br The quantification of CBZ in blood plasma clearly show
The quantification of CBZ in blood plasma clearly show a longer circulation time when the drug was incorporated in the NPs, with at least a 10-fold higher concentration in mice receiving PEBCA-CBZ compared free CBZ (Fig. 4A). When evaluating such data it is important to remember that the CBZ incorporated in NPs does not have a ther-apeutic effect before being released. Thus, release of the drug by dif-fusion and/or biodegradation of the NPs are necessary to obtain a good therapeutic effect as observed in the MAS98.12 model. The in vivo fluorescence imaging data obtained with PEBCA NPs labeled with NR668 demonstrates accumulation of the fluorescence in the same tissues as those where CBZ was found to be present. As ex-pected, these data show that most of the NPs ended up in the liver. The liver/spleen ratio of the fluorescence per pixel measured 24 h after in-jection was estimated to be 2.9 for NPs without CBZ and 2.4 for NPs with CBZ, whereas the ratio of total CBZ content in liver to spleen was calculated to 2.1 based on the MS analyses. Although one should be careful in interpreting the quantitative data from the fluorescent ima-ging (based upon per pixel measurements) the biodistribution data, obtained with two different methods, showed similar results. Our ob-servations that injection of free NR668 did not give any measurable in vivo signal, the report that NR668 did not leak from NPs with a similar composition , and the different kinetics observed for PEBCA-CBZ and CBZ (Fig. 4A), indicate that most of the CBZ and NR668 are en-closed within the NPs 24 h after injection. Therefore, the biodistribution of these low molecular substances seems to represent well the dis-tribution of the PEBCA NPs at this time point. In another study with comparable NPs, but using another PEGylation and fluorescent marker, a liver/spleen ratio of 5.3 was reported 6 h after injection . In this study, the mean fluorescent signal obtained in the tumors 24 h after injection was 12% of that in liver for the NPs not containing CBZ and 3% for the NPs containing CBZ. The higher fluorescence obtained fol-lowing injection of PEBCA NPs without drug than for NPs containing CBZ (Fig. 3A,B) may in part be due to the somewhat larger size of the NPs with drug (Table S1).
Macrophages are the most abundant immune H2DCFDA in mammary tumors. Tumor associated macrophages (TAMs) were originally thought to exert anti-tumor activities, but increasing clinical and experimental evidence show that TAMs also may promote tumor progression and influence anticancer drug responses [28–30]. The pro-tumorigenic macrophages are known as alternatively activated and referred to as anti-inflammatory (M2-type), whereas the classically activated pro- Journal of Controlled Release 293 (2019) 183–192
inflammatory (M1-type) macrophages exhibit anti-tumorigenic prop-erties [31,32]. Plasticity is a hallmark of the macrophage population and dynamic changes in their phenotype define the different subtypes . A set of markers is usually recommended for a comprehensive characterization of the whole population [22,33]. In the present study we have used the well-accepted nitric oxide synthase (iNOS) for de-tection of the M1 phenotype and mannose receptor (CD206) to define the M2-type [22,33].
The TAMs are influenced by the context, e.g. by factors in the mi-croenvironment or externally added anticancer drugs. Interestingly, the effect of docetaxel has been shown to partly depend upon depletion of M2 macrophages and expansion of M1 macrophages in models of breast cancer . In contrast, we did not observe any response in the mac-rophage populations upon treatment with free CBZ, another taxane, despite efficient growth retardation. However, treatment with PEBCA encapsulated CBZ, resulted in significant improved anti-tumor efficacy, and complete remission in 75% of the tumors. Even though the number of tumors used for immunohistochemical quantification is small, our data suggest two possible mechanisms that might contribute to the good effect. First, a trend towards elevated inflammation in tumors upon treatment with PEBCA NPs (with or without drug) was observed. This may imply a role of PEBCA NPs in homing anti-tumorigenic M1 macrophages into the tumors, and thus further support the effect of CBZ. Secondly, we also showed that PEBCA-CBZ treatment significantly reduced CD206 expression in tumors compared to treatment with NPs without drug, which may point towards depletion of pro-tumorigenic macrophages.
Recently it was published that M2 macrophages show a vigorous endocytic uptake by macropinocytosis, whereas this uptake was vir-tually inactive in the M1 macrophages . Furthermore, another study showed that the M2 macrophages use endocytosis to degrade collagen and promote tumor growth in solid tumors . We speculate that the decrease in M2 macrophages observed after treatment with PEBCA-CBZ, and the concomitant pronounced effect on tumor growth, may be related to the macropinocytic uptake of PEBCA-CBZ and sub-sequent killing of these M2 macrophages. Thus, the inherent properties of M1 and M2 macrophages and selective toxic effect of drug containing particles on M2 macrophages may increase the efficacy of the treat-ment. It has earlier been published that driving TAMs towards M1 polarization (increasing the ratio of M1/M2 macrophages) has shown promising therapeutic effects in mice cancer models .