*2.2. ATR FTIR*

The ATR FTIR (attenuated total reflectance Fourier transform infrared) spectroscopy build-up and growth factor embedding experiments were performed with two independent repeats on a ZnSe IRE (internal reflection element). The spectra in Figure 3 show the PEM build-up proceeded as expected up to 5.5 bilayers (some of the later layer spectra have been o ffset vertically for clarity). The spectra show the characteristic peaks of fucoidan and chitosan (assigned previously [55–57]). The characteristic peaks assigned to chitosan are the amide I/C=O at 1633 cm<sup>−</sup><sup>1</sup> and amide II at 1535 cm<sup>−</sup>1. While the characteristic peaks attributed to the sulfate stretching vibration are at 1249 cm<sup>−</sup><sup>1</sup> and 1220 cm<sup>−</sup>1. Other peaks of interest include the overlapping peaks at 1167 cm<sup>−</sup><sup>1</sup> assigned to C–O–C stretching vibration fucoidan and 1152 cm<sup>−</sup><sup>1</sup> assigned to C–O–C/C–N stretching vibrations of chitosan. The glycosidic linkages and skeletal C–O stretching vibrations is encompassed by the peaks at 1090 cm<sup>−</sup>1, 1051 cm<sup>−</sup><sup>1</sup> and 1025 cm<sup>−</sup><sup>1</sup> for both polysaccharides. A complete list of peak positions and assignments can be found in Table 1.

**Figure 3.** ATR FTIR (attenuated total reflectance Fourier transform infrared) spectra of build-up of a 8 bilayer FUC/CS PEM on a ZnSe IRE, where FGF-2 was embedded at bilayer 6. The grey line represents the spectrum of PEI, green lines represent FUC layers, red lines represent CS, and the orange line shows the spectrum of FGF-2 after a 5 min PBS rinse.

During the individual layer adsorption steps, when fucoidan was adsorbed an increase in the sulfonate stretching band at 1238 cm<sup>−</sup><sup>1</sup> is clearly seen along with increases in the lower wavenumbers of the glycosidic linkage region (1100–950 cm<sup>−</sup>1). As chitosan adsorbs the greatest differences are increases in the entire glycosidic linkage region and in the amide I and II bands. These amide bands decrease slightly upon subsequent fucoidan adsorption. This decrease is the result of stripping of chitosan from the multilayer upon adsorption of fucoidan. Stripping of polyelectrolytes has been observed in fucoidan/chitosan multilayers in past work from this group (and is commonly observed more broadly with polyions of dissimilar molecular weights), when fucoidan of much lower molecular weight has been used (see [56] and references contained therein).

The spectrum of the PBS rinse after FGF-2 adsorption displays some significant changes. At 5.5 bilayers the PEM has been calculated to be 192 ± 10 nm thick using the Sauerbrey equation from the QCM-D measurements presented in Figure 2, panel B. When the film was exposed to PBS/FGF-2/PBS the Sauerbrey thickness decreased to 168 ± 5 nm, despite an initial spike caused by PBS. The significant decrease in absorbance after the PBS/FGF-2/PBS cycle indicates mass loss of polysaccharides from the film. The peak heights of the sulfonate bands and the glycosidic region match that of the 5th bilayer, chitosan terminating film suggesting much of the previously adsorbed fucoidan layer has been removed. In addition, increases in the amide I/II bands indicating that FGF-2 adsorbed to the multilayer.

**Table 1.** Assignment of bands observed for ATR FTIR (attenuated total reflectance Fourier transform infrared) spectra of (i) a 9.5 bilayer fucoidan/chitosan polyelectrolyte multilayer on a Ge IRE (internal reflection element) and (ii) an 8 bilayer fucoidan/chitosan polyelectrolyte multilayer with FGF-2 embedded at bilayer 6 built on a ZnSe IRE [27,30–40]. Annotations: ν is stretching vibration, νas is asymmetric stretching vibration, νs is symmetric stretching vibration, γ is out-of-plane bending vibration, δ is in-plane bending vibration.


Following the PBS/FGF-2/PBS cycle the multilayer build-up was continued. The first fucoidan layer after this cycle has the same peak heights as the preceding fucoidan layer that was diminished by the adsorption of the FGF-2 layer (and associated PBS rinse cycles). The characteristic peaks in the next chitosan layer spectra increase very little, whilst the next bilayer appears to return to a more typical build-up as seen with the early layers prior to PBS/FGF-2 exposure. There is one additional difference in the final chitosan layer; the sulfate band attributed to fucoidan increases, likely due to underlying chitosan peaks in the spectrum and the large amount of chitosan that appears to be adsorbing to this layer. It is unlikely to be a result of the penetration depth of the evanescent wave as the *d*p of the ZnSe IRE is approximately 850 nm (*ñ* = 1650 cm<sup>−</sup>1) and the Sauerbrey thickness of the multilayer at the time of formation of the eighth chitosan layer is 193 ± 5 nm for the film with embedded FGF-2 (see the supporting information of our previous work [54] for calculations of *d*p).

The spectra presented in Figure 4, show more clearly the spectral change associated with the adsorption step of the growth factor. The amide I/II bands characteristic of proteins, in this case FGF-2, are sharp and clear, and are found at peak maxima of 1642 cm<sup>−</sup><sup>1</sup> and 1541 cm<sup>−</sup>1, respectively. The amide I/II bands increase over the 15 min adsorption. It is clear that FGF-2 has adsorbed to the surface of the multilayer. The subsequent PBS rinse showed no further change in the polysaccharide peaks or the amide bands suggesting the FGF-2 remained bound onto the multilayer surface and no further mass loss of the polysaccharides occurred. The FGF-2 exposure spectra will also include some contribution from the bulk solution above the PEM as well as any FGF-2 adsorbed to the PEM.

Finally, it was important to confirm that the FGF-2 remains bound within the PEM after build-up is continued, i.e., it is not removed by polyelectrolyte stripping. The spectra of the layers added after FGF-2 were processed by subtracting the spectra of the PBS rinse after FUC6 in a 1 to 1 ratio from each. These spectra are then processed to remove the O-H bending mode of water lost during this adsorption step, by summing the spectrum with a spectrum of PBS. Representative spectra are presented in Figure 5. Negative changes in the amide bands of the spectra in this figure may show if FGF-2 was desorbing or being removed from the multilayer.

**Figure 4.** ATR FTIR individual layer spectra of the FGF-2 adsorption every 5 min in orange and the subsequent PBS rinse in blue. FGF-2 spectra are produced by 1 to 1 subtraction of the prior PBS rinse spectrum from each spectrum collected over the adsorption time, followed by subtraction of a PBS spectra to remove the O-H bending mode contribution of water.

**Figure 5.** Representative ATR FTIR spectra of the layers added after FGF-2 adsorption. These spectra were produced by subtracting the spectra of the PBS rinse after FUC6 in a 1 to 1 ratio from each, followed by adding a PBS spectra to flatten the region between 1650–1700 cm<sup>−</sup><sup>1</sup> to remove the O–H bending mode of water lost during this adsorption step. Green lines show the fucoidan layers whilst red shows the chitosan layers. The vertical lines indicate the peak maxima of the amide I/II of FGF-2.

The first FUC (seventh bilayer) adsorption after the embedded growth factor contains a clear sulfate band characteristic of fucoidan as would be expected, however, there is significant distortion in the region from 1100–900 cm<sup>−</sup><sup>1</sup> due to the overlapping nature of PBS peaks in this region. In addition, the amide I band peak maxima can be found at 1643 cm<sup>−</sup>1, whilst the amide II maxima is at 1539 cm<sup>−</sup>1, these peaks are indicative of FGF-2 remaining bound to the multilayer after fucoidan adsorption. Upon subsequent chitosan adsorption the amide I and II peaks became more rounded and the peak maxima of the amide I shifted to 1638 cm<sup>−</sup><sup>1</sup> but the amide II remained in the same position. Additionally, two peaks increase significantly at 1384 cm<sup>−</sup><sup>1</sup> and 1093 cm<sup>−</sup>1, assigned to the CH3 deformation and the C-O-C stretching mode of the glycosidic linkage overlapping with the symmetric stretching of phosphate in PBS (see electronic Supplementary Materials Figure S1). These peaks only increase with each chitosan addition. In the next fucoidan layer spectra the amide I band shifts back to 1640 cm<sup>−</sup>1, and both amide bands greatly reduce in size. This indicates that chitosan is removed from the film as was seen previously, while the shift towards the FGF-2-like amide I band suggests that the growth factor is still trapped within the film. The final adsorption of chitosan sees the amide I shift more dramatically to 1634 cm<sup>−</sup><sup>1</sup> due to significantly larger adsorbed amount.

These spectra also clearly show that the sharp peak at 1093 cm<sup>−</sup><sup>1</sup> is associated with chitosan adsorption. This sharp peak appears at the same wavenumber as the first of the glycosidic linkage peaks of the polyelectrolytes, i.e., the C-O-C and C-O stretching bands. However, this peak also overlaps with the symmetric stretching of phosphate in PBS [58], which is composed of two peaks at 1062 cm<sup>−</sup><sup>1</sup> and 1125 cm<sup>−</sup><sup>1</sup> (see electronic Supplementary Materials Figure S1).

In addition to determining that the FGF-2 does not release upon simple exposure to PBS, it is valuable to determine the likely structure and distribution of the FGF-2 within the multilayer. The presence of FGF-2 as a distinct layer within the multilayer will likely result in a di fferent interaction within a wound environment compared to FGF-2 that is evenly distributed throughout the PEM film. In recently submitted work from our group, we determined that lysozyme was able to adsorb onto and permeate into a multilayer of FUC/CS (fucoidan/chitosan) [54]. Lysozyme and FGF-2 are both small proteins, with similar molecular weights and hydrodynamic radii; for lysozyme the molecular weight is 14.7 kDa and has a hydrodynamic radius of 19.5 Å, whilst FGF-2 has values of 17.2 kDa and 28 Å, respectively [9,28]. In addition, both have an overall positive charge at physiological pH, with isoelectric points at 11.3 for lysozyme and 9.6 for FGF-2. It was therefore our initial hypothesis that FGF-2 would behave similarly when a solution of the growth factor was placed in contact with a multilayer.

To test this hypothesis, ATR FTIR spectroscopy on a Ge IRE was employed to monitor the build-up of a 9.5 bilayer FUC/CS PEM, similar to our previous experiments with lysozyme. The build-up was found to match the data from our earlier work, and is presented in Figure 6. The multilayer was then exposed to PBS solution for 5 min, and then a 25 <sup>μ</sup>g·mL−<sup>1</sup> FGF-2 solution (in PBS, which was used to maintain the secondary structure of the proteins) was injected into the flowcell and remained stagnant over the film for 15 min and a spectra collected of the bulk solution over the multilayer. The spectra of the protein exposure are presented in Figure 7 panel A, where a ratio of 1 to 1 was used to subtract the spectrum of PBS over FUC10 from the spectrum of 15 min protein exposure. The film was exposed to FGF-2 was exposed to PBS for 2 h and this spectrum is presented in Figure 7 panel B.

**Figure 6.** ATR FTIR spectra of the build-up of a 9.5 bilayer PEM on a Ge IRE. The spectra are produced by subtracting a spectrum of the background electrolyte from each spectrum collected after each polymer adsorption/rinse step. The spectrum of PEI is shown in grey, the spectra of the fucoidan layers are shown in green and the chitosan layers are shown in red.

**Figure 7.** (**A**) ATR FTIR difference spectra of a 9.5 bilayer PEM exposed to PBS for 5 min followed by either lysozyme (dark blue) or FGF-2 (orange) for 15 min. (**B**) ATR FTIR difference spectrum of a 9.5 bilayer PEM exposed to PBS for 5 min followed by FGF-2 (orange) for 120 min. (Difference spectra acquired by subtracting the spectrum of the PBS exposed multilayer from the multilayer exposed to the two biomolecules).

Upon fucoidan adsorption the sulfate peaks increase up to bilayer 4. From bilayer 5 onwards, the sulfate peak increases upon both fucoidan and chitosan adsorption. The amide I/II bands characteristic of chitosan increase upon adsorption of chitosan, but decrease upon subsequent adsorption of fucoidan throughout build-up, however, this becomes more noticeable in the latter layers. This is attributed to mass loss of chitosan via stripping by fucoidan as it adsorbs and swelling of the film. Swelling can be seen in ATR FTIR spectra where the penetration depth of the IRE is not much greater than the thickness of the film. This can also account for the increase in the sulfate peak after chitosan adsorption mentioned above.

The spectra in Figure 7 show that lysozyme can be detected within the multilayer, with amide I and amide II bands clearly visible at 1651 cm<sup>−</sup><sup>1</sup> and 1547 cm<sup>−</sup>1. In addition, the glycosidic linkage region of the polysaccharides can also be seen with a maxima at 1084 cm<sup>−</sup>1, as well as the sulfonate stretching band characteristic of FUC at 1215–1252 cm<sup>−</sup>1. In contrast, the FGF-2 spectrum shows negative bands

at 1647 cm<sup>−</sup><sup>1</sup> and 1547 cm<sup>−</sup><sup>1</sup> assigned to the amide I/II of CS, as well as the negative sulfonate stretching band and the glycosidic linkage region with minima at 1215–1252 cm<sup>−</sup>1, and 1084 cm<sup>−</sup>1, respectively.

The refractive index of the Ge prism upon which these experiments were performed was *n*Ge = 4.0, within the *ñ* = 700–3450 cm<sup>−</sup><sup>1</sup> range [59]. A high refractive index means that the penetration depth (*d*p) of the evanescent wave from the surface of the IRE in an aqueous environment is small, where *d*p = 414 nm at *ñ* = 1540 cm<sup>−</sup><sup>1</sup> and *d*p = 505 nm at *ñ* = 1250 cm<sup>−</sup><sup>1</sup> (see our previous work for all calculated *d*p values and graphs of refractive indices for ZnSe and Ge IREs in the mid-IR range). In our earlier work, AFM measurements were used to determine the thickness of the 9.5 bilayer PEM in KCl electrolyte (377 ± 10 nm) and when exposed to PBS (432 ± 14 nm) [54]. These thickness measurements show that the multilayer is thicker than the penetration depth of the evanescent wave from a Ge IRE in the region of the amide I/II bands. Therefore, the spectra in Figure 7 shows that the film allows lysozyme to penetrate, as indicated by the amide I/II bands, and the PEM deswells as expected (and visualized by the increase in the polysaccharide peaks). However, the FGF-2 does not interact with the PEM in the same manner, the spectra indicates that the PEM is continuing to swell (likely due to the PBS) and is not counteracted by protein sorption (no positive amide I/II bands can be seen).

The FGF-2 bulk solution was allowed to remain on the film for 2 h (Figure 7 panel B), after this time the characteristic peaks of chitosan could be seen at 1636 cm<sup>−</sup><sup>1</sup> and 1558 cm<sup>−</sup>1, plus the glycosidic linkage region centred around 1080 cm<sup>−</sup>1. However, there appeared to be no amide I/II bands that matched the shape/ratios of the protein or any sulfate stretching bands indicative of fucoidan. This indicates that FGF-2 did not permeate into the film over this time frame and the film de-swelled into the evanescent wave. Specifically, a region/layer with high chitosan content, or that chitosan is di ffusing through the PEM into the lower layers closer to the IRE surface. This di ffusion may be facilitated by the long exposure to PBS meaning that the film remains in the swollen state during this time. Therefore, the chitosan may be freer to di ffuse due to the higher degree of extrinsic versus intrinsic charge compensation and the 'looser' structure of the swollen PEM.

Since the our previous work determined that the PEM was able to exclude proteins based on size [54], the spectra presented in Figure 7 indicate that FGF-2 must interact via a di fferent mechanism than lysozyme (LYZ) with the multilayer components since the di fference in molecular weight and hydrodynamic radii between LYZ and FGF-2 is small. It must be also noted that heparin-like glycosaminoglycans (GAGs) (i.e., fucoidan) support dimerisation of FGF-2 which contributes to the potency of the growth factor in vivo [60]. However, our experiments could not distinguish between dimerised FGF-2 in contact with the fucoidan surface of the PEM, or whether monomers of FGF-2 were binding to the fucoidan on the films without dimerisation occurring.
