Effect of TAT-DOX-PEG irradiated gold nanoparticles conjugates on human osteosarcoma cells.

The paper aims to investigate the cytotoxic effect on tumor cells of irradiated AuNPs in green light and subsequently functionalized with HS-PEG-NH2. The toxicity level of gold conjugates after their functionalization with DOX and TAT peptide was also evaluated. The AuNPs were prepared using the modified Turkevich method and exposed to visible light at a wavelength of 520 nm prior their PEGylation. The optical properties were analyzed by UV-vis spectroscopy, the surface modification was investigated using FTIR and XPS spectroscopies and their sizes and morphologies were evaluated by TEM and DLS techniques. DOX and TAT peptide were linked to the surface of PEGylated AuNPs by reacting their amino groups with glycidyloxypropyl of PEGylated DOX or TAT conjugates under mild conditions at room temperature and in the presence of ethanol as catalyst. The conjugates containing DOX or DOX and TAT have been characterized by fluorescence and FTIR techniques. The changes of electrochemical features were observed using cyclic voltammetry, suggesting a better stability of irradiated nanoparticles. By mass spectrometry it was confirmed that the compounds of interest were obtained. The cell viability test showed that irradiated and non-irradiated nanoparticles coated with PEG are not toxic in normal cells. Tumor cell viability analysis showed that the PEGylated nanoparticles modified with DOX and TAT peptide were more effective than pristine DOX, indicating cytotoxicity up to 10% higher than non-irradiated ones.

Scheme S1. The reaction scheme between DOX and PEG500 via oxirane opening ring, in aqueous media, at room temperature, for 72 h Scheme S2. The reaction scheme between cys-TAT and PEG500 via oxirane opening ring, in aqueous media, at room temperature, for 72 h II. Ultraviolet-visible spectroscopy (UV-Vis) Experimental UV-Vis absorption spectra were recorded using a Shimadzu Spec Pharma 1800 instrument. The samples were 1/10 diluted to the initial concentration of the particle suspensions before recording the spectra. The resulting concentrations of the particle samples were calculated using Lambert-Beer law, based on the mean diameter provided by TEM imaging data 1 .
Results and discussion 1. Structure characterization of AuNPs, iAUNPs, AuPEG2000-NH2 and iAuPEG2000-NH2 nanoparticles by UV-Vis spectroscopy UV-Vis spectroscopy is well-known to assess the gold nanoparticle characteristics, principally related to size and concentration, in close connection with imaging data 2,3 . Exposing the samples in the wavelength range between 300 and 700 nm, the corresponding spectra were obtained, providing the absorption band of each compound as shown in Figure S1. One can observe that the intensity of the absorption band intensity (SPR) has increased in irradiated sample iAuNPs as compared to non-irradiated one, suggesting the continuation of nucleation process after light absorption 4 . The red shift in the SPR absorbance of the nanoparticles with polymer was largely determined by a slight change in the refractive index of the local environment of the AuNPs, indicating an increase in nanoparticle size 5 . At the same time, a slight increase in the SPR bandwidth was explained by the amplification of local plasmonic field, which may be due to the dimerization tendency of irradiated nanoparticles 6 , as confirmed by TEM images. The UV-vis data shows that the SPR intensity of nanoparticles has changed after polymer coating. The red shift in the SPR absorbance of the PEGylated nanoparticles was largely determined by a slight change in the refractive index of the local environment of the iAuPEG2000-NH2, indicating an increase in nanoparticle size 5 . According to Figure S1, the spectrum of iAuPEG2000-NH2 suspension reveals a maximum intensity of the SPR band at 528 nm, while those of AuPEG2000-NH2 at 536 nm. The red shifted absorption bands of the PEGylated nanoparticle spectra are evidence regarding the successful grafting of the polymer onto the gold nanoparticle surface, either native or irradiated. Figure S1. UV-Vis spectra of AuNPs, iAUNPs, AuPEG2000-NH2 and iAuPEG2000-NH2 nanoparticles displaying the influence of irradiation on spectral features. The spectrum of iAuPEG2000-NH2 is less shifted and shows an absorption band of higher intensity than in AuPEG2000-NH2.

iAuPEG2000-NH2using a calibration curve in UV-Vis.
After purification by centrifugation, the excess of unbound ligand and displaced citrate were removed along with a percentage of gold particles that should be quantified. The calibration curves were obtained for each product, by measuring the absorbance at 536 and 528 nm for AuPEG2000-NH2 and iAuPEG2000-NH2, having different, known concentrations (see Table S1 and S2).
The absorbance values of purified nanoparticles were found of 0.838 and 1.421 a.u. for AuPEG2000-NH2 and iAuPEG2000-NH2, respectively. Using the corresponding equations of the calibration curves (see Fig. S2 and S3), one can assess that the concentrations of the aforementioned products are around 0.2 mM in both cases, indicating a percentage of 80% recovered nanoparticles after polymer coating.  Figure S2. The absorbance calibration curve of AuPEG2000-NH2 based on the data from Table S1.

Supplementary figures and notes
To determine the mean diameter and dimensional distribution of the synthesized nanoscale entities, a series of microscopic images were analysed, measuring about 1000 particles per product. Figure   S6 illustrates TEM images of PEGylated nanoparticles (irradiated and non-irradiated), along with the starting products (AuNPs and iAuNPs), using a scale of 100 nm scale to observe the morphological details, while in the main manuscript there were presented images at a scale of 200 nm to observe the agglomeration tendency, when applicable.
The Table S3 correlates the particles size and morphology data obtained by different techniques: UV-Vis spectroscopy, TEM, DLS/ELS.  The polymer coating (e) has a slight influence on the particle size, but showing a more significant effect on aggregation behaviour. The irradiated PEGylated nanoparticles exhibit a phenomenon of uniform clustering, with the formation of entities with dimensions up to 100 nm.

Supplementary results and discussions
The variation of FTIR spectra for the intermediate products AuNPs, iAuNPs, AuPEG2000-NH2 and iAuPEG2000-NH2 are presented in Figure S7, beside sodium citrate and HS-PEG2000-NH2.
Usually, the absorption of citrate molecules by specific coordination of carboxylate is dominant, but a number of other citrate molecules are subjected to intermolecular interactions with adsorbed species, which are not in contact with metal surface 7 Table S4 summarizes all FTIR data obtained and peak assignment. Figure S7. FTIR spectra of AuNPs, iAuNPs, AuPEG2000-NH2 and iAuPEG2000-NH2 samples besides sodium citrate and PEG2000-NH2 Firstly, the mass concentration of the final products has been determined. The total amount of