Interaction of Metallic Nanoparticles With Biomimetic Lipid Liquid Crystalline Cubic Interfaces

In the past decades, events occurring at the nano-bio interface (i.e., where engineered nanoparticles (NPs) meet biological interfaces such as biomembranes) have been intensively investigated, to address the cytotoxicity of nanomaterials and boost their clinical translation. In this field, lamellar synthetic model membranes have been instrumental to disentangle non-specific interactions between NPs and planar biological interfaces. Much less is known on nano-biointeractions occurring at highly curved biological interfaces, such as cubic membranes. These non-lamellar architectures play a crucial -but far from understood-role in several biological processes and occur in cells as a defence mechanism against bacterial and viral pathologies, including coronaviruses infections. Despite its relevance, the interaction of cubic membranes with nano-sized objects (such as viral pathogens, biological macromolecules and synthetic NPs) remains largely unexplored to date. Here, we address the interaction of model lipid cubic phase membranes with two prototypical classes of NPs for Nanomedicine, i.e., gold (AuNPs) and silver NPs (AgNPs). To this purpose, we challenged lipid cubic phase membranes, either in the form of dispersed nanoparticles (i.e., cubosomes) or solid-supported layers of nanometric thickness, with citrate-stabilized AuNPs and AgNPs and monitored the interaction combining bulk techniques (UV-visible spectroscopy, Light and Synchrotron Small-Angle X-ray Scattering) with surface methods (Quartz Crystal Microbalance and Confocal Laser Scanning Microscopy). We show that the composition of the metal core of NPs (i.e., Au vs Ag) modulates their adsorption and self-assembly at cubic interfaces, leading to an extensive membrane-induced clustering of AuNPs, while only to a mild adsorption of isolated AgNPs. Such differences mirror opposite effects at the membrane level, where AuNPs induce lipid extraction followed by a fast disruption of the cubic assembly, while AgNPs do not affect the membrane morphology. Finally, we propose an interaction mechanism accounting for the different behaviour of AuNPs and AgNPs at the cubic interface, highlighting a prominent role of NPs’ composition and surface chemistry in the overall interaction mechanism.


S1.1 Small Angle X-ray Scattering
SAXS measurements on AuNPs aqueous dispersion were carried out in sealed glass capillaries of 1,5 mm diameter.
In diluted solution without interparticle interaction, the structure factor S(Q) can be approximated equal to 1, and the scattering intensity I(q) assumes the following form: I(Q)=nΔρ 2 Vp 2 P(Q) (1) Where n is the number density of the objects in the dispersion, Δρ is the contrast between the solvent and the scattering objects, Vp is the particle's volume and P(Q) is the form factor. Within the Guinier approximation 1 , valid for diluted and monodispersed particles, P(Q) can be expressed as: And substituting equation (1) we can obtained: And in logarithmic form: Then, according to the Guinier approximation, when S(Q)=1, the slope of scattering profile in the low Q region in a ln I(Q) vs Q 2 plot can be associated to the average gyration radius of the particles if the equation QRg<1 is respected. Finally, we can extract the particle's radius, assuming a spherical shape, exploiting the following relation:

Figure S1
Guinier approximation for gold nanoparticles: ln I(q) vs q 2 plot. The slope of linear fitting (solid red line) of the scattering intensity in the Guinier Region (red markers) is related to the gyration radius of the particles. The size and polydispersity obtained from the fitting procedure are summarized in the Table S1 below.

Figure S2
Guinier approximation for silver nanoparticles: ln I(q) vs q 2 plot. The slope of linear fitting (solid red line) of the scattering intensity in the Guinier Region (red markers) is related to the gyration radius of the particles. The size and polydispersity obtained from the fitting procedure are summarized in the Table S1 below.

± 1
Table S1 Nanoparticles radii obtained for AuNPs and AgNPs for the analysis of the SAXS profiles according to the Guinier approximation.
To further evaluate the AuNPs size through UV-Vis spectroscopy we exploited the following equation 2 : with diameter of gold nanoparticles, absorbance at the surface plasma resonance peak, 450 absorbance at the wavelength of 450 nm and 1 and 2 are dimensionless parameters, taken as 3 and 2,2, respectively. The diameter value obtained is of 20 nm. Wavelength (nm)

AuNPs
The concentration of citrated gold nanoparticles was determined via UV-Vis spectrometry, using the Lambert-Beer law (E(λ) = ε(λ)lc), taking the extinction values E(λ) at the LSPR maximum, i.e. λ = 520 nm. The extinction coefficient ε(λ) of gold nanoparticles dispersion was determined by the method reported in literature 3 , by the following equation: with core diameter of nanoparticles, and and dimensionless parameters ( = 3,32111 and = 10,80505). The arithmetic mean of the sizes obtained by optical and scattering analyses was selected, leading to a ε(λ) of 2.0·10 8 M -1 cm -1 . The final concentration of the citrated AuNPs is therefore ~8.3·10 -10 M.

Figure S4 UV-Vis absorption spectra of AgNPs after 1:3 dilution in water (1.6x10 -9 M). The plasmon absorption peak is at 520 nm.
To confirm the AgNPs size evaluated by SAXS we exploited the UV-vis spectroscopy according the following equation 4  where d is the diameter and λmax is the wavelength corresponding to the maximum absorption peak. Exploiting this equation, we found a nanoparticle's diameter equal to d=18.8 nm.
The concentration of citrated AgNPs was determined via UV-Vis spectrometry, using the Lambert-Beer law (E(λ) = ε(λ)lc). The extinction coefficient ε(λ) of silver nanoparticles dispersion was determined by the method reported in literature, according to the following equation: The final concentration obtained for AgNPs is 1.8x10 .8 M. Finally, before each measurement, the AgNPs dispersion was diluted to the same concentration of AuNPs (8.3x10 -10 M).

Supplementary Characterization of Cubosomes
Dynamic Light Scattering and Zeta-Potential   In order to obtain the spacing parameter of the Pn3m cubic phase we exploit the following equation 6 : Where h, k and l are the Miller indexes representing the crystallographic planes of the liquid crystallin phase and Qhkl is the q position measured for each reflex. In the case of a Pn3m phase, the characteristic values of (h 2 + k 2 + l 2 ) 1/2 are √2, √3, √4, √6, √8 and √9. Thus, plotting = ℎ (1/Å) vs =(ℎ2+ 2+ 2)1/2, the spacing parameter d can be obtained by the slope b of the liner fitting, reported in figure S7, according to the following equation: The evaluated spacing parameter is 10.4 nm.