3.2 Synthesis and characterization of tri-Dopa-PSBMA
Screening through theoretical calculation, the optimal block copolymer structure of tri-Dopa-PSBMA was determined. To visually compare block copolymers’ underwater adhesion and antifouling properties, the sin-Dopa-PSBMA and tri-Dopa-PSBMA polymers with different degrees of polymerization were synthesized (Scheme S1). The two polymers corresponded to the structures of Molecules 1 and 5 in the previous section. The characteristic peaks in the 1H NMR spectrum of each compound correspond to their chemical structures, revealing that the target polymers have been successfully synthesized (Figure S13-19 , S22-23 ). It was worth noting that the characteristic peaks of catechol were not observed in the 1H NMR spectrum of sin-Dopa-PSBMA and tri-Dopa-PSBMA (Figure S11-12), which may be attributed to the low proportion and solubility of the catechol segment in the polymer.
As above mentioned, PSBMA shows the upper critical solution temperature (UCST) phase transition behavior in an aqueous solution54. Figure S24 shows the aggregation behaviors of sin-DOPA-PSBMA and tri-DOPA-PSBMA zwitterionic polymers in aqueous solutions measured by dynamic light scatting. Both polymers show the UCST phase transition in the temperature range of 15-65 °C, while the UCST of tri-DOPA-PSBMA is higher than that of sin-DOPA-PSBMA indicating a lower water solubility of the tri-DOPA-PSBMA. Insolubility of sulfobetaine (co) polymers at low temperatures is based on an interlocking of zwitterionic side groups of different polymer chains and incorporating rigid hydrophobic functionality into sulfobetaine copolymers will decrease their water solubility55. Although there was a one-to-one correspondence between the PSBMA chain and rigid DOPA residue for both copolymers, the influence of the DOPA residue on the water solubility and the UCST of the two polymers was remarkably different. The presence of a single DOPA residue can be assumed to sterically interrupt this interlocking to a greater extent than the presence of a TREN scaffold with three DOPA residues groups, thereby a higher gain in entropy during the dissolution of a sin-DOPA-PSBMA compared to a tri-DOPA-PSBMA copolymer will induce a more soluble sin-DOPA-PSBMA56. Additionally, the UCST decreased, and the water solubility of the tri-DOPA-PSBMA increased with the increase of the hydrophilic PSBMA chain length55, 57. Since there is a balance between enthalpic polymer-substrate interaction favoring surface adsorption and mixing entropy favoring polymer dissolution, the hydrophilic-hydrophobic balance of the copolymer plays a vital role in the surface adsorption from the aqueous solution.
With a “grafting to” method, the DOPA-functionalized PSBMA was used to modify silicon wafers by a solution dip-coating method. XPS measurements indicate a change in chemical composition on the PSBMA-treated substrates (FigureS25 ). The new peak at the binding energy of 231.5 eV, assigned to S2s in PSBMA, appears. The reduction in silicon content and the appearance of sulfur element on the substrate surface confirm the successful grafting of PSBMA polymers. Figure 3b-cshow the surface silicon element content as a function of treatment time measured by XPS. It can be seen that the silicon content in untreated silicon wafer substrate is about 45.0%, while the content is significantly reduced after dipping in the DOPA-PSBMA solution. It reaches the lowest value at the treatment time only after 10 min dipping in tri-DOPA-PSBMA solution and then the values do not vary much with the treatment time prolonging from 10 min to 6 hours (Figure 3c ). Correspondingly, it is found that the surface silicon content decreases sharply after 1-hour dipping in sin-DOPA-PSBMA solution, and a gradual reduction comes until it reaches the lowest value after 11 hours of treatment (Figure 3b ). These results demonstrate the successful attachment of both sin-DOPA-PSBMA and tri-DOPA-PSBMA chains on the silicon substrates and a shorter equilibrium time of adsorption from tri-DOPA-PSBMA solution than that from sin-DOPA-PSBMA solution58. The significant enhancement of the surface adsorption of the PSBMA polymer on three dopamine anchors is consistent with the higher hydrophobic property from DLS results and the higherE ads from the calculation. Interestingly, polymer surface modification in 10 minutes by using the solution dip-coating method is of great significance to industrial applications.
It is generally accepted that the best non-fouling properties can only be achieved when surface hydration and steric repulsion work together, where surface hydration is the primary factor59, 60. Contact angle (CA) results show that the pristine silicon wafer’s initial contact angle was approximately 34°. The lower contact angle (~11°) of the cleaned substrate indicates more hydroxylated groups and stronger surface hydration, which will enhance protein resistance. All DOPA-PSBMA polymer-modified substrates show similarly low contact angles as that modified before (i.e., hydroxylated silicon wafer), which may be contributed to resistance to protein adsorption (Figure 3d ). To evaluate the protein resistant property of DOPA-PSBMA modified silicon wafers, the relative fouling of IgG on various surfaces was measured at 4 °C and 37 °C by using ELISA with a reference of untreated silicon wafer protein adsorption and laser scanning confocal microscopy (LSCM) was used for monitoring the adsorption of FITC-BSA on substrates. Figure 4a shows that the relative adsorption amounts of DOPA-PSBMA modified surfaces are approximately 2-7 % at both 4 °C and 37 °C, demonstrating a much lower protein adsorption level than that of hydroxylated surfaces (39 ± 3 and 17 ± 3%, respectively) with the similar surface hydrophilicity. The protein adsorption is reduced to an ultra-low fouling degree as compared with uncoated surfaces, comparable to the PSBMA surfaces modified by the “grafting-from” method61. However, protein adsorption on the sin-DOPA-PSBMA modified surface is still a little higher than that on the surface grafted with tri-DOPA-PSBMA. These results indicate that DOPA-PSBMA polymer can effectively adhere to the hydroxylated substrate by using a “grafting-to” method to provide a highly protein-resistant surface. Although all surfaces suffered slightly more fouling at low-temperature conditions of 4 °C than at physiological temperature of 37 °C, the adsorption level is such low to be super-low fouling even at low temperatures. On the other hand, the best non-fouling ability of DOPA-PSBMA modified surfaces achieved here has to be considered as the result of the combination of surface hydration from surface hydrophilicity and steric repulsion resulting from chain flexibility of the attached PSBMA polymer. The highly efficient protein resistance of DOPA-PSBMA coated silicon wafers was further proved by the LSCM image of FITC-BSA-adhered substrates.
The development of antifouling surfaces, especially for medical devices, requires strong anchoring of the polymer to the surface in order to withstand long-term in vivo exposure to physiological conditions62, 63. To explore the long-term stability of the DOPA-PSBMA modified substrates, the substrates were incubated in PBS buffer at different time intervals till 30 days before being verified by LSCM images of the attachment tests of FITC-BSA (Figure 3e ). As for the freshly modified substrates, the attachment of FITC-BSA was hardly observed, meanwhile FITC-BSA adsorption can be observed on the blank substrate (pristine silicon wafer inFigure S13 ). However, the adsorption for the sin-DOPA-PSBMA substrate incubated in PBS buffer for 18 days increased sharply, which is the same as that of the hydroxylated sample. In effect, the adsorption for the 30 days incubated tri-DOPA-PSBMA modified samples was still hardly observed, implying that the tri-DOPA-PSBMA was stable and has long-term antifouling properties. The relatively fouling area of the FITC-BSA adsorption substrate shows the same trend by quantitative fluorescence values (Figure S14 ). The high adsorption energy and solvation free energy of Molecule 5 provide theoretical support for the long-term adsorption stability and fouling resistance of polymer tri-DOPA-PSBMA.