#	Pagina
attuale pagina	/open-h2020/projects/195449/results.html

Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - AuNP-PhotoBioDrug (Functionalized AuNPs and their use for biological applications)

Teaser

Summary

The design of gold nanoparticles (AuNPs) and exploration of their physical and chemical properties is a topic of high interest due to their extensive use in biomedicine and new technologies. The biological function of AuNPs depends on their size, shape and surface charge and functionalisation, etc. Their reactivity in solution and within a biological media can be modified due to the interaction of biomolecules (proteins, etc.) to their surface. In connection to this, the reactivity of AuNPs in solution cannot be directly extrapolated to that of the intracellular media because after interaction in a living milieu the nanoparticle surface is instantaneously coated by proteins, generating the well-known protein corona (PC). This is a key process because PC can modify the biological reactivity of AuNPs and the function of proteins, generating in some cases adverse effects such as cytotoxicity. However, functionalisation of AuNPs with suitable ligands such as drugs, PEG, etc., may help to decrease or even eliminate the undesired side effects and to enhance the biological function, delivery and targeting of the nanomaterial. Thus, a better understanding on the parameters affecting the formation and stability of PC may clearly help in the design of new nanomaterials with enhanced biological function and safety. Moreover, it would be of potential interest to predict the photobehaviour of AuNPs before introducing them in a living cell.
The general aim of this project is the design and functionalisation of AuNPs with anti-inflammatory 2-arylpropionic acid drugs and study their photoreactivity in different environments. As the physical and chemical properties of either the nanoparticle or the drug may change after interaction, we have investigated how they vary according to different parameters such as NP-drug distance by using spacers of different length between the drug and the nanoparticle surface; besides, we have also designed NPs of different size and shape. Their photoreactivity have been investigated in different media: solvents, presence of proteins, etc. Spectroscopic techniques such as UV, steady-state and time-resolved fluorescence, and laser flash photolysis have been employed. Finally, intracellular studies using SK-Br3 human breast cancer cells as well as non-carcinogenic MCF-10A human breast cells have been performed with the aim of characterising the cytotoxicity of the nanomaterials.

Work performed

An important number of different drug-spacer conjugates have been synthesised using two different drugs and spacers of different lengths. Besides, as the employed drugs have a chiral centre, the different enantiomers have been used with the aim of investigating possible configuration effects. All new compounds have been fully characterised. On the other hand, spherical AuNPs of 4 and 15 nm size have been synthesised. Besides, gold nanorods (AuNRs) have been also designed. All nanomaterials have been functionalised with HS-PEG-COOH and with the different (S)- or (R)-drug-spacer derivatives. Their photoreactivity has been studied by means of UV, fluorescence and laser flash photolysis spectroscopies. The photoreactivity has been investigated in organic (acetonitrile and ethanol) and aqueous solutions and in the presence of transport proteins such as human and bovine serum albumins and alfa-acid glycoproteins from human and bovine plasma. Finally, we have been able to covalently attach HSA and HAG to the AuNP surface, and we have also investigated their photoreactivity.
In general, the absorption and emission spectra of all AuNP-PEG-drug derivatives is affected by the presence of proteins. These results can be attributed to the formation of the protein corona. Hypochromism and fluorescence enhancement were found to be slightly higher for the 15 nm conjugates in comparison to the 4 nm systems. Moreover, the nature of the drug and protein played a role in the photoreactivity of the NP/protein mixtures.
The fluorescence kinetics were also measured. In contrast to the steady state fluorescence enhancement, the decay traces as well as the anisotropy decays of the different protein/NP derivatives were coincident.
LFP experiments were performed in organic and aqueous solvents. In general, the triplet excited states of the drug-functionalised 4 nm were detected and characterised. Considerable differences in the triplet formation and lifetime evolution was observed. It is worth to note that the triplet yields also depends on the NP-drug distance.
Covalent binding of HSA and HAG to the different AuNP conjugates was successfully accomplished. Again, a complete UV, fluorescence (steady-state and time-resolved) and LFP characterisation was performed. Emission of the protein attached to the NP surface was clearly affected by the nature of the protein.
Finally, gold nanorods (AuNRs) were also synthesised. AuNRs coated with PEG and the different drug-spacer derivatives show a maximum absorption band around 808 nm. The importance of AuNRs lies in their use for photothermal therapy (PTT), because the temperature enhancement generated upon irradiation of their 808 nm band may induce the death of carcinogenic cells. Interestingly, it was observed that the temperature enhancement of the AuNR conjugates depends on their drug-spacer functionalization.
Biological experiments have been carried out with AuNPs and AuNRs functionalized with PEG and PEG-drug-spacer derivatives. Cytotoxic experiments have been performed with SK-Br3 human breast cancer cells and with MCF-10A human breast healthy cells. Cytotoxicity of drug-functionalized 4 nm AuNPs seems to be slightly higher for carcinogenic SK-BR3 compared to those of non-carcinogenic MCF-10A cells. Similar experiments have been done with functionalized AuNP-PEG-drug derivatives with HSA (or HAG) covalently bound to the PEG moiety, and with functionalized AuNRs.
It is worth to mention that all work packages and objectives proposed in the present project have been successfully accomplished.

Final results

The objectives proposed in the project have been successfully accomplished; an important number of new drug functionalised nanomaterials have been synthesised and characterised. Their photoreactivity have been investigated in different media by means of fluorescence and laser flash photolysis. Besides, experiments in more complex media such as in the presence of proteins have also been performed. Finally, we have also performed biological experiments to investigate the behaviour of the nanoparticles within SK-BR3 human breast cancer cells and MCF-10A human breast healthy cells. All these experiments have never been performed before. We believe that the results can be published in high impact journals and they will be exposed in international conferences.
On the other hand, during the two years duration of this project, Dr. VayÃ¡ has maintained a close collaboration with different distinguished international research groups, and he has published four papers in high impact journals. Besides, Dr. VayÃ¡ has participated as speaker in two different international conferences.
Finally, it is worth to mention that this MSC-IF project has clearly aided Dr. VayÃ¡ to be awarded with the â€œRamÃ³n y Cajalâ€ contract (Spanish Ministry of Science and Technology). This is a five years contract within the Spanish research system and constitutes the natural follow up step to finally get a permanent position at the University.