**1. Introduction**

644 Non-Viral Gene Therapy

Wiradharma, N., Tong, Y. W. & Yang, Y.-Y. (2009). Self-assembled oligopeptide

Wojcieszyn, J. W., et al. (1983). Studies on the mechanism of polyethylene glycol-mediated

Wojcieszyn, J. W., et al. (1981). Diffusion of injected macromolecules within the cytoplasm of living cells. *Proceedings of the National Academy of Sciences*, Vol. 78, pp. 4407-4410. Won, Y.-Y., Sharma, R. & Konieczny, S. F. (2009). Missing pieces in understanding the

Wood, K. C., et al. (2008). Tumor-targeted gene delivery using molecularly engineered

Wood, K. C., et al. (2005). A family of hierarchically self-assembling linear-dendritic hybrid

Wu, C. H., Wilson, J. M. & Wu, G. Y. (1989). Targeting genes: delivery and persistent

Wu, G. Y. & Wu, C. H. (1987). Receptor-mediated in vitro gene transformation by a soluble DNA carrier system. *Journal of Biological Chemistry*, Vol. 262, pp. 4429-4432. Wyman, T. B., et al. (1997). Design, synthesis, and characterization of a cationic peptide that

Yang, S., et al. (2009). Cellular uptake of self-assembled cationic peptide–DNA complexes:

Ye, J., et al. (2010). Determination of penetratin secondary structure in live cells with raman microscopy. *Journal of the American Chemical Society*, Vol. 132, pp. 980-988. Yukawa, H., et al. (2010). Quantum dots labeling using octa-arginine peptides for imaging of adipose tissue-derived stem cells. *Biomaterials*, Vol. 31, pp. 4094-4103. Zauner, W., et al. (1997). Glycerol and polylysine synergize in their ability to rupture

Zauner, W., Ogris, M. & Wagner, E. (1998). Polylysine-based transfection systems utilizing receptor-mediated delivery. *Advanced Drug Delivery Reviews*, Vol. 30, pp. 97-113. Ziegler, A., et al. (2005). The cationic cell-penetrating peptide CPPTAT Derived from the HIV-

gene transfer. *Experimental Cell Reasearch*, Vol. 232, pp. 137-145.

metabolic evidence. *Biochemistry*, Vol. 44, pp. 138-148.

*Biomaterials*, Vol. 30, pp. 3100-3109.

*Biology*, Vol. 96, pp. 151-159.

*Chemistry*, Vol. 19, pp. 403-405.

*International Edition*, Vol. 44, pp. 6704-6708.

*Journal of Biological Chemistry*, Vol. 264, pp. 16985-16987.

Vol. 139, pp. 88-93.

3017.

135, pp. 159-165.

nanostructures for co-delivery of drug and gene with synergistic therapeutic effect.

cell fusion using fluorescent membrane and cytoplasmic probes. *Journal of Cell* 

intracellular trafficking of polycation/DNA complexes. *Journal of Controlled Release*,

hybrid polymers functionalized with a tumor-homing peptide. *Bioconjugate* 

polymers for highly efficient targeted gene delivery. *Angewandte Chemie* 

expression of a foreign gene driven by mammalian regulatory elements in vivo.

binds to nucleic acids and permeabilizes bilayers. *Biochemistry*, Vol. 36, pp. 3008-

multifunctional role of the enhancer chloroquine. *Journal of Controlled Release*, Vol.

vesicular membranes: a mechanism for increased transferrin-polylysine-mediated

1 protein tat is rapidly transported into living fibroblasts: optical, biophysical, and

Mono-disperse silica nano-particles with pending functional acid groups lying on their surface were reacted with coupling agents and, then, with lysozyme, to get proteinfunctionalized entities. The synthetic procedure reported therein gives tiny amounts of protein functionalized sites; surface coverage by the protein is, thus, moderate. The amount of covalently bound lysozyme was estimated from *UV*-vis methods and resulted to be about <5> molecules per nano-particle. Electro-phoretic mobility experiments indicate the occurrence of significant variations in surface charge density of functionalized nanoparticles compared to the original ones and ensure a significant binding efficiency onto reconstructed, or synthetic, vesicles.

Protein-functionalized nano-particles form clusters and are readily re-dispersed by application of shear methods. Thereafter, they remain in disperse form for long times. According to *DLS*, protein-functionalized nano-particles interact with either cationic or cat-anionic synthetic vesicles. Care was made to ensure that nano-particles and vesicles have comparable sizes. The above procedure ensures to determine the fate of the reactive pathways by *DLS*. At room temperature and moderate ionic strength, the binding of protein-functionalized entities onto the aforementioned vesicles is completed in about one hour. The nano-particle vesicle complexes precipitate as fine powders, or form large floating objects, depending on vesicle size, relative concentrations of proteinfunctionalized particles and their net charge (which is related to the *pH* of the dispersing medium).

The binding efficiency for the above processes is controlled by the overlapping of repulsive and attractive interactions between particles and vesicles. The kinetic pathways relative to the interactions between vesicles and nano-particles were investigated, and significant differences were met in the two cases. Some technological implications of the above systems are preliminarily discussed. For instance, it is stated that interactions between nano-particles and vesicles mimic those occurring between cells and solid particles, or viral vectors, located in the medium surrounding vesicles.

Binding of Protein-Functionalized Entities onto Synthetic Vesicles 647

Silica was used as an anchoring site because of its good bio-compatibility, which is substantially higher compared to polymer-based nano-particles. It is particularly useful since blood and other bio-fluids contain markers adsorbing on the surface of hydrophobic carriers, and indicating to *RES* (the *Reticulum Endothelial System*) the urgency to remove them from the target tissue (5). Conversely, silica nano-particles (*NP*s) are of friendly use with biological tissues. As most inorganic oxides, *SiO2* is strongly hydrophilic in character. Such property increases its compatibility and ensures a safe circulation in bio-fluids. In addition, it is possible to anchor efficiently proteins or other biologically active substances

Lysozyme was chosen to test the covalent binding efficiency onto nano-particles because of its ubiquitary nature. In the following, we report on anchoring efficiency and do not explicitly account for the effect that *pH*, salts or other substances have on the fate of proteinfunctionalized *NP*s in biological matrices. The present communication only focuses on the

In the second part of this contribution the interactions between protein-functionalized silica *NP*s and synthetic vesicles were experienced. The synthetic vesicles dealt with in this contribution are also relatively mono-dispersed, thermodynamically, or kinetically, stable and are characterized, in some cases, by a bi-layer structure (6). Hence, they can be considered the synthetic analogues of cell membranes. Were the interactions between vesicles and *NP*s effective, the possibility of surface adhesion, or encapsulation, can be realized. Perspectives of these complex systems as composite drug-delivery carriers are,

We choose different vesicle-forming materials based on a synthetic double-chain surfactant and mixtures of different surface active species. The performances of the former class, based on quaternary ammonium salts, were extensively characterized by Barenholz and coworkers (7,8). Such species, however, are of questionable utility for biomedical applications, since most quaternary ammonium salts have strong anti-bacterial character. In addition, the vesicles they form are intrinsically meta-stable and progressively coagulate into large

Recently, alternatives to the above lipido-mimetic systems were proposed; they rely on systems obtained by mixing oppositely charged surfactants, or lipids, in due amounts. Such mixtures are currently defined by the acronym cat-anionic. These systems came in use since when Kaler and Khan independently characterized some of them (9,10). In cat-anionic systems the vesicle size and net charge are tuned by modulating the ratio between the two components, provided one of them is in excess. (N.B. If not, the 1/1 mixtures precipitate out.) It is possible, thus, to get negatively or positively charged vesicles. This can be relevant in case vesicles should selectively interact through electrostatic interactions with proteins (11)

In the following we report on the synthetic procedures we have followed, on the optimization required getting stable dispersions of hybrid protein-silica colloids, and on their interactions with vesicles. For reasons to be discussed later, the characterization of the above protein-silica composite material is based on the combination of optical (*UV-vis* or *CD*), dynamic light scattering, *DLS*, and electro-phoretic mobility methods. Some relevant results are briefly reported in the forthcoming sections. Biomedical applications, which are

or *DNA* (12) and form complexes, or lipo-plexes, with the above substances.

i. the surface area of the resulting nano-composites can be properly controlled, ii. the number of covalently bound proteins can be modulated accordingly, and, iii. the conformation of proteins adsorbed therein can be somehow predicted (4).

onto mono-dispersed silica.

thus, at hand.

entities.

synthesis and characterization of such materials.

#### **Graphical Abstract**

**Left**, Dependence of the size of nano-particle/vesicle clusters on reaction time, inferred by DLS methods.

**Right**, Clusters of vesicles and protein-functionalized nano-particles obtained by mixing DDAB vesicles and nano-particles in number ratio 300/1. Images were visualized through a Zeiss optical microscope using normal light. The bar size in the left bottom of the figure is 100 mm large.

Nanotechnologies deal with the synthesis, the characterization, the production and the application of objects and devices operating at the nano-scale level (1). As a consequence of substantial interest in this field, many scientific journals and books are progressively addressing attention to nano-technologically oriented items. The same holds for the number of patents relative to these subjects. This is because the demand on the possible applications of the above materials is drastically increasing in the last few years. Practical applications are manifold and find use in electronics, in the preparation of magnetic devices, in chemistry (mostly in the field of heterogeneous catalysis), but also in biotechnology and biomedicine.

Materials at the nano-scale are widely different each from the other in chemical composition, size and surface functionalities. Particles properties depend on the preparation procedures and can be tailored accordingly. Nano-particles can be made of metals, oxides, polymers and/or a combination thereof. Properly functionalized particles, such as quantum dots, spheres, disks, filaments, tubes and composite objects (all in the nano-meter size range) find substantial application in the aforementioned fields.

In this contribution, we report on silica nano-particles, onto which a protein, lysozyme, was covalently bound (2,3). The synthetic part of the work is reported below. The same holds for the characterization of the resulting hybrid composites. A substantial amount of work was needed to ensure the required performances to nano-particles, which were tailored in terms of state of the dispersion, size, stability and net charge. Obviously, the above effects are strongly interrelated each other. The reasons for using protein-functionalized entities arise by the need to have objects at the nano-scale level, characterized by a significant number of bound proteins. Apart from intrinsic interest towards the structural properties of hybrid nano-composites, the advantages of protein-functionalized particles are manifold compared to other bio-medical formulations, since:

 **Left**, Dependence of the size of nano-particle/vesicle clusters on reaction time, inferred by

**Right**, Clusters of vesicles and protein-functionalized nano-particles obtained by mixing DDAB vesicles and nano-particles in number ratio 300/1. Images were visualized through a Zeiss optical microscope using normal light. The bar size in the left bottom of the figure is

Nanotechnologies deal with the synthesis, the characterization, the production and the application of objects and devices operating at the nano-scale level (1). As a consequence of substantial interest in this field, many scientific journals and books are progressively addressing attention to nano-technologically oriented items. The same holds for the number of patents relative to these subjects. This is because the demand on the possible applications of the above materials is drastically increasing in the last few years. Practical applications are manifold and find use in electronics, in the preparation of magnetic devices, in chemistry (mostly in the field of heterogeneous catalysis), but also in biotechnology and

Materials at the nano-scale are widely different each from the other in chemical composition, size and surface functionalities. Particles properties depend on the preparation procedures and can be tailored accordingly. Nano-particles can be made of metals, oxides, polymers and/or a combination thereof. Properly functionalized particles, such as quantum dots, spheres, disks, filaments, tubes and composite objects (all in the nano-meter size range)

In this contribution, we report on silica nano-particles, onto which a protein, lysozyme, was covalently bound (2,3). The synthetic part of the work is reported below. The same holds for the characterization of the resulting hybrid composites. A substantial amount of work was needed to ensure the required performances to nano-particles, which were tailored in terms of state of the dispersion, size, stability and net charge. Obviously, the above effects are strongly interrelated each other. The reasons for using protein-functionalized entities arise by the need to have objects at the nano-scale level, characterized by a significant number of bound proteins. Apart from intrinsic interest towards the structural properties of hybrid nano-composites, the advantages of protein-functionalized particles are manifold compared

find substantial application in the aforementioned fields.

to other bio-medical formulations, since:

**Graphical Abstract** 

DLS methods.

100 mm large.

biomedicine.


Silica was used as an anchoring site because of its good bio-compatibility, which is substantially higher compared to polymer-based nano-particles. It is particularly useful since blood and other bio-fluids contain markers adsorbing on the surface of hydrophobic carriers, and indicating to *RES* (the *Reticulum Endothelial System*) the urgency to remove them from the target tissue (5). Conversely, silica nano-particles (*NP*s) are of friendly use with biological tissues. As most inorganic oxides, *SiO2* is strongly hydrophilic in character. Such property increases its compatibility and ensures a safe circulation in bio-fluids. In addition, it is possible to anchor efficiently proteins or other biologically active substances onto mono-dispersed silica.

Lysozyme was chosen to test the covalent binding efficiency onto nano-particles because of its ubiquitary nature. In the following, we report on anchoring efficiency and do not explicitly account for the effect that *pH*, salts or other substances have on the fate of proteinfunctionalized *NP*s in biological matrices. The present communication only focuses on the synthesis and characterization of such materials.

In the second part of this contribution the interactions between protein-functionalized silica *NP*s and synthetic vesicles were experienced. The synthetic vesicles dealt with in this contribution are also relatively mono-dispersed, thermodynamically, or kinetically, stable and are characterized, in some cases, by a bi-layer structure (6). Hence, they can be considered the synthetic analogues of cell membranes. Were the interactions between vesicles and *NP*s effective, the possibility of surface adhesion, or encapsulation, can be realized. Perspectives of these complex systems as composite drug-delivery carriers are, thus, at hand.

We choose different vesicle-forming materials based on a synthetic double-chain surfactant and mixtures of different surface active species. The performances of the former class, based on quaternary ammonium salts, were extensively characterized by Barenholz and coworkers (7,8). Such species, however, are of questionable utility for biomedical applications, since most quaternary ammonium salts have strong anti-bacterial character. In addition, the vesicles they form are intrinsically meta-stable and progressively coagulate into large entities.

Recently, alternatives to the above lipido-mimetic systems were proposed; they rely on systems obtained by mixing oppositely charged surfactants, or lipids, in due amounts. Such mixtures are currently defined by the acronym cat-anionic. These systems came in use since when Kaler and Khan independently characterized some of them (9,10). In cat-anionic systems the vesicle size and net charge are tuned by modulating the ratio between the two components, provided one of them is in excess. (N.B. If not, the 1/1 mixtures precipitate out.) It is possible, thus, to get negatively or positively charged vesicles. This can be relevant in case vesicles should selectively interact through electrostatic interactions with proteins (11) or *DNA* (12) and form complexes, or lipo-plexes, with the above substances.

In the following we report on the synthetic procedures we have followed, on the optimization required getting stable dispersions of hybrid protein-silica colloids, and on their interactions with vesicles. For reasons to be discussed later, the characterization of the above protein-silica composite material is based on the combination of optical (*UV-vis* or *CD*), dynamic light scattering, *DLS*, and electro-phoretic mobility methods. Some relevant results are briefly reported in the forthcoming sections. Biomedical applications, which are

Binding of Protein-Functionalized Entities onto Synthetic Vesicles 649

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.

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 is known on the latter ones.
