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  • br Scheme Synthesis route of MSN SS KLA A


    Scheme 2. Synthesis route of MSN-SS-KLA (A) and the structure of KLA (B).
    respectively. In theory, there should be no KLA released from MSN-SS-KLA in the absence of DTT, but released KLA was detected in our ex-periments under this condition. The reason may be that a little KLA-FITC is adsorbed on the surface of MSN-SS-KLA instead of covalently bonding to MSN. This part of KLA-FITC is hard to be removed by washing but can be slowly released during drug release process. These results prove that release of KLA from MSN-SS-KLA could be achieved under reductive conditions, such as Puromycin environment.
    As already stated, KLA can be released by stimuli of reductive agent, accompanied by de-shielding of the BSA layer. This would result in the uncapping of pores of MSN and release of DOX. Thus, we tried to in-vestigate DOX release profiles of [email protected]/BSA under re-ductive environment. Also, we used DTT as substitute of glutathione to provide the reductive environment. As shown in Fig. 4, at the begin-ning, the DOX release rate of [email protected]/BSA in presence of DTT is significantly faster than that without DTT. And the drug release rate increases with DTT concentration increases. These results are consistent with those provided in Fig. 3. Because release rate of KLA increases, the caps of pores open more quickly which result in the in-crease of release rate of DOX. At 144 h, about 9.9%, 56.0% and 83.3% 
    of DOX was released at mM DTT, 0.5 mM DTT and 5 mM DTT, re-spectively. Theoretically, the pores of MSN are completely uncapped at high concentration of DTT, and the drug should be completely released. However, about 83.3% of the total drugs was released actually. This may be ascribed to the adsorption of a small part of drugs on BSA by electrostatic interactions. As BSA was trapped in the dialysis bags, those part of DOX absorbed on BSA did not penetrate through the bags and was not detected in the release experiments. Previous study have re-ported similar results. Cheng et al. designed and constructed a tumor-targeted and enzyme-induced drug-delivery system based on multi-functional MSN ([email protected]/α-CD) [43]. In the presence of cathepsin B, about 80% of the loaded DOX has been released from the [email protected]/α-CD nanoparticles incubated in pH 7.4 PBS buffer for 60 h, which is also attributed to the absorbtion of DOX on the protein.
    3.3. Enzyme-induced drug release
    In this study, BSA was used as the outer layer of MSN based drug carrier, thus we tried to study Puromycin the effect of enzyme on drug release
    kinetics. As reported, trypsin is capable of degrading BSA [44]. In vitro release experiments with different concentrations of trypsin were car-ried out. From Fig. 5, it is found out that in the first few hours, the rate of drug release from the [email protected]/BSA with trypsin is sig-nificantly faster than that without trypsin. About 10.0%, 46.1% and 51.1% of DOX were released at trypsin concentration of U, 30 U and 60 U during observing time. Due to the high activity of trypsin, there is no obviously difference between the drug release behavior at 30 U and that at 60 U trypsin. The results indicate that the degradation of BSA coating on the surface of [email protected]/BSA could accelerate the release of DOX. Thus, we can conclude that a dual-sensitive drug- 
    Fig. 3. Release profiles of KLA from MSN-SS-KLA under different conditions.
    delivery system ([email protected]/BSA) which could achieve ex-pected off-on release behavior has been constructed, and holds great potential for antitumor application as smart carriers.
    3.4. Cellular uptake and intracellular drug release
    Smart drug carriers need to enter into the tumor cells to perform their function. Thus the cellular uptake of [email protected]/BSA by cells was evaluated. HeLa cells were chosen as model cell lines and incubated with [email protected]/BSA. The detailed cellular uptake of [email protected]/BSA nanoparticles is exhibited in Fig. 6. The [email protected]/BSA nanoparticles could be internalized by HeLa cells and release the loaded DOX inside the cells, proved by the red fluorescence which is attributed to DOX (Fig. 6A2–A3). To further