The pronounced cytotoxicity of ATV against glioblastoma in 2D cell culture (Figure , 5) as well as 3D spheroid models (Figure ) suggests its potential for GBM chemotherapy. However, in order to unfold its therapeutic potential in vivo, ATV needs to reach tumor site first. This includes permeation through the BBB after e.g. parenteral administration. Several sophisticated in vitro BBB models were developed in recent years to resemble the situation in vivo. [80, 81] One promising approach are models based on human induced pluripotent stem cells (hiPSCs) derived BBB endothelial cells. [52, 82] Using such a hiPSC-derived model, physiological characteristics like high transendothelial electrical resistances (TEER) up to 2,500 Ω•cm 2 , distinct upregulation of typical BBB genes as well as the formation of an in vivo-like tight junction (TJ) network could be achieved. The hiPSC-derived BBB endothelial cells were seeded on top of a transwell insert and subsequently the permeability of ATV from the apical to the basolateral compartment was investigated (Figure ). A ratio of A-pBuOzi-A/ATV = 10/1 g/L was chosen due to the small size of the respective ATVloaded micelles (Figure ,f) potentially facilitating transcellular permeability, assuming integrity of the drug loaded micelles. The latter is not necessarily given due to e.g. potential transfer of incorporated ATV to proteins present in solution as observed for A-pBuOx-A/PTX in vitro and in vivo . ATV was either solubilized with A-pBuOzi-A or dissolved in DMSO and applied at 90 µM (50 mg/L) as well as 150 µM (83 mg/L). For comparison, oral administration of 40 mg ATV in hypercholesterolaemic haemodialysis patients resulted in ATV peak plasma concentrations of 28.6 ± 15.2 µg/L (51 nM; reached 1-2 hours after initial administration), together with the active metabolites o-and p-OH ATV (8.1 and 0.6 µg/L) as well as inactive 3-lactone metabolites (44.2 µg/L). This clearly shows the need of ATV-formulation to achieve necessary ATV concentrations potentially suitable for glioblastoma therapy. At both concentrations, no pronounced cytotoxic effects of ATV on the endothelial cells of the artificial BBB were observed after 4 h incubation, ensuring the integrity of the monolayer during the course of the experiment (Figure ). After 24 h incubation, again no cytotoxicity up to ATV = 90 µM occurred (Figure ). Only at ATV = 150 µM, cell viability decreased to 41 % and 49 % for DMSO/ATV and A-pBuOzi-A/ATV, respectively. ATV was quantified in the apical and basolateral chamber after 4 h and 24 h incubation. To ensure the permeability analysis of A-pBuOzi-A/ATV and DMSO/ATV through the BBB endothelial cell layer itself, transwells without cells were always included to correct the permeation values for the barrier formed by the membrane support. In all cases, a higher ATV concentration occurred on the basolateral side of the latter compared to wells containing cells (Figure ). Furthermore, diffusion through the blank membrane was more or less the same for A-pBuOzi-A/ATV and DMSO/ATV therefore excluding variances due to different diffusion profiles. With respect to the wells without cells, the ATV transport through the BBB was less efficient at apical ATV feed of 150 µM, compared to 90 µM (Figure ). Nevertheless, absolute basolateral ATV concentrations increased from 4 h to 24 h incubation and from [AT-V feed ] = 90 µM to 150 µM in all samples (Figure ). For both ATV concentrations and time-points, ATV permeability was slightly higher for A-pBuOzi-A/ATV than for DMSO/ATV, however, the differences were within the standard deviations (Figure ). This suggests that our nanoformulation does not alter the permeation profile of ATV through this BBB in vitro model significantly. Whether this is due to a rapid release of ATV from the micelles to e.g. proteins present in medium or e.g. the disintegration of the ATVloaded micelles at the cell-membrane -not further improving endocytosis or transcellular delivery -remains to be elucidated. However, rapid disintegration of drug-loaded micelles within the blood stream is not necessarily detrimental regarding therapeutic efficacy, as evidenced by POx/PTX nanoformulations, which exhibit an excellent therapeutic efficacy in vivo. Interestingly, the proportional basolateral ATV concentration with respect to the wells without cells increased from 4 h to 24 h incubation for both, DMSO/ATV and A-pBuOzi-A/ATV (Figure ). Taking a closer look at the TEER values showed a decrease in resistance after 24 h at both ATV feed concentrations of 90 and 150 µM (Figure ). Even though we observed no ATV cytotoxicity up to 90 µM (Figure ), the integrity of the BBB is apparently affected, probably through a loosening of the tight junctions. This may be an early indication of cytotoxic effects. Although the transport across the BBB is not significantly improved, the presented novel POzi/ATV formulation may allow much higher ATV serum concentrations, compared to oral administration or alternative drug delivery systems, as much higher ATV concentrations can potentially be administered. Therefore, therapeutic doses may be reached, even without improved transport across the BBB. Furthermore, co-administration with agents temporarily permealizing the BBB could help to increase the ATV concentration across the BBB. Such agent could be SP1049C or small molecules known to permealize the BBB. Possibly, such agent could be coformulated with ATV and therefore improve co-delivery of both APIs.