br In vivo safety and tumor penetration

3.6. In vivo safety and tumor penetration
To evaluate the safety and the tumor penetration in vivo, [email protected]&PTX and [email protected] were investigated by intravenous administration into HepG2 subcutaneous tumor-bearing nude mice. DOX + PTX, free DOX, and saline were used as controls. The mice were sacrificed 24 h after the final injection (day 28), and the major organs, including the heart, liver, spleen, lung, kidney, and HepG2 subcutaneous tumor, were excised for in vivo fluores-cence of DOX using a PerkinElmer IVIS instrument. As shown in Figs. 7A and S17A, the [email protected]&PTX rendered significant 
tumor targeted accumulation compared with the control group of DOX + PTX. This result indicated that FA-CM is capable of tumor tar-geted delivery of anti-cancer drugs because of the function of FA.
To systematically investigate the toxicity of drugs-loaded FA-CM versus free drugs in vivo, H&E staining of the main organs was carried out (Fig. 7B). The administration of either free DOX or DOX + PTX exhibited a direct side effect on cardiac myocyte, which was marked by myocardial fiber disorder, widened gap of intercellular space, and myocardial fiber rupture. Hepatic lesions were observed as mild hepatic cord loss, increased vacuolation, nuclear condensation in hepatocytes, or even disappearance of hepatocytes after intravenous administration of free DOX/PTX or DOX + PTX. Furthermore, a large amount of red blood Imipenem appeared in the splenic pulp. Renal tubular epithelial cells were vacuolated and dilated. Atrophy in the glomerulus was observed. The group of free PTX displayed slight inflammation in the lung compared with the other groups. By contrast, damages to the heart, liver, spleen, lung, and kidney were remarkably reduced in the groups of [email protected], [email protected], and [email protected]&PTX. In addition, body weight change was also considered as indicator of systemic toxicity. As shown in Fig. 7C, the body weight of HepG2 subcutaneous tumor-bearing nude mice treated with saline (blank control), [email protected], [email protected], and [email protected]&PTX showed continuous and gradual increase. By contrast, mice treated with free DOX/PTX or DOX + PTX exhibited drastic loss in body weight. In addition, as shown in Fig. S17B, the FA-CM treatment also had no significant effect on the loss body weight of mice com-pared with the control group of saline. Therefore, all these results demonstrated cystic fibrosis FA-CM is biosafe and could reduce the systemic toxicity of free drugs to several organs, which is capable of deliver-ing anticancer drugs in vivo.
Fig. 8. The antitumor effect of [email protected]&PTX on HepG2 tumor bearing BALB/C nude mice compared with the other intravenous administrations: (A) tumor volume, (B) tumor weight, (C) tumor morphology, (D) histology of tumor and (E) TUNEL assay. Scale bar: 50 lm. *p < 0.05.
3.7. In vivo anti-tumor efficacy in HepG2 subcutaneous bearing nude mice
In vivo antitumor activity of [email protected]&PTX against HepG2 subcutaneous tumors was further examined. HepG2 subcutaneous bearing nude mice were randomized into seven treatment groups, namely, saline, free DOX, [email protected], free PTX, [email protected], DOX + PTX, and [email protected]&PTX. Different formulations of drugs were intravenously injected into the HepG2 subcutaneous tumor-bearing nude mice. The nude mice were sacrificed two days after the final intravenous administration. The tumors were harvested, weighted and photographed. The growth volume ratios of tumor and tumor weight were calculated. As shown in Fig. 8A, [email protected]&PTX exhibited remarkably higher inhibition effect toward tumor growth compared with DOX + PTX and better antitu-mor growth curve compared with [email protected] and [email protected]. These results demonstrated the synergistic effect of combination therapy based on [email protected]&PTX. Meanwhile, [email protected] and [email protected] exhibited enhanced inhibition effect toward tumor growth compared with free DOX and free PTX, respectively, because of the targeted drug delivery of FA-CM. In addition, the mice treated with [email protected]&PTX showed dramatically reduced tumor weight and tumor size compared with other groups (Fig. 8B and C). These results again confirm the synergistically enhanced effect of [email protected]&PTX on suppressing tumor growth. More-over, the tumor weight and tumor size of the mice treated with [email protected] or FA-[email protected] were lower than those from free DOX and free PTX.