**3. Materials preparation**

*LYS-ABOP nano-particles*. 20 mg of *APOB* in 50 millimolar kg-1 (*mmol*) aqueous Borax were added with 13.0 mg *EDAC*, 10.0 mg *NHS* and7.27 mg *N(Et)3*, at 25°C (7), and homogenized upon stirring. 44.13 mg solid *LYS* was added and the reaction proceeded for 5 hours, at 40°C. The reaction between *ABOP* and *LYS* is concomitant to an increase in opalescence of the dispersions, due to both a decreased surface charge density (because of the reaction of *COOH* groups with *EDAC)*, and to protein covalent binding. The final dispersion was centrifuged at 12000 rpm for 10 minutes, at 4°C. Thereafter, 10 mmol glycine was added, to quench un-reacted groups that were eventually bound onto *NP*s. The latter procedure significantly reduces the dispersion turbidity. A summary of the whole synthetic procedure, in four stages, is sketched in *Scheme 1*, where are indicated the different preparation steps.

Prolonged sonication, dialysis, with pH and/or ionic strength adjustment significantly tailors the average size of *NP*s and reduces their size. Substantial characterization is required to ensure the attainment of equilibrium conditions, since the size of particles increases with aging, *Figure 1*. The dispersions were extensively dialyzed against the buffer, until no more lysozime, *EDAC* and/or *NHS* was determined in the supernatant. To avoid the occurrence of clusters, the dispersions must be sheared (18) or forced to flow in tilted long syringe steel needles (19), as indicated in *Figure 2A*.

surely relevant, require dedicated formulation work and need substantial studies on the cyto-toxicity of vesicles, lysozyme-bound *NP*s and of the related adducts. The former systems were previously characterized on this regard by some of us (13), but almost nothing

*Materials*. 3-aminopropyl-(3-oxobutanoic acid)-functionalized silica *NP*s, termed *APOB*, contain significant amounts of carboxylic acids on their surface. They are given from the purveyor (Sigma Aldrich) as 2.5% (w/v) dispersions in dimethylformamide, *DMF*. Their nominal density is 0.927 g ml-1 at 25°C (14). *NP*s were dialyzed against aqueous Borax (50 millimol, pH 8.5) under stirring, sonicated and recovered. The resulting dispersions are relatively stable, since *APOB* nano-particles bear negative charges on their

Hen yolk lysozyme, *LYS*, (Sigma Aldrich) was dialyzed, crystallized, lyophilized and dried over *P2O5*. Its purity was confirmed by ionic conductance, density and viscosity of the

Sodium dodecylsulfate, *SDS*, cetyltrimethylammonium bromide, *CTAB*, and didodecyldimethylammonium bromide, *DDAB*, (Sigma Aldrich) were individually dissolved in ethanol and precipitated by addition of cold acetone. The products were vacuum dried at 70°C. Their purity was confirmed by conductometric determination of the critical micellar concentration, *CMC*, at 25.00 °C. *Pluronic F-127* (Sigma Aldrich), a surface active block copolymer, forming micelles at high temperatures (17), was used as dispersant, when

A water soluble carbodiimide, termed *EDAC*, hydroxysuccynimide, *NHS*, triethylamine,

*LYS-ABOP nano-particles*. 20 mg of *APOB* in 50 millimolar kg-1 (*mmol*) aqueous Borax were added with 13.0 mg *EDAC*, 10.0 mg *NHS* and7.27 mg *N(Et)3*, at 25°C (7), and homogenized upon stirring. 44.13 mg solid *LYS* was added and the reaction proceeded for 5 hours, at 40°C. The reaction between *ABOP* and *LYS* is concomitant to an increase in opalescence of the dispersions, due to both a decreased surface charge density (because of the reaction of *COOH* groups with *EDAC)*, and to protein covalent binding. The final dispersion was centrifuged at 12000 rpm for 10 minutes, at 4°C. Thereafter, 10 mmol glycine was added, to quench un-reacted groups that were eventually bound onto *NP*s. The latter procedure significantly reduces the dispersion turbidity. A summary of the whole synthetic procedure, in four stages, is sketched in *Scheme 1*, where are indicated the different

Prolonged sonication, dialysis, with pH and/or ionic strength adjustment significantly tailors the average size of *NP*s and reduces their size. Substantial characterization is required to ensure the attainment of equilibrium conditions, since the size of particles increases with aging, *Figure 1*. The dispersions were extensively dialyzed against the buffer, until no more lysozime, *EDAC* and/or *NHS* was determined in the supernatant. To avoid the occurrence of clusters, the dispersions must be sheared (18) or forced to flow in tilted long

is known on the latter ones.

**2. Experimental section** 

corresponding aqueous solutions, at 25.00 *°C* (15,16).

*N(Et)3*, and glycine, (Sigma Aldrich), were used as such.

syringe steel needles (19), as indicated in *Figure 2A*.

surface.

required.

**3. Materials preparation** 

preparation steps.

Fig. 1. Plots of the correlation coefficient (in arbitrary units) versus the measuring time, in μs, for a 0.20 wt/vol % dispersion of Lysozyme-ABOP nano-particles (in 50 mmol Borax buffer, pH 8.5, at 25°C) one day after preparation, in blue, one week, in orange, and one month, in cyan.

Binding of Protein-Functionalized Entities onto Synthetic Vesicles 651

25.0° or 37.0°C for some minutes. Correlation fits of the light scattering intensity were elaborated by *CONTIN* algorithms (22). The auto-correlation decay function, *g1(*

determined the self-diffusion coefficient, and the hydrodynamic radii were evaluated by the

particles moving under the effect of an applied electric field, *Ē* (23). A laser-Doppler utility performed measurements, at 25.0°C, in cells equipped with gold electrodes. The scattered light passing in the medium, subjected to the action of *Ē*, shifts in frequency compared to

πη

ε°


impedance bridge, at 1 *KHz*, using a Daggett-Krauss cell thermostated to 25.00+0.01°C. *CD*. Measurements were run on a Jasco J-715 unit, working with 1 nm resolution. 0.100 cm quartz cells were used. Spectra are the average of three independent runs in the 190-300 nm

range. Signals due to native *LYS*, at 208 and 222 nm, respectively, were determined.

κ (*S cm-1*)

*UV-*V*is Light Absorbance*. Light absorption spectra, *A*, were recorded in the range 190-300 nm

*Microscopy*. Optical microscopy, in normal or polarized light, was performed through a Zeiss

*Density*. The particles density was determined by a DMA-60 Anton Paar vibrating densimeter, and thermally controlled by a water circulation bath working at 25.00 + 0.01 °C.

Fig. 3. Plot of ζ-potential values for different dispersions of nominal concentration in LYS-ABOP nano-particles equal to 0.12 w/v %. Measurements were run in 50 mmol Borax, at 25.0°C. Data are reported as number of counts (\*105) vs. measured ζ-potential value.

*Nano-particles characterization*. Nano-particles made of *LYS* and *APOB* (termed as *LYS*-*ABOP*)

ζ

μ


*m* Millipore filters and equilibrated at

*RH*). The uncertainty on vesicles sizes is to 10-20

ζ


the static dielectric constant of the medium). The


, was determined by a Wayne-Kerr

ζ = 4πστ/ε°,

τ*)*,

σ, of

25.0°C. The dispersions were passed through 0.80

. ζ

the double layer thickness and

*Ionic Conductivity*. The electrical conductance,

unperturbed conditions. Data manipulation of the signal gives the

by a Jasco V-570 unit, at 25.0+0.1 °C; the cell path length was 0.100 cm.

Stokes-Einstein equation (*Dapp* = *KBT*/*6*

nm, depending on their size.

ζ

**5. Results and discussion** 

were characterized by *DLS*, *CD*, *UV-vis* and

*-potential measurements*

ζ

where τ

uncertainty on

optical microscope.

When particles are subjected to shear forces during flow, large aggregates break down. This procedure decreases the size of *LYS*/*ABOP NP*s, *Figure 2B*.

Cat-anionic vesicles were prepared by mixing 6.00 *mmol* aqueous *SDS* with 6.00 *mmol CTAB*, in due proportions. Optimal sizes and surface charge density occur when the mole ratio between *SDS* and *CTAB* is in the range 1.5-2.5. The dispersions are milky, because multilamellar, and size-poly-disperse, vesicles occur. It was formerly observed, however, that heating them to temperatures close to 50°C reduces the average size of multi-lamellar vesicles, with formation of truly bi-layered entities (20). Thereafter, vesicles remain in such state for over two months, even when they are kept at room temperature.

Fig. 2. A; View of iron steel needles, tilted to increase the number of turns. B; Reduction in size of 0.12 w/v % LYS-ABOP nano-particles obtained by coupling (and dispersed in 50 mmol Borax, pH 8.5, at 25°C) when forced to flow in tilted needles. Data are reported as average particles size (nm) versus number of turns in the needle. Each sample was forced to flow in the needles for 50 times.

*DDAB* vesicles are multi-layered entities and their properties were tailored by extrusion and/or thermal cycling. Both vesicular dispersions were thermally equilibrated at 25.0°C soon after the preparation procedures and controlled over a long-time scale. Temperatures lower than 20°C were avoided, since they imply partial surfactant precipitation in the *SDS*-*CTAB* system.
