"INTRODUCTION: The use of biomaterials inside the body always entails the risk of infection. This risk might even be higher in in situ tissue engineering applications. Since the porous scaffold materials can form a niche for invading bacteria, the intended in situ production of novel tissue may be severely compromised by infection. Therefore, we aim to develop a new polymeric supramolecular scaffold material, exerting two important functions: preventing microbial adhesion and thereby preventing biofilm formation, and inducing endogenous (eukaryotic) cells to regenerate the body.
METHODOLOGY: In our research, supramolecular contact-killing materials based on antimicrobial peptides (AMP) are developed. A special class of supramolecular biomaterials are based on fourfold hydrogen bonding 2-ureido-4[1H]-pyrimidinone (UPy) moieties. The supramolecular base material consists of an UPy end-functionalization polycaprolactone (i.e. PCLdiUPy). These UPy-materials can be functionalized with bioactive compounds, either via a modular approach in which the UPy-base material is mixed with UPy-modified additives1, or via a post-modification strategy to specifically functionalize the surface of the biomaterial using click chemistry2. The antimicrobial activity is introduced via UPy-functionalized AMPs, using SAAP-148, a synthetic derivative of LL-373. The regenerative activity is introduced via an UPy-functionalized heparin binding peptide (UPy-HBP). The peptides were synthesized by manual Fmoc-based solid phase peptide synthesis. Solid polymer films were prepared by drop-casting PCLdiUPy with UPy-SAAP-148 or UPy-TC84 on glass coverslips. The antimicrobial activity of the UPy-AMPs in solution and when incorporated in the drop-casted samples was evaluated against Escherichia coli ESBL and Staphylococcus aureus JAR060131 and LUH14616 (MRSA) and Acinetobacter baumannii RUH875 using the LC99.9 (i.e. the lowest concentration killing at least 99.9% of the inoculum) and the JISZ2801 surface antimicrobial assay, respectively. Moreover, the cytotoxicity of these AMPs was tested against human dermal fibroblasts.
RESULTS: Coupling of the UPy-linker to SAAP-148 did not influence its antimicrobial activity in solution. For the solid drop-casted materials, incorporation of 5 mol% UPy-SAAP-148 is sufficient for killing all 4 bacterial strains tested. This indicates that the peptide remains active after immobilization in the materials. Unfortunately, TC84 loses its antimicrobial activity upon UPy-coupling, both in solution and as a solid. QCM-D adsorption studies revealed that heparin adsorbed to spin coated material films of PCLdiUPy with 5 mol% UPy-HBP mixed via the modular strategy.
Current studies focus on characterization of the UPy-SAAP-148/TC84 and multifunctional biomaterial with XPS, AFM, WCA, zeta potential and leakage experiments to investigate the material properties. Moreover, we assess the in vivo efficacy of dip-coated titanium implants with 5% UPy-SAAP-148 in the experimental biomaterial-associated infection mouse model.
CONCLUSIONS: In conclusion, this modular approach will enable a stable but dynamic incorporation of AMPs, and control of cell adhesion by using cell-adhesive peptides. Ultimately, we aim to use such materials for in situ infection-free tissue engineering.
REFERENCES: 1. Dankers, P.Y.W. et al., Nat. Mater. 4 (7), 568-574 (2005), 2. Goor, O.J.G.M. et al., Adv. Mater. 29 (5), 1604652 (2017), 3. de Breij A. & Riool, M. et al., Sci. Transl. Med. 10 (423), eaan4044 (2018)."