br PTX and FA CM DOX PTX and
+ PTX, and [email protected]&PTX, and the treatment of saline were served as vehicle controls. The major organs, including the heart, liver, spleen, lung, kidney, and HepG2 subcutaneous tumor were collected and checked in vivo fluorescence of DOX by using a Per-kinElmer IVIS instrument with a 465 nm excitation wavelength and a 600 nm emission wavelength.
2.13. Histology analysis
The tumor tissues in HepG2 tumor bearing BALB/C nude mice were randomized into seven groups each consisting of five ani-mals. The bearing mice were intravenously injected with saline (vehicle control), DOX, [email protected], PTX, [email protected], DOX
+ PTX, and [email protected]&PTX with a DOX dose of 4.2 mg/kg bw and a PTX dose of 1.4 mg/kg bw twice a week for a consecutive 4-week. The mice were euthanized, and the tumors, hearts, livers, spleens, lungs, and kidneys were collected and fixed in Bouin’s fix-ative buffer for <12 h, and then embedded in paraffin and sectioned at 7 lm. Sections were deparaffinized with xylene, rehydrated with degrading alcohol 100%, 95%, and 70%, and rinsed in water. The sections were subjected to hematoxylin and eosin (H&E) stain-ing. After dehydrating, the sections were mounted on a coverslip with neutral gum. Paraffin sections of tumor, cardiac, liver, kidney, and lung were visualized under the light microscopy.
2.14. TdT-Mediated dUTP Nick-End labeling (TUNEL) staining
Apoptotic Angiotensin I in tumor tissue of HepG2 tumor bearing BALB/C nude mice after intravenous administration of drugs were detected using TUNEL staining. Briefly, the 5 lm thick paraffin-embedded tumor tissue sections were subjected to a TUNEL Bright Red Apop-tosis Detection Kit (Vazyme Biotech, Nanjing, China) according to the manufacturer’s protocol. The sections were visualized on a flu-orescent microscope.
2.15. Statistical analyses
Data are presented as the mean ± SEM. Statistical values were defined using unpaired Student’s t-test, with p < 0.05 considered to be statistically significant.
3. Results and discussion
3.1. Synthesis and characterization of FA-CM
CoAl-LDHs, MnO2, CM, FA-CM and drug-loaded FA-CM ([email protected], DOX was chosen as a model here) were fabricated step-by-step and their physical/chemical properties were charac-terized in detail. Electron microscopy provides visualized informa-tion. As shown in Fig. 1, CoAl-LDHs nanosheets exhibited nearly hexagonal platelets with a lateral size, which is the typical mor-phology of LDHs samples . MnO2 nanosheets showed the mor-phology of two-dimensional ultrathin planes with an average diameter of approximately 100 nm . SEM and TEM images of CM confirmed the overlapping of the CoAl-LDHs and MnO2 nanosheets. DLS revealed that the average hydrodynamic size of CM was about 130 nm (Fig. S1 in the Supporting Information). Faint self-assembly happened between CM after the modification of FA, because of the change in surface charge. This result is consis-tent with previous report . The loading of DOX almost dis-played no changes on the morphology of CM (Fig. S2). EDX analysis further showed the presence of elemental Co, Al, and Mn signals, as can be seen in Fig. S3. Elemental mapping in the scan-ning TEM mode (Fig. 1I) confirmed a homogeneous distribution of CoAl-LDHs and MnO2 phase throughout the hierarchical structure.
Further details were determined using XRD analysis, FT-IR spec-troscopy, XPS spectra and zeta potential. As shown in Fig. 2A, all diffraction peaks corresponded to the pure hydrotalcite structure (JCPDS: 38-0487) with characteristics peaks of (0 0 3), (0 0 6),
(0 1 2), (0 1 5), and (0 1 8) planes . The interlayer spacing between the CoAl-LDHs nanosheets was calculated to be 0.75 nm (d003), which is identical with the equivalent reflection in a pristine CO3LDHs sample . The MnO2 nanosheets showed an obvious layered structure with a basal spacing of 0.72 nm, which is agreed well with the previous reports . Through LBL, the pattern of CM gave characteristic peaks of both CoAl-LDHs and MnO2, indicating that the stacked nanosheets of CoAl-LDHs and MnO2 were almost delaminated and rearranged to yield CM. In addition, the interlayer spacing of CM was calculated to be 0.73 nm. This result also demonstrated the rearrangement of CoAl-LDHs and MnO2 nanosheets. However, a new peak appeared at approximately 2h = 17.9L with a basal spacing of 0.49 nm. This peak was most likely related to the scattering from un-rearranged dispersed single nanosheets of CoAl-LDHs or MnO2 . After the modification of FA, the XRD peak of CM was barely identifiable, and the interlayer spacing was decreased because the faint self-assembly happened between CM nanosheets. This finding was also in line with the SEM and TEM results (Fig. 1). After the loading of DOX, the corre-sponding basal spacing of FA-CM slightly expanded from 0.33– 0.42 nm to 0.34–0.43 nm, indicating the intercalation of DOX . FT-IR spectroscopy was conducted to characterize the chem-ical bonds and surface organic groups in each sample (Fig. 2B). The spectrum of bare CoAl-LDHs showed a broad band (3100– 3700 cm 1) from the O–H stretch of the absorbed molecule H2O . The band at 1633 cm 1 was presumably caused by H2O defor-mation . The band at 1343 cm 1 was due to the m3 vibration and the bending modes of CO23 . Those bands at 792, 687, and 550 cm 1 were attributed to the vibration of (M–O) and (M– O–M) (M = Co or Al) . In the MnO2 sample, the vibration at 1046 cm 1 was ascribed to the band of Mn3+–O . Through LBL, the CM sample showed the characteristic bands of CoAl-LDHs and MnO2. Compared with that of CM, additional absorption bands at 1694 cm 1 and 1608 cm 1 were observed after FA modification, and these bands were assigned to the symmetrical ms([email protected]) and asymmetric mas([email protected]) vibration of FA respectively