**4. Methods**

*Dynamic Light Scattering*. A Zeta Nanosizer unit, Malvern, performed measurements at 632.8 nm, in back scattering mode (*BSM*), at 173°. This configuration minimizes multiple scattering and allows measuring poly-disperse systems. The unit performances were checked by standard procedures (21). Thermal equilibrium was controlled by a Peltier unit, at

When particles are subjected to shear forces during flow, large aggregates break down. This

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

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

*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*-

*Dynamic Light Scattering*. A Zeta Nanosizer unit, Malvern, performed measurements at 632.8 nm, in back scattering mode (*BSM*), at 173°. This configuration minimizes multiple scattering and allows measuring poly-disperse systems. The unit performances were checked by standard procedures (21). Thermal equilibrium was controlled by a Peltier unit, at

flow in the needles for 50 times.

*CTAB* system.

**4. Methods** 

procedure decreases the size of *LYS*/*ABOP NP*s, *Figure 2B*.

state for over two months, even when they are kept at room temperature.

25.0°C. The dispersions were passed through 0.80 μ*m* Millipore filters and equilibrated at 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(*τ*)*, determined the self-diffusion coefficient, and the hydrodynamic radii were evaluated by the Stokes-Einstein equation (*Dapp* = *KBT*/*6*πη*RH*). The uncertainty on vesicles sizes is to 10-20 nm, depending on their size.

ζ*-potential measurements*. ζ-potential methods determined the surface charge density, σ, of 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 unperturbed conditions. Data manipulation of the signal gives the ζ-potential (ζ = 4πστ/ε°, where τ the double layer thickness and ε° the static dielectric constant of the medium). The uncertainty on ζ-potentials is 0.5-1.0 mV. Data are reported in *Figure 3*.

*Ionic Conductivity*. The electrical conductance, κ (*S cm-1*), was determined by a Wayne-Kerr 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.

*UV-*V*is Light Absorbance*. Light absorption spectra, *A*, were recorded in the range 190-300 nm by a Jasco V-570 unit, at 25.0+0.1 °C; the cell path length was 0.100 cm.

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

*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.

#### **5. Results and discussion**

*Nano-particles characterization*. Nano-particles made of *LYS* and *APOB* (termed as *LYS*-*ABOP*) were characterized by *DLS*, *CD*, *UV-vis* and ζ-potential methods. A substantial amount of

Binding of Protein-Functionalized Entities onto Synthetic Vesicles 653

Fig. 4. In the top is reported a schematic representation of vesicle structure. On the upper left is reported a picture relative to multi-lamellar DDAB vesicles (in red/cyan colors, to distinguish the different layers), on the upper right the one related to bi-layered SDS/CTAB vesicles (in blue color). Sizes scale with the average hydrodynamic vesicle radius, inferred from DLS. In the bottom is indicated a cartoon of ABOP nano-particles with surface-bound LYS molecules. As before, regions in red indicate the dominance of positive charges (around the protein), when the blue color of the corona indicates an excess of negative charges.

The above approach helps determining the kinetic features of interactions between different *LYS*-*ABOP* particles. It will also be used to account for the interactions taking place between vesicles and *LYS*-*ABOP*. It is expected that attractions are experienced in the interaction between *LYS*-*ABOP* and cat-anionic vesicles (bearing a negative charge), and repulsions in

*DLS* alone does not allow to asses any firm statement on lysozyme binding, since the sizes of functionalized *NP*s is only slightly higher than before the reaction. That's why combination

The former method quantifies the amount of *ABOP*-bound protein (in native or denatured state) and whether binding is effective. The second gives information on the conformational

of optical absorbance with *DLS*, *CD* and electro-phoretic mobility is helpful.

the other case, *Figure 4*.

work was required to stabilize the resulting dispersions, because of their tendency to agglomerate and/or phase separate. The optimal conditions for an effective stabilization (i.e. stirring, temperature, *pH* and/or ionic strength) were determined. The surface charge density, σ, plays a pivotal role in the stability of these dispersions and depends on the medium ionic strength. In buffers with 50 mmol Borax and 10 mmol *NaCl* the dispersions remain stable for about two weeks; usually, they were used one day after preparation. Care was taken to get macroscopic homogeneity and to avoid the presence of clusters or sediments.

In the characterization by *DLS*, a proper selection of the elaborating functions, taking into account the particles sizes and poly-dispersity, was necessary. The cumulant method was applied and the scattering equation, *g1*(*t*), was expressed as a power-law series, according to the well-known relation

$$\ln \mathbf{g}\_1(t) = \sum\_{n=1}^{\bullet} \Gamma\_n \frac{(-t)^n}{n!} = -\Gamma\_1 t + \frac{1}{2!} \Gamma\_2 t^2 - \frac{1}{3!} \Gamma\_3 t^3 + \dots \dots \tag{1}$$

*Eq. (1)* gives information on the average value of the distribution function, (that is Γ*1* = <Γ> = *q2*<*Dapp*>), and on poly-dispersity index, *PdI* (Γ*2*/Γ*12*). Minor terms are also present.

Even when the particles number is moderate, a prolonged aging of the dispersions must be avoided, since a significant shift of the correlation functions is observed, *Figure 1*. That is, both Γ*1* (related to the particles self-diffusion, *Dapp*) and Γ*<sup>2</sup>* (related to *PdI*) depend on aging. This implies the presence of reactive terms. Data were analyzed accounting for the diffusive contributions pertinent to nano-particles and for the respective reactive terms, respectively. The results are expressed in terms of the relation

$$I\_{\mathcal{D}}(t) = \left[ \left| I\_1(0) \right|^2 + \left| I\_1(t) \right|^2 \right] \tag{2}$$

where *I2*(*t*), *I1*(0) and *I1*(*t*) are the scattering intensity at time *t,* the original value at time zero and the reactive part, respectively.

*DLS* data were interpreted according to Berne and Pecora (24). Attractive terms, due to particles coagulation, and electrostatic ones, due to the presence of repulsive or attractive forces between colloid entities, are introduced in the time-dependent scattering functions. The relation contains a reactive term, related to the formation of large particles, and is balanced by a flux, in which mobility and diffusive terms are accounted for (24). Accordingly,

$$\left(\frac{\partial c\_a}{\partial t}\right) + \nabla \bullet J\_a = k\_b c\_b - k\_a c\_a \tag{3}$$

where (*dca*/*dt*) indicates the production, or disappearance, of particles with time. The second term in the right hand side of the equation is expressed as

$$J\_a = \mu\_a E c\_a - D\_{app,a} \nabla c\_a \tag{4}$$

where μ*<sup>a</sup>* is the electro-phoretic mobility of a class of particles, at concentration *ca*, under the effect of and applied electric field, *Ē*, and *Dapp* is the self diffusion times the concentration gradient, ∇*ca*. *Ka* and *Kb* are the kinetic constants of reactants, *a*, and products, *b*, respectively.

work was required to stabilize the resulting dispersions, because of their tendency to agglomerate and/or phase separate. The optimal conditions for an effective stabilization (i.e. stirring, temperature, *pH* and/or ionic strength) were determined. The surface charge

medium ionic strength. In buffers with 50 mmol Borax and 10 mmol *NaCl* the dispersions remain stable for about two weeks; usually, they were used one day after preparation. Care was taken to get macroscopic homogeneity and to avoid the presence of clusters or

In the characterization by *DLS*, a proper selection of the elaborating functions, taking into account the particles sizes and poly-dispersity, was necessary. The cumulant method was applied and the scattering equation, *g1*(*t*), was expressed as a power-law series, according to

( ) ( ) ......... 3!

 (Γ*2*/Γ

Even when the particles number is moderate, a prolonged aging of the dispersions must be avoided, since a significant shift of the correlation functions is observed, *Figure 1*. That is,

This implies the presence of reactive terms. Data were analyzed accounting for the diffusive contributions pertinent to nano-particles and for the respective reactive terms, respectively.

> ( )

where *I2*(*t*), *I1*(0) and *I1*(*t*) are the scattering intensity at time *t,* the original value at time zero

*DLS* data were interpreted according to Berne and Pecora (24). Attractive terms, due to particles coagulation, and electrostatic ones, due to the presence of repulsive or attractive forces between colloid entities, are introduced in the time-dependent scattering functions. The relation contains a reactive term, related to the formation of large particles, and is balanced by a flux, in which mobility and diffusive terms are accounted for (24). Accordingly,

*<sup>a</sup> <sup>b</sup> <sup>b</sup> <sup>a</sup> <sup>a</sup> <sup>a</sup> <sup>J</sup> <sup>k</sup> <sup>c</sup> <sup>k</sup> <sup>c</sup>*

where (*dca*/*dt*) indicates the production, or disappearance, of particles with time. The second

*<sup>a</sup> <sup>a</sup> <sup>a</sup> app <sup>a</sup> <sup>a</sup> J* = *Ec* − *D* ∇*c*

*<sup>a</sup>* is the electro-phoretic mobility of a class of particles, at concentration *ca*, under the effect of and applied electric field, *Ē*, and *Dapp* is the self diffusion times the concentration

*ca*. *Ka* and *Kb* are the kinetic constants of reactants, *a*, and products, *b*, respectively.

μ

*<sup>c</sup>* <sup>+</sup> <sup>∇</sup> • <sup>=</sup> <sup>−</sup>

*t*

 ∂

term in the right hand side of the equation is expressed as

2

= +

<sup>1</sup> <sup>=</sup> −Γ <sup>+</sup> <sup>Γ</sup> <sup>−</sup> <sup>Γ</sup> <sup>+</sup> <sup>−</sup> <sup>=</sup> <sup>Γ</sup>

! ln <sup>3</sup>

*n*

*Eq. (1)* gives information on the average value of the distribution function, (that is

*n t*

1

*1* (related to the particles self-diffusion, *Dapp*) and

=

*n*

∞

*g t*

= *q2*<*Dapp*>), and on poly-dispersity index, *PdI*

The results are expressed in terms of the relation

and the reactive part, respectively.

2! 1

1 2

1

2

Γ

2 1

<sup>2</sup> <sup>1</sup> *<sup>I</sup> <sup>t</sup> <sup>I</sup>* (0) *<sup>I</sup>* (*t*) (2)

∂ (3)

, (4)

*t t t*

3

*n* (1)

*12*). Minor terms are also present.

*<sup>2</sup>* (related to *PdI*) depend on aging.

Γ*1* = <Γ>

, plays a pivotal role in the stability of these dispersions and depends on the

density,

sediments.

both Γ

where μ

gradient,

∇

σ

the well-known relation

Fig. 4. In the top is reported a schematic representation of vesicle structure. On the upper left is reported a picture relative to multi-lamellar DDAB vesicles (in red/cyan colors, to distinguish the different layers), on the upper right the one related to bi-layered SDS/CTAB vesicles (in blue color). Sizes scale with the average hydrodynamic vesicle radius, inferred from DLS. In the bottom is indicated a cartoon of ABOP nano-particles with surface-bound LYS molecules. As before, regions in red indicate the dominance of positive charges (around the protein), when the blue color of the corona indicates an excess of negative charges.

The above approach helps determining the kinetic features of interactions between different *LYS*-*ABOP* particles. It will also be used to account for the interactions taking place between vesicles and *LYS*-*ABOP*. It is expected that attractions are experienced in the interaction between *LYS*-*ABOP* and cat-anionic vesicles (bearing a negative charge), and repulsions in the other case, *Figure 4*.

*DLS* alone does not allow to asses any firm statement on lysozyme binding, since the sizes of functionalized *NP*s is only slightly higher than before the reaction. That's why combination of optical absorbance with *DLS*, *CD* and electro-phoretic mobility is helpful.

The former method quantifies the amount of *ABOP*-bound protein (in native or denatured state) and whether binding is effective. The second gives information on the conformational

Binding of Protein-Functionalized Entities onto Synthetic Vesicles 655

positively charged vesicles. It is expected that the kinetic features inherent to vesicle-*LYS-*

The analysis of kinetic data, performed by *DLS* methods, is essentially based on the supposed dominance of electrostatic interactions between particles. Were this hypothesis absolutely unrealistic, different kinetic approaches should be considered. For instance the elecctro-phoretic term in the flux equation should be critically reconsidered. Very presumably, however, binding is due to the combined effect of dominant electrostatic contributions plus hydrophobic and ancillary (osmotic?) ones into an as yet undefined

*Kinetic features*. Processes related to vesicles-*NPs* interactions are dealt with in this part. Dispersions of *DDAB* and (*SDS*-*CTAB*), at the same nominal concentration in lipid, were mixed with tiny amounts of *LYS-ABOP* particles. The number ratio between the latter and vesicles is moderate. This allows measuring the kinetic features inherent to the interactions between *LYS-ABOP NP*s and vesicles. An eye-view to the results, *Figure 6* and *Figure 7*, indicates that the interactive processes can be rationalized on volume fraction statistics. The kinetic pathways scale with the *VES*-*NP* number ratio. Vesicles sizes are different each from the other, but are reasonably close to *NP*s. That means that changes in size of the scattering entities are mostly due to vesicles-*NPs* adducts. The respective kinetic pathways and the size of particles obtained at the end of the respective reactive processes are different in the two cases. In the [*DDAB*+(*LYS*-*ABOP*)] system sizes at equilibrium are lower compared to the [(*SDS*-*CTAB*)+(*LYS*-*ABOP*)] one. These effects are a sound indication of clustering between

Fig. 6. Kinetics of vesicle-NP interaction inferred from DLS plots of average particles size, DH, as a function of measuring time, t (min), for two different vesicles to LYS-ABOP nanoparticles number ratios. Data refer to systems buffered with 50 mmol Borax, at 25°C. Black symbols refer to a number ratio between NP's and cat-anionic SDS-CTAB vesicles (having mole ratio between the two of 1.7/1.0) equal to 1/60 and 1/150 in the other case. In the inset

is reported the long term behavior observed in the former system.

*ABOP* particles interactions should behave accordingly.

mechanism.

state of *LYS*. Electro-phoretic mobility, finally, indicates an effective surface modification of silica upon protein binding. Binding will reduce in modulus the ζ-potential values, as observed.

Fig. 5. CD spectrum, in molal ellipticity (θ, in deg cm2 mol-1) vs. the measuring wavelength (λ, in nm) of LYS-ABOP nano-particles, in grey color, compared to the corresponding value for bulk LYS, in black. The signal intensities were rescaled to allow for a comparison. In both cases data refer to pH 8.5, with 50.0 mmol Borax buffer, and 25.0° C. The former was upward shifted, to avoid overlapping.

Optical absorbance, performed in the 220-230 nm range, determined the presence and number of *LYS* molecules bound onto *ABOP* particles. The average value is <5+1> molecules. The uncertainty is high since the number of *NP*s in the medium is low, and scattering effects reduce the datum quality. *CD* data, *Figure 5*, indicate that the conformation of covalently bound protein is very close to its native form. Thus, the presence of bound protein was inferred by optical absorbance (and elemental analysis, as well), whereas a conformation close to the native one was inferred by *CD*.

Finally, the ζ-potential of *LYS-ABOP* particles decreases from -36 mV of the bare ones to -20, in case of protein-bound entities. Data indicate a significant *LYS* binding and surface modification, but a significant reduction of surface charge density compared to the native particles. This is because some *COOH* units are linked with *LYS* and the charge density of the particle as a whole is substantially reduced. The reduction in ζ-potential also explains why *LYS*-*ABOP* adducts are less kinetically stable compared to bare *ABOP*.

The optimal conditions leading to stabilization are fulfilled for *pH* values between 7.0 and 8.5. Below *pH* 7.0 the particles sizes increase significantly and such conditions were avoided. Substantial amounts of salt must be used to stabilize the dispersions in such *pH* conditions.

A scheme representing all particles considered here is in *Figure 4*. There is indicated the structure of vesicles and *LYS-ABOP* complexes, with charge distribution in evidence. It is expected that the charge distribution is responsible for interactions with both negatively or

state of *LYS*. Electro-phoretic mobility, finally, indicates an effective surface modification of

Fig. 5. CD spectrum, in molal ellipticity (θ, in deg cm2 mol-1) vs. the measuring wavelength (λ, in nm) of LYS-ABOP nano-particles, in grey color, compared to the corresponding value for bulk LYS, in black. The signal intensities were rescaled to allow for a comparison. In both

Optical absorbance, performed in the 220-230 nm range, determined the presence and number of *LYS* molecules bound onto *ABOP* particles. The average value is <5+1> molecules. The uncertainty is high since the number of *NP*s in the medium is low, and scattering effects reduce the datum quality. *CD* data, *Figure 5*, indicate that the conformation of covalently bound protein is very close to its native form. Thus, the presence of bound protein was inferred by optical absorbance (and elemental analysis, as well), whereas a

in case of protein-bound entities. Data indicate a significant *LYS* binding and surface modification, but a significant reduction of surface charge density compared to the native particles. This is because some *COOH* units are linked with *LYS* and the charge density of

The optimal conditions leading to stabilization are fulfilled for *pH* values between 7.0 and 8.5. Below *pH* 7.0 the particles sizes increase significantly and such conditions were avoided. Substantial amounts of salt must be used to stabilize the dispersions in such *pH*

A scheme representing all particles considered here is in *Figure 4*. There is indicated the structure of vesicles and *LYS-ABOP* complexes, with charge distribution in evidence. It is expected that the charge distribution is responsible for interactions with both negatively or


ζ


cases data refer to pH 8.5, with 50.0 mmol Borax buffer, and 25.0° C. The former was

upward shifted, to avoid overlapping.

Finally, the

conditions.

ζ

conformation close to the native one was inferred by *CD*.

the particle as a whole is substantially reduced. The reduction in

why *LYS*-*ABOP* adducts are less kinetically stable compared to bare *ABOP*.

ζ


silica upon protein binding. Binding will reduce in modulus the

observed.

positively charged vesicles. It is expected that the kinetic features inherent to vesicle-*LYS-ABOP* particles interactions should behave accordingly.

The analysis of kinetic data, performed by *DLS* methods, is essentially based on the supposed dominance of electrostatic interactions between particles. Were this hypothesis absolutely unrealistic, different kinetic approaches should be considered. For instance the elecctro-phoretic term in the flux equation should be critically reconsidered. Very presumably, however, binding is due to the combined effect of dominant electrostatic contributions plus hydrophobic and ancillary (osmotic?) ones into an as yet undefined mechanism.

*Kinetic features*. Processes related to vesicles-*NPs* interactions are dealt with in this part. Dispersions of *DDAB* and (*SDS*-*CTAB*), at the same nominal concentration in lipid, were mixed with tiny amounts of *LYS-ABOP* particles. The number ratio between the latter and vesicles is moderate. This allows measuring the kinetic features inherent to the interactions between *LYS-ABOP NP*s and vesicles. An eye-view to the results, *Figure 6* and *Figure 7*, indicates that the interactive processes can be rationalized on volume fraction statistics. The kinetic pathways scale with the *VES*-*NP* number ratio. Vesicles sizes are different each from the other, but are reasonably close to *NP*s. That means that changes in size of the scattering entities are mostly due to vesicles-*NPs* adducts. The respective kinetic pathways and the size of particles obtained at the end of the respective reactive processes are different in the two cases. In the [*DDAB*+(*LYS*-*ABOP*)] system sizes at equilibrium are lower compared to the [(*SDS*-*CTAB*)+(*LYS*-*ABOP*)] one. These effects are a sound indication of clustering between

Fig. 6. Kinetics of vesicle-NP interaction inferred from DLS plots of average particles size, DH, as a function of measuring time, t (min), for two different vesicles to LYS-ABOP nanoparticles number ratios. Data refer to systems buffered with 50 mmol Borax, at 25°C. Black symbols refer to a number ratio between NP's and cat-anionic SDS-CTAB vesicles (having mole ratio between the two of 1.7/1.0) equal to 1/60 and 1/150 in the other case. In the inset is reported the long term behavior observed in the former system.

Binding of Protein-Functionalized Entities onto Synthetic Vesicles 657

According to *Figure 8*, for instance, it is evident that entities made of *DDAB* and *LYS-ABOP* particles do form small aggregates, held together by significant forces, presumably electrostatic in nature. According to optical microscopy, it results that these composite objects are made of different sub-domains, differing each from the other in optical appearance and color. Apparently, there is no significant relation between the average stoichiometry of adducts made by vesicles and nano-particles and that pertinent to mother solution. In words, there is no direct proportionality between number of particles

Fig. 8. Vesicle- PFNP clusters, obtained by mixing DDAB and PFNP in number ratios 300/1 and recovering the precipitates. They are visualized through a Zeiss Optical Microscope

The results reported here indicate the possibility to get "*hybrid*" colloid composites from the interactions between *LYS-NPs* complexes and vesicles. The reported results refer to the phenomenological aspects of the interaction process, as it was inferred from *DLS*. Very

using normal light. The bar size in the bottom is 100 μm large.

**6. Conclusions** 

in the medium and composition of the precipitates.

objects similar in size, driven by electrostatic interactions between the two entities. Differences in the size of super-colloids formed in these mixtures are the consequences of the interaction mechanisms and result in significant changes in size and shape (presumably) of the resulting entities. Similar features were observed in the [*DDAB*+(*LYS*-*ABOP*)] system, *Figure 7*. There a marked tendency to sedimentation is observed after the interactions took place. Presumably, part of the observed decrease in size may be due to sedimentation processes, occurring at the early stages of the process.

The interactions between *DDAB* and *LYS*-*ABOP NP'*s show a different kinetic behavior compared to the former system, with a significant increase in size at low times, followed by a substantial decrease at long ones. This behavior is controlled by the net charge of the reacting objects and indicates that electrostatic terms are significant. According to *Figure 7*, there is a pronounced redistribution of particles sizes after about 40 minutes. Such features are, very presumably, related to a structural rearrangement and eventual rupture of vesicle-*NP* adducts with time.

Fig. 7. Plot of the average vesicle/Lys-ABOP adducts size (in nm) with time, in minutes, upon interaction between DDAB vesicles and LYS-ABOP nano-particles. The nominal number ratio between the colloidal objects is indicated in the figure. Experimental conditions refer to the same pH and temperature values reported above.

It is also possible that adducts sedimentation takes place after some time. In words, the formation of composites colloids implies a progressive saturation mechanism in case of *SDS*-*CTAB* vesicles. Conversely, a significantly different behavior occurs in the *DDAB*  containing system. This is put in evidence by the macroscopic appearance of the supercolloid entities formed in this way. In some instances sediments are found at the bottom of vials, in others large floating particles are observed in the medium. That means that the super-colloids formed upon interaction between vesicles and nano-particles differ each from the other in packing density and surface charge, to mind but a few effects.

objects similar in size, driven by electrostatic interactions between the two entities. Differences in the size of super-colloids formed in these mixtures are the consequences of the interaction mechanisms and result in significant changes in size and shape (presumably) of the resulting entities. Similar features were observed in the [*DDAB*+(*LYS*-*ABOP*)] system, *Figure 7*. There a marked tendency to sedimentation is observed after the interactions took place. Presumably, part of the observed decrease in size may be due to sedimentation

The interactions between *DDAB* and *LYS*-*ABOP NP'*s show a different kinetic behavior compared to the former system, with a significant increase in size at low times, followed by a substantial decrease at long ones. This behavior is controlled by the net charge of the reacting objects and indicates that electrostatic terms are significant. According to *Figure 7*, there is a pronounced redistribution of particles sizes after about 40 minutes. Such features are, very presumably, related to a structural rearrangement and eventual rupture of vesicle-

Fig. 7. Plot of the average vesicle/Lys-ABOP adducts size (in nm) with time, in minutes, upon interaction between DDAB vesicles and LYS-ABOP nano-particles. The nominal number ratio between the colloidal objects is indicated in the figure. Experimental

It is also possible that adducts sedimentation takes place after some time. In words, the formation of composites colloids implies a progressive saturation mechanism in case of *SDS*-*CTAB* vesicles. Conversely, a significantly different behavior occurs in the *DDAB*  containing system. This is put in evidence by the macroscopic appearance of the supercolloid entities formed in this way. In some instances sediments are found at the bottom of vials, in others large floating particles are observed in the medium. That means that the super-colloids formed upon interaction between vesicles and nano-particles differ each from the other in packing density and surface charge, to mind but a few

conditions refer to the same pH and temperature values reported above.

processes, occurring at the early stages of the process.

*NP* adducts with time.

effects.

According to *Figure 8*, for instance, it is evident that entities made of *DDAB* and *LYS-ABOP* particles do form small aggregates, held together by significant forces, presumably electrostatic in nature. According to optical microscopy, it results that these composite objects are made of different sub-domains, differing each from the other in optical appearance and color. Apparently, there is no significant relation between the average stoichiometry of adducts made by vesicles and nano-particles and that pertinent to mother solution. In words, there is no direct proportionality between number of particles in the medium and composition of the precipitates.

Fig. 8. Vesicle- PFNP clusters, obtained by mixing DDAB and PFNP in number ratios 300/1 and recovering the precipitates. They are visualized through a Zeiss Optical Microscope using normal light. The bar size in the bottom is 100 μm large.
