**5. Conclusion**

This chapter started by discussing the challenges and intracellular barriers associated with the delivery of nucleic acids. Strategies used to overcome these hurdles were next examined, mainly in the context of peptide-derived vectors. It is clear that peptide carriers are not characterised by any typical sequences, although the majority of current designs rely on cationic residues to bind nucleic acids. This strategy, nonetheless, may be problematic during *in vivo* applications. An area that deserves more attention is the development of vectors that can bind nucleic acids using non-electrostatic forces, e.g., by including more hydrophobic residues. The stability and *in vivo* behaviour of such vectors then need to be thoroughly evaluated. Another challenge is to further improve strategies that are already in place to shield the cationic charges, e.g., by attaching PEG chains that are intracellularly cleavable so as to increase the circulation time of the complexes without compromising excessively on transfection efficiency. Advances in either will enable peptides to further realise their potential as a class of non-viral vector.

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**27** 

*1,3Italy 2Spain* 

**Binding of Protein-Functionalized** 

**Entities onto Synthetic Vesicles** 

Federica De Persiis1, Ramon Pons2, Carlotta Pucci1,

*3SOFT-INFM-CNR Research Centre, La Sapienza University, Rome* 

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

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

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

**1. Introduction** 

medium).

reconstructed, or synthetic, vesicles.

in the medium surrounding vesicles.

*1Dept. of Chemistry, La Sapienza University, Rome, Italy 2Institut de Química Avançada de Catalunya, IQAC-CSIC* 

Franco Tardani1 and Camillo La Mesa1,3

