Membrane Phase Drives the Assembly of Gold Nanoparticles on Biomimetic Lipid Bilayers

In recent years, many efforts have been devoted to investigating the interaction of nanoparticles (NPs) with lipid biomimetic interfaces, both from a fundamental perspective aimed at understanding relevant phenomena occurring at the nanobio interface and from an application standpoint for the design of novel lipid–nanoparticle hybrid materials. In this area, recent reports have revealed that citrate-capped gold nanoparticles (AuNPs) spontaneously associate with synthetic phospholipid liposomes and, in some cases, self-assemble on the lipid bilayer. However, the mechanistic and kinetic aspects of this phenomenon are not yet completely understood. In this study, we address the kinetics of interaction of citrate-capped AuNP with lipid vesicles of different rigidities (gel-phase rigid membranes on one side and liquid-crystalline-phase soft membranes on the other). The formation of AuNP–lipid vesicle hybrids was monitored over different time and length scales, combining experiments and simulation. The very first AuNP–membrane contact was addressed through molecular dynamics simulations, while the structure, morphology, and physicochemical features of the final colloidal objects were studied through UV–visible spectroscopy, small-angle X-ray scattering, dynamic light scattering, and cryogenic electron microscopy. Our results highlight that the physical state of the membrane triggers a series of events at the colloidal length scale, which regulate the final morphology of the AuNP–lipid vesicle adducts. For lipid vesicles with soft membranes, the hybrids appear as single vesicles decorated by AuNPs, while more rigid membranes lead to flocculation with AuNPs acting as bridges between vesicles. Overall, these results contribute to a mechanistic understanding of the adhesion or self-assembly of AuNPs onto biomimetic membranes, which is relevant for phenomena occurring at the nano–bio interfaces and provide design principles to control the morphology of lipid vesicle–inorganic NP hybrid systems.


Supplementary Characterization of Gold Nanoparticles S2
Small Angle X-Ray Scattering S2 Dynamic Light Scattering and Z-Potential S3 UV-Vis Spectroscopy S4

Supplementary Characterization of Liposomes S5
Dynamic Light Scattering and Z-Potential S6 Evaluation of liposomes concentration S6 Small Angle X-Ray Scattering S7

Supplementary Characterization of Gold Nanoparticles
Small Angle X-ray Scattering SAXS measurements on AuNPs aqueous dispersion were carried out in sealed glass capillaries of 1,5 mm diameter.
The structural parameters (Table S1) of citrated gold nanoparticles were evaluated from the SAXS profile of their diluted water dispersion ( Figure S1), according to a spherical form factor and a Schulz size distribution. In this concentration range, we can safely assume that there are no interparticle interactions are present, and that the structure factor S(Q) equals in the whole range of scattering vectors. Thus, the scattering profile of the particles derives from their form factor, P(Q). The SAXS spectrum reported in Figure S1 is fully consistent with the characteristic P(Q) of spherical particles with an average diameter of about 5.8 nm. The clear presence of P(Q) oscillations in the high Q Agarose gel electrophoresis S13 Bibliography S14 S3 region is consistent with a relatively low polydispersity of the synthesized AuNPs.  Table S2.

UV-vis Spectroscopy
To further evaluate the AuNPs size through UV-Vis spectroscopy we exploited the following equation 1 : with diameter of gold nanoparticles, absorbance at the surface plasma resonance peak, absorbance at the wavelength of 450 nm and and are 450 1 2 dimensionless parameters, taken as 3 and 2,2, respectively. The diameter value obtained is of 12,3 nm.

S6
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 = 521 nm. The extinction coefficient ε(λ) of gold nanoparticles dispersion was determined by the method reported in literature 2 , by the following equation: with core diameter of nanoparticles, and and dimensionless parameters ( and ). The arithmetic mean of the sizes obtained by = 3,32111 = 10,80505 optical and scattering analyses was selected, leading to a ε(λ) of 2.0·10 8 M -1 cm -1 .

Evaluation of Liposomes concentration
The lipid concentration in the starting colloidal dispersion was estimated to be 4 mg/mL from the initial lipid and water amounts employed in the formation and swelling of lipid films, assuming the absence of lipid loss due to the extrusion procedure. The liposomes concentration in the final dispersion was subsequently calculated considering the hydrodynamic diameter of each liposomal batch (  The liposomal dispersions were diluted to reach a final concentration of 1,2·10 -8 M before use.

Preparation of liposomes/AuNPs hybrids
The hybrid samples were prepared as follow: 10 µL of liposome dispersions (12 nM) were incubated with 300 µL of AuNPs 6,3 nM, in order to have a S9 liposomes/AuNPs number ratio of ~1/16. This liposomes/AuNPs number ratio was selected on the basis of our previous publication 3,4 which highlights that the aggregation of AuNPs on zwitterionic vesicles is promoted by low liposome amounts within the mix.

Small-Angle X-Ray Scattering
To characterize the NPs-vesicles hybrids' formation, 10 µL of 12 nM DOPC or DPPC liposomal dispersions were challenged with 300 µL of 6.3 nM citratestabilized AuNPs. In order to gain information on the kinetic of AuNPs S12 aggregation, the SAXS profiles have been collected after 1s, 30s, 5m and 10 minutes of the incubation.
In the low q region, plotting log 10 (I(q)) vs log 10 (q) it's possible to obtain the fractal dimension of the aggregates by the slope of the scattering profile 5 , according to: Where B is the background and p the Porod exponent. Generally, p=1 represents the fractal dimension of a linear aggregate and p=2 represents the fractal dimension of a 2D object. 5 The SAXS profiles of DOPC liposomes/AuNPs and DPPC liposomes/AuNPs in Figure 3 were fitted through a linear fit in the 0,1-0,3 nm -1 q-range, to obtain the slope values reported in table S5. The Structure factors for the scattering profiles reported in the insets of figure 4 (main text) were obtained as follow.

Incubation time
The scattering intensity (I(q)) is defined by the following equation: With k instrumental constant, N p scattering nanoparticles' number per unit volume, V p nanoparticle's volume, contrast of the experiment, B background ∆ intensity, P(q) e S(q) form and structure factors, respectively.
In order to obtain the structure factor of the liposome/AuNPs complex, we divided the scattering intensity of the liposomes/AuNPs hybrid by the scattering intensity of the neat AuNPs dispersion: For a diluted AuNPs dispersion the structure factor can be considered equal to 1.
In addition, in the high-q region (0,1-1,6 nm -1 ), the form factor of liposomes/AuNP hybrids can be approximated to the one of neat AuNPs, leading to the following equation: The mean interparticle distance between the AuNPs within the aggregates (d) can be obtained from the S(q) vs q (nm -1 ) plot (see inset of Figure  With q max value corresponding to the maximum of the correlation peaks reported in the insets of figure 4 (main text).

Agarose gel electrophoresis
In order to perform gel electrophoresis, the AuNPs and DOPC or DPPC