A. Change of vesicle size and bilayer thickness induced by low and high MW HA

Figure 1 shows the scattering curves obtained for DPPC vesicles, with the selected concentrations of low and high MW HA (Table I). The curves in Fig. 1 have been stacked on the y axis to enhance clarity. No scattering was measured for pure HA solutions. All scattering curves depict the characteristic form factor of spherical vesicles with a high polydispersity (PDI).3434. V. Nele, M. N. Holme, U. Kauscher, M. R. Thomas, J. J. Doutch, and M. M. Stevens, Langmuir 35, 6064 (2019). https://doi.org/10.1021/acs.langmuir.8b04256 The absence of the characteristic lamellar peak at high q further confirms the unilamellarity of vesicles.3535. H. Schmiedel, L. Almasy, and G. Klose, Eur. Biophys. J. 35, 181 (2006). https://doi.org/10.1007/s00249-005-0015-9 Additionally, a structure factor was not observed in the scattering data, which is expected for DPPC vesicles at low concentrations,3636. S. Qian, V. K. Sharma, and L. A. Clifton, Langmuir 36, 15189 (2020). https://doi.org/10.1021/acs.langmuir.0c02516 as well as HA solutions with added salt.3737. E. Geissler, A. M. Hecht, and F. Horkay, in Macromolecular Symposia (Wiley Online Library, New York, 2010), Vol. 291–292, Issue 1, pp. 362–370. While no visibly distinct changes in the vesicle form factor are observed in Figs. 1(a) and 1(b), fits of the model [Eq. (1)] reveal subtle changes in the size and membrane thickness of vesicles as a function of the MW and concentration of HA.Scattering intensities from unilamellar vesicles were modeled using a spherical core-shell form factor with smoothly varying interfacial functions; details of the model can be found in the supplementary material.6666. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002502 for details about the model to fit SANS spectra, DLS measurements of the reference samples, QCM and AFM results for the reference samples, and AFM cross sections of the films. Since the vesicle concentration can be assumed to be well within the dilute concentration regime, scattering intensity can be written aswhere n is the number density of vesicles, Iinc is the incoherent background, P(q) is the form factor of the vesicle of interest, and S(q) is the structure factor describing the interaction between vesicles. Here, the structure factor can be approximated to be 1, since the concentration of vesicles is low enough.The form factor of a spherical particle with a symmetric scattering length density (SLD) profile can be calculated by PSLD(q)=1Vparticle|4π∫0∞ρ(r)sin(qr)qr2r2dr|2,(2)where ρ(r) is the scattering length intensity (SLD) profile that can be defined as the linear sum of the core, two interfaces, and shell regions. The core represents the inner of the vesicle, where water is located. The shell represents the hydrophobic region of the bilayer, which has two boundaries (the two interfaces) and is defined by power-law functions. For the results discussed here, the PDI of the inner core was obtained to be 0.4 by initially using the PDI as one of the fitting parameters, along with all other structural parameters. In the next round of fittings, the PDI value was fixed at 0.4 to determine the values of the structural parameters; see all fitting parameters in Tables S1 and S2.6666. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002502 for details about the model to fit SANS spectra, DLS measurements of the reference samples, QCM and AFM results for the reference samples, and AFM cross sections of the films. This approach led to better fits in the experimental data. The SLD of DPPC and D2O were determined to be 0.2 × 10−6 Å2, and 6.35 × 10−6 Å2, respectively, based on their known molecular compositions and density.Figure 2 summarizes the changes in vesicle radius and bilayer thickness as a function of HA concentration for the two investigated MWs, obtained by fitting Eqs. (1) and (2) to the scattering data. With no HA, the extruded DPPC vesicles have an average radius of ∼36.8 ± 0.3 nm. The fits suggest that the vesicle radius decreases first with increasing concentration of high MW HA, until a minimum size is observed at a concentration of 1 mg/ml, with a radius of 34.7 ± 0.6 nm. The vesicle radius then increases with an increase in HA concentration to 1.5 mg/ml, yielding a maximum vesicle radius of 37.5 ± 1 nm. The addition of high MW HA induces a slight shrinkage of the lipid bilayer, in agreement with previous works.17,1817. M. Herzog, L. Li, H.-J. Galla, and R. Winter, Colloids Surf. B: Biointerfaces 173, 327 (2019). https://doi.org/10.1016/j.colsurfb.2018.10.00618. D. C. F. Wieland, P. Degen, T. Zander, S. Gayer, A. Raj, J. An, A. Dedinaite, P. Claesson, and R. Willumeit-Romer, Soft Matter 12, 729 (2016). https://doi.org/10.1039/C5SM01708D Here, the bilayer thickness decreases from ∼79.9 Å to ∼66.1 Å, as the concentration of high MW HA increases from 0 to 1.5 mg/ml. The results with low MW HA are quite different. First, the radius of vesicles increases with concentration, with the largest radius measured at 0.5 and 1 mg/ml (∼38.8 nm), and then it decreases to ∼37 nm with a further increase in concentration to 1.5 mg/ml, yielding opposite changes compared to high MW HA. The bilayer thickness with low MW HA decreases down to 63 Å with a concentration up to 0.33 mg/ml, i.e., more notably than with high MW HA, and it increases to ∼86 Å with a further increase in concentration. Interestingly, the calculated bilayer thickness at a concentration of 0.5 mg/ml low MW HA and above is, thus, larger than that of the reference DPPC vesicles. These findings together show that the structural response due to the interaction with HA is clearly dependent on the molecular weight (or the length) of HA.

B. High MW HA leads to the aggregation of vesicles

Size measurements of lipid-HA mixtures using DLS are summarized in Fig. 3 as a function of HA MW and its concentration; the DLS results for pure HA solutions are displayed in Fig. S3,6666. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002502 for details about the model to fit SANS spectra, DLS measurements of the reference samples, QCM and AFM results for the reference samples, and AFM cross sections of the films. for reference. The radius of pure lipid vesicles, i.e., with no added HA, was measured to be ∼38.4 ± 2.3 nm, with a PDI of 0.16. Figure 3(a) shows the average values of the three detected peaks; the error bars show the standard deviation and the size of the bubble gives the relative intensity of each peak. The discussion is focused on the peak with the highest intensity [peak 2 in Fig. 3(a) and peak 3 in Fig. 3(b); see details in the caption].The addition of low MW HA led to a small increase in the radius of vesicles; that is, 45.9 ± 2.7 nm, 42.1 ± 3.5 nm, 46.7 ± 3.4 nm, 44.7 ± 5.5 nm, and 46.6 ± 3.8 nm, for 0.1, 0.33, 0.5, 1, and 1.5 mg/ml of low MW HA, respectively. The PDI also increased from 0.16 for the vesicles to 0.32, 0.26, 0.30, 0.25, and 0.31, respectively (see labels). It should be noted that the equilibrium size of the low MW HA in the reference stock solution in the NaCl buffer is ∼9 nm,3838. R. Mendichi, L. Soltes, and A. Giacometti Schieroni, Biomacromolecules 4, 1805 (2003). https://doi.org/10.1021/bm0342178 and hence, notably smaller than the size measured by DLS.The addition of high MW HA to the vesicle solution leads to a more significant increase in the size of the assembled structures [peak 3 in Fig. 3(b)]. The average radius of peak 3 increases in direct proportion to the HA concentration. That is, the radius of the HA-lipid structures is 50.1 ± 4.8, 70.5 ± 8.8, 128.3 ± 30.3, 132.3 ± 14.4, and 182.5 ± 34.4 nm for HA concentrations of 0.1, 0.33, 0.5, 1, and 1.5 mg/ml, respectively. The PDI depends on high MW HA concentration and is very high (0.41, 0.34, 0.32, 0.33, and 0.35 for 0.1, 0.33, 0.5, 1, and 1.5 mg/mL, respectively). The reference DLS measurements for high MW HA in the absence of lipid show a wide size distribution with two broad peaks, one with an average around 30–40 nm, and a second one pointing at much larger structures with an average ranging from 350 to 550 nm, for the investigated concentrations; see Fig. S3.6666. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002502 for details about the model to fit SANS spectra, DLS measurements of the reference samples, QCM and AFM results for the reference samples, and AFM cross sections of the films. Hence, the size distribution for the high MW HA-lipid mixtures is much narrower, with one distinct and prominent peak, whose average radius increases proportionally with the HA concentration. Peak 2 in Fig. 3(b) is most likely due to high MW HA chains dispersed in the solution, while peak 1 (The DLS results for high MW HA, along with the inferred vesicle and bilayer structure from SANS, suggest that the radii detected by DLS (peak 3) can be a representative of aggregated vesicles via high MW HA. Indeed, USANS measurements also show the formation of a traversing network of HA with vesicles in solution at 1.5 mg/ml (Fig. S2).6666. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002502 for details about the model to fit SANS spectra, DLS measurements of the reference samples, QCM and AFM results for the reference samples, and AFM cross sections of the films. This is expected as the polymer solution changes from a dilute to a semidilute regime4040. B. Nystrom, A.-L. Kjoniksen, N. Beheshti, A. Maleki, K. Zhu, K. D. Knudsen, R. Pamies, J. G. Hernandez Cifre, and J. Garcia de la Torre, Adv. Colloid Interface Sci. 158, 108 (2010). https://doi.org/10.1016/j.cis.2009.05.003 and it points to a critical change of behavior at 1.5 mg/ml high MW HA, which needs further investigation.The intricate changes of the vesicle size for high MW HA revealed by SANS differ significantly from the DLS results [cf. Figs. 2 and 3(b)]. In DLS, the vesicle size consistently increases with the addition of HA. Instead of sharp multimodal size distributions, DLS shows broad peaks with large polydispersity, supporting the formation of HA-lipid vesicles and aggregates of varying sizes, where the increasing size reflects the increasing content of HA. In addition to this, we cannot exclude the presence of free HA polymers in the solution. SANS, however, only detects comparatively smaller single vesicles. This discrepancy is attributed to the different measuring principles, where DLS measures the average aggregate size, whereas SANS probes the contrast between individual vesicles and bulk. Importantly, only slight changes of the individual vesicle and bilayer size were observed per SANS analysis, indicating that even in the HA/lipid aggregates implied by DLS, the individual vesicles greatly maintain their initial structure. We also note that the DLS radii of the low MW HA-lipid structures are larger than the values determined by SANS [cf. Figs. 2 and 3(a)]. We associate this with the fact that SANS is not sensitive to the protruding HA chains from vesicles; see the proposed assembly in .

C. Amorphous layer resulting from the self-assembly of high MW HA-DPPC on gold sensors

Figure 4 shows representative QCM-D measurements of DPPC with 0, 0.1, 0.33, 0.5, 1, and 1.5 mg/ml high MW HA. This plot also shows the QI-mode AFM images of the QCM gold sensors after adsorption, specifically, the topography, surface stiffness, and adhesion maps. Multiple images (at least 5) on each sensor were taken to confirm reproducibility. For reference, Fig. S4 in the supplementary material6666. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002502 for details about the model to fit SANS spectra, DLS measurements of the reference samples, QCM and AFM results for the reference samples, and AFM cross sections of the films. shows the QI images of the bare gold sensors, with a roughness of ∼1.29 nm RMS, an average surface stiffness of ∼52 nN/μm, and an average adhesion of ∼7.3 nN to a sharp silicon tip. The average stiffness and adhesion values were determined by fitting a gaussian distribution to the histograms extracted from the respective QI images. QCM and AFM studies were performed in the NaCl buffer with H2O to mimic biological conditions, but the results with the samples prepared in D2O led to similar conclusions, as described later.Figure 4(a) shows the results for DPPC vesicles. The initial frequency decrease with a concurrent large increase of dissipation indicates fast vesicle adsorption. Adsorption rapidly slows down and equilibrium is not achieved after ∼1h. This is inconsistent with the classical fingerprint of vesicle rupture, where the frequency would display a sharp decrease followed by a rapid increase in frequency during adsorption, equilibrating at Δf ∼ 25 Hz and ΔD ∼ 1–2 × 10−6 for a lipid bilayer on a QCM sensor of the characteristics used here.4141. B. Seantier, C. Breffa, O. Felix, and G. Decher, J. Phys. Chem. B 109, 21755 (2005). https://doi.org/10.1021/jp053482f Thus, while there may be some rupture, the vast majority of vesicles remain intact on the surface. While rinsing with buffer (see gray arrow), dissipation decreases to ΔD7 ∼ 38 × 10−6 and frequency increases to Δf7 ∼ −164 Hz, which reflects that some adsorbed vesicles may either desorb or rupture, but the majority of them stay intact on the surface. Figure S5 in the supplementary material6666. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002502 for details about the model to fit SANS spectra, DLS measurements of the reference samples, QCM and AFM results for the reference samples, and AFM cross sections of the films. shows a plot of dissipation versus frequency (D−f) for DPPC vesicles. The fact that the D−f plot essentially follows the same path after rinsing tends to suggest a reversible process, i.e., the reversibly bound material from the latter part of the adsorption phase is rinsed off.Stiffness maps corresponding to the adsorbed DPPC vesicles show very soft and deformable domains occupying 50% of the total surface, with high adhesion to the tip compared to the surrounding surface. The surface-immobilized vesicles exhibit a wide height distribution and different extents of lateral spread, which leads to a large RMS roughness (average∼19.2 nm). Most of the domains exhibit a height of ∼80 nm and a width of ∼120–150 nm or 35 nm height with a larger lateral spread (see cross sections in Fig. S6).6666. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002502 for details about the model to fit SANS spectra, DLS measurements of the reference samples, QCM and AFM results for the reference samples, and AFM cross sections of the films. Note that imaging these domains can also induce a deformation due to the applied force by the tip (0.7 nN). The images also show flat domains with a height of ∼14 nm, which may be associated with the partial rupture of DPPC vesicles. Because this height is much larger (around a factor of two) than the thickness of a bilayer measured by SANS, it is more likely to be associated with the assembly of two bilayers on the surface. These AFM results also support that most of the DPPC vesicles do not rupture on the surface.At the concentration of 0.1 mg/ml high MW HA [Fig. 4(b)], there is an initial fast drop in frequency, yet slower than for DPPC vesicles [cf. Fig. 4(a)], which indicates that the affinity of HA-DPPC to the surface is decreased in the presence of HA at a small concentration. This is not surprising considering the expected steric and electrostatic repulsion for high MW (negatively charged) HA. This is followed by a step, during which the dissipation increases slowly from ΔD7 ∼ 14 to ∼20 × 10−6 and the frequency decreases from Δf7 ∼ −105 to ∼ −123 Hz in ∼1.2h. The slow frequency change as a function of time has been previously reported to be indicative of a reorganization of the adsorbed layer,4242. J.-H. Choi, S.-O. Kim, E. Linardy, E. C. Dreaden, V. P. Zhdanov, P. T. Hammond, and N.-J. Cho, J. Colloid Interface Sci. 448, 197 (2015). https://doi.org/10.1016/j.jcis.2015.01.060 which could be happening here, as well. There are no clear signs of vesicle rupture in QCM experiments. Equilibrium is not achieved even after ∼2h (Fig. S7).6666. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002502 for details about the model to fit SANS spectra, DLS measurements of the reference samples, QCM and AFM results for the reference samples, and AFM cross sections of the films. AFM shows the presence of a soft film (average stiffness ∼4.3 nN/μm) with a smaller RMS roughness of 4.6 nm, and a much smaller average adhesion (∼37 pN) compared to DPPC on gold surfaces, consistent with the full coverage of the surface with a soft film. The cross sections (Fig. S8)6666. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002502 for details about the model to fit SANS spectra, DLS measurements of the reference samples, QCM and AFM results for the reference samples, and AFM cross sections of the films. reveal mainly flat domains with a height of ∼12 nm and a width of ∼100–150 nm [Fig. S8(A)], which might result from the assembly of one or two bilayers on the surface. At some spots, adhesion is much higher than the average value [see white spots in Fig. S8(B)], while the surface stiffness is much smaller than the surrounding region, which is attributed to the presence of some intact vesicles. The maximum film thickness at these locations is ∼35 nm, i.e., similar to the vesicle radius as determined by SANS (∼37 nm), which indicates that they undergo significant deformation upon interaction with the surface and the tip.

The initial adsorption of HA-DPPC vesicles becomes faster upon an increase in concentration to 0.33 and 0.5 mg/ml high MW HA, as shown by the steeper decrease in frequency compared to 0.1 mg/ml, perhaps because the diffusion rate to the surface increases with concentration and/or because a higher near-surface concentration enhances the probability of adsorption. Also here, a plateau is not achieved during the first hour of adsorption. The frequency (Δf7 = −108 and −103 Hz for 0.33 and 0.5 mg/ml, respectively) and dissipation (ΔD7 = 13 × 10−6 and 21 × 10−6 (−), respectively) after rinsing with buffer are similar to that at 0.1 mg/ml, but the film morphology differs significantly.

Figures 4(c) and 4(d) show films with much larger domains, larger average thickness as, well as greater RMS roughness (7.7 and 7 nm, respectively) at 0.33 and 0.5 mg/ml compared to 0.1 mg/ml. The average surface stiffness (2.9 and 2.8 nN/μm, respectively) and average adhesion (47 and 28.5 pN, respectively) remain small, consistent with the full coverage of the surface with a film. At 0.33 mg/ml, there are many flat domains with a height of ∼15 nm and a width of several 100s nanometers [Fig. S8(D)],6666. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002502 for details about the model to fit SANS spectra, DLS measurements of the reference samples, QCM and AFM results for the reference samples, and AFM cross sections of the films. but also a few domains resembling deformed vesicles [Fig. S8(C)]. A further increase of HA concentration to 0.5 mg/ml yields both types of domains but are now similarly distributed [Figs. S8(E) and S8(F)]. The cross sections show vesicles with a height of ∼30–50 nm and a width of 150–200 nm at HA concentrations at 0.33 and 0.5 mg/ml. They are highly deformable and often unstable; so they rupture during imaging, especially with an increase in concentration. Interestingly, at a concentration of 0.33 mg/ml, they correlate with high adhesion, whereas at 0.5 mg/ml, they correspond to low-adhesive domains (Fig. S9).6666. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002502 for details about the model to fit SANS spectra, DLS measurements of the reference samples, QCM and AFM results for the reference samples, and AFM cross sections of the films. This might point toward the compositional change of these regions with the increase of HA content, which reduces the interaction with the (negatively) charged tip. (However, we cannot completely exclude the possibility that the lipids released during vesicle rupture coated the tip, which would also influence the interaction with the surface film.) QCM and AFM experiments were also performed with only high MW HA (in the absence of lipids) for reference. In this case, the adsorption of HA is poor at concentrations ≤0.5 mg/ml [Figs. S10(A) and S10(B)],6666. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002502 for details about the model to fit SANS spectra, DLS measurements of the reference samples, QCM and AFM results for the reference samples, and AFM cross sections of the films. [Δf7 7 −6 (−)], and hence, it significantly differs from the HA-lipid films that form in the same concentration range.A further increase in HA concentration leads to significantly different results for HA-lipid vesicles. At 1 mg/ml, the frequency plateaus are at Δf7 ∼ −31 Hz and ΔD7 ∼ 6.1 × 10−6 (−), which indicates the reduction of the adsorbed mass of HA-DPPC on the gold surface [Fig. 4(e)]. Consistent with QCM results, QI images at 1 mg/ml high MW HA show a significant reduction of the film thickness and RMS roughness (4.5 nm), while the average adhesion and surface stiffness remain small (average values ∼85 pN and 2 nN/μm), supporting the full coverage of the surface with a soft film. The cross sections in Figs. S8(G) and S8(H) show the presence of deformed vesicles on the surface, with heights of ∼40 nm and widths of 200 nm or more, but less crowded on the surface than at lower concentrations. Interestingly, the adsorption of high MW HA at 1 mg/ml (reference, no lipid) is promoted compared to lower HA concentrations [Δf7 ∼ −10 Hz and ΔD7 ∼ 2 × 10−6 (−)], the adsorption rate is faster [Fig. S10(C)], and leads to a homogeneous thin film.Figure 4(f) shows that a further increase of the HA concentration to 1.5 mg/ml leads to a smaller decrease (increase) of frequency (dissipation) in QCM experiments [Δf7 ∼ −17 Hz and ΔD7 ∼ 3.2 × 10−6 (−)], which indicates that the adsorption of lipid-HA vesicles is further hindered compared to 1 mg/ml. This coincides with a decrease of the average film thickness and the RMS roughness (∼3.3 nm) and, notably, a slight increase in the average adhesion and surface stiffness (124 pN and 6 nN/μm, respectively), which can be justified by the adsorption of a thinner film on the surface. Furthermore, the regions with high adhesion correspond here to high stiffness and low height, consistent with the characteristics of the gold substrate, and hence, with the partial coverage of the surface. The morphology of the films is also clearly different from that at smaller concentrations (0.33–1 mg/ml), as now, a finer network can be observed. The domains are flat and exhibit a height of ∼50 nm, but there are also a few domains with a smaller height of ∼14 nm [Figs. S8(I) and S8(J)].Figure 5(a) summarizes the changes in frequency and dissipation for the investigated concentrations of high MW HA. The results are close to a stepwise reverse isotherm, i.e., an increase in concentration leads to a decrease in adsorbed mass (less negative Δf), with a step at around 0.5 mg/ml. This isotherm type suggests the action of competitive adsorption. Figure 5(b) displays the rate of frequency change during the initial adsorption step and also reveals nonmonotonic trends, suggesting the presence of at least three, if not more, mechanisms governing the adsorption of HA-lipid mixtures. Based on the DLS results, it is most likely that there is competition between all the present structures (vesicles and their aggregates as well as free HA), and at the very highest concentration, free HA is able to dominate adsorption, leading to the observed final decrease in the adsorption rate and low adsorbed mass.Samples prepared in NaCl buffer with deuterated water were also investigated by QCM and AFM at HA concentrations of 0, 0.1, 0.33, and 1.5 mg/ml to confirm that the film structure is not affected by the solvent used in the sample preparation or during the rinsing step; representative results are shown in Fig. S11.6666. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002502 for details about the model to fit SANS spectra, DLS measurements of the reference samples, QCM and AFM results for the reference samples, and AFM cross sections of the films. The adsorption behavior was reproduced in the buffer with deuterated water, except at the concentration of 0.1 mg/ml. In this case, most of the adsorbate exhibits heights of 30–50 nm and widths between 100 and 200 nm, but there is also a significant aggregation of vesiclelike structures on several spots with much greater heights (see Figs. S12 and S13).6666. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0002502 for details about the model to fit SANS spectra, DLS measurements of the reference samples, QCM and AFM results for the reference samples, and AFM cross sections of the films. The adsorption behavior of these large aggregates is not represented by the QCM results in Fig. S11(B), which suggests that aggregation could happen after adsorption due to vesicle motion on the surface. In contrast, Fig. 4(b) shows the formation of a continuous but rough film on the surface of the samples prepared in a buffer with water. The origin of this difference has not been deciphered yet, and hence, we consider that both scenarios are possible at low concentrations of high MW HA.