br the source of cancer cell lysate
the source of cancer cell lysate and cancer cell membrane (Fig. 2C). The zeta potential of DOX/[email protected] was determined to be -9.6 mV. After further cloaked with the negative charged cell membrane, the zeta potential of these nanoparticles further decreased to -19.8 mV (Figure S3). Furthermore, to demonstrate the enhanced colloid stability of na-noparticles after CM cloaking, the hydrodynamic sizes of DOX/ [email protected] and DOX/[email protected]@CM in water or mimic physio-logical environment (PBS + 10% FBS) were measured by dynamic light scanning (DLS). As shown in Fig. 2D and E, the size of DOX/ [email protected] dramatically increased from 130.6 nm (in water) to 942.1 nm (in PBS + 10% FBS), while the diameter of DOX/[email protected]@CM only slightly increased from 154.1 nm to 160.1 nm. Mean-while, no significant aggregation or obvious size alteration of DOX/ [email protected]@CM was observed during storage in RPMI-1640 medium even for 8 days (Figure S4). These results indicated that DOX/ [email protected]@CM have good stability for biological application.
To evaluate pH-triggered drug release of DOX/[email protected]@CM, the cumulative release profiles were recorded under various conditions of pH 7.4, 6.5, and 5.0, which mimicked physiological condition, tumor microenvironment and intracellular endosome/lysosomes, respectively (Fig. 3) [35,36]. At pH 7.4, only a slight amount of DOX released from DOX/[email protected]@CMsystem even after incubation for 48 h, with a drug release percentage of 2.7%, which is much lower than DOX/ [email protected] with the release percentage of 14.3%, suggesting that CaCO3 could efficiently prevent the premature release in physiological condi-tion. At pH 6.5, the released amount of DOX from DOX/[email protected]@CM dramatically increased to above 20% only 8 h of incubation. However, the highest drug release rate was observed at pH 5.0 and near 70% of encapsulated drug was found to release, which is much higher than that at pH 7.4 and pH 6.5. The good pH sensitivity of our system
might be ascribed to elaborate structure which involve two steps for controlled drug release: 1) CaCO3 detachment from MSNs by acid-in-duced dissolution in aqueous solution ; 2) the excess H+ in acidic solution will make the interaction of DOX and MSNs weaker through the competitive adsorption [38,39].
3.3. Cell uptake and intracellular drug release
To verify the internalization and intracellular drug release of DOX/ [email protected]@CM, CLSM was used to track the DOX red fluorescence in the WAY316606 with the nuclei exhibiting blue fluorescence after stained by Hoechst 33342 (Fig. 4). After 2 h of incubation, a clear DOX signal was found mainly in the cytoplasm. However, with the time increased to 8 h, the red fluorescence became dominant in the nuclei rather than in
the cytoplasm. Furthermore, we compare the cellular uptake of DOX/ [email protected]@CM in LNCaP-AI cells and MCF-7 cells to demonstrate their homologous targeting properties. As shown in Figure S5, the fluorescence intensity of DOX/[email protected]@CM in the corresponding source cells of LNCaP-AI was much higher than those in the heterotypic cells of MCF-7. These results suggested the highly specific self-re-cognition aﬃnity of DOX/[email protected]@CM to the source cells.
Considering nanoparticles with diameter of 100–200 nm usually enter into the cells by endocytosis to form intracellular acidic endosome in cytoplasm, and that free DOX easily intercalation with DNA back-bone in the nuclei for exerting cell inhibition eﬀect [40,41], thus the red fluorescence in the nuclei might be resulted from the released DOX rather than from the DOX/[email protected]@CM itself. To further
demonstrate the intracellular drug release can be regulated by the en-dosome/lysosome acidic environment, the endosome/lysosome pH was neutralized by NH4Cl (50 μM) for 45 min at 37 °C, and then the cells were incubated with DOX/[email protected]@CM for 8 h, while the cells without NH4Cl pretreatment were used as a control. As expected, the drug release in the cells pretreated with NH4Cl was significantly in-hibited (Figure S6). These results indicate that the CaCO3 cap can be rapidly dissociated from MSNs under endosome/lysosome acidic en-vironment for triggering drug release and subsequent nucleic accumu-lation.
3.4. Cytotoxicity of [email protected]@CM and DOX/[email protected]@CM
Before investigating the anti-cancer eﬀect of DOX/ [email protected]@CM, the human normal liver cell line QSG-7701 cells were used to assess their cytotoxicity. As shown in Figure S7, the cell viabilities still remained over 90% at the equivalent DOX concentra-tions up to 16 μg/mL for 24 h or 48 h of incubation, indicating low cytotoxicity of our drug vehicles ([email protected]@CM). Furthermore, the hemolysis test of [email protected]@CM was also carried out and the results were depicted in Figure S8. [email protected]@CM couldn’t damage erythrocyte cells, demonstrating their good blood compatibility. Next, the cytotoxicity of DOX/[email protected]@CM at diﬀerent DOX con-centrations and the corresponding amount of blank nanovehicles of [email protected]@CM towards LNCaP-AI cells were investigated by CCK8 assay. As shown in Fig. 5A, free DOX and DOX/[email protected]@CM as chemotherapeutic agents displayed a dose- and time-dependent cyto-toxicity to prostate cancer cells. The cell viability decreased sig-nificantly with the DOX concentration above 2 μg/mL, especially after incubation for 48 h. Moreover, the cytotoxicity of DOX/[email protected]@CM was even higher than free DOX with DOX concentration in-creased to 8 μg/mL or 16 μg/mL. In the case of drug-free vehicle of [email protected]@CM, the cell viability of all corresponding DOX amount was above 90% after 24 h or 48 h. Therefore, [email protected]@CM has great potency as a biocompatible, pH-responsive drug delivery vehicle for anticancer therapy.