Gold nanorods (NR) and asymmetric nanoparticles (ANP) capped with a biocompatible polymer bearing binding groups for molecules and metal cations: pharmacological and thermal antimicrobial action activated by near-IR irradiation Nanorods (NR) e nanoparticelle asimmnetriche (NPA) d'oro ricoperte di polimero biocompatibile con funzioni leganti per molecole e ioni metallici: aziona antimicrobica farmacologica e termica attivata da irraggiamento nel vicino IR.

The project will be realized in the follorwing steps: i) We will prepare gold NR and ANP with convenient shape, capable of absorbing in the NIR interval; ii) Gold NR e ANP must be stabilized against in-vivo aggregation and must be biocompatible. To this aim we will cover them with multifunctional poly(hydroxyethylaspartamide) (PHEA) polymers. These polymers, known for their bicompatibility, can be prepared with a versatile functionalization/purification step-by-step procedure, to obtain PHEA backbones bearing a tunable number of different functions. We plan to append polyethyleneglycol (PEG) chains, n-alkyl thiols and ligands for Mn+ and/or receptors for antibiotics. The –SH terminated alkyl chains will serve as anchoring point to gold nano-objects, the long hydrophilic PEG polymers will impart solubility and bicompatibility, the binding functions will allow the upload of antimicrobial metal cations and molecules. We plan to obtain a hyerarchical disposition: a core made of gold nanoobjects, an internal hydrophobic layer made by n-alkyl chains on the PHEA backbone, where also binding units are positioned, and an external hydrophilic PEG layer; iii) The binding functions that we will append to PHEA will be mainly chelating polyamines, aza-crown ethers, pyridines or combinations of the three. These ligands exothermalally interact with Ag+ and Cu2+, and the decrease of stability with increasing T (ΔT) will switch on their release. We will bind in the polymer layer antiobiotics such as Ampicillin, Vancomycin, Cyprofloxacin (bearing –COOH groups), or Gentamycin, Kanamycin, Neomycin (bearing several –NH2 groups), by appending to PHEA coordinatively unsaturated and kinetically inert metal complexes (like Cu2+ or Zn2+ complexes with small tetraazamacrocycles), that offer one or more free sites on the metal for carboxylate and amine coordination. Moreover, we will exploit less specific multipoint interactions for antibiotics, such as H-bond and electrostatic carboxylate/protonated amine interactions, and hydrophobic interactions (for the less hydrophilic antibiotics, interacting with the hydrophobic layer around the NR and ANP); iv) The ΔT by photoirradiation will be induced using NIR laser. v) We will measure the uptake and ΔT-induced release of Ag+, Cu2+ and antibiotics by means of an arsenal of separation, analytical, spectroscopic, imaging, and optical techniques, merging the know-hows of the chemical, physical-optical and biophysical components of the research team; vi) At every stage of the research, we will check the biocompatibility of our products with cell viability and cyotoxicity tests, and their anti-bacterial activity with in-vitro tests on several bacterial strains, including MDR. The rationale is that without light irradiation the studied nano-objects are ineffective against bacteria and not cytotoxic. Uncovered NR and ANP, the same covered with simple polymer (no binding functions), the same covered with polymers bearing binding functions and finally charged with Mn+ and antibiotics will be checked with and without light irradiation. We want to understand the separate contribution of heat, metal cations and antibiotics to bactericidal effect, as we plan to fine tuning the combination of the three to obtain maximum efficiency. We will also study the internalization and diffusion in the cell of NR and ANP by means of by means of fluorescence imaging and dynamic spectroscopy (including Fluorescence Recovery after Photobleaching, FRAP), and correlate the chemical features of the nano-objects, their shape and their antibacterial efficiency with their ability to be internalized.