αvβ3-Specific Gold Nanoparticles for Fluorescence Imaging of Tumor Angiogenesis

This paper reports on the development of tumor-specific gold nanoparticles (AuNPs) as theranostic tools intended for target accumulation and the detection of tumor angiogenesis via optical imaging (OI) before therapy is performed, being initiated via an external X-ray irradiation source. The AuNPs were decorated with a near-infrared dye, and RGD peptides as the tumor targeting vector for αvβ3-integrin, which is overexpressed in tissue with high tumor angiogenesis. The AuNPs were evaluated in an optical imaging setting in vitro and in vivo exhibiting favorable diagnostic properties with regards to tumor cell accumulation, biodistribution, and clearance. Furthermore, the therapeutic properties of the AuNPs were evaluated in vitro on pUC19 DNA and on A431 cells concerning acute and long-term toxicity, indicating that these AuNPs could be useful as radiosensitizers in therapeutic concepts in the future.


Introduction
In recent years, gold nanoparticles (AuNPs) have gained serious attention since their first use as radioactive 198 Au-nanocolloid in the early 1950s for nanobrachytherapy [1][2][3]. Since then, the focus has shifted to the development of ultra-small target-specific AuNPs with a very narrow size distribution and, ultimately, tailored shapes for use in various imaging modalities such as CT [4], Raman [5], or photoacoustic imaging [6]. On the one hand AuNPs represent a perfect platform for multimerization of target-specific effectors on their surface and on the other hand they offer the possibility of detection using multimodal imaging techniques by surface modification [7], as well as for theranostic purposes [8][9][10][11]. Many approaches of AuNPs with a size of >10 nm are based on a phenomenon typically known as 'enhanced permeability and retention' (EPR) effect due to passive extravasation of nanoparticles across the perforated vasculature of tumors [12]. Rapid renal clearance is preferable for radioactive diagnostic nanoparticles to avoid a high radiation burden on healthy organs and tissues, which can be achieved for AuNPs smaller than 6 nm in diameter [13]. The development of methods for the synthesis of ultrasmall (<5 nm) AuNPs [14] followed by surface-modification for enhanced stability and homogenization [15][16][17] paved was used for the fusion of the images. All injections and measurements with mice were performed under anesthesia (2-3% isoflurane/O2, 2-3 mL/min).

Synthesis and Functionalization of Gold Nanoparticles
Integrin αvβ3, a transmembrane protein expressed on endothelial cells, and binds the Arg-Gly-Asp (RGD) triple peptide motif of extracellular matrix proteins. Growing malignant tumors continuously requires angiogenesis, and for this purpose the integrin αvβ3 is overexpressed. As a result, αvβ3 is preferentially expressed in tumor angiogenesis and is a potential target for AuNPs decorated with RGD peptides [47]. Therefore, ultra-small AuNPs (3 ± 2 nm) were synthesized by the Brust-Schiffrin-method [15] with thiol-PEG3-OH used as the stabilizing ligand and to achieve an increased biocompatibility. The AuNPs were further functionalized via ligand exchange with furan-protected maleimide-PEG4-thiol ( Figure 1). Afterwards, the furan-maleimide-AuNPs 2a were deprotected at 95 °C in DMSO for 2 h (2b), followed by the attachement with the thiol-functionalized αvβ3specific ligand thiol-c(RGDfK) 6 ( Figure A1) and the thiol-functionalized fluorescent dye 3 ( Figure A2) [43,51]. For the use in OI, the AuNPs were functionalized with 3, which was performed at a ratio of 40:1 (AuNPs:dye). Using more NIR-dye resulted in the aggregation of the AuNPs as well as decreased fluorescence signals due to self-quenching. The purification of the AuNPs was performed via dialysis and size-exclusion chromatography. The AuNPs 7 and 8 were incubated with PBS, rat plasma, and cell media for at least 24 h at 37 °C, and no aggregation was found. Their stability was confirmed by UV/Vis spectroscopy and HPLC. The AuNP-RGD-NIR-dye 8 exhibited very low photobleaching (<3% within 24 h at 37 °C) and enabled biocompatible fluorescence in the optical window of tissue, with absorption of 600-800 nm (AbsMax = 750 nm) and emission at 750-820 nm (EmMax = 780 nm) ( Figure A13). The loss of mass of the AuNPs was determined by thermogravimetric analyses for each functionalization step. The measurements were compared between the product and educt to determine the additional loading of the respective functionalization step. Therefore, the difference in the loss of mass of product and educt gave the total mass of newly The loss of mass of the AuNPs was determined by thermogravimetric analyses for each functionalization step. The measurements were compared between the product and educt to determine the additional loading of the respective functionalization step. Therefore, the difference in the loss of mass of product and educt gave the total mass of newly attached molecules. The mean number of newly attached molecules could be estimated by dividing the mass difference by the molar mass of the respective molecule. After knowing the number of the newly attached molecules, a formula from Zhu et al. was used to calculate the total molar mass of the AuNPs [42] (Table 1). A brief description of the synthesis and characterization can be found in the appendix. All AuNPs were fully characterized by  (Table 1), dynamic light scattering (DLS) ( Table 2), and NMR ( Figures A12-A15). The dynamic light scattering was performed on a Malvern Zetasizer ZS90. AuNPs were dissolved in distilled H 2 O at a concentration of 1 µg/mL. All values are the mean value of at least three different measurements. If the polydispersity index (PDI) is >0.5 the size by number is more relevant than the size by volume. The size by number is best comparable to the size measured by EM. The AuNPs could be stored in lyophilized form for >12 months at −20 • C without losing their integrity. In contrast, if stored in solution at room temperature, aggregation in form of precipitation occurred within weeks, especially for peptide-or NIR-decorated particles [33]. AuNP-RGD-NIR-dye~50 RGD, 10 NIR-dye 413 6.20

Cell Experiments
Several different IC 50 values for RGD derivatives are already available in literature, ranging from 0.1 nM up to 6.7 µM. The main reason for the observed differences is the assay method used to determine the IC50 values. IC 50 values of 0.1-1 nM can be found for RGD peptides having been determined by ELISA assays [45] and IC 50 values around 20 nM were reported for solid-phase α v β 3 binding assays for monomeric RGD derivatives [44]. Those IC 50 values were derived by non-living experiments. Cell experiments are closer to in vivo condition. Therefore, for the AuNPs 7 and 8, the α v β 3 -avidities were determined by competitive displacement experiments on α v β 3 -expressing A431 cells using 125 I-Echistatin as α v β 3 -specific radioligand and competitor ( Figure 2a). As internal reference, the RGD monomer c(RGDfK) was evaluated. With the evaluation of RGD derivatives by displacement experiments, IC 50 values comparable with those in existing literature were found [48]. For c(RGDfK) an IC 50 value of (1.75 ± 0.84) µM was found. The multi-RGD decoration at the surface of AuNP 7 led to a lower IC 50 value of (1.07 ± 0.74) µM compared to the isolated RGD monomer. Further functionalization of the AuNPs with cyanine dye 3 led to a slightly higher IC 50 value of (3.37 ± 0.73) µM for AuNP 8.
Next, the cellular uptake of AuNPs 8 was evaluated on A431 cells at different timepoints from 1-24 h. An at least partly receptor-specific uptake was found for the AuNPs (Figure 2b), as the cellular uptake of the dually modified particles could be partly blocked by pre-incubation using a 10-fold excess of c(RGDfK) 1 h before incubation with AuNPs.
Furthermore, the AuNPs 8 were tested for their behavior in vitro on A431 cells via fluorescence microscopy. First, the concentration for optimal microscopy was evaluated in a concentration range between 10-100 µmol/mL after an incubation time of 24 h. These initial cell studies revealed an optimized concentration of 50 µg/mL of AuNPs 8 for cell imaging. Next, via confocal fluorescence microscopy, the cell internalization, and the fate of the AuNPs were observed at different time points from 1-24 h ( Figure A16). The confocal microscopy images revealed several findings: The number of observed fluorescence foci in the cells rises within the observation time from 1-24 h indicating an accumulation of AuNPs within the cell plasma. Further, these foci become bigger after 1 h of incubation. The AuNPs accumulated most probably in vesicles within the cells. At later time points, the vesicles appeared to become smaller containing less particles, whereas several new and much smaller foci showed up outside the cells. This is perhaps explained by the formation (and their later excretion) of vesicles containing the AuNPs. The conclusion that the mentioned dots represent vesicles is confirmed by the fact that the structures can be stained with membrane Orange, but not with the nuclei marker Sytox Green. This means that the vesicles should consist of excreted cell plasma compartments, underlining that the AuNPs are not accumulating within the cell nuclei. Moreover, the vesicles within in the cells showed a higher fluorescence signal for both membrane Orange and NIR dye. Next, the cellular uptake of AuNPs 8 was evaluated on A431 cells at different timepoints from 1-24 h. An at least partly receptor-specific uptake was found for the AuNPs (Figure 2b), as the cellular uptake of the dually modified particles could be partly blocked by pre-incubation using a 10-fold excess of c(RGDfK) 1 h before incubation with AuNPs.
Furthermore, the AuNPs 8 were tested for their behavior in vitro on A431 cells via fluorescence microscopy. First, the concentration for optimal microscopy was evaluated in a concentration range between 10-100 μmol/mL after an incubation time of 24 h. These initial cell studies revealed an optimized concentration of 50 μg/mL of AuNPs 8 for cell imaging. Next, via confocal fluorescence microscopy, the cell internalization, and the fate of the AuNPs were observed at different time points from 1-24 h ( Figure A16). The confocal microscopy images revealed several findings: The number of observed fluorescence foci in the cells rises within the observation time from 1-24 h indicating an accumulation of AuNPs within the cell plasma. Further, these foci become bigger after 1 h of incubation. The AuNPs accumulated most probably in vesicles within the cells. At later time points, the vesicles appeared to become smaller containing less particles, whereas several new and much smaller foci showed up outside the cells. This is perhaps explained by the formation (and their later excretion) of vesicles containing the AuNPs. The conclusion that the mentioned dots represent vesicles is confirmed by the fact that the structures can be stained with membrane Orange, but not with the nuclei marker Sytox Green. This means that the vesicles should consist of excreted cell plasma compartments, underlining that the AuNPs are not accumulating within the cell nuclei. Moreover, the vesicles within in the cells showed a higher fluorescence signal for both membrane Orange and NIR dye.

Radiosensitizing Experiments
The developed AuNPs are useful for diagnosis but could also serve as therapeutic agents in form of radioactive [ 198 Au]AuNPs or as radiosensitizer for X-ray irradiation. In this work, their ability as radiosensitizer was evaluated by irradiation of AuNP-incubated DNA and subsequent gel electrophoresis. AuNPs are effective secondary electron emitters when irradiated with X-rays due to their high photoelectric absorption [36] and their application as radiosensitizers in nuclear medicine is lively discussed [37,52,53]. If they are exaggerated by an external radiation source, Auger-Meitner electrons (AMEs) are

Radiosensitizing Experiments
The developed AuNPs are useful for diagnosis but could also serve as therapeutic agents in form of radioactive [ 198 Au]AuNPs or as radiosensitizer for X-ray irradiation. In this work, their ability as radiosensitizer was evaluated by irradiation of AuNP-incubated DNA and subsequent gel electrophoresis. AuNPs are effective secondary electron emitters when irradiated with X-rays due to their high photoelectric absorption [36] and their application as radiosensitizers in nuclear medicine is lively discussed [37,52,53]. If they are exaggerated by an external radiation source, Auger-Meitner electrons (AMEs) are emitted. This property was tested at the most prominent cell damage: double strand breaks (DSBs) of DNA. The induced radiation damage to pUC19 plasmid DNA was investigated as a function of dose and concentration of AuNPs. Indirectly induced single strand breaks (SSBs) were confirmed by using DMSO. DMSO can capture OH-radicals to form methanesulfinic acid (MSA) and is therefore a very prominent radical scavenger [54]. The concentration of DMSO is high enough to reach a scavenging effect for several weeks.
Two different AuNP derivatives, non-targeted AuNP-PEG 1 and targeted AuNP-RGD 7, were tested for their radiosensitizing properties. The pUC19 plasmid DNA (280 kDa, 10 ng/µL per sample, BioLabs, New England) served as a biological model. A semiquantitative analysis was performed to prove the therapeutic efficiency of the AuNPs. To distinguish between DNA damage caused directly (e.g., by AE) and indirectly (especially by OH-radicals), all experiments were repeated in the presence of DMSO (2 M, applied as radical scavenger). DNA damage was quantified by agarose gel electrophoresis and compared with 1 Kb Plus DNA ladder protein (Thermo Fisher) and linear plasmid derived from pUC19 by BamH1-kit. Three different conformation states were evaluated: Supercoiled (native form, sc), open circular (according to SSBs, oc), and linear (after DSBs, lin) ( Figure 2). After irradiation, 10 µL samples were mixed with 1.25 µL 10 × BlueJuice gel loading buffer for tracking of DNA migration. After gel electrophoresis (2 h at 120 V, 400 mA, 120 W) the gels were stained with ethidium bromide and the relative fluorescence intensities of the fractions were calculated (BioRad Fluorescence Analyzer) ( Figures A17 and A18).
In Figure 3, SSBs without AuNPs are found in form of oc-DNA of 17-40% between 25-100 Gy X-ray irradiation (Maxishot Y.TU, Yxlon). DNA incubated with AuNP 1 (1.5 µg/µL) showed oc-DNA of 31-46% between 25-100 Gy X-ray irradiation, indicating a 14% higher SSB induction at a dose of 25 Gy and 6% more SSBs at a dose of 100 Gy. At lower doses, the higher radiosensitizing effect of AuNP 1 was partially quenchable with DMSO to <10% oc-DNA. The radiosensitizing effect was highest at 25 Gy. DSBs were induced in every experiment to the same extent (3 ± 2%) and were not quenchable by DMSO. Therefore, no direct DNA damage was observable, but a higher indirect DNA damage at AuNP-incubated DNA was found. radical scavenger). DNA damage was quantified by agarose gel electrophoresis and compared with 1 Kb Plus DNA ladder protein (Thermo Fisher) and linear plasmid derived from pUC19 by BamH1-kit. Three different conformation states were evaluated: Supercoiled (native form, sc), open circular (according to SSBs, oc), and linear (after DSBs, lin) ( Figure 2). After irradiation, 10 μL samples were mixed with 1.25 μL 10 × BlueJuice gel loading buffer for tracking of DNA migration. After gel electrophoresis (2 h at 120 V, 400 mA, 120 W) the gels were stained with ethidium bromide and the relative fluorescence intensities of the fractions were calculated (BioRad Fluorescence Analyzer) (Figures A17 and A18).
In Figure 3, SSBs without AuNPs are found in form of oc-DNA of 17-40% between 25-100 Gy X-ray irradiation (Maxishot Y.TU, Yxlon). DNA incubated with AuNP 1 (1.5 μg/μL) showed oc-DNA of 31-46% between 25-100 Gy X-ray irradiation, indicating a 14% higher SSB induction at a dose of 25 Gy and 6% more SSBs at a dose of 100 Gy. At lower doses, the higher radiosensitizing effect of AuNP 1 was partially quenchable with DMSO to <10% oc-DNA. The radiosensitizing effect was highest at 25 Gy. DSBs were induced in every experiment to the same extent (3 ± 2%) and were not quenchable by DMSO. Therefore, no direct DNA damage was observable, but a higher indirect DNA damage at AuNPincubated DNA was found. Next, the radiosensitizing effect at the same dose but different AuNP concentrations was evaluated ( Figure 4). Further, we wanted to test whether it is possible to induce more DSBs by more production of AMEs, when the radiation source is in the direct vicinity of the AuNPs. For this experiment, Re-188-solution as inducer for radiosensitizing effects was chosen with its characteristic 2.12 MeV βemission and 155 keV γ-coemission. The highest effect for radiosensitizing was determined for 27.5 Gy as incubation dose. To reach this dose, 0.5 MBq Re-188 within 18 h incubation time in a 50 μL volume in a 1.5 mL Eppendorf vial were calculated using Formula 1. AuNP concentrations from 0.1-12.0 μg/μL were tested. Interestingly, an optimal radiosensitizing effect for AuNP-concentrations between 1.0-1.5 μg/μL was found. At these AuNP-concentrations the direct DNA damage in form of DSBs was 4.8-11.7%. This effect was completely quenchable by 2 M DMSO for Re-188 alone, meaning that there are only nascenting OH-radicals responsible for the DNA damage. In contrast, by the combination of Re-188 with AuNPs 1, this effect was only partially quenchable by 2 M DMSO, meaning that additional AMEs from the AuNPs Next, the radiosensitizing effect at the same dose but different AuNP concentrations was evaluated ( Figure 4). Further, we wanted to test whether it is possible to induce more DSBs by more production of AMEs, when the radiation source is in the direct vicinity of the AuNPs. For this experiment, Re-188-solution as inducer for radiosensitizing effects was chosen with its characteristic 2.12 MeV βemission and 155 keV γ-coemission. The highest effect for radiosensitizing was determined for 27.5 Gy as incubation dose. To reach this dose, 0.5 MBq Re-188 within 18 h incubation time in a 50 µL volume in a 1.5 mL Eppendorf vial were calculated using Formula 1. AuNP concentrations from 0.1-12.0 µg/µL were tested. Interestingly, an optimal radiosensitizing effect for AuNP-concentrations between 1.0-1.5 µg/µL was found. At these AuNP-concentrations the direct DNA damage in form of DSBs was 4.8-11.7%. This effect was completely quenchable by 2 M DMSO for Re-188 alone, meaning that there are only nascenting OH-radicals responsible for the DNA damage. In contrast, by the combination of Re-188 with AuNPs 1, this effect was only partially quenchable by 2 M DMSO, meaning that additional AMEs from the AuNPs have a direct impact to the DNA damage ( Figure A18). From these experiments it can be concluded that Re-188 in combination with AuNPs have a higher radiosensitizing effect than X-rays in combination with AuNPs. This effect has to be studied further.
Formula (1) Calculation of ground dose in a 6-well-plate or Eppendorf vial for Re-188 by Geant4-simulation [55]. D: energy dose, S: S-value, A: activity, T 1/2 : half-live of radionuclide, T: irradiation time. have a direct impact to the DNA damage ( Figure A18). From these experiments it can be concluded that Re-188 in combination with AuNPs have a higher radiosensitizing effect than X-rays in combination with AuNPs. This effect has to be studied further.

Colony Formation Assay (CFA)
The radiosensitizing effect found for the DNA experiments was repeated in vitro by irradiation of AuNP-incubated and non-incubated A431 cells and subsequent CFA [52,53]. AuNPs within cell plasma are discussed to damage certain plasma compartments by additionally produced Auger-Meitner-electrons during X-ray irradiation [56]. This hypothesis was proven in a pilot experiment. First, AuNPs 1 were tested for cell toxicity with different concentrations on A431 cells by CFA. No cell toxicity was found up to a concentration of 1 mg/mL (2.9 μM) ( Figure A21) comparable with the literature data [57][58][59]. Therefore, A431 cells were incubated with and without AuNPs 1 and 7 24 h before irradiation. After incubation, the cells were washed and subsequently treated with doses of 0-10 Gy by X-ray irradiation. Afterwards, the cells were seeded for CFA. Indeed, a low difference in survival fractions for AuNP-incubated and non-incubated cells was found for a dose of 2 Gy ( Figure 5). A slightly lower survival fraction for AuNP-incubated cells was found at 4 Gy for AuNP-RGD 7 (8.0 ± 1.5)% but not for AuNP-PEG 1 (15.0 ± 2.6)% compared to cells in absence of AuNPs (14.9 ± 2.1)%. A clearly much lower survival fraction at a dose of 6 Gy was found for AuNPs 7 (1.9 ± 0.7)% and AuNP 1 (2.9 ± 0.7)% compared to cells in absence of AuNPs (7.2 ± 0.8)% ( Figures 5 and A22), indicating a radiosensitizing effect. Further, at a dose of 8 Gy, no colony formation was found for AuNP-incubated cells. Furthermore, at a dose of 10 Gy, no colony formation was found for cells in absence of AuNPs. Interestingly, the specific AuNPs 7 showed a stronger effect at a dose of 4 and 6 Gy compared to non-specific AuNPs 1. At a dose of 2 Gy, the cells were also incubated with 0.2 M DMSO as radical scavenger during irradiation. The factor 10 less DMSO concentration is important for cell life. These fractions showed lower cell damage for AuNP 7 (59.3 ± 4.3)% and much lower cell damage for AuNP 1 (78.9 ± 6.8). Non-incubated cells showed cell survival of (98.9 ± 5.2)% with 0.2 M DMSO at a dose of 0 Gy, indicating no toxic effects at these conditions.

Colony Formation Assay (CFA)
The radiosensitizing effect found for the DNA experiments was repeated in vitro by irradiation of AuNP-incubated and non-incubated A431 cells and subsequent CFA [52,53]. AuNPs within cell plasma are discussed to damage certain plasma compartments by additionally produced Auger-Meitner-electrons during X-ray irradiation [56]. This hypothesis was proven in a pilot experiment. First, AuNPs 1 were tested for cell toxicity with different concentrations on A431 cells by CFA. No cell toxicity was found up to a concentration of 1 mg/mL (2.9 µM) ( Figure A21) comparable with the literature data [57][58][59]. Therefore, A431 cells were incubated with and without AuNPs 1 and 7 24 h before irradiation. After incubation, the cells were washed and subsequently treated with doses of 0-10 Gy by X-ray irradiation. Afterwards, the cells were seeded for CFA. Indeed, a low difference in survival fractions for AuNP-incubated and non-incubated cells was found for a dose of 2 Gy ( Figure 5). A slightly lower survival fraction for AuNP-incubated cells was found at 4 Gy for AuNP-RGD 7 (8.0 ± 1.5)% but not for AuNP-PEG 1 (15.0 ± 2.6)% compared to cells in absence of AuNPs (14.9 ± 2.1)%. A clearly much lower survival fraction at a dose of 6 Gy was found for AuNPs 7 (1.9 ± 0.7)% and AuNP 1 (2.9 ± 0.7)% compared to cells in absence of AuNPs (7.2 ± 0.8)% (Figures 5 and A22), indicating a radiosensitizing effect. Further, at a dose of 8 Gy, no colony formation was found for AuNP-incubated cells. Furthermore, at a dose of 10 Gy, no colony formation was found for cells in absence of AuNPs. Interestingly, the specific AuNPs 7 showed a stronger effect at a dose of 4 and 6 Gy compared to non-specific AuNPs 1. At a dose of 2 Gy, the cells were also incubated with 0.2 M DMSO as radical scavenger during irradiation. The factor 10 less DMSO concentration is important for cell life. These fractions showed lower cell damage for AuNP 7 (59.3 ± 4.3)% and much lower cell damage for AuNP 1 (78.9 ± 6.8). Non-incubated cells showed cell survival of (98.9 ± 5.2)% with 0.2 M DMSO at a dose of 0 Gy, indicating no toxic effects at these conditions.

In Vivo Experiments
In order to prove the concept of receptor-specific tumor accumulation of the peptidefunctionalized particles and their suitability for optical imaging in vivo, two A431-tumor bearing mice were injected with AuNPs 8 according to previously established protocols [43]. In brief, the mice were injected with 75 μg AuNPs 8 in 100 μL sterile PBS. The injected amount of AuNPs 8 corresponded to 1.5 μg (1.75 nmol) of NIR-dye per animal as AuNPs 8 contain 2% dye (see Table 1). This amount is comparable to literature values of 1-50 μg/g for cyanine-dye conjugates for in vivo fluorescence imaging of tumors [10,49,[60][61][62]. The mice were measured repeatedly over a period of up to 72 h post injection (p.i.) with a fluorescence imager (In Vivo Xtreme, Bruker) ( Figure 6) by using 730 nm as excitation wavelength and 790 nm as emission wavelength. A very low background signal was found, which is assumed to derive from the chlorophyll-containing diet of the mice. After injection of AuNPs 8, the background signal faded to the underground and a fast renal clearance of an excess of AuNPs 8 was observed in vivo. AuNPs 8 were found ex vivo in the collected urine and showed no degradation when analyzed by HPLC. The tumor-tomuscle ratio increased during the time of observation. The accumulation was measured by region-of-interest (ROI) interpretation ( Figure A23), and after 72 h, an ex vivo biodistribution (Figure 7) was performed and compared with the obtained in vivo data. The accumulation values detected by in vivo measurements and biodistribution experiments were found to be comparable for tumor, muscle, and kidney but different for the liver, since in biodistribution experiments, the weight of the organs was also considered and the organs could be measured directly ( Figure A24, Table A1), whereas in live imaging, the signal of the liver was found to be relatively lower, since the organ was measured from the back of the mice. However, the animal experiments revealed a higher accumulation of AuNPs 8 in the A431-tumor in comparison to muscle at 3-72 h post i.v. injection.

In Vivo Experiments
In order to prove the concept of receptor-specific tumor accumulation of the peptidefunctionalized particles and their suitability for optical imaging in vivo, two A431-tumor bearing mice were injected with AuNPs 8 according to previously established protocols [43]. In brief, the mice were injected with 75 µg AuNPs 8 in 100 µL sterile PBS. The injected amount of AuNPs 8 corresponded to 1.5 µg (1.75 nmol) of NIR-dye per animal as AuNPs 8 contain 2% dye (see Table 1). This amount is comparable to literature values of 1-50 µg/g for cyanine-dye conjugates for in vivo fluorescence imaging of tumors [10,49,[60][61][62]. The mice were measured repeatedly over a period of up to 72 h post injection (p.i.) with a fluorescence imager (In Vivo Xtreme, Bruker) ( Figure 6) by using 730 nm as excitation wavelength and 790 nm as emission wavelength. A very low background signal was found, which is assumed to derive from the chlorophyll-containing diet of the mice. After injection of AuNPs 8, the background signal faded to the underground and a fast renal clearance of an excess of AuNPs 8 was observed in vivo. AuNPs 8 were found ex vivo in the collected urine and showed no degradation when analyzed by HPLC. The tumor-to-muscle ratio increased during the time of observation. The accumulation was measured by region-of-interest (ROI) interpretation ( Figure A23), and after 72 h, an ex vivo biodistribution (Figure 7) was performed and compared with the obtained in vivo data. The accumulation values detected by in vivo measurements and biodistribution experiments were found to be comparable for tumor, muscle, and kidney but different for the liver, since in biodistribution experiments, the weight of the organs was also considered and the organs could be measured directly ( Figure A24, Table A1), whereas in live imaging, the signal of the liver was found to be relatively lower, since the organ was measured from the back of the mice. However, the animal experiments revealed a higher accumulation of AuNPs 8 in the A431-tumor in comparison to muscle at 3-72 h post i.v. injection. Nanomaterials 2021, 11, x FOR PEER REVIEW 10 of 29

Discussion
Stable αvβ3-specific AuNPs 7 were successfully synthesized with a slightly better avidity compared to the monomeric peptide ligand c(RGDfK). AuNPs additionally decorated with an NIR-dye had a slightly lower avidity as compared with the monomeric RGD-ligand, since in this case, a lower number of RGD ligands is located on the surface of the AuNPs (Table A1).
Via confocal fluorescence microscopy, the fate of AuNPs 8 on A431 cells was observed within 1-24 h. The AuNPs started to concentrate within the cell plasma but not within the nuclei as confirmed by confocal microscopy. Additionally, the formation of vesicles after 24 h and their excretion could be verified. The fluorescence signal started to concentrate in small dots within the cell plasma and in the extracellular medium those small foci could be observed too, indicating the excretion of the AuNPs in form of vesicles. This vesicle formation and excretion from cells within 24 h after incubation is perhaps a certain property of the AuNPs, when healthy tissue should expel therapeutic AuNPs. Within tumor tissue, the vesicles may stick in the interstitial cell region because of the lower blood supply and lower nutrition exchange with the surrounding tissues, presupposed that the AuNP would penetrate the deep tumor tissue.

Discussion
Stable αvβ3-specific AuNPs 7 were successfully synthesized with a slightly better avidity compared to the monomeric peptide ligand c(RGDfK). AuNPs additionally decorated with an NIR-dye had a slightly lower avidity as compared with the monomeric RGD-ligand, since in this case, a lower number of RGD ligands is located on the surface of the AuNPs (Table A1).
Via confocal fluorescence microscopy, the fate of AuNPs 8 on A431 cells was observed within 1-24 h. The AuNPs started to concentrate within the cell plasma but not within the nuclei as confirmed by confocal microscopy. Additionally, the formation of vesicles after 24 h and their excretion could be verified. The fluorescence signal started to concentrate in small dots within the cell plasma and in the extracellular medium those small foci could be observed too, indicating the excretion of the AuNPs in form of vesicles. This vesicle formation and excretion from cells within 24 h after incubation is perhaps a certain property of the AuNPs, when healthy tissue should expel therapeutic AuNPs. Within tumor tissue, the vesicles may stick in the interstitial cell region because of the lower blood supply and lower nutrition exchange with the surrounding tissues, presupposed that the AuNP would penetrate the deep tumor tissue.

Discussion
Stable α v β 3 -specific AuNPs 7 were successfully synthesized with a slightly better avidity compared to the monomeric peptide ligand c(RGDfK). AuNPs additionally decorated with an NIR-dye had a slightly lower avidity as compared with the monomeric RGD-ligand, since in this case, a lower number of RGD ligands is located on the surface of the AuNPs (Table A1).
Via confocal fluorescence microscopy, the fate of AuNPs 8 on A431 cells was observed within 1-24 h. The AuNPs started to concentrate within the cell plasma but not within the nuclei as confirmed by confocal microscopy. Additionally, the formation of vesicles after 24 h and their excretion could be verified. The fluorescence signal started to concentrate in small dots within the cell plasma and in the extracellular medium those small foci could be observed too, indicating the excretion of the AuNPs in form of vesicles. This vesicle formation and excretion from cells within 24 h after incubation is perhaps a certain property of the AuNPs, when healthy tissue should expel therapeutic AuNPs. Within tumor tissue, the vesicles may stick in the interstitial cell region because of the lower blood supply and lower nutrition exchange with the surrounding tissues, presupposed that the AuNP would penetrate the deep tumor tissue.
The activation of AuNPs by X-rays to emit AMEs was tested at pUC19 DNA incubated with AuNPs 1 [36][37][38]. No additional DSBs were found for all doses (25-100 Gy). Interestingly, additional SSBs (14%) in the presence of AuNP 1 were found at 25 Gy compared to non-AuNP-incubated DNA. Those SSBs were not inhibitable by DMSO, indicating the emission of AMEs from the AuNPs by external X-ray irradiation due to direct DNA damage without influence of OH-radicals. It is known that AuNPs can degrade DNA to little extend (1-2%) but does not increase significantly for longer time (weeks) and with organic solvents or no-neutral pH [63,64]. To further investigate this phenomenon, an experiment with AuNP 1 and 7 at concentrations of 1 and 10 µg/mL was performed to evaluate the impact of the AuNPs to the degradation of DNA (Figures A19 and A20). The DNA degradation was not significantly different for all experimental conditions, and the formation of oc-DNA was at (5.48 ± 0.95)%. As for our experimental setting (2 h incubation, neutral pH), we can exclude that the >10% DNA damage is from the AuNP itself, but most prominent due to the induction of AMEs [65]. Further, an optimum for the AuNP concentration between 0.5-1.5 mg/mL was determined by incubation of DNA with Re-188 solution. The reason for this observation may be guessed by taking into account that at lower AuNP concentrations the number of additionally produced AMEs are too low for a visible DNA damage, whereas at higher concentrations, the AuNPs could shield the AMEs from a neighboring AuNP and Re-188. With DMSO as radical scavenger, the DNA damage was not completely quenchable.
The Auger-Meitner effect has a very short effective range of~2 nm 3 in vivo and is most effective when directly incorporated into the DNA [66]. Therefore, a nanoparticle not exceeding a diameter of 5 nm could in theory be an effective Auger-emitter in a volume of~9 nm 3 when activated with X-rays [36]. This is a very short range within a cell, which has a typical diameter of 120-200 µm. In vitro fluorescence microscopy confirmed ( Figure A16) the accumulation of AuNPs in the cell plasma but not in the nuclei. For this reason, direct DNA damage can be excluded. Due to their behavior, the AuNPs could reach other cell compartments in the cell plasma that are also important for cell life [67]. Auger-Meitner electrons could then damage those compartments and the cell is about to die [56]. This hypothesis was evaluated in the following experiment: First, we carried out in vitro radiosensitizing experiments with AuNP-PEG 1 and AuNP-RGD 7 on A431 cells showing a similar effect as compared with literature data [37,38]. In the CFA experiments ( Figure 5), AuNPs 1 and 7 did not have a much higher effect at a dose of 2 Gy compared to reference experiments in absence of AuNPs and had a slightly higher effect at 4 Gy compared to the untreated cells. AuNPs 7 had a higher effect at 6 Gy compared to AuNPs 1. These findings could be explained by a faster accumulation of the targeted AuNPs 7 from the medium into the cell plasma compared to unspecific AuNPs 1 and other AuNPspecies [43]. Perhaps bigger AuNPs could produce more Auger electrons or remain within the cells for a longer period of time, thereby allowing a higher effect to be observed. In addition, bigger gold nanorods (AuNRs) functionalized with RGD peptides could be an interesting Auger-Meitner emitter [68].
Initial in vivo experiments with AuNP-RGD-NIR-dye 8 showed a similar tumor accumulation and biodistribution within 72 h as compared to other NIR-dye-functionalized AuNPs [43]. In fluorescence live-imaging the organ-to-muscle ratios were 2.40 ± 0.12 (kidneys), 1.47 ± 0.07 (tumor), and 2.20 ± 0.11 (liver) ( Figure A7). In biodistribution experiments, a significantly higher uptake in the tumor was found compared to the muscles (Figure 7). There was a relatively large difference in tumor uptake due to the different growth rate of the tumors. It is known that growing tumors have a high angiogenesis level and therefore a higher α v β 3 -integrine expression [47]. Hence, a higher uptake of RGD-functionalized AuNPs in a fast-growing tumor is explainable.

Conclusions
These initial results prove that the dually modified particles show potential as imaging tools for α v β 3 -expressing tumors in vivo via optical imaging. Especially for long-term observations of in vivo processes, the AuNPs could be useful. Since their accumulation is from slower nature (>1 d), the AuNPs cannot be used for short-term investigations such as Ga-68-PET. Further, the AuNPs could also serve as a tool for characterization of cell cycles and behavior by confocal fluorescence microscopy or live-cell microscopy. Interestingly, different modifications lead to different cell answer such as vesiculation and excretion. In vitro experiments for the assessment of the radiosensitizing ability of the AuNPs showed limited effects for the ultra-small AuNPs. However, in vivo, a major amount of AuNPs is excreted very fast renally. The rest of AuNPs accumulate in target organs and have a high retention there. Therefore, these AuNPs could serve as tool for radiotherapy as radiosensitizer or as activated [ 198 Au]AuNPs. Further in vivo experiments for the determination of radiosensitizing effects for bigger AuNPs and AuNRs and also for [ 198 Au]AuNPs are underway.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to institutional data protection.

Acknowledgments:
The authors are grateful to Kerstin Wetzig and Katja Tietze for her excellent technical assistance during radiosensitizing experiments. We like to thank Karsten Richter from the German Cancer Research Center (DKFZ) for measuring the EM in the Central Unit Electron Microscopy. We also want to thank Ulrich Scherer for using the TGA and Thorsten Röder for using the DLS at Mannheim University of Applied Sciences. Moreover, the authors would like to thank Jörg Kotzerke for fruitful discussions throughout the radiosensitizing experiments. Moreover, the authors would like to thank Trina Irico for the extensive proofreading of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.

Appendix A. Organic Syntheses
c(RGDfK) peptide: The cyclic pentapeptide c(RGDfK) was synthesized in 0.2 mmol scale by solid-phase peptide synthesis on solid support using the standard Fmoc strategy on 250 mg H-Asp(tBu)-2-chlortrityl-resin (loading: 0.8 mmol/g). For amino acid conjugation, 3.9 eq. HBTU (N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate), 4.0 eq. of Fmoc-protected amino acids, and 4.0 eq. of DIPEA were used in DMF as solvent. Coupling times were 30 min for each amino acid. After coupling of the last amino acid and Fmoc removal, the linear, protected peptide was cleaved from the resin using 1% TFA in CH 2 Cl 2 . The crude intermediate was isolated by evaporation of the volatile components of the mixture and then dissolved in dry DMF (120 mL). To this solution, DIPEA (64 µL, 0.70 mmol, 3.5 eq.) was added and the solution was cooled to 0-4 • C before DPPA (44 µL, 0.25 mmol, 1.25 eq.) was added. The mixture was allowed to warm to ambient temperature and reacted for 96 h until the cyclization was complete. The volatile components of the mixture were removed in vacuo, relyophilized in acetonitrile:water 1:1, and the residue was treated for 3 h with a mixture of TFA/TIS (triisopropylsilane) 97.5:2.5 (5 mL) to completely deprotect the peptide. After concentration in vacuo and precipitation of the product in cooled diethyl ether (25 mL), the crude product was obtained by centrifugation, washing with Et 2 O (5 mL) twice, and drying of the obtained solid. Purification of the product was performed by semi-preparative HPLC (t R = 3.75 min; gradient: 0-10% MeCN in H 2 O with 0.1% formic acid in 6 min at 4 mL/min). The product was obtained after lyophilization as a colorless solid in 67% overall yield (81 mg, 134 mmol). MALDI-MS  Thiol-hexyl-cyanine 3 ( Figure A2): Bis-1,1′-(4-sulfobutyl)indotricarbocyanine-5,5′-dicarboxylic acid sodium salt (SIDAG precursor) (50 mg, 68 μmol, Chess, Mannheim) was dissolved in DMF (6 mL). Thiol-functionalization was performed according to Licha et al. [69]. In brief, HBTU (10.2 mg, 27 μmol) and 6-aminohexane-1-thiol 5.3 mg, 31 μmol) were Thiol-hexyl-cyanine 3 ( Figure A2): Bis-1,1 -(4-sulfobutyl)indotricarbocyanine-5,5dicarboxylic acid sodium salt (SIDAG precursor) (50 mg, 68 µmol, Chess, Mannheim) was dissolved in DMF (6 mL). Thiol-functionalization was performed according to Licha et al. [69]. In brief, HBTU (10.2 mg, 27 µmol) and 6-aminohexane-1-thiol 5.3 mg, 31 µmol) were added and the solution was cooled to 0 • C. Then, DIPEA (18 µL, 102 µmol) was added and the reaction was stirred for 3 h at ambient temperature in the dark. Et 2 O (50 mL) was added to the solution and centrifuged. The Et 2 O was decanted and the residue was washed 3 × with Et 2 O, dried, and stored at −20 • C. The resulting green powder (54 mg) was used without further purification. The successful conjugation of 6-aminohexane-1-thiol was verified by Ellmans reagent and comparison of UV/Vis spectra at 412 nm. The purity was verified by HPLC (>80% General procedure to prepare PEGylated AuNP 1: Briefly, hydrogen tetrachloroaurate(III) trihydrate (560 mg, 1.44 mmol, ≥99.9% trace metal basis) was dissolved in 30 mL of tracepure water resulting in a bright yellow solution and then extracted by mixing with 300 mL of a tetraoctylammonium bromide (TOABr, 1011 mg, 1.85 mmol) toluene solution. The contents were stirred vigorously for 20 min at room temperature to facilitate the phase transfer of the Au(III) into the toluene layer, which resulted in the organic layer turning to a dark orange color and the aqueous layer becoming clear colorless. If the aqueous layer was not colorless, further TOABr (50 mg, 0.09 mmol) and toluene (100 mL) were added. After complete phase transfer, the aqueous layer was removed. The organic layer was dried with MgSO4 and filtered to remove excess of water. The solution was cooled to 0 °C in an ice bath. Then, 2.7 eq. of freshly prepared HO-PEG3-thiol (640 mg, 3.85 mmol) in 20 mL of dichloromethane was added and allowed to stir until the orange solution faded to colorless (~1 h). A fresh solution of tetrabutylammonium borohydride (TBABH) (3659 mg, 14.22 mmol) in 20 mL dichloromethane was then added to the rapidly stirring toluene solution over 5 s. The solution turned dark black instantly. The PEG-AuNP 1 started to precipitate from toluene after 1 h. After stirring the mixture for 16 h from 0 °C to 20 °C, 50 mL of tracepure water were added under slow stirring to extract the PEGylated AuNPs for 120 min. The organic layer was decanted, and the aqueous layer was washed alternatingly with 3 × 50 mL toluene/5 mL MeCN and 3 × 50 mL toluene/5 mL isopropanol. The black aqueous layer was transferred into a visking cellulose dialysis tube (molecular cutoff 14000 Da) with 3 × 10 mL tracepure water, and dialysis was performed in 3 × 10 L of distilled water for 1 h, 2.5 h, and 16 h. Afterwards, the AuNP 1 was lyophilized to yield 407 mg (44%) of black powder. These PEGylated AuNPs are relatively small (3.0 ± 2.0 nm), exhibit excellent stability, and can be repeatedly dried and dissolved in water.
General procedure for the preparation of furan-protected maleimide AuNP 2a: The preparation of AuNP 2a was performed by a place-exchange reaction of a freshly prepared furan-masked maleimide-PEG-thiol ligand with the PEGylated AuNPs. Maleimide-PEGthiol ligand (120 mg, 0.34 mmol) was dissolved in 6 mL 1:1 tracepure H2O:MeOH and was added to PEG-AuNP 1 (300 mg) in 30 mL tracepure H2O and stirred for 90 min. A 1:1 mole ratio of maleimide ligand to PEG ligand is crucial. The mixed ligand AuNP 2a sample was then purified by dialysis. Yield: 313 mg (99%).
General procedure of the Michael addition reaction: 20 mg of AuNP 2a were dissolved in dry DMSO (3 mL) under argon atmosphere and stirred for 2 h at 95 °C to remove Figure A2. Cyanine derivative (SIDAG precursor [49]) and thiol-functionalized derivative 3 for coupling reactions via Michael addition.
General procedure to prepare PEGylated AuNP 1: Briefly, hydrogen tetrachloroaurate(III) trihydrate (560 mg, 1.44 mmol, ≥99.9% trace metal basis) was dissolved in 30 mL of tracepure water resulting in a bright yellow solution and then extracted by mixing with 300 mL of a tetraoctylammonium bromide (TOABr, 1011 mg, 1.85 mmol) toluene solution. The contents were stirred vigorously for 20 min at room temperature to facilitate the phase transfer of the Au(III) into the toluene layer, which resulted in the organic layer turning to a dark orange color and the aqueous layer becoming clear colorless. If the aqueous layer was not colorless, further TOABr (50 mg, 0.09 mmol) and toluene (100 mL) were added. After complete phase transfer, the aqueous layer was removed. The organic layer was dried with MgSO 4 and filtered to remove excess of water. The solution was cooled to 0 • C in an ice bath. Then, 2.7 eq. of freshly prepared HO-PEG 3 -thiol (640 mg, 3.85 mmol) in 20 mL of dichloromethane was added and allowed to stir until the orange solution faded to colorless (~1 h). A fresh solution of tetrabutylammonium borohydride (TBABH) (3659 mg, 14.22 mmol) in 20 mL dichloromethane was then added to the rapidly stirring toluene solution over 5 s. The solution turned dark black instantly. The PEG-AuNP 1 started to precipitate from toluene after 1 h. After stirring the mixture for 16 h from 0 • C to 20 • C, 50 mL of tracepure water were added under slow stirring to extract the PEGylated AuNPs for 120 min. The organic layer was decanted, and the aqueous layer was washed alternatingly with 3 × 50 mL toluene/5 mL MeCN and 3 × 50 mL toluene/5 mL isopropanol. The black aqueous layer was transferred into a visking cellulose dialysis tube (molecular cut-off 14000 Da) with 3 × 10 mL tracepure water, and dialysis was performed in 3 × 10 L of distilled water for 1 h, 2.5 h, and 16 h. Afterwards, the AuNP 1 was lyophilized to yield 407 mg (44%) of black powder. These PEGylated AuNPs are relatively small (3.0 ± 2.0 nm), exhibit excellent stability, and can be repeatedly dried and dissolved in water.
General procedure for the preparation of furan-protected maleimide AuNP 2a: The preparation of AuNP 2a was performed by a place-exchange reaction of a freshly prepared furan-masked maleimide-PEG-thiol ligand with the PEGylated AuNPs. Maleimide-PEGthiol ligand (120 mg, 0.34 mmol) was dissolved in 6 mL 1:1 tracepure H 2 O:MeOH and was added to PEG-AuNP 1 (300 mg) in 30 mL tracepure H 2 O and stirred for 90 min. A 1:1 mole ratio of maleimide ligand to PEG ligand is crucial. The mixed ligand AuNP 2a sample was then purified by dialysis. Yield: 313 mg (99%).
General procedure of the Michael addition reaction: 20 mg of AuNP 2a were dissolved in dry DMSO (3 mL) under argon atmosphere and stirred for 2 h at 95 • C to remove the furan-protecting group. Afterwards, the resulting AuNP 2b solution was cooled to below The thermogravimetric analyses were performed with a Mettler Toledo TGA/SDTA851 e . AuNPs (2-5 mg) were weighed into 70-μL-aluminum oxide crucibles (Mettler Toledo, Gießen, Germany) and heated from 25-750°C (10 K/min) in a N2 or CO2 stream (30 mL/min). The loading of the different AuNPs is shown in table 1 and was calculated by the different mass losses, which increase the more the AuNPs are functionalized. Therefore, the quantity of the different ligands per particle can be calculated.

•
The mass loss of the AuNP-PEG 1 was 24.27%. This accounts for ~500 PEG ligands at the AuNP surface. M ~ 345 kDA.

•
The mass loss of AuNP-RGD 7 was 27.89% and the RGD accounts for 5.91% mass loss (60 RGD ligands per AuNP). Hence, we can calculate the molar mass for example for AuNP-RGD 7 to be ~405 kDa.

Appendix B.2. UV/Vis Spectra and Size Determination
UV/Vis measurements were performed with an Eppendorf BioSpektrometer Kinetic. We measured the absorption of the AuNPs at a concentration of 1 μg/mL to estimate the particle size before performing electron microscopy. The absorption at the surface-plasmon resonance (maximum) divided by the absorption at 450 nm (minimum) gives a factor that can be compared with tables from literature [70]. Emission scans were performed on a Tecan infinite M200 with excitation wavelength of 690 nm.

Appendix B.2. UV/Vis Spectra and Size Determination
UV/Vis measurements were performed with an Eppendorf BioSpektrometer Kinetic. We measured the absorption of the AuNPs at a concentration of 1 µg/mL to estimate the particle size before performing electron microscopy. The absorption at the surface-plasmon resonance (maximum) divided by the absorption at 450 nm (minimum) gives a factor that can be compared with tables from literature [70]. Emission scans were performed on a Tecan infinite M200 with excitation wavelength of 690 nm.

Appendix B.2. UV/Vis Spectra and Size Determination
UV/Vis measurements were performed with an Eppendorf BioSpektrometer Kinetic. We measured the absorption of the AuNPs at a concentration of 1 μg/mL to estimate the particle size before performing electron microscopy. The absorption at the surface-plasmon resonance (maximum) divided by the absorption at 450 nm (minimum) gives a factor that can be compared with tables from literature [70]. Emission scans were performed on a Tecan infinite M200 with excitation wavelength of 690 nm.

Appendix B.3. Electron Microscopy
AuNP samples were diluted in deionized water at convenience (fade red solution), particles adsorbed onto glow-discharged carbon-coated EM-grids and directly observed by TEM (Zeiss EM912, Carl Zeiss Oberkochen or Titan 80-300, FEI Company, HZDR). Images were digitally registered via a CCD camera (Sharp eye, TRS, Moorenweiss or US1000 der Firma Gatan). The number and size of particles was measured by FIJI software (v1.50e).

Appendix B.3. Electron Microscopy
AuNP samples were diluted in deionized water at convenience (fade red solution), particles adsorbed onto glow-discharged carbon-coated EM-grids and directly observed by TEM (Zeiss EM912, Carl Zeiss Oberkochen or Titan 80-300, FEI Company, HZDR). Images were digitally registered via a CCD camera (Sharp eye, TRS, Moorenweiss or US1000 der Firma Gatan). The number and size of particles was measured by FIJI software (v1.50e).

Appendix B.3. Electron Microscopy
AuNP samples were diluted in deionized water at convenience (fade red solution), particles adsorbed onto glow-discharged carbon-coated EM-grids and directly observed by TEM (Zeiss EM912, Carl Zeiss Oberkochen or Titan 80-300, FEI Company, HZDR). Images were digitally registered via a CCD camera (Sharp eye, TRS, Moorenweiss or US1000 der Firma Gatan). The number and size of particles was measured by FIJI software (v1.50e).

Appendix B.3. Electron Microscopy
AuNP samples were diluted in deionized water at convenience (fade red solution), particles adsorbed onto glow-discharged carbon-coated EM-grids and directly observed by TEM (Zeiss EM912, Carl Zeiss Oberkochen or Titan 80-300, FEI Company, HZDR). Images were digitally registered via a CCD camera (Sharp eye, TRS, Moorenweiss or US1000 der Firma Gatan). The number and size of particles was measured by FIJI software (v1.50e).          in [51]) can be found.

Fluorescence Microscopy In Vitro
Initial cell studies revealed an optimized concentration of 50 μg/mL of AuNP-RGD-NIR-dye 8 for cell imaging (scheme 5). Via confocal fluorescence microscopy, the cell internalization of the particles, vesicle formation, and excretion were verified after 1, 3, 6, and 24 h. The cells start to excrete AuNPs after 3 h. The particles are located in the cytoplasm and not inside the nuclei. Fluorescence microscopy was performed on a Leica TCS SP8 confocal microscope with lasers at λ = 488 (Sytox), 552 (memOrange), and 638 (NIRdye) nm.

Fluorescence Microscopy In Vitro
Initial cell studies revealed an optimized concentration of 50 µg/mL of AuNP-RGD-NIR-dye 8 for cell imaging. Via confocal fluorescence microscopy, the cell internalization of the particles, vesicle formation, and excretion were verified after 1, 3, 6, and 24 h ( Figure A16). The cells start to excrete AuNPs after 3 h. The particles are located in the cytoplasm and not inside the nuclei. Fluorescence microscopy was performed on a Leica TCS SP8 confocal microscope with lasers at λ = 488 nm (Sytox), 552 nm (memOrange), and 638 nm (NIR-dye).