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.