*2.1. QCM-D*

Gorouhi et al. found that epidermal growth factor was still e ffective at promoting cell proliferation when covered by two bilayers in a PEM [32], thus, FGF-2 was placed at bilayer 6 of 8 in our study. Quartz crystal microbalance with dissipation (QCM-D) monitoring experiments were performed to

monitor the build-up of the multilayers, either with, or without, the inclusion of the growth factor. In the case of a growth factor-embedded film, the PEM was constructed with sequential exposure of the substrate to the two polysaccharides until bilayer 5.5 (fucoidan terminating). The PEM was then exposed to PBS (phosphate buffer solution) for 5 min followed by 15 min FGF-2 25 <sup>μ</sup>g·mL−<sup>1</sup> solution, then a 5 min rinse with PBS to remove any unbound FGF-2. This PBS/FGF-2/PBS cycle substituted as the 6th CS (chitosan) layer. The PEM construction was continued as normal from the fucoidan terminating seventh bilayer, until 8 bilayers were deposited. For the non-embedded/blank system (i.e., without growth factor incorporation), the multilayer was exposed to PBS at the 5.5. bilayer formation point, and then chitosan was added in place of the growth factor, prior to continuing the formation until 8 bilayers were formed. Two independent experiments were performed with a total of two sensors for each condition. A representative measurement is shown in Figure 1, whilst the average Sauerbrey thickness, average hydrated mass and average dissipation can be found in Figure 2.

**Figure 1.** A representative QCM-D (Quartz crystal microbalance with dissipation) plot of frequency (**A**) and dissipation (**B**) for build-up of 8 bilayers for FUC/CS (fucoidan/chitosan) multilayers on gold sensors (5th overtone) where the PEM (polyelectrolyte multilayer) exposed to FGF-2 (fibroblast growth factor-2) are indicated by an orange line, whilst the control PEM exposed to only PBS (phosphate buffer solution) is marked with a blue line. The vertical dashed lines indicate the start of the adsorption step of each polymer. \* Indicates were FGF-2 adsorption began for the data set presented in orange.

**Figure 2.** QCM-D calculations for (**A**) the average hydrated Sauerbrey mass, (**B**) the average Sauerbrey thickness; and (**C**) the average dissipation of the 8 bilayer FUC/CS PEM with embedded FGF-2 (orange with square markers–and highlighted with an asterisk on the x-axis) and PBS without FGF-2 (blue with circle markers).

The QCM-D data presented in Figure 1 indicate that the fucoidan layers show an increase in both frequency magnitude and dissipation, which becomes larger with increasing layer number, reflecting the supra-linear growth of the multilayer. The chitosan layers show a sharp decrease in both frequency magnitude and dissipation. In both panels of Figure 1 a sharp spike can be seen when the multilayer is initially exposed to PBS. The frequency magnitude and dissipation then decrease to values less than the 6 bilayer fucoidan-terminating PEM within 5 min for both multilayers exposed to FGF-2 in PBS and PBS only. There is a small but continual decrease in the frequency when the film is exposed to PBS (both with FGF-2 and without) indicating a mass loss during this time. However, QCM-D cannot distinguish between polymer mass and water mass loss. Our previous work has shown that 10 bilayer fucoidan/chitosan multilayers experience both a degree of mass loss and swelling when exposed to PBS [54]. However, any swelling occurring here (Figure 1) cannot be seen in the frequency measurements after the initial spike, in fact, a decrease in thickness is observed (see Figure 2).

The pattern seen in the early layers, of lower frequency and dissipation for chitosan layers, and a sharp increase in both frequency magnitude and dissipation for fucoidan layers, continues after PBS exposure with some di fferences, the first fucoidan layer after PBS exposure shows the same frequency as the previous fucoidan layer. The dissipation increases sharply with the fucoidan adsorption after PBS. The seventh and eighth chitosan layers show a decrease from the previous reported fucoidan layers in the frequency magnitude and dissipation values. The hydrated mass calculated using the Sauerbrey relation and the Sauerbrey thickness (panel A and B of Figure 2), show that the incorporation of FGF-2 does not have a significant impact on these two attributes of the multilayer compared to a PEM only exposed to PBS. The hydrated mass can be seen to follow a linear profile up to bilayer 3 where a saw-tooth profile emerges, with the chitosan layers having a lower hydrated mass than the previous fucoidan layers. Upon exposure to PBS the hydrated mass can be seen to decrease, following this exposure the continued build-up of the multilayer again shows a saw-tooth profile similar to before the PBS/FGF-2/PBS cycle. The PEM that was only exposed to PBS appears to show a marginally higher adsorbed mass, however, this was present prior to PBS exposure so is likely caused by a variation in the samples themselves.

Similarly, the thickness data in panel B of Figure 2, show a similar profile to the mass calculations prior to PBS exposure, where the linear profile ends at bilayer 3 and the saw-tooth begins. Again, when chitosan is adsorbed to the fucoidan-terminating multilayer the film becomes thinner, when the next layer of fucoidan is adsorbed the entire multilayer become thicker. When exposed to PBS, the thickness of the multilayer increases, upon the adsorption of the first subsequent bilayer pair, consisting of fucoidan then chitosan, the thickness decreases. These changes in thickness and mass show a swelling and deswelling profile. No polymer is added during the PBS only rinse and very little mass is added during the PBS/FGF-2/PBS cycle, ye<sup>t</sup> the mass and thickness both increase.

The average dissipation data is presented in panel C of Figure 2, the PEMs with and without growth factor have very similar dissipations after being exposed to FGF-2 in PBS or only PBS. Though, this small di fference may be due to variation in the samples that exists prior to the PBS exposure. For the seventh layer of fucoidan, there was a significant di fference in the dissipation with the PBS only system having a much greater dissipation than the multilayer with embedded FGF-2, the eighth fucoidan layer shows a similar di fference. However, the dissipation of the seventh and eighth chitosan layers are the same for both the multilayer with embedded FGF-2 and the multilayer that was only exposed to PBS.
