**1. Introduction**

'Nanotheranostics'—referring to the use of nanotechnology for combined imaging and treatment of diseases—is currently an active research field as combining diagnosis with therapy has several advantages. The knowledge of the biodistribution of therapeutic agents through imaging can improve the guidance and initiation of cancer therapy. This can, for example, help to decide on the best time point for applying photothermal therapy. In addition, therapy success can be assessed at earlier time points and follow-up of therapy efficiency can be improved drastically. In the clinic, this will ultimately result in earlier intervention, better patient management, and improved prognosis [1]. Within the field

of nanotheranostics, especially gold nanoparticles (AuNPs) that show a localized surface plasmon resonance (LSPR) can have a significant impact, as the LSPR effect can be used for both imaging and therapy [2]. The LSPR effect can be explained by the collective oscillation of the conduction band electrons due to light. This collective oscillation induces an enhanced absorption and scattering of the light [3]. The scattering properties of the light can be exploited by several imaging techniques including surface enhanced Raman scattering [4,5] and darkfield microscopy [2]. The light energy that is not scattered by the particles but absorbed, is converted into heat. This specific heat generation is the basis for both photoacoustic imaging (PAI) and photothermal therapy (PTT) [6,7].

PAI is based on the acoustic waves generated by the thermo-elastic expansion that occurs when a specific compound absorbs a pulsed electromagnetic wave [8]. Exogenous contrast (e.g., carbon nanotubes, AuNPs) have shown to generate higher PAI contrast compared to endogenous molecules (e.g., hemoglobin) [9]. Due to the negligible scattering of ultrasound in tissue compared to light, a relatively high imaging depth of approximately 5 cm is possible with PAI [10]. PTT on the other hand is also based on the heat conversion of AuNPs during irradiation with a continuous laser [11]. Since cancer cells are more sensitive to heat than other cell types, a temperature increase above 43 ◦C is lethal due to the inability to remove the heat in poorly vascularized tumor tissue [12].

For PAI and PTT, the ratio between the light scattering and absorption properties per AuNP is crucial. Hereto, several shapes of AuNPs have been studied ranging from nanorods, nanoshells, nanocages to nanostars [8]. Usually, nanostars have higher absorption vs. scattering coefficients compared to nanorods and nanoshells, but similar to nanocages [11,13]. This higher absorption coefficient is the crucial parameter for efficient heat conversion, important for PAI and PTT. Several studies have examined the use of gold nanorods as a contrast agent for PAI in tumors [14–18]. For nanostar-shaped AuNPs, mainly non-quantitative in vitro data [19] and in vivo mapping of the lymphatic system and first results on tumor imaging have been shown [20,21]. We have recently demonstrated their tumor-targeting ability using PAI [22]. For PTT of tumors, several groups have reported on nanostars as an effective in vitro PTT agent [13,19,21,23,24], but many questions remain regarding the PTT efficiency using these nanostars in vivo [23,25].

Alternatively, AuNPs can also be imaged with computed tomography (CT), since they absorb X-rays more efficiently compared to frequently used contrast agents such as iodine-based compounds [26,27]. In general, heavy atoms are frequently used as a contrast agent for X-ray based CT for diagnostic imaging in the clinic and for preclinical research [26]. Nonetheless, relatively high local AuNP concentrations are needed to generate sufficient contrast for CT, in particular for small voxel sizes as required for preclinical imaging applications [28]. AuNPs, such as nanorods or spheres, have shown their effectiveness as blood-pool and tumor-targeting contrast agent using CT [26,29]. However, for nanostars, quantitative in vitro and in vivo studies regarding their potential as contrast agents for CT are lacking [19].

Several routes have been explored to administer AuNPs as a theranostic agent against cancer, but many questions remain. Intratumoral administration is the most straight forward way for theranostic application, while i.v. injection has shown mixed results in terms of tumor accumulation [30,31]. Few reports showed reasonable tumor accumulation where the charge of the nanoparticles played a crucial role for tumor targeting [26,32]. Even functionalizing the nanoparticles with biological ligands gave different outcomes in terms of intra-tumor accumulation [33,34]. Active targeting, suggested to overcome problems with low intra-tumor accumulation after intravenous delivery, may have no influence on tumor uptake but does on the distribution within the tumor [33]. To improve biocompatibility and active targeting, AuNPs are frequently coated through an Au-S-bond. These non-covalent bonds are subject to potential thermal instability, releasing part of the coating material [35].

AuNPs have to fulfill some essential requirements to be applicable for in vivo theranostic photothermal approaches. First, the absorption band of the AuNP must be tuned to the near infrared region (NIR) frequency range for having maximal contrast generation and therapy efficiency due to its relative high depth penetration [36]. Second, the diameter of the AuNPs has to be around or below 100 nm to cross leaky blood vessels and being retained in the tumor. Third, charged particles are

favored since such particles show better retention after intra-tumor injection because of immediate interaction with the tumor cells [37].

Exploiting the advantage of the specific high absorption capacity of nanostars, we have studied the potential of nanostars for PAI/CT and PTT in vitro and in vivo. In this study, we first optimized the synthesis and functionalization of nanostars for efficient uptake by tumor cells and assessed their PAI and PTT capabilities in vitro using an ovarian cancer cell line (SKOV3). For in vivo validation, gold nanostars were intratumorally injected in a xenograft mouse model and their local distribution in the tumor assessed with CT and PAI. Finally, photothermal therapy was performed and evaluated using bioluminescence imaging (BLI), magnetic resonance imaging (MRI), and histology.
