- Integration of cell penetrating peptides into gold nanoparticles by host-guest interactions: We have prepared gold nanoparticles bearing host anchored to the surface (beta-cyclodextrins). We have synthesized monovalent and divalent guest-bearing cell penetrating peptides. By combination of the building blocks, we have developed multicomponent nanoparticles bearing different surface coverage of peptide and stabilizers (PEGs). Their properties and morphology was studied by several techniques (DLS, TEM, zeta potential measurements, UV). We have observed that the binding of the CPP to guest nanoparticles have a mayor host-guest contribution and a minor electrostatic contribution. In this way, we were able to avoid nanoparticle aggregation by using statistical/multivalent interactions.
The following items indicate concrete points developed during the grant:
• Preparation of host-bearing cell penetrating peptides: We have developed efficient synthetic strategies towards the preparation of a variety of cell penetrating peptides that include several functionalities (e.g. guest motives, fluorophores anchored). This has been accomplished by synthetic strategies including solid phase and homogeneous solution synthesis.
• Demonstration of the functionalization of CPPs in nanoparticles by host-guest chemistry, including the use of multivalent interactions, and demonstrate their release: We have demonstrated the formation of supramolecular host-guest complexes between β -CD@AuNPs and adamantane-peptides and adamantane-PEG by several techniques, such as DLS, zeta-potential measurements and fluorescence measurements. The release of the cargo has been demonstrated with competition experiments. We also have studied the importance of the molecular design towards multivalent recognition between nanoparticles and peptides.
• Determination of the critical parameters for the stabilization of nanoparticles in different conditions: We carried out a detailed investigation concerning the the stability of the assemblies in conditions which are biologically relevant. Overall, we have observed a more important stabilizing effect due to electrostatic factors rather than by steric stabilization (e.g. like the stabilization achieved by adding polyethyleneglycol stabilizers).
• Obtaining information about binding to cell membranes: We have evaluated the interaction between CPP anchored to nanoparticles in cell models (HeLa) and demonstrated efficient uptake and cytosolic delivery of the peptide after host/guest release.
- Peptide exchange within cells: We have further evaluated the dynamic exchange of peptides within cells, for this we have developed nanoparticles loaded with one cell type that were delivered into cells. The addition of a second peptide that penetrated the cells and reached the nanoparticle allowed the exchaThe results of this propoposal have already given rise to two publications up to date and we have attended to two international symposiums to present these results (including one poster prize). Aditionally, three more maniscripts are currently being prepared with the most promising results in cells and delivery that will be pu blished in the best multidisciplinary chemistry journals.nge of one peptide for another. This could be employed for therapeutic strategies.
