Synthesis of novel hybrid nanosystems composed by core-shell SiC/SiOx nanowires conjugated with porphyrins for X-ray-excited PDT

Abstract

My PhD thesis dealt with the preparation and characterization of hybrid nanosystems for possible application in nanomedicine, in particular for the treatment of deep solid tumours by X-Ray activated PhotoDynamic Therapy, according to the “self lighting photodynamic therapy” recently proposed in the literature. The activity has been performed in the framework of the BioNiMED Project funded by the CARIPARMA foundation and coordinated by IMEM-CNR (Parma). The project aims to develop a nanosystem capable of causing oxidative stress through the production of singlet oxygen, a cytotoxic species, when exposed to a source of X-Ray. During my PhD work, I functionalized cubic SiC/SiOx nanowires, grown at IMEM (CNR), and prepared nanosystems consisting of “core shell” cubic SiC@SiOx nanowires conjugated with porphyrins. SiC@SiOx nanowires work as scintillators: indeed, when they are irradiated with X-Ray, they emit light in the wavelength range corresponding to the porphyrin absorption. This energy transfer excites the porphyrin, which in turns produces singlet oxygen. Indeed, porphyrins are a wide class of photosensitizers, largely used in conventional PDT. Cubic SiC is known to be a biocompatible material, employed in biomedical field, and the biocompatibility of the SiC/SiOx nanowires was also assessed and previously published. In a previous PhD work developed in our laboratory the tetra-(4- carboxyphenyl)porphyrin (H2TCPP ) was covalently linked to the SiC/SiOx NWs by a ‘click’ reaction, giving a novel nanosystem able to promote X-ray- excited PDT, as evidenced by in vitro studies. During my thesis work, I conjugated the selected porphyrin to the nanowires by the formation of the covalent amide bond and introduced different ending chains in the porphyrin moiety in order to increase the dispersion of the nanosystem in aqueous medium. The formation of the amide bonds required the previously functionalization of the nanowire surface with amino groups reacting the silica hydroxyl groups with APTES (aminopropyltriethoxysilane). To bind the porphyrin to the nanowire surface, the carboxylic groups of H2TCPP porphyrin were previously activated with typical condensation agents (EDC, HOBt, and DMAP) and then reacted with the amino groups to give the amide bond formation. This conjugation approach resulted to give a higher degree of porphyrin loading, as evidenced by fluorescence spectra, and occurred under very mild conditions (r.t. vs. high temperature used in the thermal click reaction). In addition, it was possible to bind polar chains to the conjugated porphyrin. It was planned to introduce short PEG chains to modulate the polarity of the nanosystem. In particular, two different ending NH2-PEG chains were introduced, PEG6-CH2COOH and –PEG8-OH. In the first case, I prepared NH2CH2CH2(OCH2CH2)5OCH2CO2H by a multistep synthesis starting from hexaethylene glycol. Then this chain was bound to the residual activated carboxylic groups of the porphyrin conjugated to the nanowires. Finally, deprotection with trifluoroacetic acid gave free acid carboxylic functions at the end of the PEG- chains. The nanosystem was characterized by fluorescence spectroscopy that confirmed that functionalization occurred successfully. Last, the nanowires were detached from the support using an ultrasound microtip. In vitro experiments (clonogenic tests) performed on the adenocarcinoma human alveolar basal epithelial (A549) cell line evidenced the ability to significantly reduce the survival fraction with respect to simple radiotherapy. To prepare the second chain, NH2-PEG8-OH, without the ionizable acid group, I started from tetraethylene glycol by a multistep synthesis. The residual activated carboxylic groups of the H2TCPP porphyrin conjugated to the nanowires were reacted with this chain giving a less polar nanosystem. The activity of the nanowires, after detachment from the support, was tested by in vitro experiment on A549 tumoural cell line. The lower activity observed could be attributed to lower internalization due to the formation of boundles in the biological medium. To increase the cytotoxic activity, a further aim was to obtain a thicker porphyrin coating. Thus, it was planned to link a second different porphyrin on the conjugated H2TCPP porphyrin. In particular, tetra(4- aminophenyl)porphyrin could be successfully reacted and in vitro experiments are in progress. To face the open problem of the number of porphyrin arms involved in the conjugation, we planned to apply XPS spectroscopy to study the nanosystem conveniently modified by the presence of bromine atom. To check the possibility of evaluating the C=O/Br ratio in XPS spectra, I synthesized the bromophenyl tetra-derivative of H2TCPP porphyrin as reference compound. Then H2TCPP and the bromine derivative porphyrins were deposited on Pt-metalized wafer by drop casting. XPS spectra will give us information on the utility of this approach. Last, to evaluate the porphyrin loading, I synthesized the Cu-TCPP porphyrin, a metal derivative stable enough to be conjugated to the nanowires. Indeed, the complete removal of copper from the porphyrin requires the treatment with conc. sulfuric acid. The determination of Cu amount by atomic absorption was performed. To explore a different type of linker to anchor a porphyrin to a solid support, I successfully synthesized a metal tetra-phosphonated porphyrin. In particular, starting from the tetra(4-hydroxyphenyl)porphyrin (H2THPP) it was possible to obtain the Zn-THPP functionalized with four phosphonic acid chain ( –CH2)6PO3H), compound that is not reported in the literature. This type of porphyrins is of great interest for both energy transfer process and electron transfer process. The anchorage of this porphyrin on a flat silicon support will be useful to study the porphyrin position on the surface, parallel or not, by X-ray-excited optical luminescence (XEOL).