br In order to verify
In order to verify the molecular mechanism of the anti-tumor effects induced by [email protected] NPs, we assessed three apoptosis-related protein o-Phenanthroline levels (PARP, PTEN and p53) in MDA-MB-231 tumor-bearing mice. We found (Fig. 9) that when mice were treated with OA and OA-containing NPs, an increase in the expression of all three was observed. Notably, [email protected] NPs could significantly upregulate the levels of PARP, PTEN and p53 compared with OA and the other formulations, demonstrating the enormous potential of [email protected] NPs to induce tumoral cell apoptosis . The effect of FA-CS-g-OA on the levels of PARP, PTEN and p53 was slightly greater than
Fig. 8. Histopathology data for tumor-bearing mice. (A) Sections of heart, liver, spleen, lung, kidney and tumor stained with hematoxylin and eosin. (B) TUNEL staining and Ki67 immunohistochemistry of tumor sections. (C) Statistical analysis of the TUNEL and Ki67 positive rates (n = 8, results shown as mean ± S.D.; * indicates P < 0.05, ** indicates P < 0.01). Scale bars are 100 μm in the H&E assay and are 50 μm in the TUNEL and Ki67 assays.
that of free OA, which may arise from the active targeting effects of the FA, and acidic microenvironment-triggered OA release. All these data suggest that OA could be a good choice in the treatment of MDR tu-mors, either as an adjuvant or as a chemotherapeutic itself . With fast tumor growth, the extracellular matrix (ECM) will create growth-induced solid stress, impeding the penetration of both macro-molecules and nanomedicines [53,54]. The transforming growth factor (TGF-β) autocrine pathway plays a major role in ECM proliferation, which can also stimulate collagen I and matrix metalloproteinase (MMP) production, eventually exacerbating fibrosis in tumors. There-fore, the effects of OA on breast tumors was estimated through the change in expression of TGF-β, MMP-2 and collagen I  (Fig. 9). There are small reductions in TGF-β, MMP-2 and collagen I expression with free DOX and free OA. These are much more noticeable for the FA-CS-g-OA and [email protected] NPs, which significantly inhibited the expression of TGF-β, MMP-2 and collagen I. These results illustrate that the [email protected] NPs were able to downregulate the expression of these proteins in tumoral cells, and thus can have an anti-fibrosis effect through the TGF-β/MMP/collagen I signal pathway .
3.10. Evaluation of side effects
DOX is widely associated with serious side effects, including liver and kidney damage, oxidative stress and inflammation reactions . Previous work suggests that OA can have a number of biological ac-tions, with antioxidant, anti-inflammatory, and hepatoprotective properties . The serum levels of key clinical biomarkers were thus assessed to determine if the OA in the [email protected] NPs could mitigate against these effects of DOX. An increase in the levels of bio-chemical markers including aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine (CRE) would be indicative of organ failure. A dramatic increase in AST, ALT, BUN, and CRE levels was observed in DOX-treated mice (Fig. 10A,
B), indicating DOX-induced tissue damage. After administration of OA and OA containing NPs, however, the activity of ALT and AST is sig-nificantly reduced compared to DOX alone (P < 0.01) (Fig. 10A). There was no significant difference between free OA and the OA-loaded NPs (P > 0.05). The CRE and BUN levels induced by [email protected] DOX NPs are also significantly lower than those of mice treated with free DOX (Fig. 10B), and close to those in the saline group, confirming that the NPs greatly reduce the renal toxicity of free DOX.