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    2020-08-28

    
    to approx. 0.5% at 96 h after injection. The amount of CBZ found in tumors after injection of free CBZ was approximately half of that ob-tained after injection of PEBCA-CBZ at both time points. When com-paring the CBZ concentrations in the tissue samples obtained 24 and 96 h after injection of PEBCA-CBZ, there is a decrease in tumor, spleen and kidney, and an increase in liver and EPZ6438 nodes from 24 to 96 h. The only significant difference is the decrease in the MAS98.12 tumor tissue (p = .02). Also, the CBZ concentration after injection of free CBZ was significantly lower in tumors after 96 h when compared to 24 h (p < .001). Plasma and tissue samples were also analyzed for CBZ following injection of PEBCA NPs without CBZ; all these samples (si-milar to those samples analyzed after injection of PEBCA-CBZ) were below the LOQ of the analytical method.
    3.5. Macrophage infiltration in treated MAS98.12 tumors
    The infiltration of CD68 antibody stained macrophages into control and treated MAS98.12 tumors was estimated using automatic quanti-fication of scanned slides. An elevated number of tumor infiltrating macrophages was observed in mice injected with PEBCA-CBZ or PEBCA without drug compared to mice receiving free CBZ or saline, but the differences did not reach statistical significance (Fig. 5A). Also the marker of pro-inflammatory M1 macrophages (iNOS) demonstrated increased macrophage infiltration into tumors of mice receiving PEBCA-CBZ or PEBCA (Fig. 5B). However, the anti-inflammatory M2 macrophages subset, measured as CD206 positive cells, demonstrated
    Fig. 4. CBZ concentrations in plasma and organs measured with LC-MS/MS after administration of 15 mg CBZ/kg. Plasma concentration measured as function of time (A). CBZ concentrations in tumors and organs measured after 24 h (B) and 96 h (C). LNs: lymph nodes. Data shown are mean values ± SD (n = 3). Note that logarithmic scales are used on all y-axes.
    increased infiltration compared to the saline control only in tumors receiving PEBCA (Fig. 5C). Furthermore, the tumors of mice receiving PEBCA-CBZ showed significant lower levels (mean value of 23%) of this pro-tumorigenic macrophage population than tumors receiving PEBCA alone (p < 0.001; Fig. 5C).
    We tested cellular toxicity of PEBCA-CBZ, free CBZ and PEBCA without drug in three cell lines, specifically MDA-MB-231, MDA-MB- 
    Fig. 5. Macrophage infiltration in treated MAS98.12 tumors. The macrophage infiltration was measured in the MAS98.12 tumors 96 h after injection of saline (control), PEBCA NPs (without drug), free CBZ, and PEBCA-CBZ. (A): The total population of infiltrated macrophages was quantified using an antibody to CD68. (B): The population of anti-tumorigenic (pro-inflammatory) macro-phages was quantified using an antibody to iNOS. (C): The population of pro-tumorigenic (anti-inflammatory) macrophages was quantified with an antibody to CD206. Data for the whole tumors are shown as mean ± SEM (n = 3 for control samples; n = 4 for PEBCA and n = 5 for CBZ and PEBCA-CBZ). Asterisks indicate statistical significance obtained by unpaired parametric t-test, where p < 0.0005 is marked with *** and p < 0.0001 is marked with ****.
    468 and MCF-7, measuring both cell proliferation by incorporation of [3H]thymidine after 24 h and cell viability after 72 h by using the MTT assay. PEBCA-CBZ and CBZ were significantly more toxic than PEBCA without drug for all cell lines (range 130–350 fold), but there was no difference in the toxic effect of PEBCA-CBZ and CBZ in any of these test systems in any cell line (Fig. S5). It was also apparent that a small
    fraction (10–20%) of the cells in all cell lines tested survived even very high CBZ concentrations when using the MTT assay after incubation for 72 h. The toxicity of PEBCA-CBZ and free CBZ were also tested in all three cell lines using two other test systems. The effect on protein synthesis (incorporation of [3H]leucine) was measured following 24 h of incubation, and the ATP levels (CellTiter-Glo®) were measured after 72 h. The results obtained with these test systems (data not shown) were very similar to those shown in Fig. S5. The MTT assay was also performed on MDA-MB-231 cells after 24 and 48 h of incubation with similar results to those shown after 72 h of incubation (Fig. S5), al-though the toxic effect was smaller after these shorter incubation times (data not shown).
    4. Discussion
    The PEBCA NPs containing CBZ demonstrated a remarkable good therapeutic effect in the triple negative PDX mouse model, where complete remission was obtained in 6 out of 8 tumors following two injections of 15 mg CBZ/kg (Fig. 2A). Several possible explanations for the advantageous effect of PEBCA-CBZ versus free CBZ can be envi-sioned, and the obtained data point at least to the following factors: The longer circulation in blood and higher concentration of the drug in the tumor (Fig. 4) and the higher ratio of anti-tumorigenic (anti-iNOS la-beled) macrophages to pro-tumorigenic (anti-CD206 labeled) macro-phages [22,25] in the treated tumors (Fig. 5).