Active topography with mucin coating for biofilm control

Introduction: In the innate immune system of mammals, mucins cover the epithelial tissues and help prevent the colonization of microbial pathogens. There are two types of mucins: secreted mucin, which forms a mucus gel to trap pathogens, and membrane-associated mucins, on epithelial tissues that prevent pathogens from attaching to the cilia. Abiotic biomaterials used for biomedical implants lack such protection and thus are susceptible to microbial colonization, leading to biofilm formation and chronic infections with high-level tolerance to antibiotics. Previous studies demonstrated that mucin coatings can reduce bacterial colonization, and the primary mucin component glycan can repress virulence gene expression in Pseudomonas aeruginosa (PAO1). However, the studies to date have focused on mucin coating of static biomaterials. To engineer better infection-resisting biomaterials, we coated the um-size pillars of our recently developed active topography with denatured mucin. We hypothesize that this bioinspired design will provide effective protection from bacterial colonization by mimicking the host’s innate immunity with beating cilia of mucin-coated epithelial tissues.

Materials and

Methods: We coated polydimethylsiloxane (PDMS) with denatured porcine mucin (Sigma). Briefly, plasma was used to activate PDMS surfaces and convert the methyl group to the hydroxyl group, which was then precoated with silane. After activation with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide-hydrochloride (EDC) and N-Hydroxysulfosuccinimide (sulfo-NHS), denatured porcine mucin was covalently conjugated to the carbonylated surface through an amide bond. Fourier-transform Infrared Spectroscopy (FTIR) was used to detect the functional group of each step and confirm the covalent binding between mucin and PDMS. The hydrophilicity of the surface was evaluated by measuring the water contact angle. Biofilm of PAO1, Staphylococcus aureus, and Escherichia coli were grown on PDMS surfaces at 37ºC for six hours. Biofilms were stained with LIVE/DEAD staining and the biomass was quantified using COMSTAT2 after gentle wash to remove planktonic cells. Besides for the flat PDMS surface, mucin was also coated on the pillar surfaces, which were loaded with superparamagnetic Fe3O4 nanoparticles at the pillar tips. Pillars were actuated with 1mT electromagnetic field at 10Hz frequency.

Results, Conclusions, and Discussions: Coating with mucin reduced the contact angle of PDMS from 110° to 59°, rendering the surface more hydrophilic. And the N-H amine bonds were detected on mucin-coated surfaces by FTIR. The pyrazole ring and sulfonate group peaks were only found on the surface before mucin coating, which confirms that the sulfo-NHS was replaced by mucin. Mucin coatings strongly inhibit the attachment of both the wildtype PAO1 and its mucoid mutant, PDO300, by 95.7±0.8% and 71.3±11.2%, respectively, compared to the uncoated controls’ biofilm. The combination of mucin coating and active topography showed further reduction effect. Compared reduction with uncoated beating pillars, actuated of coated pillars showed additional 88.3±7.0% of biofilm biomass reduction after 6 hours of incubation. In flow cell systems, engineered surfaces also showed better antifouling effects for long-term biofilm control. For 3-day biofilm of PAO1, actuation of mucin-coated pillar showed 99.7±0.2% less biofilm than surfaces with uncoated static pillars, and 80.2±14.5% less than surfaces with actuated but uncoated pillars. Abiotic biomaterials lack the host defense and are susceptible to microbial colonization. In this study, we demonstrate that coating with denatured mucin can further enhance the antifouling activities of active topography. Bioinspired design can be leveraged for long-term biofilm control and the design of safer medical devices.

Acknowledgements (Optional):