Lignin-Cobalt Nano-Enabled Poly(pseudo)rotaxane Supramolecular Hydrogel for Treating Chronic Wounds

Abstract

Chronic wounds (CWs) are a growing issue for the health care system. Their treatment requires a synergic approach to reduce both inflammation and the bacterial burden. In this work, a promising system for treating CWs was developed, comprising cobalt-lignin nanoparticles (NPs) embedded in a supramolecular (SM) hydrogel. First, NPs were obtained through cobalt reduction with phenolated lignin, and their antibacterial properties were tested against both Gram-negative and Gram-positive strains. The anti-inflammatory capacity of the NPs was proven through their ability to inhibit myeloperoxidase (MPO) and matrix metalloproteases (MMPs), which are enzymes involved in the inflammatory process and wound chronicity. Then, the NPs were loaded in an SM hydrogel based on a blend of α-cyclodextrin and custom-made poly(ether urethane)s. The nano-enabled hydrogel showed injectability, self-healing properties, and linear release of the loaded cargo. Moreover, the SM hydrogel's characteristics were optimized to absorb proteins when in contact with liquid, suggesting its capacity to uptake harmful enzymes from the wound exudate. These results render the developed multifunctional SM material an interesting candidate for the management of CWs.

Keywords: bacterial infection; chronic wounds; cobalt-lignin nanoparticles; inflammation; matrix metalloproteases; myeloperoxidases; poly(ether urethane); poly(pseudo)rotaxane; supramolecular hydrogels; α-cyclodextrin.

Figure 1

Comparison of ATR-FTIR spectra of lignin, tannic acid, and lignin conjugated with TA and LTH (Lig-TA-LTH). The arrows indicate relevant peaks proving the conjugation of TA with lignin.

Figure 2

CoLig NPs’ morphology as assessed through SEM imaging. The CoLig NPs have an irregular round shape that is the result of cluster formation after Co and Lig-TA-LTH interaction.

Figure 3

MPO and MMP inhibition of CoLig NPs. *** refers to p < 0.001 and ** to p < 0.01.

Figure 4

SEM images of bacteria after treatment with CoLig NPs. The bacteria species usead are (a) P. aeruginosa and (b) S. aureus. Arrows indicate cells with damaged membranes.

Figure 5

Cytotoxicity of CoLig NPs against human fibroblasts (BJ-5ta) and keratinocytes (HaCaT). *** refers to p < 0.001.

Figure 6

Comparison between the CoLig NPs’ cytotoxicity toward BJ-5ta and inhibitory capacity toward S. aureus.

Figure 7

BSA uptake test in the SM-gels with a CHP407:SHF68 volume ratio of 80:20, an αCDs concentration of 10% w/v, and different overall polymeric concentrations (1, 3, and 5% w/v). (a) % of dry weight reduction in the SM-gels at 1, 3, and 5% w/v polymer concentrations. (b) % of BSA absorbed in the SM-gels from the aqueous medium. After 72 h, the differences between the three conditions remained stable, presenting statistically relevant differences. *** refers to p < 0.001.

Figure 8

Stability in PBS of the SM-gel loaded with CoLig NPs. An unaltered SM-gel was also tested as control condition.

Figure 9

Rheological properties of the SM-gel loaded with CoLig NPs. An unaltered SM-gel was also characterized for comparison. (a) Frequency sweep test and (b) amplitude sweep test. The addition of CoLig NPs increased the system’s rigidity.

Figure 10

CryoSEM images of the SM-gel with or without CoLig NPs. The addition of CoLig NPs increased the SM-gel’s degree of crosslinking. The numbers of branches of the SM-gels were calculated using the Skeleton tool of ImageJ.

Figure 11

Comparison of ATR-FTIR spectra of SHF68, CoLig NPs, and their combination. The arrows indicate the peak shift proving the interaction between CoLig NPs and SHF68 chains.

Figure 12

Quantification of CoLig NPs’ release from SM-gel through two different methods: ICP and fluorescence analysis. The CoLig NPs’ release appeared to be close to the 0-order and matched SM-gel dissolution. Moreover, the two analysis methods were in accordance, confirming the NPs’ integrity.

Figure 13

SEM images of bacteria after treatment with SM-gel with and without CoLig NPs. Arrows indicate cells with a damaged membrane.

Figure 14

Cytotoxicity of SM-gel with and without CoLig NPs against human fibroblast (BJ-5ta) and keratinocytes (HaCaT). The * refers to p < 0.05, and n.d. refers to non-significative differences.