ability, remoldability, and injectability.53 As shown in Figure 5E, the cut hydrogel blocks healed within 30 min and integrated together even after the application of a certain tensile force. In addition, mAMP-Gel has excellent injectable properties due to its shear thinning behavior (Figure 5F and Video S1). Rheological tests were further performed to examine the dynamic mechanism of mAMP-Gel.54 Dynamic oscillatory sweep tests revealed that the hydrogel storage modulus (G') was higher than the loss modulus (G''), indicating its elastic gel-like properties (Figure 5G). As shown in Figure 5H, the strain sweeps indicated that the hydrogels had a typical elastic response. The value of G′ decreased rapidly to below that of G″ above the critical strain region (γ = 66%), indicating a collapse of the gel to the sol state. We further found that the hydrogel very rapidly recovered its mechanical properties when it was applied with an alternating strain between 1% and 100% (Figure 5I). In addition, after repeated alternating strain sweeps, the values of G′ and G″ recovered to their initial values, further confirming the dynamic but stable structure of mAMP-Gel. The above tests demonstrated the dynamic structure and mechanism of mAMP-Gel.

Video S1. The injectability and moldability of a representative mAMP hydrogel
Download video file (880.5KB, mp4)

Since dynamic hydrogels possess great potential in biomedicine, some critical parameters (swelling rate, degradation rate, antibacterial activity, and biocompatibility) of the resultant mAMP-Gel were further studied. We found that mAMP-Gel exhibited excellent swelling properties and was almost completely degraded within 2 weeks in vitro (Figures 5J and S10). Note that the antibacterial activity of mAMP-Gel was slightly compromised during the hydrogel formation process,55 probably due to sterics restricting the contact between the AMP ligand and bacteria (Figures 5K and S11). Nevertheless, bacteria could be completely killed by mAMP-Gel after an extended incubation time. PEG-mAMP6 also still exhibited excellent antibacterial properties. The number of bacteria after treatment was significantly reduced after 6 h, and the residual bacteria mostly showed collapsed and fused states (Figures 5L and 5M). It is also worth mentioning that the dynamic mAMP-Gel exhibited excellent biocompatibility. In addition to negligible hemolytic toxicity (Figures 5N and 5O), cells cocultured with the dynamic hydrogel maintained high viability for 12 h (Figures 5P and S12), and their proliferation rate was nearly equivalent to that of the cells in the control group without treatment (Figure 5Q). The above results implied the great potential of this dynamic mAMP-Gel hydrogel for use as a biocompatible and anti-infective scaffold biomaterial, particularly for the treatment and repair of infection-prone tissues.

Using the dynamic hydrogel as a wound dressing

With this dynamic antibacterial hydrogel in hand, we further established a full-thickness infected wound model on rat skin to investigate the potential of the hydrogel as an advanced wound dressing material for infected skin repair and regeneration (Figure 6A). Two different groups, treated with PBS (control) or with the dynamic mAMP-Gel, were used to evaluate wound healing efficacy. As shown in Figure 6B, great suppuration was observed on the infected wound bed in the control group on day 3, indicating the severity of wound infection. In contrast, such pyogenic phenomena were never observed in the group injected with mAMP-Gel hydrogel, suggesting that mAMP-Gel treatment could effectively kill bacteria and inhibit infection. Re-culture of the wound exudate on agar plates confirmed that there were significantly fewer bacteria in the mAMP-Gel group on day 3 (Figures 6D and S13), verifying the efficient anti-infective ability of the hydrogel. Due to the rapid elimination of bacteria, the tissue reconstruction process in the mAMP-Gel group was initiated in a timely manner, and the wounds in this group healed within 14 days (Figure 6C). However, no wounds in the control group healed completely within the same period.

Figure 6.

Figure 6

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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.

H&E and Masson’s trichrome staining were further employed to evaluate tissue healing efficacy. As shown in Figures 6E and 6F, the skin defects in the mAMP-Gel group were almost closed with a highly regular epithelium, numerous hair follicles, extensive collagen deposition, and almost complete regeneration of dermal tissue on day 14. In contrast, epithelialization in the control group was incomplete, and a large area of immature granulation tissue was deposited around the wound. Quantitative analysis showed that the number of hair follicles, granulation length, and collagen deposition were significantly improved in the mAMP-Gel group (Figures 6G–6I). Moreover, the expression levels of TNF-α and IL-1β were also significantly reduced due to the anti-infection ability of mAMP-Gel, which could significantly reduce inflammatory responses (Figure 6J). Finally, histological assessment of major organs (e.g., heart, liver, spleen, lung, and kidney) was performed to examine the long-term biosafety of the mAMP-Gel hydrogel (Figure S14). Similar to the control group, no obvious lesions or other histopathological abnormalities were observed in tissue sections in the mAMP-Gel group.

Although the in vivo anti-infection abilities of the mAMP polymer and mAMP-Gel were not significantly different, the superiority of mAMP-Gel during the wound repair stage was clear. Infected wounds treated with mAMP-Gel healed and had a better tissue structure (Figures 4B and 6B). The H&E staining images revealed that although the wounds in both the mAMP polymer group and mAMP-Gel group could completely epithelialize in 14 days, the epithelial layer in the mAMP-Gel group was notably more regular and thicker. In addition, some elongated hair follicles grew in the mAMP-Gel group (Figures S7 and S6E). This is probably due to the protective effect of the hydrogel, which can provide structural support and keep the wound moist, thus facilitating the growth and migration of endothelial cells and fibroblasts.56 The above positive results clearly demonstrated that the multivalency-supported dynamic hydrogel might be used as a biocompatible, easy to handle, and antibacterial scaffold material for infected tissue repair and regeneration.

Conclusion

In summary, we herein reported a precise strategy to construct multivalency by combining controlled polymerization and click chemistry and then applied the generated multivalent molecules to fabricate dynamic biomaterials. We first synthesized a series of well-defined linear polymers with different alkynyl side groups through RAFT polymerization. Subsequently, the alkynyl polymers were clicked with azido-AMP to obtain precisely controlled multivalent AMP polymers (mAMPs). Due to the aggregation of cationic AMP ligands and locally enhanced disturbance of the bacterial membrane, the mAMPs significantly amplified the bactericidal effect (8- to about 10-fold). The mAMPs were further employed for dynamic crosslinking with negatively charged CNSs via multiple electrostatic interactions. As an ECM-like dynamic biomaterial, the mAMP-derived hydrogel exhibited typical dynamic properties, including self-healing ability, injectability and remoldability. Furthermore, this hydrogel could be used for treating infected open skin wounds due to its inherent antibacterial activity. The high adaptability to wound geometry, efficient resistance to stress-induced fragmentation, and enhanced bactericidal effect enabled the dynamic hydrogel to efficiently disinfect infected sites and continuously support skin wound repair in vivo. In summary, the precision chemistries used in this study may provide an efficient means for multivalency construction, and the multivalency-assisted fabrication of ECM-mimicking dynamic hydrogels will also provide new insights for the biomimetic design of dynamic and highly bioactive materials in tissue engineering and regenerative medicine.

Material and methods

See supplemental information for details.

Acknowledgments

We acknowledge the National Natural Science Foundation of China (32222041, 21875092 and 82272157), the National Natural Science Foundation of Jiangsu Province (BK20220059), the National Key Research and Development Program of China (2019YFA0112000), the Innovation and Entrepreneurship Program of Jiangsu Province, and the “Jiangsu Specially-Appointed Professor” Program.

Author contributions

X.C., L.T., H.W., and G.P. conceived the idea and designed the experiments. G.P. supervised the project. X.C., X.L., and W.H. carried out the experiments. X.C. and G.P. analyzed the data with the help of M.W., A.G., and S.G. X.C., H.W., and G.P. wrote the manuscript.

Declaration of interests

The authors declare no competing interests.

Published Online: July 13, 2023

Footnotes

Contributor Information

Liping Tong, Email: lp.tong@siat.ac.cn.

Huaiyu Wang, Email: hy.wang1@siat.ac.cn.

Guoqing Pan, Email: panguoqing@ujs.edu.cn.

Lead contact website

http://faculty.ujs.edu.cn/panguoqing/zh_CN/index.htm.

Supplemental information

Document S1. Supplemental material and methods, Figures S1–S14, and Tables S1–S5
mmc1.pdf (3MB, pdf)
Document S2. Article plus supplemental information
mmc3.pdf (12.2MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Video S1. The injectability and moldability of a representative mAMP hydrogel
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Document S1. Supplemental material and methods, Figures S1–S14, and Tables S1–S5
mmc1.pdf (3MB, pdf)
Document S2. Article plus supplemental information
mmc3.pdf (12.2MB, pdf)

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