ERA NanoSci - Multifunctional Gold Nanoparticles for Gene Therapy
Theoretical Chemistry: Electronic Structure, Dynamics, Simulation
Final Report Abstract
Gold nanoparticles have emerged as novel safe and biocompatible tools for manifold applications, including biological imaging, clinical diagnostics and therapeutics. PEG derivatives to increase the stability of the AuNPs, a cell penetrating peptide (TAT peptide) able to improve the cellular uptake of the AuNPs, a cell adhesion peptide as tumoral marker candidate probably due to ÿvÿ3 integrin interactions (RGD peptide), fluorescent dyes to follow where the nanoparticles go, and a siRNA sequence able to silence c-myc protooncogene expression. We designed two methods to link siRNA on the surface of the AuNPs: 1) ionic approach, where the siRNA was linked to positively charged AuNPs just by ionic interactions; 2) covalent approach, where thiolated siRNA sequences were used to allow further Au-S bonds between the NPs and the RNA. All the AuNPs were fully characterized by different chemical and physical techniques, such as TEM, SEM, DLS, z-potential, FTIR, UV/Vis. The chemical functionalization were also characterized and quantified to provide a number of PEG, peptides or siRNA molecules per AuNP, mainly using colorimetric methodologies. In order to evaluate the efficiency of the 18 different prepared AuNPs, a hierarchical approach including three biological systems of increasing complexity were used: in vitro cultured human cells, in vivo invertebrate (freshwater polyp, Hydra) and in vivo vertebrate (mouse) models. Our synthetic methodology involved fine-tuning of multiple structural and functional moieties. Selection of the most active functionalities was assisted step by step through functional testing adopting this hierarchical strategy. Moreover, we have also characterized the dynamics and kinetics of the events occurring at the bio/non-bio interface, from the first interaction nanoparticle/cell membrane, to the intracellular trafficking and final extracellular clearance. By treating a simple water invertebrate (the cnidarian Hydra polyp) with functionalized gold nanoparticles, multiple inwards and outwards routes were imaged by ultrastructural analyses, including exosomes as novel undescribed carriers to shuttle the nanoparticles in and out the cells. From the time course imaging a highly dynamic picture emerged in which nanoparticles are rapidly internalized (from 30min onwards), recruited into vacuoles/endosome (24h onwards), which then fuse, compact and sort out the internalized material either to storage vacuoles or to late-endosome/lysosomes, determining almost complete clearance within 48h from challenging. Beside classical routes, new portals of entry/exit were captured, including exosome-like structures as novel undescribed nanoparticle shuttles. The conservation of the endocytic/secretory machinery through evolution extends the value of our finding to mammalian systems providing dynamics and kinetics clues to take into account when designing nanomaterials to interface with biological entities. From the 18 different AuNPs prepared along this project, only one was able to induce c-myc silencing in the three biological systems used. This AuNP obtained by the covalent approach and containing RGD and siRNA is for instance, a safe, non-pathogenic, self-tracking and universally valid nanocarrier that could be exploited for therapeutic RNAi. In order to validate this nanosystem as antitumoral gene therapy drug, the selected AuNP was used in two lung cancer xenograft mouse models resulting in successful and significant c-myc oncogene downregulation followed by tumor growth inhibition and prolonged survival of the animals. Our results suggests that RGD gold nanoparticles-mediated delivery of siRNA by intratracheal instillation in mice leads to successful supression of tumor cells and tumor size decreasing, followed by concomitant inflammatory response in neutrophils and macrophages. In addition, RGD-functionalization of the therapeutic nanoparticles promotes siRNA targeting in LA-4 cancer cell possibly via ÿvÿ3 integrin interactions leading to downregulation c-myc oncogene. These results reiterate the capability of gold nanoparticles for targeted delivery of siRNA to cancer cells towards effective silencing of the specific target oncogene. What is more, we demonstrate that the gold-nanoconjugates trigger a complex inflammatory and immune response that might assist the therapeutic effect of the RNAi to downsize tumor size with clinically compatible doses of siRNA.
Publications
- Macrophage cellular adaptation, localization and imaging of different size polystyrene particles. Nano Biomedicine and Engineering 2009, 1, 19-38
Tian F, Prina-Mello A, Estrada G, Beyerle A, Möller W, Schulz H, Kreyling W, Stoeger T
- Improved visualisation of internalised carbon nanotubes by maximising cell spreading on nanostructured substrates. Nano Biomedicine and Engineering 2010. 201-207
Bao C, Tian F, Estrada G
- Multifunctional Nanocarriers for diagnostics, drug delivery and targeted treatment across blood-brain barrier: perspectives on tracking and neuroimaging. Particle and Fibre Toxicology 2010, 7:3
Bhaskar S, Tian F, Stoeger T, Kreyling W, de la Fuente J M, Grazú V, Borm P, Estrada G, Ntziachristos V and Razansky D
- (2012) Design of Multifunctional Gold Nanoparticles for In Vitro and In Vivo Gene Silencing. ACS Nano. 6 (9), pp 8316–8324
Conde J, Ambrosone A, Sanz V, Hernandez Y, Marchesano V, Tian F, Child H, Berry CC, Ibarra MR, Baptista PV, Tortiglione C, de la Fuente JM
(See online at https://doi.org/10.1021/nn3030223) - Gold nanoprisms as optoacoustic signal nanoamplifiers for in vivo bioimaging of gastrointestinal cancers. Small. 2013 Jan 14;9(1):68-74
Bao C, Beziere N, Del Pino P, Pelaz B, Estrada G, Tian F, Ntziachristos V, de la Fuente JM, Cui D
(See online at https://doi.org/10.1002/smll.201201779)
