Bio-Inspired Active Topography for Antifouling Biomaterials

In the innate immune system of mammalians, mucins cover the epithelial tissues and prevent the colonization of microbial pathogens. Abiotic biomaterials for biomedical implants lack such protection and thus are susceptible to microbial attachment, which leads to biofilm formation and chronic infections with high-level tolerance to antibiotics. We were motivated to engineer infection-resisting biomaterials mimicking active surfaces of host tissues. Specifically, we aimed to coat the mm-size pillars of our recently developed active topography with denatured porcine mucin. We hypothesize that this bioinspired design will provide effective protection from bacterial colonization and remove established biofilms by mimicking the host innate immunity with beating cilia of mucin-coated epithelial tissues.

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, EDC and sulfo-NHS, to activate the surface first. Denatured porcine mucin was covalently conjugated to the carbonylated surface through the amide bond. FT-IR was used to detect the functional group of each step to confirm the covalent binding between mucin and PDMS. Mucin coating was tested for both flat and pillared (with superparamagnetic Fe₃O₄nanoparticles at the tip) surfaces. Antifouling effects were tested by growing early-stage and long-term biofilms of Pseudomonas aeruginosa on pillar surfaces with/without mucin coating. Active topography was actuated with a external oscillating magnetic field (1-5 mT).. The synthesis of c-di-GMP in attached cells was monitored by measuring fluorescence intensity of the reporter strain PAO1/pCdrA::gfp^s using fluorescence microscopy and and flow cytometry. The swarming and twitching motilities of P. aeruginosa were studied using agar plates with or without mucin coating. Polyethylene glycol (PEG) was also tested as a control to determine if mucin has any biological effects on P. aeruginosa motilities in addition to chemical/physical effects.

Results

Coating with mucin reduced the contact angle of pillared PDMS surface from 148° to 40°, rendering the nearly superhydrophobic surface more hydrophilic. During the 16 days incubation period, no substantial change in contact angle was observed. In addition, the N-H amine stretching bond peaks were seen in FT-IR throughout the 16 days, which confirmed the mucin coating stability.

Mucin coatings exhibited strong inhibition of attachment of both the wild-type P. aeruginosa PAO1 and its mucoid mutant, e.g., by PDO300, 95.7 ± 0.3 % and 71.0 ± 5.6 %, respectively, compared to the uncoated control surfaces. With actuation (1 mT, 6 h, 10 Hz), mucin coating further prevented biofilm formation by 88.3 ± 4.0% compared to uncoated but actuated pillars. In total, 99.1 ± 0.3 % reduction of the original biofilm was achieved. Mucin coated active topography also exhibited strong removal of established biofilms, e.g., 1.9-log removal of 3-day biofilms in flow chambers. SEM analysis revealed that mucin coating significantly reduced bacterial attachment near pillar bases, the areas that are protected from the shear of beating pillars. Collectively, these results show that mucin coating enhanced the antifouling effects of active topography.

To understand the underlying mechanism of fouling control by mucin coating, we tested the effects of mucin on the synthesis of the secondary messenger cyclic di-GMP (c-di-GMP), a master regulator of the switch between planktonic growth and biofilm formation using the reporter strain PAO1/pCdrA::gfps Results showed that both coated mucin on the surface and mucin added as free molecules reduced c-di-GMP synthesis in P. aeruginosa. Besides, P. aeruginosashowed enhanced twitching motility and inhibited swarming motility in the presence of mucin. These effects all negatively affect the transition of bacterial cells from planktonic stage to surface attachment, leading to inhibition of biofilm formation. Unlike mucin, the antifouling agent polyethylene glycol (PEG, Mw~300kDa, similar Mw to tethered mucin MUC1) did not affect cell motilities, indicating that the antifouling effects of mucin coating involve biological factors that directly affect bacterial biofilm formation.

Implications

In this study, we covalently modified PDMS pillars with denatured mucin to mimic cilia with tethered mucin in the mammalian innate immune systems. This bioinspired design improved biofilm control by active topography and thus provides a new strategy to engineer infection resisting biomaterials. With strong activities against biofilms, this strategy can help engineer safer medical devices, such as urinary catheters, to prevent biofilm formation and catheter blockage.