AVS 66 Session BI+AS-WeM: Microbes and Fouling at Surfaces

Hydrophilic building blocks like polyethylene glycols are powerful ingredients in modern fouling-release coatings as they are capable to reduce the attractive hydrophobic interactions of microbes with hydrophobic matrix materials such as acrylates, silicones or polyurethanes. We explored how polysaccharides with known antiadhesive and anti-inflammatory properties in medical applications reduce the adhesion of marine fouling organisms. Among the advantages of polysaccharides is their availability, biocompatibility and degradability. Based on previous work on hydrophilic coatings [1] we focused on well hydrated hyaluronans, alginates, chitosans and chondroitin sulfate building blocks. The response of marine organisms and the ability of such components to reduce attachment and facilitate easy removal is explored on grafted monolayers of polysaccharides [2], their amphiphilic derivatives [3], and in more complex coatings such as polysaccharide containing polyelectrolyte multilayers and hybrid polymers. Lab and field experiments will be compared and discussed in the light of the previous notion that uptake of soil particles frequently challenge hydrophilic polymers when used in the real ocean environment [4].

[1] A. Rosenhahn, S. Schilp, J. Kreuzer, M. Grunze, PCCP 2010, 12, 4275

[2] X. Y. Cao, M. E. Pettit, S. L. Conlan, W. Wagner, A. D. Ho, A. S. Clare, J. A. Callow, M. E. Callow, M. Grunze, A. Rosenhahn, Biomacromol. 2009, 10, 907-915.

[3] V. Jakobi, J. Schwarze, J. A. Finlay, K. A. Nolte, S. Spöllmann, H.-W. Becker, A. S. Clare, A. Rosenhahn, Biomacromol. 2018,19, 402-408.

[4] J. Koc, T. Simovich, E. Schönemann, A. Chilkoti, H. Gardner, G.W. Swain, K. Hunsucker, A. Laschewsky, A. Rosenhahn, Biofouling 2019, DOI:10.1080/08927014.2019.1611790

Microfluidic environments with laminar flow are a useful tool to quantify attachment and removal of marine biofilm formers and cells^1–3. We present results on the microfluidic attachment of the marine bacterium Cobetia marina (C. marina), formerly named Halomonas marina, as it is frequently found in marine biofilms^4,5. To identify a suitable shear stress for the microfluidic attachment assays, the attachment behavior of C. marina was investigated at different shear forces on hydrophobic and hydrophilic surfaces, whereby C. marina tends to adhere best on hydrophobic coatings. Among the optimized assay parameters are the relevance of the growth state of C. marina. The optimized assay parameters will be presented as well as selected examples how coating chemistries like different self-assembling monolayers, amphiphilic alginates⁶ and different thicknesses of PG coatings⁷ alter the attachment of the marine bacterium C. marina under dynamic shear conditions.

1. Christophis, C., Grunze, M. & Rosenhahn, A. Quantification of the adhesion strength of fibroblast cells on ethylene glycol terminated self-assembled monolayers by a microfluidic shear force assay. Phys. Chem. Chem. Phys. (2010), 12, 4498-4504. doi:10.1039/b924304f
2. Alles, M. & Rosenhahn, A. Microfluidic detachment assay to probe the adhesion strength of diatoms. Biofouling (2015), 31:5, 469-480. doi:10.1080/08927014.2015.1061655
3. Nolte, K. A., Schwarze, J. & Rosenhahn, A. Microfluidic accumulation assay probes attachment of biofilm forming diatom cells. Biofouling (2017), 33 (7): 531-543. doi:10.1080/08927014.2017.1328058
4. Ista, L. K., Fan, H., Baca, O. & López, G. P. Attachment of bacteria to model solid surfaces’ oligo(ethylene glycol) surfaces inhibit bacterial attachment. FEMS Microbiol. Lett. (1996), 142, 59-63. doi:10.1016/0378-1097(96)00243-1
5. Arpa-Sancet, M. P., Christophis, C. & Rosenhahn, A. Microfluidic assay to quantify the adhesion of marine bacteria. Biointerphases (2012), 7, 26. doi:10.1007/s13758-012-0026-x
6. Jakobi, V. et al. Amphiphilic Alginates for Marine Antifouling Applications. Biomacromolecules (2018), 19, 402-408. doi:10.1021/acs.biomac.7b01498
7. Wanka, R. et al. Fouling-Release Properties of Dendritic Polyglycerols against Marine Diatoms. ACS Appl. Mater. Interfaces (2018), 10, 34965–34973. doi:10.1021/acsami.8b12017

Bacterial biofilms are microscopic assemblages of bacterial cells usually attached to a surface and held together by a self-produced extracellular polymeric slime (EPS) matrix. Biofilms are ubiquitous in the natural environment and are highly problematic in industry and medicine where they cause corrosion, fouling, contamination and chronic medical and dental infections. The basic biology of bacterial biofilm development and strategies evolved to survive in the environment of the ancient earth are now used by the bacteria to survive on modern man-made materials. Diffusion limitation within the EPS matrix results in sharp gradients as nutrients are consumed by respiring bacteria on the periphery faster than they can diffuse in. Similarly, cell signals (molecules used to co-ordinate behavior between individual cells) and waste products, such as fermentation acids, build up in the interior of the biofilm. Biofilms are mechanically complex showing a range of behaviors from elastic solids to viscous liquids. These viscoelastic properties can facilitate survival on surfaces exposed to high shear stresses and can explain the high pressure drop and frictional losses in pipelines and ship hull fouling. However, the mechanical response may also be exploited to drive antimicrobials into the biofilm for control. The development of microenvironments combined with the structural versatility of the biofilm is the basis for the distinct biofilm phenotype as an emergent property of population of single cells and is a challenge to overcome in their control.

Polyethylene glycol (PEG) containing coatings show outstanding antifouling properties, which is commonly assigned to their hydrophilicity and their highly hydrated nature. A structurally related but hyperbranched version are polyglycerols (PGs) that increase the spatial density of non-fouling polymer units and decrease the defect density in coatings.^1,2 So far they were successfully applied in biomedicine against attachment by pathogenic bacteria. Using a surface initiated ring opening polymerization reaction³, we grafted dendritic PGs on surfaces. The resulting samples were characterized by spectroscopic ellipsometry, contact angle goniometry, ATR-FTIR, and by degradation experiments. The prepared surfaces show excellent protein-resistance. The fouling release properties were tested in a standardized lab assay with diatoms (Navicula incerta) and in a dynamic field assay^[4] at the FIT test site in Florida. The initial attachment of diatoms under static conditions was similar on the PGs as compared to a hydrophobic control. However, PGs show outstanding fouling release properties. Up to 94% of attached diatoms could be removed from the coatings after the exposure to a shear stress of 19 Pa. These results were confirmed in the field assays.⁵ The range of testes species was also extended to macro-fouling organisms such as zoospores of green algae (Ulva linza) and barnacle larvae (Balanus amphitrite).

(1) Wyszogrodzka, M.; Haag, R. Synthesis and characterization of glycerol dendrons, self-assembled monolayers on gold: A detailed study of their protein resistance. Biomacromolecules 2009, 10, 1043–1054.

(2) Siegers, C.; Biesalski, M.; Haag, R. Self-assembled monolayers of dendritic polyglycerol derivatives on gold that resist the adsorption of proteins. Chemistry 2004, 10, 2831–2838.

(3) Weber, T.; Gies, Y.; Terfort, A. Bacteria-Repulsive Polyglycerol Surfaces by Grafting Polymerization onto Aminopropylated Surfaces. Langmuir 2012, 28, 15916–15921.

(4) Nolte, K. A.; Koc, J.; Barros, J. M.; Hunsucker, K.; Schultz, M. P.; Swain, G. E.; Rosenhahn, A. Dynamic field testing of coating chemistry candidates by a rotating disk system. Biofouling 2018, 49, 1–12.

(5) Wanka, R.; Finlay, J. A.; Nolte, K. A.; Koc, J.; Jakobi, V.; Anderson, C.; Clare, A. S.; Gardner, H.; Hunsucker, K.; Swain, G. W. et al. Fouling-Release Properties of Dendritic Polyglycerols against Marine Diatoms. ACS applied materials & interfaces 2018, DOI: 10.1021/acsami.8b 12017 .

Nearly all methods of introducing bioactive compounds to the surface of a substrate rely on application from above or fail over time due to depletion. In this work, we use a bio-inspired approach to deliver target molecules to an interface from below, making use of both theoretical modeling and experimental validation to rationally design customizable patterns and gradients. Mimicking the vascular systems of living organisms, networks of empty 3D-printed channels are filled with liquid containing the compound of interest, which flows through the vascular network and diffuses through the polymer, eventually reaching the substrate surface. In proof-of-principle experiments using Escherichia coli and Staphylococcus epidermidis as model organisms, we demonstrate both theoretically and experimentally that the concentration of antibiotic and duration over which it is delivered to the surface can be controlled by varying the location of the vascular channels and concentration of the antibiotic solution inside. The result is a well-defined and predictable patterned response from the bacteria growing on the surface, a first step toward developing new types of adaptive antifouling surfaces and cell culture tools.

Silver nanoparticles (Ag NPs) possess remarkable antibacterial properties that are widely recognized. The emergence of antibiotic-resistant bacteria has motivated the interest of Ag NPs as an alternative for antimicrobial protection, in a wide range of applications [1]. However, Ag NPs have also shown toxicity and low biocompatibility. In addition, their synthesis usually involves toxic compounds, further limiting their applicability as a biomaterial. Research on Ag NPs has largely focused on increasing their biocompatibility. Properties such as NPs size, dispersity, and stability have shown to play an important role on their biocompatibility [2]. Green synthesis methods that require non-toxic agents while giving control over these properties are of high interest.

Chitosan (CS) is a deacetylated derivative of chitin, a widely available polymer. Its properties include biodegradability, biocompatibility and non-toxicity, making it an attractive alternative for biomaterials. CS has been used as a bioactive coating (for proteins, drugs and antibiotics and as a stabilizing agent in the production of Ag NPs [3]. Approaches to synthesize CS-based Ag NPs include γ irradiation and sonochemical methods [4].

In this work, Directed Liquid-Plasma Nanosynthesis (DLPNS) was used to drive Ag NPs synthesis without the need of other reagents. CS membranes were used to immobilize the NPs, to explore their application as an antibacterial coating for biomaterials. The Ag NPs precursor concentration and irradiation time were used as control parameters. Surface topography and chemistry were studied by SEM, Contact Angle, XRD and EDS. Antimicrobial properties of the membranes were evaluated against gram-positive (S. aureus) and gram-negative (E. coli) bacteria. Life and death assays revealed the antibacterial activity of the membranes. To study their biocompatibility and cytotoxicity, mammalian cell cultures were used. Cell viability, adhesion and metabolism were evaluated via Alamar blue and immunostaining tests. SEM images were used to assess the presence of Ag NPs in the CS matrix, and observe the bacteria and cell morphology on the surface of the membranes.

[1] D. Wei, W. Sun, W. Qian, Y. Ye, X. Ma, Carbohydr. Res. 344 (2009) 2375–2382.

[2] E.I. Alarcon, M. Griffith, K.I. Udekwu, eds., Silver Nanoparticle Applications, Springer International Publishing, Cham, 2015.

[3] A. Civantos, E. Martínez-Campos, V. Ramos, C. Elvira, A. Gallardo, A. Abarrategi, ACS Biomater. Sci. Eng. 3 (2017) 1245–1261.

[4] N.M. Huang, S. Radiman, H.N. Lim, P.S. Khiew, W.S. Chiu, K.H. Lee, A. Syahida, R. Hashim, C.H. Chia, Chem. Eng. J. 155 (2009) 499–507.