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  • br Fig A In vivo fluorescence images of


    Fig. 6. (A) In vivo fluorescence images of MDA-MB-231 xenograft nude mice after i.v. injection of free DiR and [email protected] NPs. The arrows indicate the tumor foci of mice. (B) Ex vivo distribution and (C) statistical analysis of hearts (H), livers (Li), spleens (S), lungs (Lu), kidneys (K) and tumors (T) harvested after sacrifice (n = 3, results are shown as mean ± S.D.).
    3.8. Ex vivo anti-tumor efficacy and systemic toxicity evaluation
    The major organs (heart, liver, spleen, lung and kidney) and tumor were excised from the treated mice after 30 days and imaged using H&E staining. The organ tissue sections (Fig. 8A) revealed no obvious pa-thological abnormalities, except for the free DOX group. In this case, extensive hepatocellular vesicular steatosis, some focal inflammation of the liver, and slight congestion in the glomerular T-5224 of the kidney can be seen. The [email protected] NPs caused severe necrosis in the tumor tissue, with minimal damage to the liver and no damage ob-served in the other organs, indicating their selectivity and good histo-compatibility [45,46]. The H&E staining (Fig. 8A) further showed that the area of necrosis in the tumor tissues was larger in the [email protected] 
    To reveal the underlying mechanism of tumor growth inhibition by the NPs, the proliferation and apoptosis levels in the tumor sections were further analyzed by immunohistochemistry using the Ki67 and TUNEL assays. Photographs of tumor sections are illustrated in Fig. 8B.
    Fig. 7. In vivo anti-tumor therapeutic efficacy results. (A) Representative photos of tumor tissues after treatment for 30 days. (B) Real-time measurements of tumor size. (C) Time course average body weights of mice after each treatment. (D) Kaplane-Meier survival curves of tumor-bearing mice.
    Almost no cell apoptosis was observed in groups treated with saline. The tissues of mice treated with free DOX and free OA demonstrated moderate cell apoptosis, while apoptosis most extensive in the [email protected] group. Quantitative analyses of the TUNEL and Ki67 data are displayed in Fig. 8C. The proliferation percentage determined by Ki67 im-munohistochemistry following treatment with [email protected] NPs (3.8 ± 0.6%) was far less than that with free DOX (25.7 ± 3.1%), free OA (46.4 ± 4.2%) and the saline control group (90.7 ± 8.6%). Moreover, the percentage of apoptotic cells measured by the TUNEL assay after treatment with [email protected] NPs (82.8 ± 7.9%) was much higher than that with free DOX (55.8 ± 5.1%) and OA (21.8 ± 1.6%). Therefore, the NPs act to both induce apoptosis and inhibit cell proliferation in tumor tissues. All these findings demonstrate the effectiveness of the dual-drug-loaded NPs for targeted combination therapy in breast tumors [47].
    3.9. Effects of [email protected] NPs on protein expression
    MDR is often associated with the overexpression of the drug trans-porters P-gp and MRP1 [48], which confer resistance to cancer cells by active excretion of a wide range of drugs from the cytosol [49]. Con-siderable efforts have been devoted to the development of specific molecules that can inhibit the function of P-gp and MRP1 in order to sensitize tumor cells to conventional chemotherapeutics [50], and OA is known to have such properties [27]. The expression of MRP1 and P-gp 
    and cancer suppressive factors including PARP, PTEN and p53 were assessed by western blot. As can be seen in Fig. 9, there is a significant increase of P-gp expression in animals treated with DOX compared to those receiving saline. This indicates that DOX induced MDR and thus resulted in an increase of P-gp levels. Compared to the group treated with DOX, levels of P-gp expression are decreased by 61.4%, 36.9% and 25.3% upon treatment with [email protected] NPs, FA-CS-g-OA and free OA, respectively. Although free OA should be potent in down-regulating P-gp it has no targeting ability, and its inclusion in NPs se-lective for FA-over-expressing cells led to greater OA accumulation in the tumor site and greater reductions in P-gp expression. A similar trend was displayed in the expression levels of MRP1 (Fig. 9). MDA-MB-231 cells originally expressed a high level of MRP1. When treated with [email protected] NPs, the expression of MRP1 was almost undetectable, suggesting that the inclusion of OA in the particles is able to shut down at least some MDR mechanisms.