br We also performed a Live Dead detection
We also performed a Live/Dead detection to intuitively observe the cytotoxic eﬀect of DOX/[email protected]@CM on cancer cells. Dead 5Azacytidine were stained with PI (red fluorescence) while living cells were stained by Calcein-AM (green fluorescence). As shown in Fig. 6, the proportion of live cells decreased gradually with the increase of nanoparticles concentration. Therefore, the minimal number of the living cells (green fluorescence) along with the maximal dead cells (red fluorescence) was observed at the DOX concentration of 16 μg/mL. Meanwhile, the green and red fluorescence intensities in Fig. 6 were also quantified by ImageJ
software, and the result was shown in Figure S9. With the DOX/ [email protected]@CM concentration increased from μg/mL to 16 μg/mL, the PI red fluorescence intensity gradually increased from 1157 to 11073, while the Calcein-AM green fluorescence intensity decreased from 80,575 to 16,923. This result is consistent with that obtained from CCK8 assay, indicating the good anticancer eﬃcacy of our DOX/ [email protected]@CM.
3.5. Pro-apoptosis eﬀect of DOX/[email protected]@CM
To determine whether cell apoptosis contribute to the inhibitory eﬀect of DOX/[email protected]@CM toward cancer cells, we performed an Annexin V-FITC/PI assay to analysis the cells incubated with DOX/ [email protected]@CM at diﬀerent concentration for 48 h. The result was shown in Fig. 7, apoptotic ratio increased significantly with drug con-centration. It was worth noting that the proportion of apoptosis in-creased from 10.93% to 31.52% when DOX concentration increased from 2 μg/mL to 4 μg/mL. Most significantly, the total percentages of late apoptotic cells (26.6%) and necrotic cells (41.0%) at the DOX concentration of 16 μg/mL, were much higher than the cells incubated with DOX/[email protected]@CM at lower concentrations. The result sug-gested that DOX/[email protected]@CM treatment significantly induced cell apoptosis of LNCaP-AI cells. r> 3.6. In vivo antitumor eﬀect of DOX/[email protected]@CM
Encouraged by the good anticancer eﬀect in vitro, we next evaluated the antitumor eﬀect of DOX/[email protected]@CM on LNCaP-AI tumor-bearing nude mice in vivo. To this end, the mice divided into four groups were intravenously injected with PBS (group i), [email protected]@CM (group ii), DOX/[email protected]@CM (group iii), and free DOX (group iv) at the equivalent DOX dosage of 5 mg/kg. As shown in Fig. 8A, the mice received with [email protected]@CM treatment (group ii) showed nearly the same rapid tumor growth in comparison with
Fig. 6. Live/Dead cell analysis. LNCaP-AI cells treated with DOX/[email protected] at diﬀerent DOX concentrations ranged from μg/mL to 16 μg/mL for 48 h.
control group (group i), demonstrating the negligible eﬀect on delaying tumor growth. However, the tumor progression could be significantly inhibited after DOX/[email protected]@CM administration (group iii), and the tumor suppression in this group is even better than free DOX group (group iv). At the end of treatment at day 14, the tumors were resected and weighed (Fig. 8B). The average tumor weight in group iii sig-nificantly reduced by 71% as compared with the control group, which was much higher than the data in other groups (group ii: 4%; group iv: 38%). To further confirm the therapeutic eﬃcacy, the dissectedtumor tissues from diﬀerent groups were analyzed by hematoxylin and eosin
(H&E) staining (Fig. 8C), immunohistochemicalstaining for antigen Ki67 (Fig. 8D). From the pathological images of tumor tissues with H&E staining, a large part of nucleus shrinkage and necrosis representing cell destrction were found after the treatment with DOX/[email protected]@CM (group iii), which is much higher than in other groups. As for Ki67 staining, the nucleus of proliferative cells with positive staining as brown granules in this group was much less than other groups. The in vivo antitumor activity is well consistent with the in vitro cytotoxicity, confirming the eﬀectiveness of the DOX/[email protected]@CM.
Although cancer cell membranes can enhance the biocompatibility,
the MSN itself used in our work is non-degradable, which might be a serious concern for clinical usage. Therefore, based on the present na-nosystem, further incorporation with other biodegradable nanopaticle, such as mesoporous organo-silica nanoparticles [42,43], will be more valuable for cancer treatment.
In conclusion, we have successfully constructed a biocompatible, simple structured and tumor acidic environment responsive drug de-livery system by successive capping the CaCO3 and cloaking cancer cell membrane on mesoporous silica nanoparticles (DOX/MSN/ [email protected]). The synthesized MSNs are monodisperse nanoparticles with a size of about 100 nm, and the modification of the surface does not aﬀect the morphology of nanoparticles. The in vitro investigations demonstrated that the drug release from the obtained DOX/MSN/ [email protected] was confined by CaCO3 layer, but would be triggered by tumor microenvironment and intracellular endosome/lysosomes for delivering DOX into cancer cells like LNCaP-AI cells, and ultimately induced cell death. Moreover, DOX/MSN/[email protected] also showed a favorable anti-tumor eﬀect in the LNCaP-AI tumor model, evidenced by the significant tumor growth delay, tumor cells destruction, as well as reduced tumor cells proliferation. Overall, this study provides a pro-mising alternative as acid responsive drug delivery for eﬃcient cancer therapy.